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Grün, Christoph, Brigitte Altmann, and Eric Gottwald. "Advanced 3D Cell Culture Techniques in Micro-Bioreactors, Part I: A Systematic Analysis of the Literature Published between 2000 and 2020." Processes 8, no. 12 (December 15, 2020): 1656. http://dx.doi.org/10.3390/pr8121656.
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Bioreactors have proven useful for a vast amount of applications. Besides classical large-scale bioreactors and fermenters for prokaryotic and eukaryotic organisms, micro-bioreactors, as specialized bioreactor systems, have become an invaluable tool for mammalian 3D cell cultures. In this systematic review we analyze the literature in the field of eukaryotic 3D cell culture in micro-bioreactors within the last 20 years. For this, we define complexity levels with regard to the cellular 3D microenvironment concerning cell–matrix-contact, cell–cell-contact and the number of different cell types present at the same time. Moreover, we examine the data with regard to the micro-bioreactor design including mode of cell stimulation/nutrient supply and materials used for the micro-bioreactors, the corresponding 3D cell culture techniques and the related cellular microenvironment, the cell types and in vitro models used. As a data source we used the National Library of Medicine and analyzed the studies published from 2000 to 2020.
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Xing, Zizhuo, Brian M. Kenty, Zheng Jian Li, and Steven S. Lee. "Scale-up analysis for a CHO cell culture process in large-scale bioreactors." Biotechnology and Bioengineering 103, no. 4 (July 1, 2009): 733–46. http://dx.doi.org/10.1002/bit.22287.
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Allen, Leah M., John Matyas, Mark Ungrin, David A. Hart, and Arindom Sen. "Serum-Free Culture of Human Mesenchymal Stem Cell Aggregates in Suspension Bioreactors for Tissue Engineering Applications." Stem Cells International 2019 (November 7, 2019): 1–18. http://dx.doi.org/10.1155/2019/4607461.
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Mesenchymal stem cells (MSCs) have the capacity to differentiate towards bone, fat, and cartilage lineages. The most widely used culture and differentiation protocols for MSCs are currently limited by their use of serum-containing media and small-scale static culture vessels. Suspension bioreactors have multiple advantages over static culture vessels (e.g., scalability, control, and mechanical forces). This study sought to compare the formation and culture of 3D aggregates of human synovial fluid MSCs within suspension bioreactors and static microwell plates. It also sought to elucidate the benefits of these techniques in terms of productivity, cell number, and ability to generate aggregates containing extracellular matrix deposition. MSCs in serum-free medium were either (1) inoculated as single cells into suspension bioreactors, (2) aggregated using static microwell plates prior to being inoculated in the bioreactor environment, or (3) aggregated using microwell plates and kept in the static environment. Preformed aggregates that were size-controlled at inoculation had a greater tendency to form large, irregular super aggregates after a few days of suspension culture. The single MSCs inoculated into suspension bioreactors formed a more uniform population of smaller aggregates after a definite culture period of 8 days. Both techniques showed initial deposition of extracellular matrix within the aggregates. When the relationship between aggregate size and ECM deposition was investigated in static culture, midsized aggregates (100-300 cells/aggregate) were found to most consistently maximize sGAG and collagen productivity. Thus, this study presents a 3D tissue culture method, which avoids the clinical drawbacks of serum-containing medium that can easily be scaled for tissue culture applications.
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Koller, MR, SG Emerson, and BO Palsson. "Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures." Blood 82, no. 2 (July 15, 1993): 378–84. http://dx.doi.org/10.1182/blood.v82.2.378.378.
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Abstract There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.
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Koller, MR, SG Emerson, and BO Palsson. "Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures." Blood 82, no. 2 (July 15, 1993): 378–84. http://dx.doi.org/10.1182/blood.v82.2.378.bloodjournal822378.
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There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.
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Claessen, Maria, Eszter Varga, Steven Heshusius, Esther Heideveld, Martin Hansen, Anna Visser, Marijke Thiel-Valkhof, et al. "Large Scale Culture and Differentiation of Erythroblasts from PBMC and iPSC." Blood 132, Supplement 1 (November 29, 2018): 2319. http://dx.doi.org/10.1182/blood-2018-99-118882.
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Abstract Transfusion of donor-derived red blood cells to aleviate anemia is the most common form of cellular therapy. In addition, red blood cells hold great promise as delivery agents of e.g. specific drugs or enzymes. However, the source depends on donor availability and carries a potential risk of alloimmunization and blood borne diseases. More than 30 bloodgroup systems encode >300 bloodgroup antigens and bloodgroup matching becomes increasingly challenging in a multiethnic society. Particularly the chronically transfused patients are at risk for alloimmunisation. In vitro cultured, customizable red blood cells (cRBC) would negate these concerns and introduce precision medicine both in transfusion medicine as well as in drug delivery applications. We aim to produce human cRBC at large-scale and cost effective, for which we need to optimize culture conditions and reduce cost-drivers. We adapted our protocols to GMP culture requirements, which reproducibly provided pure human erythroid cultures within 25 days with a 3.4x107 times expansion from peripheral blood mononuclear cells without prior CD34+ isolation. This expansion depended on the serum free medium we produce, which is supplemented with erythropoietin (Epo, 1 U/ml), stem cell factor (SCF) and glucocorticoids. Expanded erythroblasts CD71 highCD235low/- were differentiated for 10 days in medium supplemented with 5% human plasma, heparin and a higher concentration of Epo (10U/ml) yielding CD71dimCD235a+CD44+CD117-DRAQ5- cRBC. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Passaging cRBC through a leukodepletion filter yielded 100% enucleated, stable cRBC. Deformability was measured by an Automated Rheoscope and Cell Analyser (ARCA), and oxygen equilibrium curves were measured with a Hemox analyzer. Both parameters were similar in cRBC and freshly isolated reticulocytes. RNA sequencing was performed daily during differentiation and revealed expression dynamics of important erythroid processes, e.g. increased expression of genes involved in blood group expression, globin regulation, and erythroid specific metabolic enzymes, concommittant with loss of expression of genes involved in the formation of organelles, and cell proliferation. The culture process is compatible with upscaling using 5L G-Rex bioreactors., Currently we are preparing a clinical study using biotinylated cRBC. Ultimately, however, large scale production requires an immortal source, for which we aim to use human induced pluripotent stem cells (iPSC) established from rare donors that lack most blood group antigens. Using single cell passaging of iPSC and differentiation in colonies, we generate at average 2x105 cRBC per single iPSC. However, the cRBC cultured from iPSC were less stable following enucleation, and expressed embryonic type globins. Comparison of transcriptome data from iPSC-derived erythroid cells at distinct differentiation stages with erythroid cells at similar stages that were cultured from adult- or cord blood mononuclear cells, or from fetal liver confirmed that most iPSC-derived erythroid cells largely express an embryonic RNA profile. In conclusion, our current protocols enable us to test cRBC cultured from adult peripheral blood for their stability after transfusion. Concurrently, we develop novel bioreactors to upscale the production, and we optimise the protocol to generate cRBC from immortal iPSC lines with near 'universal donor' genotypes. Disclosures No relevant conflicts of interest to declare.
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Gerlach, J. C., K. Klöppel, C. MÜller, N. Schnoy, M. D. Smith, and P. Neuhaus. "Hepatocyte Aggregate Culture Technique for Bioreactors in Hybrid Liver Support Systems." International Journal of Artificial Organs 16, no. 12 (December 1993): 843–46. http://dx.doi.org/10.1177/039139889301601210.
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Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.
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Chen, Keda, Chaonan Li, Ying Wang, Zhenwei Shen, Yikai Guo, Xiaoping Li, and Yanjun Zhang. "Optimization of Vero Cells Grown on a Polymer Fiber Carrier in a Disposable Bioreactor for Inactivated Coxsackievirus A16 Vaccine Development." Vaccines 9, no. 6 (June 7, 2021): 613. http://dx.doi.org/10.3390/vaccines9060613.
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At present, there are no vaccines available for hand, foot, and mouth disease, which is caused by Coxsackie virus A16 (CVA16) infection. In the present study, we isolated epidemic strains of CVA16 and optimized the production of the virus in Vero cells. The system comprised growing the infected cells on polymer fiber paper carriers in a serum-free medium containing 0.5% (w/v) lactalbumin hydrolysate a mini bioreactor. Disposable Bioflo310 and AmProtein Current perfusion bioreactors were used to monitor virus infection and Vero cell culture. The total number of cells increased from 1.5 × 109 to 3.0 × 1010. In our optimized culture process, the virus titer reached 7.8 × 107 TCID50/mL at three days after infection. The inactivated CVA16 prepared from our optimized culture procedure elicited a slightly higher neutralizing antibody titer compared with that derived from routine culture procedures. These results will promote the large-scale production of inactivated CVA16 vaccines using nonwoven polymer fiber paper cell cultures.
