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1
First, NL, MM Sims, SP Park, and MJ Kent-First. "Systems for production of calves from cultured bovine embryonic cells." Reproduction, Fertility and Development 6, no. 5 (1994): 553. http://dx.doi.org/10.1071/rd9940553.
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The development of totipotent bovine embryonic cell cultures has great value in cattle breeding. They provide: (1) a mechanism for making large numbers of clonal offspring by nuclear transfer; (2) an efficient gene transfer system through the use of selectable markers to select transgenic cells; and (3) a mechanism for site-specific gene transfer or deletion by homologous DNA sequence recombination. Bovine embryonic cell cultures have been established from blastocyst inner cell mass (ICM) cells, morulae and the precompaction 16-20-cell stage. All have exhibited similar morphology to mouse embryonic stem (ES) cells, pluripotency on differentiation and proliferation in culture. Culture systems have consisted of microdrop loose suspension short-term cultures or long-term cultures on bovine or murine fibroblast feeder layers, in either a microdrop or a culture dish. The relative merit of culture systems or media requirements for mitosis and prevention of differentiation have not been determined. At present, totipotency is also unknown for cultured cells of the 16-20-cell stage. For cultured ICM cells, totipotency was demonstrated by the birth of four calves from ICM cells cultured 27 days or less in a loose suspension microdrop. Advanced pluripotency and perhaps totipotency was demonstrated in one fetus in a recently reported study where morulae cells cultured in vitro were chimaerized with non-cultured cells. DNA fingerprinting to associate cell lines with offspring and karyotyping to ascertain chromatin normalcy is important in ES cell research. Data pertaining to the use of each are presented.
2
Huang, Xiaosong, L. Jeanne Pierce, Paul A. Cobine, Dennis R. Winge, and Gerald J. Spangrude. "Copper Modulates the Differentiation of Mouse Hematopoietic Progenitor Cells in Culture." Cell Transplantation 18, no. 8 (August 2009): 887–97. http://dx.doi.org/10.3727/096368909x471152.
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Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2 treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2 decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture.
3
Agius, L. "Metabolic interactions of parenchymal hepatocytes and dividing epithelial cells in co-culture." Biochemical Journal 252, no. 1 (May 15, 1988): 23–28. http://dx.doi.org/10.1042/bj2520023.
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When parenchymal hepatocytes isolated from adult liver are co-cultured with other epithelial cells, the production of various plasma proteins by the hepatocytes is preserved for much longer than in conventional culture. This study examines some of the metabolic interactions between parenchymal hepatocytes and epithelial cells maintained in co-culture. The leakage of lactate dehydrogenase by hepatocytes co-cultured with epithelial cells was lower than in conventional hepatocyte culture. The epithelial cells have a high glycolytic rate and provide the hepatocytes with a continual supply of lactate. The [lactate] was lower in co-cultures of hepatocytes and epithelial cells than in pure epithelial cultures of similar density, suggesting lactate clearance by the hepatocytes. Alanine uptake was higher in conventional hepatocyte cultures, which lack an exogenous supply of lactate, than in parenchymal hepatocytes in co-culture. Studies with pure parenchymal hepatocytes incubated with increasing [lactate] suggest that lactate is utilized in preference to alanine as a gluconeogenic substrate by hepatocytes co-cultured with epithelial cells. Ketogenesis and carnitine palmitoyltransferase activity declined more slowly in hepatocytes co-cultured with epithelial cells than in conventional culture. It is concluded that the co-culture model has potential for long-term studies of carbohydrate and lipid metabolism.
4
Sandstrom, CE, JG Bender, ET Papoutsakis, and WM Miller. "Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells." Blood 86, no. 3 (August 1, 1995): 958–70. http://dx.doi.org/10.1182/blood.v86.3.958.958.
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Abstract Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.
5
Sandstrom, CE, JG Bender, ET Papoutsakis, and WM Miller. "Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells." Blood 86, no. 3 (August 1, 1995): 958–70. http://dx.doi.org/10.1182/blood.v86.3.958.bloodjournal863958.
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Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.
6
Eswaramoorthy, Sindhuja D., Nandini Dhiman, Gayathri Korra, Carlo M. Oranges, Dirk J. Schaefer, Subha N. Rath, and Srinivas Madduri. "Isogenic-induced endothelial cells enhance osteogenic differentiation of mesenchymal stem cells on silk fibroin scaffold." Regenerative Medicine 14, no. 7 (July 2019): 647–61. http://dx.doi.org/10.2217/rme-2018-0166.
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Aim: We investigated the role of induced endothelial cells (iECs) in mesenchymal stem cells (MSCs)/iECs co-culture and assessed their osteogenic ability on silk fibroin nanofiber scaffolds. Methods: The osteogenic differentiation was assessed by the ALP assay, calcium assay and gene expression studies. Results: The osteogenic differentiation of the iECs co-cultures was found to be higher than the MSCs group and proximal to endothelial cells (ECs) co-cultures. Furthermore, the usage of isogenic iECs for co-culture increased the osteogenic and endothelial gene expression. Conclusion: These findings suggest that iECs mimic endothelial cells when co-cultured with MSCs and that one MSCs source can be used to give rise to both MSCs and iECs. The isogenic MSCs/iECs co-culture provides a new option for bone tissue engineering applications.
7
Stano, J., K. Mičieta, E. Tokhtaeva, M. Valšíková, M. Koreňová, and V. Blanáriková. "Demonstration of lactase activity in culture medium of melon cells." Horticultural Science 31, No. 4 (November 25, 2011): 132–35. http://dx.doi.org/10.17221/3806-hortsci.
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Lactase activity was detected in a culture medium of the cell suspension culture of watermelon (Citrullus vulgaris L.). A simple, rapid and reproducible procedure for identification of extracellular lactase is described using callus cultures of seedlings from the tested plant, hairy roots of 2.5 days old seedlings of watermelon germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of intracellular activities of lactase, 6-bromo-2-naphthyl-β-D-galactopyranoside and p-nitrophenyl-β-D-galactopyranoside were used as synthetic substrates. The extracellular lactase activity was determined by evaluating the day-zone in agar medium. The enzyme from watermelon callus cultures and seedling roots, cultivated on agar plates supplemented with 6-bromo-2-naphthyl-2-bromo-β-D-galactopyranoside, hydrolyzed this substrate releasing 6-bromo-naphthyl. By simultaneous coupling with hexazonium p-rosaniline or Fast Blue BB the corresponding azo dye was formed. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 43.8% intracellular and 54.2% extracellular distribution of lactase activity. The described agar plate method enables a rapid, simple and specific detection of plant processes of extracellular lactase.
8
Culp, D. J., and L. R. Latchney. "Mucinlike glycoproteins from cat tracheal gland cells in primary culture." American Journal of Physiology-Lung Cellular and Molecular Physiology 265, no. 3 (September 1, 1993): L260—L269. http://dx.doi.org/10.1152/ajplung.1993.265.3.l260.
