1,105 results on '"Perfusion Culture"'
Search Results
152. Medium Optimization for Perfused Culture to Provide High Product Titer
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Ray, N. G., Ozturk, S. S., Tung, A. S., Pang, R. H. L., Young, M. W., Runstadler, P. W., Kaminogawa, S., editor, Ametani, A., editor, and Hachimura, S., editor
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- 1993
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153. Kinetics of Cell Metabolism and Antibody Production in High Concentration Perfusion Cultures
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Zhang, S., Handa-Corrigan, A., Spier, R. E., Kaminogawa, S., editor, Ametani, A., editor, and Hachimura, S., editor
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- 1993
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154. Amino Acid Metabolism of a High Density Perfusion Culture
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Hansen, Henrik Albahn, Damgaard, Bo, Emborg, Claus, Kaminogawa, S., editor, Ametani, A., editor, and Hachimura, S., editor
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- 1993
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155. Tissue Engineering of Meniscal Cartilage
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Neves, André A., Medcalf, Nick, Brindle, Kevin M., Lindner-Olsson, Elisabeth, editor, Chatzissavidou, Nathalie, editor, and Lüllau, Elke, editor
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- 2001
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156. The One Step Inoculation Concept: A New Seed-Train Expansion for Recombinant Mammalian Cell Lines
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Heidemann, Rüdiger, Mered, Mokhtar, Wang, D. Q., Gardner, Bruce, Zhang, Chun, Michaels, James, Henzler, Hans-Jürgen, Konstantinov, Konstantin, Lindner-Olsson, Elisabeth, editor, Chatzissavidou, Nathalie, editor, and Lüllau, Elke, editor
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- 2001
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157. Optimizing cell seeding and retention in a three-dimensional bioengineered cardiac ventricle: The two-stage cellularization model.
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Patel, Nikita M., Yazdi, Iman K., Tasciotti, Ennio, and Birla, Ravi K.
- Abstract
ABSTRACT Current cell seeding techniques focus on passively directing cells to a scaffold surface with the addition of dynamic culture to encourage cell permeation. In 3D tissue engineered constructs, cell retention efficiency is dependent on the cell delivery method, and biomaterial properties. Passive cell delivery relies on cell migration to the scaffold surface; biomaterial surface properties and porosity determine cell infiltration capacity. As a result, cell retention efficiencies remain low. The development of an effective two-stage cell seeding technique, coupled with perfusion culture, provides the potential to improve cellularization efficiency, and retention. This study, uses a chitosan bioengineered open ventricle (BEOV) scaffold to produce a two-stage perfusion cultured ventricle (TPCV). TPCV were fabricated by direct injection of 10 million primary rat neonatal cardiac cells, followed by wrapping of the outer scaffold surface with a 3D fibrin gel artificial heart muscle patch; TPCV were perfusion cultured for 3 days. The average biopotential output was 1.731 mV. TPCV cell retention following culture was approximately 5%. Cardiac cells were deposited on the scaffold surface and formed intercellular connections. Histological assessment displayed localized cell clusters, with some dissemination, and validated the observed presence of intercellular and gap-junction interactions. The study demonstrates initial effectiveness of our two-stage cell delivery concept, based on function and biological metrics. Biotechnol. Bioeng. 2016;113: 2275-2285. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]
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- 2016
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158. Investigation of the Influence of Glucose Concentration on Cancer Cells by Using a Microfluidic Gradient Generator without the Induction of Large Shear Stress.