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Eghbali, Hadis, Michele M. Nava, Gabriella Leonardi, Davod Mohebbi-Kalhori, Roberto Sebastiano, Abdolreza Samimi, and Manuela T. Raimondi. "An Experimental-Numerical Investigation on the Effects of Macroporous Scaffold Geometry on Cell Culture Parameters." International Journal of Artificial Organs 40, no. 4 (April 2017): 185–95. http://dx.doi.org/10.5301/ijao.5000554.
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Introduction Perfused bioreactors have been demonstrated to be effective in the delivery of nutrients and in the removal of waste products to and from the interior of cell-populated three-dimensional scaffolds. In this paper, a perfused bioreactor hosting a macroporous scaffold provided with a channel is used to investigate transport phenomena and culture parameters on cell growth. Methods MG63 human osteosarcoma cells were seeded on macroporous poly(ε-caprolactone) scaffolds provided with a channel. The scaffolds were cultured in a perfused bioreactor and in static conditions for 5 days. Cell viability and growth were assessed while the concentration of oxygen, glucose and lactate were measured. An in silico, multiphysics, numerical model was set up to study the fluid dynamics and the mass transport of the nutrients in the perfused bioreactor hosting different scaffold geometries. Results The experimental and numerical results indicated that the specific cell metabolic activity in scaffolds cultured under perfusion was 30% greater than scaffolds cultured under static conditions. In addition, the scaffold provided with a channel enabled the shear stress to be controlled, the initial seeding density to be retained, and adequate mass transport and waste removal. Conclusions We show that the macroporous scaffold provided with a channel cultured in a macroscale bioreactor can be a robust reference experimental model system to systematically investigate and assess crucial culture parameters. We also show that such an experimental model system can be employed as a simplified “representative unit” to improve the performance of both perfused culture systems and hollow, fiber-integrated scaffolds for large-scale tissue engineering.
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DEAN, ROBERT C., SABHASH B. KARKARE, NITYA G. RAY, PETER W. RUNSTADLER, and K. VENKATASUBRAMANIAN. "Large-Scale Culture of Hybridoma and Mammalian Cells in Fluidized Bed Bioreactors." Annals of the New York Academy of Sciences 506, no. 1 Biochemical E (November 1987): 129–46. http://dx.doi.org/10.1111/j.1749-6632.1987.tb23815.x.
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Thanh, Nguyen Trung, Hosakatte Niranjana Murthy, and Kee Yoeup Paek. "Optimization of ginseng cell culture in airlift bioreactors and developing the large-scale production system." Industrial Crops and Products 60 (September 2014): 343–48. http://dx.doi.org/10.1016/j.indcrop.2014.06.036.
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Park, Soonyong, Ji Yul Kim, Kyoung-Hwa Ryu, Ah-Young Kim, Jaemun Kim, Young-Joon Ko, and Eun Gyo Lee. "Production of a Foot-and-Mouth Disease Vaccine Antigen Using Suspension-Adapted BHK-21 Cells in a Bioreactor." Vaccines 9, no. 5 (May 13, 2021): 505. http://dx.doi.org/10.3390/vaccines9050505.
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The baby hamster kidney-21 (BHK-21) cell line is a continuous cell line used to propagate foot-and-mouth disease (FMD) virus for vaccine manufacturing. BHK-21 cells are anchorage-dependent, although suspension cultures would enable rapid growth in bioreactors, large-scale virus propagation, and cost-effective vaccine production with serum-free medium. Here, we report the successful adaptation of adherent BHK-21 cells to growth in suspension to a viable cell density of 7.65 × 106 cells/mL on day 3 in serum-free culture medium. The suspension-adapted BHK-21 cells showed lower adhesion to five types of extracellular matrix proteins than adherent BHK-21 cells, which contributed to the suspension culture. In addition, a chemically defined medium (selected by screening various prototype media) led to increased FMD virus production yields in the batch culture, even at a cell density of only 3.5 × 106 cells/mL. The suspension BHK-21 cell culture could be expanded to a 200 L bioreactor from a 20 mL flask, which resulted in a comparable FMD virus titer. This platform technology improved virus productivity, indicating its potential for enhancing FMD vaccine production.
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Jossen, Valentin, Cedric Schirmer, Dolman Mostafa Sindi, Regine Eibl, Matthias Kraume, Ralf Pörtner, and Dieter Eibl. "Theoretical and Practical Issues That Are Relevant When Scaling Up hMSC Microcarrier Production Processes." Stem Cells International 2016 (2016): 1–15. http://dx.doi.org/10.1155/2016/4760414.
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The potential of human mesenchymal stem cells (hMSCs) for allogeneic cell therapies has created a large amount of interest. However, this presupposes the availability of efficient scale-up procedures. Promising results have been reported for stirred bioreactors that operate with microcarriers. Recent publications focusing on microcarrier-based stirred bioreactors have demonstrated the successful use of Computational Fluid Dynamics (CFD) and suspension criteria (NS1u,NS1) for rapidly scaling up hMSC expansions from mL- to pilot scale. Nevertheless, one obstacle may be the formation of large microcarrier-cell-aggregates, which may result in mass transfer limitations and inhomogeneous distributions of stem cells in the culture broth. The dependence of microcarrier-cell-aggregate formation on impeller speed and shear stress levels was investigated for human adipose derived stromal/stem cells (hASCs) at the spinner scale by recording the Sauter mean diameter (d32) versus time. Cultivation at the suspension criteria providedd32values between 0.2 and 0.7 mm, the highest cell densities (1.25 × 106cells mL−1hASCs), and the highest expansion factors (117.0 ± 4.7 on day 7), while maintaining the expression of specific surface markers. Furthermore, suitability of the suspension criterionNS1uwas investigated for scaling up microcarrier-based processes in wave-mixed bioreactors for the first time.
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Paul, Katrin, and Christoph Herwig. "Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors." Engineering in Life Sciences 20, no. 5-6 (February 5, 2020): 197–204. http://dx.doi.org/10.1002/elsc.201900162.
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Zhao, Ashley G., Kiran Shah, Julien Freitag, Brett Cromer, and Huseyin Sumer. "Differentiation Potential of Early- and Late-Passage Adipose-Derived Mesenchymal Stem Cells Cultured under Hypoxia and Normoxia." Stem Cells International 2020 (September 18, 2020): 1–11. http://dx.doi.org/10.1155/2020/8898221.
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With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.
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Souza, Marta Cristina de Oliveira, Marcos da Silva Freire, and Leda dos Reis Castilho. "Influence of culture conditions on Vero cell propagation on non-porous microcarriers." Brazilian Archives of Biology and Technology 48, spe (June 2005): 71–77. http://dx.doi.org/10.1590/s1516-89132005000400009.
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Animal cell cultures are widely employed for the production of viral vaccines and for recombinant protein expression. The cell line Vero is a continuous, adherent cell line, which has been recommended by the World Health Organization for the production of human vaccines. For the large-scale production of vaccines, microcarriers, which are microspheres that serve as support for the cells, are being increasingly used. The use of microcarriers in stirred bioreactors allows high cell densities and, consequently, high virus titres to be achieved. With the aim of selecting appropriate culture conditions for the cultivation of Vero cells at high cell densities, in this work the influence of several variables (agitation rate, ratio of inoculated cells to microcarrier mass and fetal bovine serum concentration) on cell growth on Cytodex 1 microcarriers was studied. Under the best conditions determined, a comparison with Vero cell cultivation on Cytodex 3 microcarriers was carried out.
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Gounari, Eleni, Anastasia Papadopoulou, Minas Yiangou, Angeliki E. Xagorari, Achilles Anagnostopoulos, Evangelia Yannaki, and Panayotis Kaloyannidis. "An Optimized, Ex Vivo Log-Scale Expansion of UCB-Derived Myeloid Dcs, Towards Efficient Cancer Immunotherapy." Blood 124, no. 21 (December 6, 2014): 2441. http://dx.doi.org/10.1182/blood.v124.21.2441.2441.