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In a recent study (D. J. Culp, D. K. P. Lee, D. P. Penney, and M. G. Marin. Am. J. Physiol. 263: L264-275, 1992), we reported that primary cultures of cat tracheal gland cells expressed histological, ultrastructural, and immunological characteristics of mucous cells when cultured on floating gels of rat tail collagen (released-gel cultures) compared with cells cultured on glutaraldehyde-fixed collagen gels (fixed-gel cultures). We therefore collected culture medium from gland cells grown under both culture conditions for determination and comparison of glycoconjugates with characteristics of mucin glycoproteins. Cells were cultured in the presence of [3H]glucosamine, and material of high molecular weight and density (HMD material) was isolated. HMD material from both culture conditions were each resistant to heparitinase and heparinase, whereas 72 and 25% of the radiolabel in released-gel and fixed-gel HMD material, respectively, was resistant to chondroitinase ABC. Material resistant to chondroitinase ABC was analyzed further. Both samples contained a single broad glycoprotein band [relative molecular weight (M(r)) > 250,000] after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had amino acid profiles similar to airway mucin. The sample from fixed-gel cultures had nearly equal amounts of carbohydrate and protein, was highly enriched in N-acetylglucosamine, contained mannose, displayed little blood group A immunoreactivity, and had few O-linked oligosaccharides. Conversely, the sample from released-gel cultures contained 80% carbohydrate, was composed of monosaccharides characteristic of airway mucins, displayed blood group A immunoreactivity, and contained oligosaccharides O-linked via N-acetylgalactosamine.(ABSTRACT TRUNCATED AT 250 WORDS)
9
Park, Yong H., Joshua D. Snook, Iris Zhuang, Guofu Shen, and Benjamin J. Frankfort. "Optimized culture of retinal ganglion cells and amacrine cells from adult mice." PLOS ONE 15, no. 12 (December 7, 2020): e0242426. http://dx.doi.org/10.1371/journal.pone.0242426.
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Cell culture is widely utilized to study the cellular and molecular biology of different neuronal cell populations. Current techniques to study enriched neurons in vitro are primarily limited to embryonic/neonatal animals and induced pluripotent stem cells (iPSCs). Although the use of these cultures is valuable, the accessibility of purified primary adult neuronal cultures would allow for improved assessment of certain neurological diseases and pathways at the cellular level. Using a modified 7-step immunopanning technique to isolate for retinal ganglion cells (RGCs) and amacrine cells (ACs) from adult mouse retinas, we have successfully developed a model of neuronal culture that maintains for at least one week. Isolations of Thy1.2+ cells are enriched for RGCs, with the isolation cell yield being congruent to the theoretical yield of RGCs in a mouse retina. ACs of two different populations (CD15+ and CD57+) can also be isolated. The populations of these three adult neurons in culture are healthy, with neurite outgrowths in some cases greater than 500μm in length. Optimization of culture conditions for RGCs and CD15+ cells revealed that neuronal survival and the likelihood of neurite outgrowth respond inversely to different culture media. Serially diluted concentrations of puromycin decreased cultured adult RGCs in a dose-dependent manner, demonstrating the potential usefulness of these adult neuronal cultures in screening assays. This novel culture system can be used to model in vivo neuronal behaviors. Studies can now be expanded in conjunction with other methodologies to study the neurobiology of function, aging, and diseases.
10
Bowers, W. E., and M. R. Berkowitz. "Differentiation of dendritic cells in cultures of rat bone marrow cells." Journal of Experimental Medicine 163, no. 4 (April 1, 1986): 872–83. http://dx.doi.org/10.1084/jem.163.4.872.
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Although dendritic cells (DC) originate from bone marrow, they were not observed in fresh preparations of bone marrow cells (BMC). Likewise, accessory activity was barely measurable in a sensitive assay for this potent function of DC. However, both DC and accessory activity developed when BMC were cultured for 5 d. Based on fractionation before culture, nearly all of the accessory activity could be attributed to only 5% of the total BMC recovered in a low-density (LD) fraction. The LD-DC precursors differed from mature DC in a number of important respects. Removal of Ia+ cells from the LD fraction by panning did not decrease the production of DC when the nonadherent cells were cultured. Thus, the cell from which the DC is derived does not express or minimally expresses Ia antigens, in contrast to the strongly Ia+ DC that is produced in bone marrow cultures. Irradiation of LD cells before culture prevented the development of DC. When irradiation was delayed by daily intervals, progressive increases in the number of DC resulted, up to the fifth day. These findings, together with preliminary autoradiographic data, indicate that cell division has occurred, in contrast to the DC, which does not divide. We conclude that bone marrow-derived DC arise in culture from the division of LD, Ia- precursors.
11
Blennerhassett, M. G., M. S. Kannan, and R. E. Garfield. "Density-dependent hyperpolarization in cultured aortic smooth muscle cells." American Journal of Physiology-Cell Physiology 256, no. 3 (March 1, 1989): C644—C651. http://dx.doi.org/10.1152/ajpcell.1989.256.3.c644.
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The membrane potential (Em) of cultured aortic smooth muscle cells from Sprague-Dawley (SD), Wistar-Kyoto (WKY), and spontaneously hypertensive (SHR) rats was measured in proliferating primary cultures. Em of SD cells in high-density cultures was -51 to -58 mV, whereas that of low-density cultures (1-2 days) was -30 mV. This difference was due to a continuous process of hyperpolarization during proliferation in culture. Em of WKY and SHR hyperpolarized similarly, from -12 to -42 and -38 mV, respectively. Hyperpolarization of Em of SD, WKY, and SHR cells was related to cell density rather than time in culture. Em may be a sensitive and significant indicator of the changes in the differentiated state expressed by proliferating smooth muscle in vitro.
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Li, Cheng. "Potentiation of Bio Repositories In Personalized Medicine: Tumor Cells Establishment." Cancer Research and Cellular Therapeutics 1, no. 1 (December 8, 2017): 01–03. http://dx.doi.org/10.31579/2640-1053/003.
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The introduction of three-dimensional (3D) tumor cultures has revolutionized anticancer drug research as these cultures allow for the study of drug resistance mechanisms that cannot be explored in traditional two dimensional (2D) monolayer cultures. Discoveries in the 3D tumor culture field suggest that individualized drug sensitivity testing of solid tumor specimens through the establishment and use of 3D tumor cell cultures following tissue collection will become a routine service offered by modern tissue repositories as they expand from their traditional research role to active participation in personalized medicine. Unfortunately, most information related to 3D tumor cultures comes from studies using established tumor cell lines rather than primary tumor cultures. However, accumulation of genetic aberrations in cancer cell lines occurs with increasing number of passages severely limiting their usefulness for personalized medicine. There is only very limited information available concerning technologies and standard operating procedures for the efficient and routine isolation and processing of primary tumor cells for the establishment of 3D tumor cultures from solid tumor specimens. The purpose of this work was to review experimental data from the literature that may provide relevant information concerning the isolation and processing of primary tumor cells for the establishment of 3D tumor cultures. Information reviewed here may help bio repositories in the development and standardization of technologies and standard operating procedures related to the use of 3D tumor cultures.
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Skottman, Heli, and Outi Hovatta. "Culture conditions for human embryonic stem cells." Reproduction 132, no. 5 (November 2006): 691–98. http://dx.doi.org/10.1530/rep.1.01079.
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Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.
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Engel, F. E., A. G. Khare, and B. D. Boyan. "Phenotypic Changes of Rabbit Mandibular Condylar Cartilage Cells in Culture." Journal of Dental Research 69, no. 11 (November 1990): 1753–58. http://dx.doi.org/10.1177/00220345900690110801.
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The present study describes the behavior of mandibular condylar cartilage (MCC) cells as a function of time in primary culture, since it is not yet clear whether these cells maintain their phenotype in culture. MCC cells from New Zealand white rabbits were seeded at high density and cultured in DMEM containing 50 μg/mL ascorbic acid and 10% fetal bovine serum. These cells appeared as a heterogeneous population and changed their shape, size, and refractivity as cultures aged. Cartilage-like cells, which always dominated the culture, were infiltrated with a minority of fibroblast-like cells. Cell number increased progressively, and cultures reached confluence at nine days. Antibody activity for cartilage-specific glycosaminoglycan was determined by ELISA assay. This reaction reached a maximum at six days and decreased thereafter. Cultures stained with Alcian blue (pH 1.0) supported these results. Cytoplasmic mRNA analysis indicated that the transcription of type II collagen gene was present at all time points. Type I collagen and alkaline phosphatase mRNA levels showed progressive increases from 12 h to nine days, with significantly higher values in cells cultured for six, nine, and 12 days than in cells collected from earlier time points. These results suggest that in our present culture system, MCC cells undergo phenotypic changes that resemble their maturation processes in vivo.