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Tadashi Ishida, Takuya Shimamoto, Nobuya Ozaki, Satoshi Takaki, Takahiro Kuchimaru, Sinae Kizaka-Kondoh, and Toru Omata
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GLUCOSE ,CANCER cells ,MICROFLUIDIC devices - Abstract
A microfluidic device capable of precise chemical control is helpful to mimic tumor microenvironments in vitro, which are closely associated with malignant progression, including metastasis. Cancer cells under a concentration gradient of oxygen and other sustenance materials inside a tumor in vivo have recently been reported to increase the probability of metastasis. The influence of glucose concentration on cancer cells has not been measured well, whereas that of oxygen concentration has been thoroughly examined using microfluidic devices. This is because glucose concentrations can be controlled using microfluidic concentration gradient generators, which trade off temporal stability of the glucose concentration and shear stress on the cells; by contrast, oxygen concentration can be easily controlled without microfluidic device-induced shear stresses. To study cell division and migration responses as a function of glucose concentration, we developed a microfluidic device to observe cell behaviors under various chemical conditions. The device has small-cross-section microchannels for generating a concentration gradient and a large-cross-section chamber for cell culture. With this design, the device can achieve both a cell culture with sufficiently low shear stress on cell activity and a stable glucose concentration gradient. Experiments revealed that a low glucose concentration increased the total migration length of HeLa cells and that HeLa cells under a glucose concentration gradient exhibit random motion rather than chemotaxis. [ABSTRACT FROM AUTHOR]
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- 2016
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159. Characteristics of human cell line, F2N78, for the production of recombinant antibody in fed-batch and perfusion cultures.
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Seo, Joon Serk, Min, Byung Sub, Kwon, Young-Bum, Lee, Soo-Young, Cho, Jong-Moon, Park, Keun-Hee, Yang, Yae Ji, Maeng, Ki Eun, Chang, Shin-Jae, and Kim, Dong-Il
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CELL lines , *RECOMBINANT antibodies , *PERFUSION , *CELL culture , *BIOPHARMACEUTICS , *PROTEIN expression - Abstract
A human hybrid cell line, F2N78, was developed by somatic fusion of HEK293 and Namalwa cells for the production recombinant biopharmaceutical proteins. In this study, we performed perfusion culture to verify its potential in culture process used for human cell expression platform. Cell viability could be maintained over 90% and high viable cell density was obtained at higher than 1.0 × 10 7 cells/mL by bleeding process in perfusion culture. The cells were adapted well in both culture modes, but there were apparent differences in protein quality. Compared to fed-batch culture, degalactosylated forms such as G0F and G0 as well as Man5 showed no significant increases in perfusion culture. In terms of charge variants, acidic peaks increased, whereas main peaks constantly decreased according to the length of culture period in both methods. [ABSTRACT FROM AUTHOR]
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- 2016
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160. Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system.
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Minuth, Will and Denk, Lucia
- Abstract
To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written. [ABSTRACT FROM AUTHOR]
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- 2016
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161. An understanding of potential and limitations of alginate/PLL microcapsules as a cell retention system for perfusion cultures.
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Demont, Aurelie, Cole, Harriet, and Marison, Ian W.
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MOLECULAR capsules , *ALGINATES , *LYSINE , *POLYMER research , *GELATION - Abstract
Microcapsules for high cell density culture of mammalian cells have found an increasing interest, however, the poor stability of the microcapsules and the lack of characterisation methods led to few quantitative results. Alginate-poly-L-lysine (PLL) microcapsules have been studied in detail in order to form a basis for comparison of capsules made from different polymers. Since the microcapsules can be easily retained in the bioreactor without the need for a cell separation device, high cell densities were achieved with a maximum of 4 × 107 cell/mlmicrocapsules, corresponding to a colonisation of 5% of the internal capsule volume. Measurement of microcapsule integrity and mechanical resistance showed that alginate-PLL microcapsules are not suitable for perfusion cultures since they are very sensitive to media composition, mainly the presence of non-gelling ions that have a higher affinity for alginate than PLL and Ca2+, leading to the leakage of PLL and Ca2+, and to microcapsule rupture. [ABSTRACT FROM AUTHOR]
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- 2016
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162. Influence of Culture Conditions on Cell Proliferation in a Microfluidic Channel
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Sato, Kae, Sato, Miwa, Yokoyama, Mizuho, Hirai, Mai, and Furuta, Aya
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- 2019
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163. Enhancing Effect of Interleukin-6 on Antibody Productivity of a Hybridoma Cell Line
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Terada, Satoshi, Makishima, Fusao, Takamatsu, Hiroyuki, Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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164. A Novel Protein- and Hormone-Free Medium: Its Application to Hybridoma Culture and Monoclonal Antibody Production
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Inouye, Kuniyo, Toyoda, Kazuhisa, Kondo, Masahide, Maki, Koji, Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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165. Design and Characterization of Electrically Controlled Bioreactor.