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Abstract Background: Dendritic cells (DCs) have received much attention as a therapeutic tool for infections and immunotherapy of advanced malignancies, due to their unique ability of antigen-presentation and initiation of the T-cell-dependent immune response. However, the broader application of vaccine- or antitumor specific T cell–based immunotherapy is limited by DC’s scarcity in the peripheral blood and the complex and costly methodology required for their production. To date, peripheral blood monocytes are the most common source for DCs generation and recently hematopoietic stem cell (CD34+)-derived DCs have also been produced. However, the accessibility to stem cell sources is limited and the number of generated DCs are barely enough for wide clinical use. Aims: The non-transplantable umbilical cord blood (UCB) units could be a readily available and promising source for large scale DC production, by offering adequate number of CD34+ cells. In the present study, we developed a new culture method for the generation of high numbers of myeloid DCs from non-transplantable UCB units by using new generation bioreactors (G-rex) and estimated the minimum required volume of a UCB unit to produce adequate number of DCs for clinical application. Methods: CD34+cells from non-transplantable UCBs were purified by immunomagnetic separation, cultured in the presence of a cytokine cocktail [stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin-4 (IL-4)) for 10 days in tissue culture plates and subsequently expanded in G-rex bioreactors for 25 additional days. Control cultures were completed in conventional plates. DCs were matured either with a combination of Toll-like receptor ligands [(TLR-Ls), Poly I:C and R848] or with commonly used vaccines containing TLR-Ls [Act-HIV (bacterial form of influenza), Influvac (viral form of influenza), Typhim Vi, HBV and BCG ]. DCs phenotype was analyzed by flow cytometry (CD40, HLA-DR, CD83 and CD86) and the cytokine secretion values were measured by ELISA. The endophagocytic activity of DCs was assessed by a dead yeast engulfment assay. Results: DCs derived from UCB CD34+ cells with more than 85% purity, were cultured in bioreactors or in conventional plates. DCs cultured in Grexes reached a median fold expansion 20.875 and a median absolute number of 2 billions, in sharp contrast to a median 100-fold increase of conventionally cultured DCs. The expanded DCs had the typical morphology and surface markers of myeloid DCs (CD33+/CD11+: 70.47%).To address whether those highly expanded DCs are functional and have the ability, upon maturation, to induce Th1 response, the cells were matured with either a cocktail of TLR-Ls or commonly used vaccines, and tested for the expression of molecules needed for antigen presentation, endophagocytic activity and cytokine production. Similar to conventional DCs, the highly-expanded and matured with vaccines or TLR-Ls DCs, showed dendrites and endocytotic capacity. The expanded and matured with either way DCs, expressed also HLA-DR and co-stimulatory molecules (CD40, CD86 and CD83) whereas they produced increased levels of IL-12p70, TNF-α and IL-6 but undetectable IL-10, thus suggesting a strong potential for Th1 response. We tested different volumes and UCB units for CD34+cell-derived DCs and showed that the minimum volume of a UCB unit that could be used for large scale DCs generation with our optimized culture system, is 9ml, corresponding to approximately 10% of the volume of a transplantable UCB unit. Conclusion: We established an optimized and simple culture system to expand at clinically relevant numbers, the production of human, myeloid DCs from UCB-derived CD34+ cells. These DCs have the functional properties that are necessary to obtain therapeutic gains against malignancies and viral infections. Billions of DCs were derived from a minimum of 10% of the volume of an average transplantable UCB unit. This new method could be one step closer to a broader clinical application of DC- and T-cell based immunotherapy for various malignancies. Disclosures No relevant conflicts of interest to declare.
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Reichardt, Anne, Bianca Polchow, Mehdi Shakibaei, Wolfgang Henrich, Roland Hetzer, and Cora Lueders. "Large Scale Expansion of Human Umbilical Cord Cells in a Rotating Bed System Bioreactor for Cardiovascular Tissue Engineering Applications." Open Biomedical Engineering Journal 7, no. 1 (June 14, 2013): 50–61. http://dx.doi.org/10.2174/1874120701307010050.
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Widespread use of human umbilical cord cells for cardiovascular tissue engineering requires production of large numbers of well-characterized cells under controlled conditions. In current research projects, the expansion of cells to be used to create a tissue construct is usually performed in static cell culture systems which are, however, often not satisfactory due to limitations in nutrient and oxygen supply. To overcome these limitations dynamic cell expansion in bioreactor systems under controllable conditions could be an important tool providing continuous perfusion for the generation of large numbers of viable pre-conditioned cells in a short time period. For this purpose cells derived from human umbilical cord arteries were expanded in a rotating bed system bioreactor for up to 9 days. For a comparative study, cells were cultivated under static conditions in standard culture devices. Our results demonstrated that the microenvironment in the perfusion bioreactor was more favorable than that of the standard cell culture flasks. Data suggested that cells in the bioreactor expanded 39 fold (38.7 ± 6.1 fold) in comparison to statically cultured cells (31.8 ± 3.0 fold). Large-scale production of cells in the bioreactor resulted in more than 3 x 108 cells from a single umbilical cord fragment within 9 days. Furthermore cell doubling time was lower in the bioreactor system and production of extracellular matrix components was higher. With this study, we present an appropriate method to expand human umbilical cord artery derived cells with high cellular proliferation rates in a well-defined bioreactor system under GMP conditions.
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Yang, Ying, Feng He, Longjiang Yu, Jiaxing Ji, and Yezhen Wang. "Flavonoid Accumulation in Cell Suspension Cultures of Glycyrrhiza inflata Batal under Optimizing Conditions." Zeitschrift für Naturforschung C 64, no. 1-2 (June 1, 2009): 68–72. http://dx.doi.org/10.1515/znc-2009-1-212.
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Cell growth and flavonoid production in cell suspension cultures of Glycyrrhiza inflata Batal were investigated under various initial inoculum densities, and sucrose and nitrogen concentrations to develop an optimization method for an improved flavonoid production. Both biomass accumulation and flavonoid production exhibited an “S” curve in one culture cycle, with the greatest value obtained on day 21, which showed that cell growth and fl avonoid biosynthesis went along isochronously. Moreover, according to the biomass and flavonoid production, the appreciate inoculum density, and the sucrose and nitrogen concentrations were 50 g FW L-1, 50 g L-1 and 120 mmol L-1, respectively. In addition, cell growth and flavonoid production showed a peak of 16.4 g DW L-1 and 95.7 mg L-1 on day 21 under the optimizing conditions, respectively. The flavonoid productivity of the cells which were cultured for 3 years is higher than that of the 3-year-old plant, which suggested that flavonoid production by cell cultures of G. inflata is a potentially profitable method. Therefore, this work is considered to be helpful for efficient large-scale bioprocessing of cell cultures in bioreactors.
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Roy, Arpita. "Hairy Root Culture an Alternative for Bioactive Compound Production from Medicinal Plants." Current Pharmaceutical Biotechnology 22, no. 1 (December 31, 2020): 136–49. http://dx.doi.org/10.2174/1389201021666201229110625.
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: Medicinal plants produce a diverse group of phytocompounds like anthraquinones, alkaloids, anthocyanins, flavonoids, saponins, and terpenes which are used in pharmaceutical, perfume, cosmetics, dye and flavor industries. Commercial source of these metabolites is field-grown plants, which are generally influenced by seasonal changes. Biotechnology possesses a significant role in production of high-value secondary metabolites. By incorporating biotechnological methods, it is feasible to manage biosynthetic pathways of the plant to enhance phytocompound production that is of pharmaceutical interest. Plant cell suspension, shoot, adventitious root and hairy root culture are considered as alternative methods for important bioactive compound production. These methods are controllable, sustainable and overcome several inconveniences for large scale secondary metabolites production. At present research on hairy root culture for valuable bioactive compound production has gained a lot of attention. Agrobacterium rhizogenes is an agent which causes hairy root disease in a plant and this leads to the neoplastic growth of root which is characterized by higher growth rate and genetic stability. Various studies explore the hairy root culture for production of a wide range of bioactive compounds. Scale-up of hairy root culture using bioreactors has provided an opportunity to enhance bioactive compound production at the commercial level. The present review discusses the role of hairy root culture in the production of valuable bioactive compounds, the effect of culture parameters on bioactive compound production and bioreactor applications.
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Luo, Xia, Yinjie Niu, Xiaozhe Fu, Qiang Lin, Hongru Liang, Lihui Liu, and Ningqiu Li. "Large-Scale Microcarrier Culture of Chinese Perch Brain Cell for Viral Vaccine Production in a Stirred Bioreactor." Vaccines 9, no. 9 (September 8, 2021): 1003. http://dx.doi.org/10.3390/vaccines9091003.