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Brenneman, D. E., E. A. Neale, G. A. Foster, S. W. d'Autremont, and G. L. Westbrook. "Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide." Journal of Cell Biology 104, no. 6 (June 1, 1987): 1603–10. http://dx.doi.org/10.1083/jcb.104.6.1603.
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The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.
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Fu, Weili, Xing Xie, Qi Li, Gang Chen, Chenghao Zhang, Xin Tang, and Jian Li. "Isolation, Characterization, and Multipotent Differentiation of Mesenchymal Stem Cells Derived from Meniscal Debris." Stem Cells International 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/5093725.
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This study aimed to culture and characterize mesenchymal stem cells derived from meniscal debris. Cells in meniscal debris from patients with meniscal injury were isolated by enzymatic digestion, cultured in vitro to the third passage, and analyzed by light microscopy to observe morphology and growth. Third-passage cultures were also analyzed for immunophenotype and ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. After 4-5 days in culture, cells showed a long fusiform shape and adhered to the plastic walls. After 10–12 days, cell clusters and colonies were observed. Third-passage cells showed uniform morphology and good proliferation. They expressed CD44, CD90, and CD105 but were negative for CD34 and CD45. Cultures induced to differentiate via osteogenesis became positive for Alizarin Red staining as well as alkaline phosphatase activity. Cultures induced to undergo adipogenesis were positive for Oil Red O staining. Cultures induced to undergo chondrogenesis were positive for staining with Toluidine Blue, Alcian Blue, and type II collagen immunohistochemistry, indicating cartilage-specific matrix. These results indicate that the cells we cultured from meniscal debris are mesenchymal stem cells capable of differentiating along three lineages. These stem cells may be valuable source for meniscal regeneration.
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Orlidge, A., and P. A. D'Amore. "Inhibition of capillary endothelial cell growth by pericytes and smooth muscle cells." Journal of Cell Biology 105, no. 3 (September 1, 1987): 1455–62. http://dx.doi.org/10.1083/jcb.105.3.1455.
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Morphological studies of developing capillaries and observations of alterations in capillaries associated with pathologic neovascularization indicate that pericytes may act as suppressors of endothelial cell (EC) growth. We have developed systems that enable us to investigate this possibility in vitro. Two models were used: a co-culture system that allowed direct contact between pericytes and ECs and a co-culture system that prevented physical contact but allowed diffusion of soluble factors. For these studies, co-cultures were established between bovine capillary ECs and the following growth-arrested cells (hereafter referred to as modulating cells): pericytes, smooth muscle cells (SMCs), fibroblasts, epithelial cells, and 3T3 cells. The modulating cell type was growth arrested by treatment with mitomycin C before co-culture with ECs. In experiments where cells were co-cultured directly, the effect of co-culture on EC growth was determined by comparing the mean number of cells in the co-cultures to the mean for each cell type (EC and modulating cell) cultured separately. Since pericytes and other modulating cells were growth arrested, any cell number change in co-cultures was due to EC growth. In the co-cultures, pericytes inhibited all EC proliferation throughout the 14-d time course; similar levels of EC inhibition were observed in SMC-EC co-cultures. Co-culture of ECs with fibroblasts, epithelial cells, and 3T3 cells significantly stimulated EC growth over the same time course (30-192% as compared to EC cultured alone). To determine if cell contact was required for inhibition, cells were co-cultured using Millicell chambers (Millipore Corp., Bedford, MA), which separated the cell types by 1-2 mm but allowed the exchange of diffusible materials. There was no inhibition of EC proliferation by pericytes or SMCs in this co-culture system. The influence of the cell ratios on observed inhibition was assessed by co-culturing the cells at EC/pericyte ratios of 1:1, 2:1, 5:1, 10:1, and 20:1. Comparable levels of EC inhibition were observed at ratios from 1:1 to 10:1. When the cells were co-cultured at a ratio of 20 ECs to 1 pericyte, inhibition of EC growth at 3 d was similar to that observed at other ratios. However, at higher ratios, the inhibition diminished so that by the end of the time course the co-cultured ECs were growing at the same rate as the controls. These results suggest that pericytes and SMCs can modulate EC growth by a mechanism that requires contact or proximity. We postulate that similar interactions may operate to modulate vascular growth in vivo.
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Verfaillie, CM, and JS Miller. "CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells." Blood 84, no. 5 (September 1, 1994): 1442–49. http://dx.doi.org/10.1182/blood.v84.5.1442.1442.
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Abstract Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.
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Verfaillie, CM, and JS Miller. "CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells." Blood 84, no. 5 (September 1, 1994): 1442–49. http://dx.doi.org/10.1182/blood.v84.5.1442.bloodjournal8451442.
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Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.
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Honda, B. D., and N. T. Glanville. "Glucose oxidation by adherent and mechanically detached cultured cells." Biochemistry and Cell Biology 69, no. 10-11 (October 1, 1991): 728–30. http://dx.doi.org/10.1139/o91-109.
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This study examined the effect of mechanical detachment from the growth surface on energy metabolism of cultured cells. Oxidation of [1-14C]glucose measured by production of 14CO2 by adherent neuroblastoma (123 ± 5 nmol/mg protein per minute), glioma (128 ± 10 nmol/mg protein per minute), and fibroblast (137 ± 5 nmol/mg protein per minute) cultures was similar. Removing cells from the culture flask by scraping reduced glucose oxidation by 62, 30, and 82% in neuroblastoma, glioma, and fibroblast cultures, respectively. Transferring cells from a culture flask to a test tube, to control for diffusional surface area, did not further reduce glucose oxidation. Detaching cells from the growth surface destroyed the extensive process formation and disrupted the normal spatial organization on the culture plate. These results indicate that it is essential to maintain these aspects of cellular architecture when evaluating metabolic properties of cultured cells.Key words: glucose oxidation, neuroblastoma, glioma, fibroblasts.
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Guo, W., K. Kamiya, J. Cheng, and J. Toyama. "Changes in action potentials and ion currents in long-term cultured neonatal rat ventricular cells." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C93—C102. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c93.
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A primary culture of neonatal ventricular myocytes isolated from day-old rats was established for investigating the changes in action potentials and ion currents over long periods. Cells at days 5 and 15 in culture were studied. These changes in vitro were compared with those in situ derived from the age-matched freshly isolated cells. During primary culture, quiescent cells demonstrated shortening of action potential durations (APD) resembling the developmental changes observed in situ. The beating cultured cells were not associated with APD shortening. Despite constant current amplitudes, the densities of Ca2+ currents (ICa) decreased in the quiescent cultures at later ages as a result of cell enlargement. ICa densities were maintained in the beating cultured and freshly isolated cells. Acceleration in the inactivation of ICa was observed during developments both in vitro and in situ. In addition, the densities of transient outward currents (Ito) tripled and doubled in the quiescent and beating cells during 15-day cultures. However, Ito in beating cultured cells made less contribution to APD in contrast to the quiescent cultured and freshly isolated myocytes. These findings demonstrate that electrophysiological properties differ between two types of long-term cultured cells. ICa densities remained constant in the beating cultures, suggesting that cell beating may be required for the maintenance of ICa density in developing cardiomyocytes.