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Kojima, J., Kobatake, E., Ikariyama, Y., Morioka, S., Nagaike, K., Aizawa, M., Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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166. Effect of Glutamine Supplementation on the Growth of Hybridoma Cells Cultivated in a Reactor Coupled with an Ammonia Removing System
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Nayve, F. R. P., Jr., Matsumura, M., Kataoka, H., Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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167. Large-Scale Mammalian Cell Cultures by Application of Macroporous Cellulose Carriers
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Ogata, Masafumi, Arikawa, Kei-ichi, Matsukura, Hideki, Yamazaki, Yoshinori, Suzuki, Akira, Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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168. High Density Perfusion Culture of Animal Cells Using a Novel Continuous Flow Centrifuge
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Jäger, V., Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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169. Oscillatory Behavior in Continuous Aggregate Cultures of Mammalian Cells
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Vits, H., Peshwa, M. V., Hu, W.-S., Murakami, H., editor, Shirahata, S., editor, and Tachibana, H., editor
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- 1992
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170. Bioreactors for Animal Cell Cultivation
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Hu, Wei-Shou, Vardar-Sukan, Fazilet, editor, and Sukan, Ş. Suha, editor
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- 1992
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171. Suspension Culture of Insect Cells in a Spin-Filter Bioreactor for Recombinant Protein Production
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Kim, H. R., Oh, J. H., Yang, J. M., Kang, S. K., Yoon, H. H., Chung, I. S., Furusaki, Shintaro, editor, Endo, Isao, editor, and Matsuno, Ryuichi, editor
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- 1992
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172. Production of Plant Secondary Metabolites from High Density Perfusion Cultures
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Su, W. Winston, Humphrey, Arthur E., Furusaki, Shintaro, editor, Endo, Isao, editor, and Matsuno, Ryuichi, editor
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- 1992
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173. Segregated growth kinetics of Escherichia coli DH5α-NH36 in exponential-fed perfusion culture for pDNA vaccine production.
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Munguía‐Soto, Rodolfo, García‐Rendón, Aurora, Garibay‐Escobar, Adriana, Guerrero‐Germán, Patricia, and Tejeda‐Mansir, Armando
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BACTERIAL genetics , *ESCHERICHIA coli , *PERFUSION , *DNA vaccines , *FLOW cytometry , *POLYMERASE chain reaction - Abstract
The clinical demand of plasmid DNA (pDNA) has been increasing constantly. An exponential-fed perfusion (EFP) culture is a new mode for plasmid production for clinical trials and commercialization. However, the culture conditions may lead to cell filamentation and growth cessation. In this study, the variation of the physiological state and the plasmid contents of Escherichia coli DH5α hosting pVAX1-NH36 in an EFP culture for application as a Leishmaniasis vaccine was investigated. The culture performance was monitored using flow cytometry (FC) and real-time quantitative PCR. The FC studies showed a high viability of cell population and a constant distribution of complexity and size. A high homogeneity of pDNA (>95 % of supercoiled) was obtained, which might be attributed to a better culture environment. The obtained plasmid specific and volumetric yields of 1.8 mg/g dcw and 36.5 mg/L represent typical values for laboratory-scale plasmid production in a defined medium. A segregated kinetic model of the perfusion system was developed and fitted to the experimental data (R² > 0.96). A practical conclusion of this work is that a space-time yield analysis of a bioprocess requires a viability evaluation. This new strategy of culture operation might help in the efficient production of pDNA for therapeutic use. [ABSTRACT FROM AUTHOR]
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- 2015
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174. Production of Recombinant Protein C in a Perfusion Culture
- Author
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Sugiura, T., Maruyama, H. B., Sasaki, Ryuzo, editor, and Ikura, Koji, editor
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- 1991
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175. Optimization of cell culture conditions for G-CSF (granulocyte colony-stimulating factor) production by genetically engineered Namalwa KJM-1 cells
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Hosoi, Shinji, Murozumi, Kazunari, Sasaki, Katsutoshi, Satoh, Mitsuo, Tamaoki, Tatsuya, Sato, Seiji, Sasaki, Ryuzo, editor, and Ikura, Koji, editor
- Published
- 1991
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176. Hybridoma Culture in the Hollow-Fiber System -The Effects of Growth Factors-
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Omasa, Takeshi, Kobayashi, Masaki, Nishikawa, Toshio, Shioya, Suteaki, Suga, Ken-ichi, Uemura, Syo-ichi, Imamura, Yoshio, Sasaki, Ryuzo, editor, and Ikura, Koji, editor
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- 1991