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Mandarin fish (Siniperca chuatsi) is one of the important cultured fish species in China. Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca Chuatsi rhabdovirus (SCRV) have hindered the development of mandarin fish farming industry. Vaccination is the most effective method for control of viral diseases, however viral vaccine production requires the large-scale culture of cells. Herein, a suspension culture system of Chinese perch brain cell (CPB) was developed on Cytodex 1 microcarrier in a stirred bioreactor. Firstly, CPB cells were cultured using Cytodex 1 microcarrier in 125 mL stirring flasks. With the optimum operational parameters, CPB cells grew well, distributed uniformly, and could fully cover the microcarriers. Then, CPB cells were digested with trypsin and expanded step-by-step with different expansion ratios from the 125 mL stirring bottle to a 500 mL stirring bottle, and finally to a 3-L bioreactor. Results showed that with an expansion ratio of 1:3, we achieved a high cell density level (2.25 × 106 cells/mL) with an efficient use of the microcarriers, which also confirmed the data obtained from the 125 mL stirring flask. Moreover, obvious cytopathic effects (CPE) were observed in the suspended CPB cells post-infection with ISKNV and SCRV. This study provided a large-scale culture system of CPB cells for virus vaccine production.
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Naik, Poornananda Madhava, and Jameel Mohammed Al-Khayri. "Cell suspension culture as a means to produce polyphenols from date palm (Phoenix dactylifera L.)." Ciência e Agrotecnologia 42, no. 5 (September 2018): 464–73. http://dx.doi.org/10.1590/1413-70542018425021118.
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ABSTRACT Date palm accumulates a wide range of secondary metabolites high in nutritional and therapeutic value. In the present study, date palm (Phoenix dactylifera L., cv. Shaishi) shoot-tip-induced callus was used to establish cell suspension cultures in Murashige and Skoog (MS) liquid medium containing 1.5 mg L-1 2-isopentenyladenine (2iP) and 10 mg L-1 naphthaleneacetic acid (NAA). To study the growth kinetics, cultures were maintained for 12 weeks during which weekly measurements were carried out to determine the biomass accumulation based on packed cell volume (%), fresh weight and dry weight (g). In addition, weekly determination of polyphenols (catechin, caffeic acid, kaempferol, and apigenin) was carried out using high performance liquid chromatography (HPLC). The 11-week-old culture was found highest in the production of biomass (62.9 g L-1 fresh weight and 7.6 g L-1 dry weight) and polyphenols (catechin-155.9 µg L-1, caffeic acid-162.7 µg L-1, kaempferol-89.7 µg L-1, and apigenin-242.7 µg L-1) from the cell suspension cultures. This is the first report on the production of polyphenols from the cell suspension culture of date palm. This study facilitates further development of large-scale production of polyphenols and the utilization of bioreactors.
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Liu, Jin Yu, Jürg Hafner, Galya Dragieva, and Günter Burg. "Bioreactor Microcarrier Cell Culture System (Bio-MCCS) for Large-Scale Production of Autologous Melanocytes." Cell Transplantation 13, no. 7-8 (October 2004): 809–16. http://dx.doi.org/10.3727/000000004783983422.
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Applegate, Mark A., and Gregory Stephanopoulos. "Development of a single-pass ceramic matrix bioreactor for large-scale mammalian cell culture." Biotechnology and Bioengineering 40, no. 9 (November 1992): 1056–68. http://dx.doi.org/10.1002/bit.260400909.
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Kalra, K., B. Banerjee, K. Weiss, and C. Morgan. "Developing efficient bioreactor microcarrier cell culture system for large scale production of mesenchymal stem cells (MSCs)." Cytotherapy 21, no. 5 (May 2019): S73. http://dx.doi.org/10.1016/j.jcyt.2019.03.468.
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Ito, Yukitaka, Sou Nakamura, Tomohiro Shigemori, Naoshi Sugimoto, Yoshikazu Kato, Kazuya Hashimoto, Naohide Watanabe, et al. "High-Vorticity with Periodic Flow Enhances in Vitro Biogenesis of Healthy Platelets from iPSC-Derived-Megakaryocytes." Blood 128, no. 22 (December 2, 2016): 2181. http://dx.doi.org/10.1182/blood.v128.22.2181.2181.
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Abstract Each transfusion requires 200-300 billion platelets in patients with thrombocytopenia. To continuously supply such a huge number of platelets by ex vivo generation, two distinct steps, megakaryopoiesis and platelet shedding, must be both considered. For the former, one approach is to increase the number of source cell, megakaryocytes. For example, the immortalized megakaryocyte cell line (imMKCL) system uses self-renewing megakaryocyte (MK) cell lines derived from induced pluripotent stem cells (iPSCs) (Nakamura et al., Cell Stem Cell, 2014). For the latter, there have been an idea of bioreactors whereby shedding of platelets from proplatelets could be promoted by flow-dependent shear force within the bone marrow in vivo (Junt et al., Science, 2007; Zhang et al., J Exp Med, 2012). Based upon this idea, we constructed a flow chamber type bioreactor recapitulating in vivo blood flow shear rate. However, this bioreactor failed to efficiently yield platelets, and moreover, the produced platelets had poor quality as indicated by high Annexin V levels (Exp Hematol, 2011 and unpublished result). Recently, we demonstrated two different kinetics of platelet biogenesis from bone marrow MKs, whereby either thrombopoietin (TPO) mostly regulates steady-state shedding of platelets from proplatelets, or interleukin-a (IL-1a) triggers inflammation-dependent rupture of MK cytoplasm contributing to a quick increase of platelet count at higher rate (Nishimura et al., J Cell Biol, 2015). However, the rupture type platelets revealed shorter half-life with relatively higher Annexin V levels. Therefore, to gain insights from platelet biogenesis in vivo, we focused on biophysical analysis of steady-state platelet biogenesis via proplatelets in bone marrow. Our observations strongly indicated that the presence of 'vorticity' defined by vortex turbulence in addition to shear-dependent 'stress' and 'strain' correlates with the efficient shedding of competent platelets. From this new finding, we developed an alternative bioreactor system, which enabled generation of 100 billion platelets from imMKCL in a 16L-scale liquid culture condition without any adherent machinery using two 10L-bioreactors. Furthermore, platelets generated via new bioreactors showed low Annexin V levels (<10-15%) and shortened bleeding time post transfusion into NOG mice and rabbits with thrombocytopenia, comparable to human blood product platelets. Regarding the platelet production using WAVE bag system (GE healthcare, UK), the system is already clinically available for cord blood cell expansion in most countries, but lacks adequate levels of vorticity and shear strain/stress. Accordingly, the produced platelets had high Annexin V levels (i.e., 50-65%) as well as diminished yield efficiency (P<0.001). In conclusion, our study has uncovered the novel biophysical aspect of platelet biogenesis. The application of the new set of physical parameters in constructing large sized bioreactors shall facilitate the industrialization of platelet production. Disclosures Eto: Megakaryon Co. Ltd.: Research Funding.
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Singh, Pritika, Rupam Guleri, Amrita Angurala, Kuldeep Kaur, Kulwinder Kaur, Sunil C. Kaul, Renu Wadhwa, and Pratap Kumar Pati. "Addressing Challenges to Enhance the Bioactives ofWithania somniferathrough Organ, Tissue, and Cell Culture Based Approaches." BioMed Research International 2017 (2017): 1–15. http://dx.doi.org/10.1155/2017/3278494.
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Withania somniferais a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues.In vitropropagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential ofin vitrocell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years.In vitroculture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.
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Nurhayati, Retno Wahyu, Yoshihiro Ojima, Takeaki Dohda, and Masahiro Kino-Oka. "Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system." Biotechnology Progress 34, no. 2 (December 28, 2017): 362–69. http://dx.doi.org/10.1002/btpr.2595.
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Pangas, SA, and TK Woodruff. "Production and purification of recombinant human inhibin and activin." Journal of Endocrinology 172, no. 1 (January 1, 2002): 199–210. http://dx.doi.org/10.1677/joe.0.1720199.
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Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion. Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers. Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein. To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein. These cells produce approximately 200 microg/ml per day total recombinant human inhibin. Conditioned cell media can be purified through column chromatography resulting in dimeric mature 32-34 kDa inhibin A and 28 kDa activin A. The purified recombinant proteins maintain their biological activity as measured by traditional in vitro assays including the regulation of FSH in rat anterior pituitary cultures and the regulation of promoter activity of the activin-responsive promoter p3TP-luc in tissue culture cells. These proteins will be valuable for future analysis of inhibin and activin function and have been distributed to the US National Hormone and Peptide Program.