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Abaci, Hasan Erbil, Rachel Truitt, Eli Luong, German Drazer, and Sharon Gerecht. "Adaptation to oxygen deprivation in cultures of human pluripotent stem cells, endothelial progenitor cells, and umbilical vein endothelial cells." American Journal of Physiology-Cell Physiology 298, no. 6 (June 2010): C1527—C1537. http://dx.doi.org/10.1152/ajpcell.00484.2009.
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Hypoxia plays an important role in vascular development through hypoxia-inducible factor-1α (HIF-1α) accumulation and downstream pathway activation. We sought to explore the in vitro response of cultures of human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), human endothelial progenitor cells (hEPCs), and human umbilical cord vein endothelial cells (HUVECs) to normoxic and hypoxic oxygen tensions. We first measured dissolved oxygen (DO) in the media of adherent cultures in atmospheric (21% O2), physiological (5% O2), and hypoxic oxygen conditions (1% O2). In cultures of both hEPCs and HUVECs, lower oxygen consumption was observed when cultured in 1% O2. At each oxygen tension, feeder-free cultured hESCs and iPSCs were found to consume comparable amounts of oxygen. Transport analysis revealed that the oxygen uptake rate (OUR) of hESCs and iPSCs decreased distinctly as DO availability decreased, whereas the OUR of all cell types was found to be low when cultured in 1% O2, demonstrating cell adaptation to lower oxygen tensions by limiting oxygen consumption. Next, we examined HIF-1α accumulation and the expression of target genes, including VEGF and angiopoietins ( ANGPT; angiogenic response), GLUT-1 (glucose transport), BNIP3, and BNIP3L (autophagy and apoptosis). Accumulations of HIF-1α were detected in all four cell lines cultured in 1% O2. Corresponding upregulation of VEGF, ANGPT2, and GLUT-1 was observed in response to HIF-1α accumulation, whereas upregulation of ANGPT1 was detected only in hESCs and iPSCs. Upregulation of BNIP3 and BNIP3L was detected in all cells after 24-h culture in hypoxic conditions, whereas apoptosis was not detectable using flow cytometry analysis, suggesting that BNIP3 and BNIP3L can lead to cell autophagy rather than apoptosis. These results demonstrate adaptation of all cell types to hypoxia but different cellular responses, suggesting that continuous measurements and control over oxygen environments will enable us to guide cellular responses.
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Dasch, J. R., and P. P. Jones. "Independent regulation of IgM, IgD, and Ia antigen expression in cultured immature B lymphocytes." Journal of Experimental Medicine 163, no. 4 (April 1, 1986): 938–51. http://dx.doi.org/10.1084/jem.163.4.938.
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Long-term cultured bone marrow cells were characterized with respect to a number of B and pre-B cell markers. Cells expressing ThB, B-220, and IgM were found within cultures set up according to the procedure of Whitlock and Witte. This culture system was modified by placing sorted pre-B cells (ThB+, IgM-) from bone marrow in culture with previously-established bone marrow adherent layers. These cultures commenced growth without the lag associated with the Whitlock cultures. These cultured nonadherent cells show a high frequency of IgM+ cells, but do not express either IgD or Ia, and we refer to them as immature B cells. Cells with a similar phenotype (IgM+, Ia-, IgD-) are found within the spleens of young but not adult mice. The phorbol ester PMA induces expression of IgD on the cultured immature B cells, but has no effect on Ia expression. This suggests that the processing of H chain RNA transcripts may be affected by protein kinase C. These results demonstrate that the appearance of IgM, IgD, and Ia are independently controlled in long-term cultured B-lineage cells.
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Babij, P., J. Zhao, S. White, J. Woodcock-Mitchell, J. Mitchell, M. Absher, L. Baldor, M. Periasamy, and R. B. Low. "Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 265, no. 2 (August 1, 1993): L127—L132. http://dx.doi.org/10.1152/ajplung.1993.265.2.l127.
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RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.
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Harrison, DE, CP Lerner, and E. Spooncer. "Erythropoietic repopulating ability of stem cells from long-term marrow culture." Blood 69, no. 4 (April 1, 1987): 1021–25. http://dx.doi.org/10.1182/blood.v69.4.1021.1021.
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Abstract Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU- S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.
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Harrison, DE, CP Lerner, and E. Spooncer. "Erythropoietic repopulating ability of stem cells from long-term marrow culture." Blood 69, no. 4 (April 1, 1987): 1021–25. http://dx.doi.org/10.1182/blood.v69.4.1021.bloodjournal6941021.
Full textAbstract:
Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU- S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.
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Ylostalo, Joni H. "3D Stem Cell Culture." Cells 9, no. 10 (September 27, 2020): 2178. http://dx.doi.org/10.3390/cells9102178.
Full textAbstract:
Much interest has been directed towards stem cells, both in basic and translational research, to understand basic stem cell biology and to develop new therapies for many disorders. In general, stem cells can be cultured with relative ease, however, most common culture methods for stem cells employ 2D techniques using plastic. These cultures do not well represent the stem cell niches in the body, which are delicate microenvironments composed of not only stem cells, but also supporting stromal cells, extracellular matrix, and growth factors. Therefore, researchers and clinicians have been seeking optimal stem cell preparations for basic research and clinical applications, and these might be attainable through 3D culture of stem cells. The 3D cultures recapitulate the in vivo cell-to-cell and cell-to-matrix interactions more effectively, and the cells in 3D cultures exhibit many unique and desirable characteristics. The culture of stem cells in 3D may employ various matrices or scaffolds, in addition to the cells, to support the complex structures. The goal of this Special Issue is to bring together recent research on 3D cultures of various stem cells to increase the basic understanding of stem cells and culture techniques, and also highlight stem cell preparations for possible novel therapeutic applications.
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Carson, D. D., J. P. Tang, J. Julian, and S. R. Glasser. "Vectorial secretion of proteoglycans by polarized rat uterine epithelial cells." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2425–35. http://dx.doi.org/10.1083/jcb.107.6.2425.
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We have studied proteoglycan secretion using a recently developed system for the preparing of polarized primary cultures of rat uterine epithelial cells. To mimic their native environment better and provide a system for discriminating apical from basolateral compartments, we cultured cells on semipermeable supports impregnated with biomatrix. Keratan sulfate proteoglycans (KSPG) as well as heparan sulfate-containing molecules (HS[PG]) were the major sulfated products synthesized and secreted by these cells. The ability of epithelial cells to secrete KSPG greatly increased in parallel with the development of cell polarity. Furthermore, KSPG secretion occurred preferentially to the apical medium in highly polarized cultures. In contrast, HS(PG) secretion did not increase along with development of polarity, although most HS(PG) (85%) were secreted apically as well. Pulse-chase studies indicated that highly polarized cultures secreted 80-90% of the sulfated macromolecules they synthesized, predominantly to the apical secretory compartment. The half-lives for KSPG and HS(PG) secretion were approximately 3 and 4 h, respectively. Parallel studies of cells cultured on tissue culture plastic-coated with biomatrix indicated that neither the state of confluency nor the biomatrix was primarily responsible for inducing the KSPG secretion observed in polarizing cultures. Experiments with uterine strips indicated that the steroid hormone, 17-beta-estradiol, markedly stimulated synthesis and secretion of sulfated macromolecules, but had no preferential effect on KSPG production. The ratio of KSPG to HS(PG) secretion from uterine strips was similar to that found in the apical medium of highly polarized cell cultures. Thus, the pattern of proteoglycan secretion observed in polarized cell cultures mimicked that observed for uterine cells, although the preferential increase in KSPG production by polarized cells could not be attributed to an estrogen response. Collectively, these studies describe the major sulfated molecules secreted by rat uterine epithelial cells under varying conditions and provide evidence for a novel influence of cell polarity on the cell's ability to secrete sulfated glycoconjugates.