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177. Effects of Growth Factors on Hybridoma Culture in the Perfusion System
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Omasa, Takeshi, Kobayashi, Masaki, Nishikawa, Toshio, Shioya, Suteaki, Suga, Ken-ichi, Uemura, Syo-ichi, Imamura, Yoshio, Sasaki, Ryuzo, editor, and Ikura, Koji, editor
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- 1991
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178. Effects of Shear Stress on the Growth of Hybridoma Cells Cultivated in Serum-Free Medium Coupled with Ammonia Removing System
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Nayve, F. R., Jr., Matsumura, M., Kataoka, H., Sasaki, Ryuzo, editor, and Ikura, Koji, editor
- Published
- 1991
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179. Fermentation and Cell Culture Processes
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Ward, Owen P. and Ward, Owen P.
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- 1991
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180. Dynamic Physiological Culture of Ex Vivo Human Tissue: A Systematic Review
- Author
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Zahir Soonawalla, Aron Hughes, Daniel Ll Hughes, Somnath Mukherjee, and Eric O'Neill
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0301 basic medicine ,Cancer Research ,0206 medical engineering ,Context (language use) ,02 engineering and technology ,Biology ,dynamic physiological culture ,03 medical and health sciences ,Tissue culture ,bioreactor ,Perfusion Culture ,In vivo ,primary human tissue culture ,cancer ,perfusion culture ,RC254-282 ,cell culture ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Human physiology ,020601 biomedical engineering ,Cell biology ,030104 developmental biology ,Oncology ,Cell culture ,organotypic tissue slice culture ,Systematic Review ,Ex vivo - Abstract
Simple Summary Within cancer research, a strong emphasis is placed on the development of models that accurately reproduce the conditions in which tumours develop and grow. A limitation of several models is that they fail to replicate the tumour’s blood supply. Our aim was to evaluate the concurrent literature regarding dynamic physiological culture techniques that have been used to successfully culture human tissue. We conducted a systematic review of the literature and identified 22 articles that described the use of different dynamic culture techniques in order to create a system that was physiologically representative. The most common method described was the use of perfusion culture. This article serves as a detailed reference of novel technologies that can be implemented within cancer research in order to improve the physiological conditions of current culture techniques. Realistic cancer models will translate into a greater understanding of the disease which will directly impact on patient outcomes. Abstract Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h–60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.
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- 2021
181. Withdrawal: Erickson, P., Houwayek, T., Teryek, M. and Parekkadan, B. (2021), A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion. Biotechnology and Applied Biochemistry. https://doi.org/10.1002/bab.2183
- Author
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Tony Houwayek, Patrick Erickson, Biju Parekkadan, and Matthew Teryek
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Computer science ,Process Chemistry and Technology ,Microfluidics ,Biomedical Engineering ,Bioengineering ,General Medicine ,Replicate ,Residence time (fluid dynamics) ,Applied Microbiology and Biotechnology ,Perfusion Culture ,Cell culture ,Drug Discovery ,Bioreactor ,Molecular Medicine ,Transient (computer programming) ,Sample collection ,Biological system ,Biotechnology - Abstract
Cells absorb and secrete substances to-and-from their surroundings as part of metabolism, signaling, and other functions. These fluxes are dynamic, changing over time in response to external cues and internal programs. Static cultures are inadequate for measuring these dynamics because the environments of the cells change as substances accumulate or are depleted from medium, which affects cell behavior in unintended ways. Static cultures also offer limited time resolution due to the impracticality of frequent or prolonged manual timepoint sampling, and cannot expose cells to smooth, transient changes in stimulus concentrations. Perfusion cultures overcome these challenges by constantly replenishing medium to maintain cellular environments, while continuously sampling the effluent stream. However, many perfusion culture implementations are microfluidic devices, which cannot culture large tissue constructs and require specialized equipment and expertise to use. Previously published macrofluidic devices often use custom parts that are difficult to replicate, do not support both solute input control and measurement, and do not account for effects of dispersion on measured signals. In this study, a multiplexed macrofluidic perfusion culture platform was developed to measure secretion and absorption rates of substances by cells in a temporally controlled environment. The modular platform can handle up to 31 streams with automated sample collection using a fraction collector. This paper presents the assembly of this dynamic bioreactor and a method for quantitatively handling the effects of dispersion using residence time distributions. The system is then applied to monitor the secretion of a circadian clock gene-driven reporter from transfected cells. This article is protected by copyright. All rights reserved.