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Petersen, Ian, Chanyarat Paungfoo-Lonhienne, Esteban Marcellin, Lars Keld Nielsen, and Axayacatl Gonzalez. "Towards Sustainable Bioinoculants: A Fermentation Strategy for High Cell Density Cultivation of Paraburkholderia sp. SOS3, a Plant Growth-Promoting Bacterium Isolated in Queensland, Australia." Fermentation 7, no. 2 (April 9, 2021): 58. http://dx.doi.org/10.3390/fermentation7020058.
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Paraburkholderia sp. SOS3 is a plant growth-promoting bacterium (PGPB) that displays pleiotropic effects and has the potential to be applied at a large scale across several agronomically important crops. The use of SOS3 is a suitable option to reduce the use of chemical fertilisers. While the benefits of SOS3 have been demonstrated in vitro, its potential applications at large scale are limited due to low biomass yield in current batch culture systems. Here, we developed a strategy for high-cell density cultivation of SOS3 in instrumented bioreactors, moving from low-biomass yield in a complex medium to high-biomass yield in a semi-defined medium. We achieved a 40-fold increase in biomass production, achieving cell densities of up to 11 g/L (OD600 = 40). This result was achieved when SOS3 was cultivated using a fed-batch strategy. Biomass productivity, initially 0.02 g/L/h in batch cultures, was improved 12-fold, reaching 0.24 g/L/h during fed-batch cultures. The biomass yield was also improved 10-fold from 0.07 to 0.71 gbiomass/gsolids. Analysis of the fermentation profile of SOS3 indicated minimal production of by-products and accumulation of polyhydroxybutyrate (PHB) during the exponential growth phase associated with nitrogen limitation in the medium. By implementing proteomics analysis in fed-batch cultures, we identified the expression of four metabolic pathways associated with growth-promoting effects, which may be used as a qualitative parameter to guarantee the efficacy of SOS3 when used as a bioinoculant. Ultimately, we confirmed that the high-cell density cultures maintained their plant growth-promoting capacity when tested in sorghum and maize under glasshouse conditions.
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Daly, Andrew C., Binulal N. Sathy, and Daniel J. Kelly. "Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions." Journal of Tissue Engineering 9 (January 1, 2018): 204173141775371. http://dx.doi.org/10.1177/2041731417753718.
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Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O2 and 3% O2. At 20% O2, dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.
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Mao, Ping, Huaxin Duan, Caixia Wang, Tingfen Deng, Changru Luo, Yanli Xu, and Yuping Zhang. "In Vitro Large-Scale Expansion of Unfractionated Mononucleated Cells of Human Umbilical Cord Blood: Investigation for Clinical-Grade Generation of Cord Blood Hematopoietic Cells." Blood 114, no. 22 (November 20, 2009): 3222. http://dx.doi.org/10.1182/blood.v114.22.3222.3222.
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Abstract Abstract 3222 Poster Board III-159 Previous studies of expansion for hematopoietic stem/progenitor cells showed progenitor cells could reach a great expansion but were mainly performed at small culture volume. The poor cell number of expanded cells even great folds of expansion in a no more than 10 ml culture system limited the application for clinic transplantation. Thereof, we expanded umbilical cord blood (UCB) hematopoietic cells at a large scale in 250 ml bioreactor culture system. The mononuclear cell isolated form fresh UCB samples were ex vivo expanded in 250 ml bioreactor with serum-free medium supplied with SCF, FL3 and TPO at concentration 20 ng/ml. Cell number, surface markers and colony forming potential after 7 days of expansion have been evaluated. The sub-lethally irradiated animal models were used to analyze engraftment capability of HSC by transplant of expanded cells in severe combined immunodeficient and nonobese diabetic (NOD/SCID) mice. The detection of human hematopoietic cells in the bone marrow of the mice was performed at 6 wk after the transplant. We have successfully expanded hematopoietic progenitor cells 5 times using the modified bioreactor. The cell viability showed no obvious variations during culture. Total cells increased from 1.76±0.50 ×108 (range 1.2-2.5) to 4.14±0.83×108 (range 3.7-5.6) after expansion, CD34+ cells from 1.25±0.31 ×106 (range 0.96-1.75) to 3.96±0.78×106 (range 2.88-5.04) and CD133+ cells from 1.55±0.69×106 (range 0.96-2.75) to 4.77±0.88×106 (range 4.00-5.04). The colonies per 105 cells of CFU-E /BFU-E, CFU-GM and CFU-Mix increased from 369.6±71.5 to 1648.5±504.3, 42.8±81.4 to 146.4±54.5, 39.1±10.3 to 144.7±38.8, respectively. The positive expression of CD34+ was 0.7%±0.28% at day 0 and 0.9%±0.34% after culture in bioreactor at day 7 and CD133+ cells was 0.8%±0.24% and 1.1%±0.35%. There was no significant survive rate difference between expanded and nonexpanded cells transplantation group. The analysis of multilineage hematopoiesis showed that transplanted human hematopoietic cells is represented in murine bone marrow cells by detection the percentage of human cells identified with human specific anti CD3/19/33/45/61/71 MoAb by FACS and the human specific gene Alu-1 and Cat-1 by PCR. Disclosures No relevant conflicts of interest to declare.
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Kochan, Ewa, Sylwia Caban, Grażyna Szymańska, Piotr Szymczyk, Anna Lipert, Paweł Kwiatkowski, and Monika Sienkiewicz. "Ginsenoside content in suspension cultures of Panax quinquefolium L. cultivated in shake flasksand stirred-tank bioreactor." Annales Universitatis Mariae Curie-Sklodowska, sectio C – Biologia 72, no. 1 (July 16, 2018): 15. http://dx.doi.org/10.17951/c.2017.72.1.15-26.
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<p>Plant suspension cultures are described as a source for the acquisition of medicinal secondary metabolites which in the future may become an alternative to traditional raw materials. This study demonstrates that the cell cultures of one of the ginseng species – Panax quinquefolium L. synthesize ginsenosides, which are triterpene saponins having a multidirectional pharmacological effects. Tested suspension cultures were run on a small scale in the shake flasksand in scale up of the process in a 10-liter stirred tank. In the shake flasks,the highest biomass yield (2.28 gl-1 for dry and 33.99 gl-1 for fresh weight) was reached on day 30 of culture, and the highest content of saponins (2.66 mg g -1 dw) was determined on day 28 of culture. In the bioreactor, nearly 2.67 and 3-fold increase of respectively dry and fresh biomass was recorded in relation to the inoculum. Large-scale cultures synthesized protopanaxatriol derivatives such as Rg1 and Re ginsenosides, however, no saponins belonging to the protopanaxadiol derivatives were reported.</p>
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Wink, Michael, A. Wilhelm Alfermann, Rochus Franke, Bernhard Wetterauer, Melanie Distl, Jörg Windhövel, Oliver Krohn, et al. "Sustainable bioproduction of phytochemicals by plant in vitro cultures: anticancer agents." Plant Genetic Resources 3, no. 2 (August 2005): 90–100. http://dx.doi.org/10.1079/pgr200575.
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Due to their complex structure with several chiral centres important anticancer agents are still extracted from plants and not synthesized chemically on a commercial scale. Sustainable bioproduction of the compounds of interest may be achieved by plant in vitro cultures. Undifferentiated callus and suspension cultures, which can be cultivated in large bioreactors easily, very often fail to accumulate the compounds of interest, whereas shoot and root cultures as well hairy roots normally produce the same compounds as in the appropriate organs. The production of anticancer compounds, such as the alkaloids vinblastine, vincristine, paclitaxel (Taxol®), camptothecin, or the lignan podophyllotoxin, by plant in vitro cultures is reviewed. Taxanes can be produced in bioreactors using cell suspensions of various Taxus species with good yields; presently paclitaxel is produced on a commercial scale by Phyton Biotech (Germany). Camptothecin has low yields in suspension cultures of Camptotheca acuminata or Nothapodytes foetida (0.0003–0.01%), but a good production (0.1–0.3% dry wt) in root and hairy root cultures of Ophiorrhiza pumila, O. mungos and C. acuminata. Podophyllotoxin can be produced in cell suspension and root as well as hairy root cultures of Podophyllum and various Linum species up to 130 mg/l (Linum album cell suspensions); its derivative 6-methoxypodophyllotoxin is accumulated in hairy roots of L. persicum up to about 500 mg/l. The in vitro production of dimeric indole alkaloids in Catharanthus roseus has failed so far both in undifferentiated and differentiated in vitro cultures. In cases where in vitro cultures show good yields, they can be employed in biotechnology for the sustainable production of valuable products.