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Sahai, A., L. A. Cole, D. L. Clarke, and R. L. Tannen. "Rocking promotes differentiated properties in LLC-PK cells by improved oxygenation." American Journal of Physiology-Cell Physiology 256, no. 5 (May 1, 1989): C1064—C1069. http://dx.doi.org/10.1152/ajpcell.1989.256.5.c1064.
Full textAbstract:
Previous studies from our laboratory indicated that in contrast to cells cultured under still conditions, LLC-PK cells cultured under conditions of continuous rocking exhibit pH-modulated ammonia production and also manifest lower levels of lactate dehydrogenase activity. In the present studies, we assessed whether other metabolic features and parameters of differentiation are modified by rocking. Rocked, as contrasted with still, cultures exhibited decreased medium lactate production and increased cellular ATP content consistent with heightened oxidative metabolism. Still cultures did not exhibit either dome formation or sodium-dependent alpha-methylglucoside uptake until the cultures reached full confluency. By contrast, rocked cultures underwent dome formation as well as enhanced alpha-methylglucoside uptake during the growth phase, and these parameters were quantitatively greater than in still culture even after full confluency was achieved. Thus rocking promotes several functions, which can reflect differentiation in LLC-PK cells. To determine whether provision of adequate oxygen accounts for the enhancement of cellular metabolism and differentiated functions by rocking, still cultures grown in a high oxygen concentration of 36% were compared with standard cultures grown in 18% oxygen. The high oxygen environment resulted in the development of pH-modulated ammonia production, a decrease in lactate dehydrogenase activity, lower medium lactate formation, increased ATP levels, more copious dome formation, and increased alpha-methylglucoside uptake. Thus rocking promotes cellular metabolism and differentiated functions by the provision of adequate oxygenation.
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Chitteti, Brahmananda Reddy, Bradley Poteat, Sonia Rodriguez Rodriquez, Nadia Carlesso, Melissa A. Kacena, and Edward F. Srour. "Interactions of the Cellular Components of the Hematopoietic Niche." Blood 112, no. 11 (November 16, 2008): 3563. http://dx.doi.org/10.1182/blood.v112.11.3563.3563.
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Abstract Hematopoietic Stem Cell (HSC) self-renewal and multilineage differentiation potential is governed by multiple intrinsic and extrinsic parameters. Collectively, these parameters dictate the fate of HSC and underscore the heterogeneity observed within phenotypically defined groups of stem cells. While cell cycle status and the genetic profile of HSCs are critical intrinsic modulators of cell fate, interactions with cytokines, growth factors, and cellular elements of the hematopoietic niche (HN) are key extrinsic regulators of stem cell function. We examined the impact of cellular elements of the HN on stem cell fate and maintenance by analyzing the combined effect of calvaria-derived osteoblasts (OB) and mesenchymal stromal cells (MSC) on cultured murine HSC. Murine bone marrow-derived KSL cells were co-cultured with OB alone, MSC alone, or with mixtures of OB and MSC at different ratios for one week. Cultures were supplemented with SCF, Fl-3, Tpo, IL-3, IL-6, IGF1 & OPN. OB alone, maintained the functional properties of cultured HSCs significantly better than MSC thus corroborating the importance of OB in the overall competence of the HN. On day 7, the fold-increase in the number of LSK cells was 1473 ± 291 in OB cultures, 561 ± 159 in MSC cultures, and 603 ± 263 in OB+MSC cultures (n= 4 for all 3 groups). During the same 7 day-period, the number of CFU in progeny cells expanded 74 ± 15 fold in OB cultures, 23 ± 2 fold in MSC cultures, and 27 ± 15 in OB+MSC cultures (n=3 for all groups). The substantial increase in KSL progeny in OB cultures on day 7 was accompanied by a high percentage of cells in active phases of cell cycle (% G0/G1 = 72.5 ± 7.0, n=3) compared to their counterparts in MSC or OB+MSC cultures. In addition, co-culture of KSL cells with OB resulted in an unexpected higher maintenance of the Sca-1+Lin- phenotype (26.5% ± 2.8%) relative to MSC cultures (4.6% ± 1.0%) and OB+MSC cultures (11.7% ± 1.8%; n=3 for all). Only some of these results were reproduced when KSL cells were cultured in OB-conditioned medium suggesting that cell-to-cell contact may be essential for the observed activities. To assess the in vivo potential of LSK cells maintained in these cultures, the 10-day expansion equivalent of 1,000 LSK cells were competitively transplanted in lethally irradiated congenic mice and chimerism was monitored for the next 4 months. At 1 and 2 months post-transplantation, the level of chimerism sustained by LSK cells maintained in OB cultures for 10 days surpassed or was slightly lower than that observed with freshly isolated LSK cells (72.7% vs 59.7% and 57.4% vs 74.7%, respectively) suggesting that OB culture conditions effectively expanded short-term repopulating cells. At 4 months post-transplantation, mice receiving freshly isolated LSK cells were 83.6% ± 1.8% chimeric compared to 53.7% ± 16.1% for mice transplanted with cells from OB cultures and 31.9% ± 21.4% for mice receiving cells from OB+MSC cultures. Overall, these data suggest that OB-LSK interactions promote the maintenance of both short-term and long-term repopulating cells while MSC suppress the OB-mediated activity. To investigate the mechanism of OB-mediated maintenance of stem cell phenotype and function, we examined Notch signaling using Real-Time Q-PCR on cells maintained in culture for 7 days. Relative to the expression in KSL cells, expression of Notch 2 was elevated in OB cultures and suppressed over 2-fold in cultures of MSC and OB+MSC. Similarly, the expression of Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, Deltex, and SKP2 was increased in OB cultures and suppressed in MSC and OB+MSC cultures. Collectively, these data illustrate that cell-to-cell contact between OB and KSL cells promotes the in vitro maintenance of long-term and short-term repopulating cells and suggest that this stem cell function-promoting activity is induced in part by the upregulation of Notch-mediated signaling between HSCs and osteoblasts. The suppressive effect imparted by MSC on stem cell maintenance compared to cultures of OB alone suggest that these two cellular elements of the HN have opposite effects on the fate and function of stem cells.
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Fukusaki, Eiichiro, Kanokwan Jumtee, Takeshi Bamba, Takehiro Yamaji, and Akio Kobayashi. "Metabolic Fingerprinting and Profiling of Arabidopsis thaliana Leaf and its Cultured Cells T87 by GC/MS." Zeitschrift für Naturforschung C 61, no. 3-4 (April 1, 2006): 267–72. http://dx.doi.org/10.1515/znc-2006-3-419.
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Cell suspension cultures are now recognized as important model materials for plant bioscience and biotechnology. Very few studies of metabolic comparisons between cell cultures and original plants have been reported, even though the biological identity of cultured cells with the normally grown plant is of great importance. In this study, a comparison of the metabolome for primary metabolites extracted from the leaves of Arabidopsis thaliana and cultured cells from an Arabidopsis suspension culture (cell line T87) was performed. The results suggest that although cell suspension cultures and Arabidopsis leaves showed similarities in the common primary metabolite profile, nonetheless, moderate differences in quantitative profile were revealed.
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Jarrahy, Reza, Weibiao Huang, George H. Rudkin, Jane M. Lee, Kenji Ishida, Micah D. Berry, Modar Sukkarieh, Benjamin M. Wu, Dean T. Yamaguchi, and Timothy A. Miller. "Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds." American Journal of Physiology-Cell Physiology 289, no. 2 (August 2005): C408—C414. http://dx.doi.org/10.1152/ajpcell.00196.2004.
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Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.