- Published
- 2021
182. Generation of a large-scale vascular bed for the in vitro creation of three-dimensional cardiac tissue
- Author
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Hidekazu Sekine, Minoru Ono, Akitoshi Inui, Kazunori Sano, Eiji Kobayashi, Katsuhisa Matsuura, Tatsuya Shimizu, Izumi Dobashi, and Azumi Yoshida
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0301 basic medicine ,Pathology ,medicine.medical_specialty ,HUVECs, human umbilical vein endothelial cells ,Angiogenesis ,Biomedical Engineering ,HE, hematoxylin/eosin ,DMEM, Dulbecco's Modified Eagle Medium ,Regenerative medicine ,Vascular bed ,Biomaterials ,03 medical and health sciences ,0302 clinical medicine ,bFGF, basic fibroblast growth factor ,Tissue engineering ,medicine.artery ,Medicine ,Superior mesenteric artery ,lcsh:QH573-671 ,Superior mesenteric vein ,hiPSC, human induced pluripotent stem cells ,PERV, porcine endogenous retrovirus ,GFP, green fluorescent protein ,lcsh:R5-920 ,hiPSCs ,lcsh:Cytology ,business.industry ,3D, three-dimensional ,medicine.disease ,NHDFs, normal human dermal fibroblasts ,VEGF, vascular endothelial growth factor ,Small intestine ,ECM, extracellular matrix ,Transplantation ,030104 developmental biology ,medicine.anatomical_structure ,Cardiac cell sheet ,Heart failure ,Original Article ,Perfusion culture ,lcsh:Medicine (General) ,business ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Introduction: The definitive treatment for severe heart failure is transplantation. However, only a small number of heart transplants are performed each year due to donor shortages. Therefore, novel treatment approaches based on artificial organs or regenerative therapy are being developed as alternatives. We have developed a technology known as cell sheet-based tissue engineering that enables the fabrication of functional three-dimensional (3D) tissue. Here, we report a new technique for engineering human cardiac tissue with perfusable blood vessels. Our method involved the layering of cardiac cell sheets derived from human induced pluripotent stem cells (hiPSCs) on a vascular bed derived from porcine small intestinal tissue. Methods: For the vascular bed, a segment of porcine small intestine was harvested together with a branch of the superior mesenteric artery and a branch of the superior mesenteric vein. The small intestinal tissue was incised longitudinally, and the mucosa was resected. Human cardiomyocytes derived from hiPSCs were co-cultured with endothelial cells and fibroblasts on a temperature-responsive dish and harvested as a cardiac cell sheet. A triple-layer of cardiac cell sheets was placed onto the vascular bed, and the resulting construct was subjected to perfusion culture in a bioreactor system. Results: The cardiac tissue on the vascular bed pulsated spontaneously and synchronously after one day of perfusion culture. Electrophysiological recordings revealed regular action potentials and a beating rate of 105 ± 13/min (n = 8). Furthermore, immunostaining experiments detected partial connection of the blood vessels between the vascular bed and cardiac cell sheets. Conclusions: We succeeded in engineering spontaneously beating 3D cardiac tissue in vitro using human cardiac cell sheets and a vascular bed derived from porcine small intestine. Further development of this method might allow the fabrication of functional cardiac tissue that could be used in the treatment of severe heart failure. Keywords: Cardiac cell sheet, Vascular bed, Perfusion culture, hiPSCs, Angiogenesis
- Published
- 2019
183. Fiber-Reinforced Silk Composite for Enhanced Urokinase Production Using High-Density Perfusion Culture and Bioactive Molecule Supplementation
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G. Janani, Biman B. Mandal, and Shivanshi Kumar