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Hutami, Sri. "TINJAUAN Penggunaan Suspensi Sel dalam Kultur In Vitro." Jurnal AgroBiogen 5, no. 2 (August 23, 2016): 84. http://dx.doi.org/10.21082/jbio.v5n2.2009.p84-92.
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<p>Cell suspension culture could be defined as a<br />process that allows rapidly dividing homogenous suspension<br />of cells to grow in liquid nutrient media. There are two main<br />types of suspension cultures: (1) Batch cultures in which<br />cells are nurtured in a fixed volume of medium until growth<br />ceases and (2) Continuous cultures in which cell growth is<br />maintained by continuous replenishment of sterile nutrient<br />media. Plant cell suspension cultures are mostly used for the<br />biochemical investigation of cell physiology, growth, metabolism,<br />protoplast fusion, transformation and for large scale<br />production of seed by bioreactor and production of secondary<br />metabolites. Contamination is one of the largest problems<br />when dealing with cell cultures. Differences between<br />the products of cell suspension culture and whole plant are<br />frequently observed. These phenomena’s may be resulted<br />from lack of differentiation and organization and cell cultureinduced<br />variation. Utilization of cell suspension culture in<br />Indonesia is still limited, some of them for mass production<br />of plantation seed with bioreactor system and for production<br />of secondary metabolites. The success of this study give the<br />opportunity for mass production of seeds from other plants<br />and also production of secondary metabolites.</p>
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Zhu, Li Kuan, Bo Yan Song, Zhen Long Wang, and Yu Kui Wang. "Optimze the Structure of Impeller for Stirred Bioreactor." Advanced Materials Research 694-697 (May 2013): 148–53. http://dx.doi.org/10.4028/www.scientific.net/amr.694-697.148.
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This paper mainly makes comparative analysis on four main types of blade in stirred bioreactor by Computational Fluid Dynamics(CFD) simulation. Firstly we establish simulation method suited for stirred bioreactor, then simulate the velocity and shear force of flow field in the bioreactor. No matter from flow field mixing or shear force aspect, Elephant Ear blades is the most suitable for cell large scale culture. At last, it optimizes the installation method and angle of Elephant Ear blades. It concludes that anticlockwise rotation and 45°installation angle is the optimum.
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Torizal, Fuad G., Ikki Horiguchi, and Yasuyuki Sakai. "Physiological Microenvironmental Conditions in Different Scalable Culture Systems for Pluripotent Stem Cell Expansion and Differentiation." Open Biomedical Engineering Journal 13, no. 1 (February 28, 2019): 41–54. http://dx.doi.org/10.2174/1874120701913010041.
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Human Pluripotent Stem Cells (PSCs) are a valuable cell type that has a wide range of biomedical applications because they can differentiate into many types of adult somatic cell. Numerous studies have examined the clinical applications of PSCs. However, several factors such as bioreactor design, mechanical stress, and the physiological environment have not been optimized. These factors can significantly alter the pluripotency and proliferation properties of the cells, which are important for the mass production of PSCs. Nutritional mass transfer and oxygen transfer must be effectively maintained to obtain a high yield. Various culture systems are currently available for optimum cell propagation by maintaining the physiological conditions necessary for cell cultivation. Each type of culture system using a different configuration with various advantages and disadvantages affecting the mechanical conditions in the bioreactor, such as shear stress. These factors make it difficult to preserve the cellular viability and pluripotency of PSCs. Additional limitations of the culture system for PSCs must also be identified and overcome to maintain the culture conditions and enable large-scale expansion and differentiation of PSCs. This review describes the different physiological conditions in the various culture systems and recent developments in culture technology for PSC expansion and differentiation.
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Sahabian, Anais, Malte Sgodda, Ortwin Naujok, Rabea Dettmer, Julia Dahlmann, Felix Manstein, Tobias Cantz, Robert Zweigerdt, Ulrich Martin, and Ruth Olmer. "Chemically-Defined, Xeno-Free, Scalable Production of hPSC-Derived Definitive Endoderm Aggregates with Multi-Lineage Differentiation Potential." Cells 8, no. 12 (December 4, 2019): 1571. http://dx.doi.org/10.3390/cells8121571.
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For the production and bio-banking of differentiated derivatives from human pluripotent stem cells (hPSCs) in large quantities for drug screening and cellular therapies, well-defined and robust procedures for differentiation and cryopreservation are required. Definitive endoderm (DE) gives rise to respiratory and digestive epithelium, as well as thyroid, thymus, liver, and pancreas. Here, we present a scalable, universal process for the generation of DE from human-induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs). Optimal control during the differentiation process was attained in chemically-defined and xeno-free suspension culture, and high flexibility of the workflow was achieved by the introduction of an efficient cryopreservation step at the end of DE differentiation. DE aggregates were capable of differentiating into hepatic-like, pancreatic, intestinal, and lung progenitor cells. Scale-up of the differentiation process using stirred-tank bioreactors enabled production of large quantities of DE aggregates. This process provides a useful advance for versatile applications of DE lineages, in particular for cell therapies and drug screening.
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Xie, Liangzhi, Christian Metallo, James Warren, Warren Pilbrough, Joseph Peltier, Tanya Zhong, Lana Pikus, et al. "Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6? cell culture under sparging conditions." Biotechnology and Bioengineering 83, no. 1 (May 5, 2003): 45–52. http://dx.doi.org/10.1002/bit.10644.
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Heathman, Thomas R. J., Alvin W. Nienow, Qasim A. Rafiq, Karen Coopman, Bo Kara, and Christopher J. Hewitt. "Agitation and aeration of stirred-bioreactors for the microcarrier culture of human mesenchymal stem cells and potential implications for large-scale bioprocess development." Biochemical Engineering Journal 136 (August 2018): 9–17. http://dx.doi.org/10.1016/j.bej.2018.04.011.
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Rungarunlert, S., N. Klincumhom, C. Nemes, M. Techakumphu, M. K. Pirity, and A. Dinnyes. "293 MASS PRODUCTION OF Nkx2.5-POSITIVE CARDIAC PROGENITOR CELLS DERIVED FROM MOUSE EMBRYONIC STEM CELLS IN SLOW-TURNING LATERAL VESSEL FOR CELL TRANSPLANTATION AND DRUG TESTING." Reproduction, Fertility and Development 23, no. 1 (2011): 244. http://dx.doi.org/10.1071/rdv23n1ab293.
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Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of cardiomyocyte production. The 3 lines were cultured in STLV bioreactor and compared with classical hanging drop (HD) and static suspension culture methods. Embryonic bodies at day 3 to 8 were collected and analysed by using fluorescence-activated cell sorting for markers of pluripotency (e.g. Oct-4, SSEA1, Nanog) and cardiac (e.g. Nkx2.5, Troponin T) lineage commitments. Data was analysed by one-way ANOVA and t-tests. The results showed that both level and kinetics of Nkx2.5 expression was dependent on culture conditions. The STLV and static suspension culture methods produced higher rates of Nkx2.5-positive cells on day 5 than that of HD (70 and 54 v. 30%, respectively). The STLV method produced a highly uniform population of efficiently differentiating EB in large quantities and resulted in the highest, 108 yield of cardiomyocytes in a single 110-mL STLV on day 4. In conclusion, the STLV method provides a technological platform for controlled large-scale generation of ES-cell-derived cardiomyocytes for clinical and industrial applications. In vivo transplantation tests of cardiomyocytes produced via STLV are currently underway. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship, No. RTA 5080010 (M.T.), and the Thailand Commission on Higher Education [CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-RG-2007 (N.K.)].
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Pais, Daniel A. M., Chris Brown, Anastasia Neuman, Krishanu Mathur, Inês A. Isidro, Paula M. Alves, and Peter G. Slade. "Dielectric Spectroscopy to Improve the Production of rAAV Used in Gene Therapy." Processes 8, no. 11 (November 13, 2020): 1456. http://dx.doi.org/10.3390/pr8111456.