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Liedtke, C. M. "Differentiated properties of rabbit tracheal epithelial cells in primary culture." American Journal of Physiology-Cell Physiology 255, no. 6 (December 1, 1988): C760—C770. http://dx.doi.org/10.1152/ajpcell.1988.255.6.c760.
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The purpose of this study was to establish whether rabbit tracheal epithelial cells, grown in primary cell culture, retain neurohormonal receptor and mediator activity. Epithelial cells were isolated by enzymatic treatment and cultured on a collagen matrix. The culture medium consisted of a 1:1 mixture of Dulbecco's modified Eagle medium and Ham's F12 supplemented with 5% fetal calf serum, epidermal growth factor, insulin, transferrin, fibronectin, hydrocortisone, and trace elements. Cultures had a population doubling time of 48 h. Mucus-containing cells and cilia were not observed after 7 days of incubation. Positive immunofluorescent staining with monoclonal antibodies to keratins established the epithelial nature of the cell cultures. Primary cell cultures responded to beta-adrenergic agonists with a dose- and time-dependent increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Propranolol, a beta-adrenergic antagonist, blocked the effects of the beta-adrenergic agonist. Adrenergic agonists also mediated a dose-dependent increase in the generation and release of prostaglandin E2 (PGE2). PGE2 caused an increase in cAMP levels. The results demonstrate that primary cultures of rabbit tracheal epithelial cells retain hormonal responses of the isolated epithelium and tracheal mucosa-submucosa.
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Kim, Jeong A., Nam D. Tran, Shur-Jen Wang, and Mark Fisher. "Pericytes Regulate Fibrinolytic Function of Brain Capillary Endothelial Cells." Stroke 32, suppl_1 (January 2001): 359. http://dx.doi.org/10.1161/str.32.suppl_1.359-a.
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P110 Our prior work has shown that astrocytes inhibit fibrinolysis of brain capillary endothelial cells (eg, Stroke 1999:30;1671–1677). The complex cellular microenvironment at the blood-brain barrier includes pericytes, which are adjacent to and share basement membrane with brain capillary endothelial cells. We analyzed the hemostatic regulatory role of pericytes in a blood-brain barrier model consisting of human brain pericytes cultured on transwell membrane inserts with human brain capillary endothelial cells. We measured fibrinolysis proteins and function in media conditioned by 48 hour co-culture of human brain capillary endothelial cells and human brain pericytes, as well as brain capillary endothelial mono-cultures. Compared to endothelial mono-cultures, pericyte-endothelial co-cultures exhibited levels of tissue plasminogen activator (tPA) protein reduced by 50±15% (p<.05). Co-culture preparations showed 32±13% (p<.05) increase in levels of plasminogen activator inhibitor-1 (PAI-1) protein, the primary inhibitor of tPA. tPA activity of co-culture preparations was 54±17% (p<.05) of endothelial mono-culture preparations. These data demonstrate that human brain pericytes have an important hemostatic regulatory role for endothelial-dependent fibrinolysis in vitro. These findings provide further support for brain-specific hemostasis, with pericytes as well as astrocytes playing key regulatory roles.
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Tanini, Annalisa, Maria Luisa Brandi, Umberto Modigliani, Carlo M. Rotella, and Roberto Toccafondi. "Transient lack of response to TSH of human cultured thyroid cells obtained from hyperfunctioning tissue." Acta Endocrinologica 113, no. 3 (November 1986): 346–54. http://dx.doi.org/10.1530/acta.0.1130346.
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Abstract. TSH-induced cAMP accumulation in cells obtained from normal and pathological thyroid tissue was studied during the first 12 days of primary culture. In normal thyroid tissue cultures (N = 7), the response of cAMP to TSH was present from the second day of culture and reached its maximum after 8 days. A similar behaviour was observed in cultures obtained from euthyroid sporadic goitres (N = 8), even if the rate of response was slightly lower than that of normal tissue. Similarly, cultured cells from euthyroid 'autonomous' nodules (N = 8) appeared to be responsive to TSH during the period of study, but the rate of response was also lower than in the controls. On the contrary, in cultures obtained from toxic adenomas (N = 5) and from diffuse toxic goitres (N = 5) the response to TSH was absent during the first 4 days of culture. The cells became sensitive to TSH from 6 and 6 day onwards, with the rate of response increasing progressively and reaching its maximum on day 12. Finally, in cultured cells obtained from different areas of multinodular toxic goitres (N = 4), the response to TSH was similar to that of euthyroid goitres in cells prepared from 'cold' areas, and to that of toxic adenomas in cells obtained from 'hot' areas. The present data demonstrate the existence of an inhibitory action of unknown factors, possibly iodothyronines or thyroglobulin, on the TSH effect in short-term cultures obtained from thyrotoxic tissues. A normal TSH responsiveness can be restored when the culture is prolonged.
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Mesel-Lemoine, Mariana, Mustapha Cherai, Sabine Le Gouvello, Maude Guillot, Virginie Leclercq, David Klatzmann, Véronique Thomas-Vaslin, and François M. Lemoine. "Initial depletion of regulatory T cells: the missing solution to preserve the immune functions of T lymphocytes designed for cell therapy." Blood 107, no. 1 (January 1, 2006): 381–88. http://dx.doi.org/10.1182/blood-2005-07-2658.
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Abstract We investigated the causes of the altered functionality of T cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T cells cultured for 6 days with anti-CD3 ± anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft-versus-host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amounts of cultured T cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and humans. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T cells (Treg's). Thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.
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Mesel-Lemoine, Mariana, Mustapha Cherai, Sabine Le Gouvello, Maude Guillot, Virginie Leclercq, David Klatzmann, Véronique Thomas-Vaslin, and Francois M. Lemoine. "Immune Dysfunctions of Cultured T Lymphocytes Are Due to a Preferential Expansion of CD4+CD25+ Suppressor Cells." Blood 106, no. 11 (November 16, 2005): 5199. http://dx.doi.org/10.1182/blood.v106.11.5199.5199.
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Abstract We investigated the causes of the altered functionality of T-cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T-cells cultured for 6-days with anti-CD3 ± anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T-cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft versus host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amount of cultured T-cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and human. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T-cells (Treg) and thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T-cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T-cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.
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Lacasse, J., C. Kreft, J. Gutkowska, J. Genest, and M. Cantin. "Cultured juxtaglomerular cells: production and localization of renin." Canadian Journal of Physiology and Pharmacology 65, no. 7 (July 1, 1987): 1409–15. http://dx.doi.org/10.1139/y87-221.
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Trypsin- or collagenase-dispersed renal cortical cells from newborn mice were filtered and cultured. The cultures comprised 25% juxtaglomerular cells as identified by immunocytochemistry. Renin was measured by radioimmunoassay in the culture medium and in the cells at various time intervals. This in vitro system was responsive to isoproterenol, which stimulated renin release in a dose-dependent manner.
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Kitano, Otome, and Kohji Nakazawa. "Neuronal Differentiation of NT2 Cells in Monolayer and Spheroid Cultures." MATEC Web of Conferences 333 (2021): 07008. http://dx.doi.org/10.1051/matecconf/202133307008.
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Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.
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Kitano, Otome, and Kohji Nakazawa. "Neuronal Differentiation of NT2 Cells in Monolayer and Spheroid Cultures." MATEC Web of Conferences 333 (2021): 07008. http://dx.doi.org/10.1051/matecconf/202133307008.
Full textAbstract:
Metabolism and differentiation of cultured cells are influenced by changes in cellular morphology. In this study, we investigated the differences in cell proliferation and neuronal differentiation of NT2 cells in monolayer (2D) and spheroid (3D) cultures. In the monolayer culture, the cells adhered and extended on a tissue culture plate. For the spheroid culture, we fabricated a microwell chip comprising 195 circular microwells (600 ìm in diameter) on a cutture plate, and the surface was modified with polyethylene glycol to promote spheroid formation. The cells were aggregated in each microwell and formed spheroids within 24 hours of culture, and the spheroid morphology was maintained thoughout the culture period. Although the cell proliferation ability in monolayer culture was higher than that in spheroid culture, the neuronal differentiation ability of NT2 spheroid culture was higher than that in monolayer culture. Furthermore, the neuronal differentiation of NT2 spheroids was dramatically enhanced by retinoic acid treatment. These results indicate that NT2 cell properties are influenced by differences in cell morphologies, and that spheroid culture is a promising technique to induce neuronal differentiation.
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Grant, M. I., K. Anderson, G. McKay, M. Wills, C. Henderson, and C. MacDonald. "Manipulation of the phenotype of immortalised rat hepatocytes by different culture configurations and by dimethyl sulphoxide." Human & Experimental Toxicology 19, no. 5 (May 2000): 309–17. http://dx.doi.org/10.1191/096032700678815936.
Full textAbstract:
The liver-specific phenotype of immortalised rat hepato-cytes is not irretrievably lost as they age in culture but can be manipulated by modifying the culture environment. Testosterone metabolism was used to investigate the profile of cytochrome P450 isoenzymes present in two immortalised cell lines, P9 and LQC, and in primary cultures of rat hepatocytes, cultured on collagen films, gels and double gel cultures (sandwich configuration). The extent of testosterone metabolism, and the range of metabolites produced, was increased in immortalised cells by the presence of collagen as a substratum film or gel but survival was poorer and the range of metabolites was reduced in sandwich culture. In contrast, testosterone metabolism was retained in primary hepatocytes in sandwich cultures at a higher level than in collagen film or gel cultures. Expression of alpha class glutathione-S-transferases (GSTs) increased and that of GSTP1 decreased (changes which indicate a recovery of normal liver GST phenotype) when the medium of immortalised cell cultures was supplemented with dimethyl sulph-oxide (DMSO). DMSO also improved ethoxyresorufin 0-deethylation (EROD) and testosterone metabolism in immortalised cells. It also markedly inhibited prolifera-tion, DNA, RNA and protein synthesis. Maximal testosterone metabolism was observed in immortalised cells cultured on collagen gels in the presence of l1o (v/v) DMSO. Development of a protocol for treating immortalised liver cells cultured on collagen gels with DMSO to switch between proliferation and differentiation may provide a convenient system expres-sing the xenobiotic metabolising enzymes required for in vitro toxicity testing.
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Betriu, Nausika, Anna Andreeva, and Carlos E. Semino. "Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells." Cancers 13, no. 18 (September 7, 2021): 4504. http://dx.doi.org/10.3390/cancers13184504.
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The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.
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Mrukowicz, Jacek Z., J. Denise Wetzel, Mehmet I. Goral, Agnes B. Fogo, Peter F. Wright, and Terence S. Dermody. "Viruses and Cells with Mutations Affecting Viral Entry Are Selected during Persistent Rotavirus Infections of MA104 Cells." Journal of Virology 72, no. 4 (April 1, 1998): 3088–97. http://dx.doi.org/10.1128/jvi.72.4.3088-3097.1998.
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ABSTRACT To better understand mechanisms of persistent rotavirus infections of cultured cells, we established independent, persistently infected cultures of MA104 cells, using rotavirus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented with fresh medium every 7 days. Viral titers in culture lysates varied from 104 to 107 PFU per ml during 350 days of culture maintenance. Trypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antibody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen. We tested the capacity of rotaviruses isolated from the persistently infected cultures (PI viruses) to infect cells cured of persistent infection. Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which indicates that viruses and cells coevolve during persistent rotavirus infections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on growth of wt and PI viruses. Trypsin pretreatment is required for postattachment penetration of rotavirus virions into cells. In contrast to the case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells. However, PI viruses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected during maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotavirus entry.
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Ichii, Michiko, Kenji Oritani, Takafumi Yokota, Isao Takahashi, Takahiro Shirogane, Norimitsu Saitoh, Rie Tanigawa, Satomi Kasai, and Yuzuru Kanakura. "Establishment of Stroma-Free Cultures for Human B Lymphopoiesis: Roles of High Cell Density Condition and Mesenchymal Stem Cell-Secreted Factors." Blood 110, no. 11 (November 16, 2007): 1420. http://dx.doi.org/10.1182/blood.v110.11.1420.1420.
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Abstract Background: Due to lacking of appropriate culture systems, it has been difficult to analyze mechanisms of human B lymphocytes. However, we recently found that human mesenchymal stem cells (MSC) have high ability to support human B lymphopoiesis. Although many investigators have believed that stromal layers are essential for supporting the differentiation of human B lymphocyte progenitors, this hypothesis is not a case. By manipulating our co-culture system, we have successfully established stroma-free suspension cultures, which produce CD10+ B cells from human umbilical cord blood (CB) CD34+ cells. Methods: Our stroma-free culture of CB CD34+ cells was performed in QBSF® medium with 10% FCS in the presence of 10 ng/ml stem cell factor (SCF) and 5 ng/ml Flt3-ligand (FL) with or without 5 ng/ml IL-7. Results: Even when co-cultures were separated from MSC with membrane filters, they could produce CD10+ cells. Moreover, addition of MSC supernatant to the cultures permitted CD34+ cells to emerge CD10+ cells in the absence of MSC. Therefore, the cell-cell contact between MSC and B lymphocyte progenitors was not essential. For stroma-free human B cell cultures with MSC supernatant, QBSF® was the most suitable and serum components were essential while depending on their lot. Although addition of thymic stromal lymphopoietin or IL-7 increased the production of CD10+ cells, neutralizing antibodies for them showed no effect. Addition of Hemokinin-1 antagonist diminished, albeit to a limited degree, the production of CD10+ cells. Addition of neutralizing antibody for CXCR4 had no effect. Therefore, MSC supernatant contains some supportive factors except for them. When cultures without MSC or their supernatant stared at 1x104 cells/ml, they could successfully produce CD10+ cells. The density of the cultured cells was critical. The production of CD10+ cells was not detected when CD34+ cells were seeded at low density (1x103/ml). Moreover, when the cultured cells were diluted and adjusted at 1x104/ml weekly, the emergence of CD10+ cells was not observed while the production of CD33+ myeloid cells was enhanced. Therefore, surrounding hematopoietic cells seemed to be required to support human B lymphopoiesis. Our suspension cultures of 1x104 CD34+ cells in the presence of SCF, FL, and IL-7 without any stromal materials generated approximately 0.5–1x106 CD10+ cells at 4 week. CD33+ cells were first expanded within 2 weeks, and then CD10+ cells appeared. When the cultured cells were transplanted into NOD/SCID/γcnull mice, they reconstituted both myeloid and B lymphoid lineages. Therefore, some cultured cells maintain stem cell character. Conclusions: We have established stroma-free suspension cultures, which effectively produce CD10+ B cells from CB CD34+ cells. High cell density condition can in part substitute for stromal layers in supporting human B lymphopoiesis although the addition of MSC supernatant enhances the production of CD10+ cells. Our suspension culture does not use any stromal cells, which produce many positive or negative regulators for human B lymphopoiesis. This simplicity proposes that this culture system is useful in a variety of fields such as the screening direct effects of drugs influencing on human B lymphocyte development and the evaluation of progenitors in patients with B-cell malignancies as well as the cloning of human B lymphocyte-supportive molecules.