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The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell concentration, infection timing, and cell harvest viabilities since they can have a significant influence on rAAV productivity and product quality. Herein we developed the use of dielectric spectroscopy as a process analytical technology (PAT) tool used to continuously monitor the production of rAAV in 2 L stirred tank bioreactors, achieving enhanced control over the production process. This study resulted in improved manufacturing robustness through continuous monitoring of cell culture parameters, eliminating sampling needs, increasing the accuracy of infection timing, and reliably estimating the time of harvest. To increase the accuracy of baculovirus infection timing, the cell growth/permittivity model was coupled to a feedback loop with real-time monitoring. This system was able to predict baculovirus infection timing up to 24 h in advance for greatly improved accuracy of infection and ensuring consistent high rAAV productivities. Furthermore, predictive models were developed based on the dielectric measurements of the culture. These multiple linear regression-based models resulted in correlation coefficients (Q2) of 0.89 for viable cell concentration, 0.97 for viability, and 0.92 for cell diameter. Finally, models were developed to predict rAAV titer providing the capability to distinguish in real time between high and low titer production batches.
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Van der Loo, Johannes C. M., William Swaney, Diana Nordling, Axel Schambach, Christopher Baum, David A. Williams, Lilith Reeves, and Punam Malik. "Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor." Blood 112, no. 11 (November 16, 2008): 3539. http://dx.doi.org/10.1182/blood.v112.11.3539.3539.
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Abstract The need for gamma-retroviral vectors with self-inactivating (SIN) long terminal repeats for clinical trials has prompted a shift in the method with which large scale GMP-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN retrovirus vectors that produced high amounts of genomic vector RNA in packaging cells, and to design scalable processes for closed system culture, transfection and virus harvest. Using improved expression plasmids, the Vector Production Facility, an academic GMP manufacturing laboratory that is part of the Translational Cores at Cincinnati Children’s Hospital, has developed such a method based on the Wave Bioreactor® production platform. In brief, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave Cell Bag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16–20 hrs post-transfection, the media is changed; virus is harvested at 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted into Cryocyte freezing containers, and frozen at or below −70°C. Several processing parameters, including the confluency of cells harvested prior to transfection, the timing of transfection, the amount of plasmid DNA, exposure of cells to PBS/TrypLESelect, and the timing of the media change post-transfection affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding (as is standard when using tissue culture plastic) increased the titer from 104 to 4 × 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 μg doubled the titer in the Wave, while it reduced titer by 20–40% in tissue culture flasks (Fig. 1). Using an optimized protocol, six cGMP-grade SIN gamma-retroviral vectors have now been produced in support of the FDA’s National Toxicology Program (NTP), with unconcentrated vector titers ranging from 1 × 106 to as high as 4 × 107 IU/mL. Using similar processing, we have produced a large scale SIN gamma-retroviral vector (GALV pseudotyped) for an international X-linked SCID trial with average unconcentrated titers of 106 IU/mL in all viral harvests. In summary, the process developed at the Cincinnati Children’s Hospital Vector Production Facility allows for large scale closed-system production of high-titer retroviral vectors for clinical trials using transient transfection. Figure Figure
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Xu, Meng-Xue, Li-Ping Liu, Yu-Mei Li, and Yun-Wen Zheng. "The Opportunities and Challenges regarding Induced Platelets from Human Pluripotent Stem Cells." Stem Cells International 2021 (May 1, 2021): 1–10. http://dx.doi.org/10.1155/2021/5588165.
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As a standard clinical treatment, platelet transfusion has been employed to prevent hemorrhage in patients with thrombocytopenia or platelet dysfunctions. Platelets also show therapeutic potential for aiding liver regeneration and bone healing and regeneration and for treating dermatological conditions. However, the supply of platelets rarely meets the rising clinical demand. Other issues, including short shelf life, strict storage temperature, and allogeneic immunity caused by frequent platelet transfusions, have become serious challenges that require the development of high-yielding alternative sources of platelets. Human pluripotent stem cells (hPSCs) are an unlimited substitution source for regenerative medicine, and patient-derived iPSCs can provide novel research models to explore the pathogenesis of some diseases. Many studies have focused on establishing and modifying protocols for generating functional induced platelets (iPlatelets) from hPSCs. To reach high efficiency production and eliminate the exogenous antigens, media supplements and matrix have been optimized. In addition, the introduction of some critical transgenes, such as c-MYC, BMI1, and BCL-XL, can also significantly increase hPSC-derived platelet production; however, this may pose some safety concerns. Furthermore, many novel culture systems have been developed to scale up the production of iPlatelets, including 2D flow systems, 3D rotary systems, and vertical reciprocal motion liquid culture bioreactors. The development of new gene-editing techniques, such as CRISPR/Cas9, can be used to solve allogeneic immunity of platelet transfusions by knocking out the expression of B2M. Additionally, the functions of iPlatelets were also evaluated from multiple aspects, including but not limited to morphology, structure, cytoskeletal organization, granule content, DNA content, and gene expression. Although the production and functions of iPlatelets are close to meeting clinical application requirements in both quantity and quality, there is still a long way to go for their large-scale production and clinical application. Here, we summarize the diverse methods of platelet production and update the progresses of iPlatelets. Furthermore, we highlight recent advances in our understanding of key transcription factors or molecules that determine the platelet differentiation direction.
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Skała, Ewa, Joanna Makowczyńska, Joanna Wieczfinska, Tomasz Kowalczyk, and Przemysław Sitarek. "Caffeoylquinic Acids with Potential Biological Activity from Plant In vitro Cultures as Alternative Sources of Valuable Natural Products." Current Pharmaceutical Design 26, no. 24 (July 21, 2020): 2817–42. http://dx.doi.org/10.2174/1381612826666200212115826.
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Background: For a long time, the researchers have been looking for new efficient methods to enhance production and obtain valuable plant secondary metabolites, which would contribute to the protection of the natural environment through the preservation of various plant species, often rare and endangered. These possibilities offer plant in vitro cultures which can be performed under strictly-controlled conditions, regardless of the season or climate and environmental factors. Biotechnological methods are promising strategies for obtaining the valuable plant secondary metabolites with various classes of chemical compounds including caffeoylquinic acids (CQAs) and their derivatives. CQAs have been found in many plant species which are components in the daily diet and exhibit a wide spectrum of biological activities, including antioxidant, immunomodulatory, antihypertensive, analgesic, anti-inflammatory, hepato- and neuroprotective, anti-hyperglycemic, anticancer, antiviral and antimicrobial activities. They have also been found to offer protection against Alzheimer’s disease, and play a role in weight reduction and lipid metabolism control, as well as modulating the activity of glucose-6-phosphatase involved in glucose metabolism. Methods: This work presents the review of the recent advances in use in vitro cultures of various plant species for the alternative system to the production of CQAs and their derivatives. Production of the secondary metabolites in in vitro culture is usually performed with cell suspension or organ cultures, such as shoots and adventitious or transformed roots. To achieve high production of valuable secondary metabolites in in vitro cultures, the optimization of the culture condition is necessary with respect to both biomass accumulation and metabolite content. The optimization of the culture conditions can be achieved by choosing the type of medium, growth regulators or growth conditions, selection of high-productivity lines or culture period, supplementation of the culture medium with precursors or elicitor treatments. Cultivation for large-scale in bioreactors and genetic engineering: Agrobacterium rhizogenes transformation and expression improvement of transcriptional factor or genes involved in the secondary metabolite production pathway are also efficient strategies for enhancement of the valuable secondary metabolites. Results: Many studies have been reported to obtain highly productive plant in vitro cultures with respect to CQAs. Among these valuable secondary metabolites, the most abundant compound accumulated in in vitro cultures was 5-CQA (chlorogenic acid). Highly productive cultures with respect to this phenolic acid were Leonurus sibiricus AtPAP1 transgenic roots, Lonicera macranthoides and Eucomia ulmoides cell suspension cultures which accumulated above 20 mg g-1 DW 5-CQA. It is known that di- and triCQAs are less common in plants than monoCQAs, but it was also possible to obtain them by biotechnological methods. Conclusion: The results indicate that the various in vitro cultures of different plant species can be a profitable approach for the production of CQAs. In particular, an efficient production of these valuable compounds is possible by Lonicera macranthoides and Eucomia ulmoides cell suspension cultures, Leonurus sibiricus transformed roots and AtPAP1 transgenic roots, Echinacea angustifolia adventitious shoots, Rhaponticum carthamoides transformed plants, Lavandula viridis shoots, Sausera involucrata cell suspension and Cichorium intybus transformed roots.
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Pandey, Puspa R., Amarel Tomney, Marites T. Woon, Nicholas Uth, Farjad Shafighi, Igor Ngabo, Haritha Vallabhaneni, Yonatan Levinson, Eytan Abraham, and Inbar Friedrich Ben-Nun. "End-to-End Platform for Human Pluripotent Stem Cell Manufacturing." International Journal of Molecular Sciences 21, no. 1 (December 21, 2019): 89. http://dx.doi.org/10.3390/ijms21010089.
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Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.
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Mulero-Cerezo, Joaquín, Álvaro Briz-Redón, and Ángel Serrano-Aroca. "Saccharomyces Cerevisiae Var. Boulardii: Valuable Probiotic Starter for Craft Beer Production." Applied Sciences 9, no. 16 (August 8, 2019): 3250. http://dx.doi.org/10.3390/app9163250.
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The use of probiotic starters remaining viable in unpasteurized and unfiltered beers could significantly increase health benefits. Here, the probiotic Saccharomyces cerevisiae var. boulardii (Scb) and a commercial Saccharomyces cerevisiae (Sc) strain, which is commonly employed in the brewing industry, are compared as single starters. The healthy value of the produced beers and growth performance in a laboratory bioreactor are analysed by determining antioxidant activity, phenolic content and profile, alcohol, biomass growth modelling by the logistic and Gompertz equations, biovolume estimation from 2D microscopy images, and yeast viability after fermentation. Thus, in this study, the craft beer produced with the probiotic yeast possessed higher antioxidant activity, lower alcohol content, similar sensory attributes, much higher yeast viability and more acidification, which is very desirable to reduce contamination risks at large-scale production. Furthermore, Scb exhibited faster growth in the bioreactor culture and larger cell volumes than Sc, which increases the probiotic volume of the final craft beer.
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Perry, Christopher, and Andrea C. M. E. Rayat. "Lentiviral Vector Bioprocessing." Viruses 13, no. 2 (February 9, 2021): 268. http://dx.doi.org/10.3390/v13020268.
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Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.
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Martinez, Andres, Richard McMahon, Kathleen Jenkins, and William M. Miller. "Using Computational Fluid Dynamics (CFD) to Enhance Ex Vivo Platelet Production Via Shear Forces within Microfluidic Bioreactors." Blood 128, no. 22 (December 2, 2016): 1352. http://dx.doi.org/10.1182/blood.v128.22.1352.1352.
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Abstract Currently, platelet transfusions are entirely dependent on human volunteer donors, and these methods are limited by a 5-day shelf life and differences in donor/recipient immunology. In vivo, platelets are formed when bone marrow megakaryocytes (Mks) extend long, cytoplasmic projections, called proplatelets (proPLTs), into the sinusoid where shear forces accelerate PPL elongation and release platelets into circulation. Methods of in vitro platelet production have yielded less than 10 platelets/Mk (plts/Mk), compared to >1000 plts/Mk in vivo. There is a need for a clinically relevant process for in vitro platelet production, but much is unknown about what initiates and regulates PPL formation and how to maximize platelet release. We are exploring the production of functional platelet-like particles (PLPs) within a microfluidic bioreactor that utilizes shear forces on Mks to generate proPLTs and PLPs. Microfluidic devices have emerged as a valuable tool for cell culture studies. Advantages include low input cell requirements, the ability to screen multiple conditions in parallel, compatibility with time-lapse imaging, and tight control of microenvironment conditions. In addition, device fabrication is straightforward and inexpensive using soft photolithography. Through the use of computational fluid dynamics (CFD) simulations and microfluidic device fabrication, a design - test - build methodology was used to develop a dual-flow microfluidic bioreactor system with uniform shear stress at levels similar to those found in the bone marrow niche. We are using this bioreactor to study the proPLT formation process and enhance in vitro PLP yields. Experimental studies were conducted to validate the simulations in terms of streamline profiles and flow patterns with and without cell capture. Microenvironment characteristics include but are not limited to extracellular matrix (ECM) protein coatings. Furthermore, the design of the bioreactor allows for a wide physiological shear rate range. Our bioreactor design yields 21 ± 3 PLPs/Mk. Bioreactor-derived PLPs are shown to exhibit functional activity, as evidenced by CD41a and CD42b surface marker expression, activation (PAC1 binding and CD62P expression) in the presence of thrombin agonist, and morphological/cytoskeletal changes upon binding to fibrinogen - before and after activation. The system can be further scaled, for example, through parallelization of reactors. Furthermore, the videos and images captured within our system show that Mks make beads-on-a-string proPLTs, thick extensions that thin out into characteristic proPLTs, what appears to be shuttling of PLPs into larger bodies, and most interestingly, a burst of single PLPs upon Mk trapping. A second round of proPLT formation and PLP release by trapped Mks can be induced by increasing the flow rate through the central channel. Future studies with small-molecule modulators of the actin and microtubule cytoskeleton will help to understand the factors that regulate the initiation of proPLT formation and PLP release and improve in vitro platelet yields. Figure (A) Schematic for bioreactor. Two different syringe pumps are used, allowing independent flow rate changes to each channel. (B) CFD simulations of shear stress through 7-µm slits at a flow rate of 1.5 µL/min in each channel. The bioreactor generates uniform shear profiles across all slits, within and above the physiological shear stress range (1 - 4 dynes/cm2 in marrow sinusoids). (C) Bioreactor is positioned over a microscope equipped with real-time imaging and green fluorescence. The entire system is placed inside an incubator at 370C and 5% CO2. Media is perfused through the system before Mk loading. Image shows a cell-free system of the fabricated bioreactor. (D) Upon Mk (Calcein AM live stain) loading and shear exposure, Mks generate proPLTs through the slits that then get fragmented by the secondary shear flow (image at 20X, scale bar 50 μm). Figure. (A) Schematic for bioreactor. Two different syringe pumps are used, allowing independent flow rate changes to each channel. (B) CFD simulations of shear stress through 7-µm slits at a flow rate of 1.5 µL/min in each channel. The bioreactor generates uniform shear profiles across all slits, within and above the physiological shear stress range (1 - 4 dynes/cm2 in marrow sinusoids). (C) Bioreactor is positioned over a microscope equipped with real-time imaging and green fluorescence. The entire system is placed inside an incubator at 370C and 5% CO2. Media is perfused through the system before Mk loading. Image shows a cell-free system of the fabricated bioreactor. (D) Upon Mk (Calcein AM live stain) loading and shear exposure, Mks generate proPLTs through the slits that then get fragmented by the secondary shear flow (image at 20X, scale bar 50 μm). Disclosures No relevant conflicts of interest to declare.
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Santos, M. R. A., M. G. R. Ferreira, M. C. M. Guimarães, R. A. Lima, and C. L. L. G. Oliveira. "Callogenesis in leaves of Kalanchoe pinnata Lam. by 2,4-D and BA action." Revista Brasileira de Plantas Medicinais 16, no. 3 suppl 1 (2014): 760–64. http://dx.doi.org/10.1590/1983-084x/13_031.
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The Kalanchoe pinnata Lam. is a bush species of the Crassulaceae that is distinguished by its important medicinal properties. Its leaves are used as cataplasm to treat headaches and wounds. There is evidence for a hypotensive and anti-inflammatory effect. Techniques of plant tissue culture have been applied to plant species that produce substances likely to be explored in pharmacology, cell suspension being the main technique. At the industrial level, this method utilizes bioreactors in order to produce secondary metabolites on a large scale. The objective of this study was to evaluate the effects of in vitro combinations of 2,4-dichlorophenoxiacetic acid (2,4-D) and benzylaminopurine (BA) on callus induction in leaf explants of K. pinnata. Leaf fragments were inoculated in MS medium supplemented with 3.0% sucrose, 0.8% agar and factorial combinations of 2,4-D (0.00, 4.52, 9.06, 18.12 µM) and BA (0.00, 4.44, 8.88, 17.76 µM). The cultures were kept in the darkness at 24±2ºC for 50 days. The percentage of callus induction and the area of explants covered by callus cells were evaluated. In the absence of growth regulators, callus induction did not occur, with necrosis of all explants. The highest percentage of callus induction was 100%, obtained with the combination of 9.06 µM 2,4-D and 8.88 µM BA, but the calluses covered only 25% of the leaf area. The most efficient combination was 4.52 µM 2,4-D and 8.88 µM BA, resulting in 91% callus induction with 50 to 100% of the explants being covered by callus cells.