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Dickman, K. G., and J. L. Renfro. "Primary culture of flounder renal tubule cells: transepithelial transport." American Journal of Physiology-Renal Physiology 251, no. 3 (September 1, 1986): F424—F432. http://dx.doi.org/10.1152/ajprenal.1986.251.3.f424.
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Renal proximal tubule cells from the winter flounder (Pseudopleuronectes americanus) were maintained in a functionally differentiated state for up to 16 days in primary culture on floating collagen gels. The cells were confluent after 7-8 days in culture, contracted the collagen gels, and exhibited ciliary activity. Electron microscopy indicated that the cultures were composed of continuous sheets of columnar epithelial cells that had established structural polarity. When mounted in Ussing chambers, the cultures exhibited a small mucosa-negative potential difference (0.6 +/- 0.10 mV) and a low transepithelial resistance (23 +/- 2.3 omega X cm2). Short-circuit current averaged 24 microA/cm2. The cultured epithelium was four times more permeable to Na than to Cl and actively secreted sulfate and p-aminohippuric acid and reabsorbed hexoses. Glucose reabsorption was rheogenic and occurred via a high-affinity (Km = 0.16 mM), low-capacity (Vmax = 5 microA/cm2), phlorizin-sensitive transport system. We concluded that the cultured cells express many of the differentiated properties of the intact flounder proximal tubule and thus provide a suitable model system for studying renal transport processes.
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Rosenzweig, M., DF Marks, H. Zhu, D. Hempel, KG Mansfield, PK Sehgal, S. Kalams, DT Scadden, and RP Johnson. "In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells." Blood 87, no. 10 (May 15, 1996): 4040–48. http://dx.doi.org/10.1182/blood.v87.10.4040.bloodjournal87104040.
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Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.
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Smith-Thomas, L. C., and J. W. Fawcett. "Expression of Schwann cell markers by mammalian neural crest cells in vitro." Development 105, no. 2 (February 1, 1989): 251–62. http://dx.doi.org/10.1242/dev.105.2.251.
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During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.
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Cardier, Jose E., Olga Wittig, Jose Alonso, and Egidio Romano. "Generation of Endothelial Progenitor Cells from Amniotic Fluid Stem Cells." Blood 112, no. 11 (November 16, 2008): 5403. http://dx.doi.org/10.1182/blood.v112.11.5403.5403.
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Abstract Recently it has been shown that human amniotic fluid contains stem cells of fetal origin (AFS) that express embryonic and adult stem cell markers. AFS can be expanded extensively in culture and give rise to multiple differentiated cells such those of myogenic, osteogenic, neurogenic and endothelial lineages, all of them with potential therapeutic value. In this study, we investigated the generation of endothelial progenitor cells (EPC) from AFS. For this purpose, remnant amniotic fluid was obtained from mothers undergoing amniocentesis for medical reasons at 18 weeks of pregnancy. Cells were collected by centrifugation and cultured in DMEM, supplemented with Chang medium and fetal bovine serum. Adherent cells were expanded and characterized by flow cytometry at various times using antibodies for CD34, CD45, CD133, CD117, KDR (VEGFR-2) and CD31. Cultures were also carried out in endothelial growth medium and analyzed for EPC markers (CD133, KDR, CD31). Adherent cells showed, at 8 and 17 days of culture, fibroblastoid features. Flow cytometry analysis, at 2 weeks of culture, showed cells expressing CD 133 (37%), CD 34 (1%), CD 117 (3%) and KDR (12%). Cultures carried out in endothelial differentiation medium (EDM), at 33 days of culture, showed a significant increase of cells expressing EPC markers: CD133 (40%), CD34 (3%) KDR (24%), KDR/CD31 (9%). These cells formed blood vessel-like structures on collagen-coated plates. The in vivo angiogenic capacity of these cells was demonstrated by inoculating them into collagen microspheres and implanting them subcutaneously into immunocompromised mice. Histologic examination revealed that these cells formed microvessels on implantation. Our data indicates that EPC can be generated and expanded from AFS, and they might be useful for therapeutic angiogenesis in ischemic tissues.
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Wilson, Timothy, Reeta Viitala, Mervi Puska, Mika Jokinen, and Risto Penttinen. "Macrophage Induced Effect of Particulate Silica on Rat Mesenchymal Stem Cells In Vitro." Key Engineering Materials 396-398 (October 2008): 123–26. http://dx.doi.org/10.4028/www.scientific.net/kem.396-398.123.
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The role of silica and macrophages in fibrosis is well documented, but in bone formation it is relatively unknown despite decades of research with bioactive glasses. In this study macrophages were isolated from rat peritoneal and then cultured for five days in the presence of two types of silica microparticles with different solubilities. After the fifth day the culture medium was collected, purified and used as an additive in bone marrow derived rat stem cell cultures. The stem cells were cultured for five days in α-mem containing only 0,5% of FCS, enabling cell survival but disrupting their proliferation. As controls, stem cells were also cultured in α-mem containing silica microparticles. At days one and five the amount of soluble collagen was assayed from the culture medium and the cells were counted. All stem cell cultures with macrophage medium additives were found to be proliferative, with statistically significant difference to controls. However, collagen was only produced in cultures containing medium from macrophages cultured with fast-dissolving silica microparticles. This suggests that silica can induce cell proliferation and extra cellular matrix protein secretion which is mediated by macrophages, and that the solubility of silica is also a major factor in this reaction.
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Smith, Stephen L. "Effect of Serum on Long-Term Cultures of Human Umbilical Cord Blood Megakaryocyte Progenitors." Blood 104, no. 11 (November 16, 2004): 4153. http://dx.doi.org/10.1182/blood.v104.11.4153.4153.
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Abstract Platelet recovery following umbilical cord blood (CB) transplants can be delayed for several months. In vitro expansion and transplantation of CB megakaryocyte progenitor cells (MPC) may be one way to increase circulating platelets thereby shortening the period of thrombocytopenia. The in vitro expansion and differentiation of CB MPC derived from enriched CB CD34+ cells was studied after immuno-magnetic bead isolation. Isolated CB CD34+ cells were incubated in serum-free culture medium supplemented with a Tpo peptide agonist (TpoA), Flt-3L, Stem Cell Factor (SCF) and Vascular Endothelial Growth Factor (VEGF). At week 2, the cultures were divided and 1) maintained in serum-free medium, 2) supplemented with 5% human serum and, 3) supplemented with 5% mouse serum. Using flow cytometry and histocytochemistry, the cultured cells were evaluated for MPC using CD41a, CD36, von Willebrand factor (VWF) and also for myeloid progenitors using CD15 and CD33. The mean total percent MPC (CD41a+ and CD36+) at week 2 was 53 ±17% and 49 ±12%, respectively. After 2–4 weeks of additional culture the %MPC declined to less than 30% in cultures with or without serum supplements. However, at weeks 5–8 of additional culture the %MPC increased in the cultures supplemented with human or mouse serum. The mean %MPC was 45 ±19% (serum-free), 55 ±16% (human serum) and, 71 ±5% (mouse serum) in cultures at week 5–8 of additional culture. The remaining cells in the cultures were myeloid progenitors (CD15+ or CD33+). Total cell numbers at week 5–8 were inhibited 3 to 5x-fold in mouse serum supplemented cultures compared to serum free and human serum supplemented cultures. In addition, VWF+ cells could be detected after 5 weeks of additional culture when either human or mouse serum was present. These data help to explain the delay in platelet recovery typically associated with cord blood transplants but, indicate that additional serum factors are present to enhance megakaryocyte differentiation. These results also suggest that the transplant of cultured CB MPC may prove beneficial for the treatment of thrombocytopenic patients and normal platelet recovery.