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Urokinase ,biology ,Chemistry ,0206 medical engineering ,Biomedical Engineering ,Fibroin ,02 engineering and technology ,021001 nanoscience & nanotechnology ,biology.organism_classification ,020601 biomedical engineering ,Cell biology ,Biomaterials ,Urokinase receptor ,Perfusion Culture ,Bombyx mori ,Cell culture ,medicine ,HT1080 ,0210 nano-technology ,Plasminogen activator ,medicine.drug - Abstract
Urokinase plasminogen activator (uPA) has been extensively used as a thrombolytic drug in cases of myocardial infarction, thromboembolism, and ischemic brain stroke. Media optimization and high-density perfusion culture are the decisive factors that facilitate enhanced urokinase production in a conditioned medium. In this study, we have aimed for a high-density perfusion culture of HT1080, a human fibrosarcoma cell line, by formulating optimal media for enhanced urokinase productivity. Four scaffold variants were fabricated from silk fibroin and microfibers of Bombyx mori (BM) and Antheraea assamensis (AA) and physico-chemically characterized. Field emission scanning electron microscopy studies revealed a heterogeneous distribution of pores with interconnected networks supporting cell infiltration, attachment, and long-term viability. AA-based fiber-reinforced scaffolds (ASAF) demonstrated superior mechanical strength, integral stability, and increased cell proliferation as compared to pure silk scaffolds. Media formulation was accomplished by limiting serum concentration (2% FBS) and supplementing with 20 μg/mL arginine and 20 ng/mL TGF-β1 to retain the stationary phase of cells and augment the urokinase production. A perfusion bioreactor culture of HT1080-laden scaffolds in the presence of formulated media was performed for improving the production of urokinase, with a maximum activity of 432 U/L. Also, gene expression analysis revealed that the individual silk scaffolds have different effects on regulating the expression of plasminogen activator urokinase and plasminogen activator urokinase receptor. In brief, our results suggest that a perfusion bioreactor culture of HT1080-laden ASAF scaffolds in formulated media promotes an increased urokinase production, such that it can be further used as a novel 3D matrix platform for industrial production of the lifesaving uPA drug.
- Published
- 2019
184. Process design and development of a mammalian cell perfusion culture in shake‐tube and benchtop bioreactors
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Hervé Broly, Andrea Müller, Massimo Morbidelli, Jonathan Souquet, and Moritz Wolf
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Cell Survival ,Chemistry ,Cell Culture Techniques ,Cell Count ,Bioengineering ,CHO Cells ,Equipment Design ,Shake ,Bleed ,Applied Microbiology and Biotechnology ,Perfusion ,Bioreactors ,Cricetulus ,Perfusion Culture ,Perfusion rate ,High productivity ,Mammalian cell ,Bioreactor ,Animals ,Biotechnology ,Biomedical engineering - Abstract
The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCDmax ), minimum cell-specific perfusion rate (CSPRmin ), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell-1 ·day-1 ) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 106 cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures.
- Published
- 2019
185. Quality by Design characterization of the perfusion culture process for recombinant FVIII
- Author
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Sang Kyul Han, Yong Jae Kim, Chan Wha Kim, Sang Hoon Paik, Yoomin Jeong, Ji youn Kim, and Sunggeun Lee
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Quality Control ,0301 basic medicine ,Time Factors ,Process (engineering) ,Cell Culture Techniques ,Bioengineering ,Applied Microbiology and Biotechnology ,Recombinant factor viii ,Quality by Design ,law.invention ,03 medical and health sciences ,Bioreactors ,0302 clinical medicine ,Perfusion Culture ,law ,Bioreactor ,030212 general & internal medicine ,Mathematics ,Pharmacology ,Factor VIII ,General Immunology and Microbiology ,Sulfates ,Temperature ,Reproducibility of Results ,General Medicine ,Hydrogen-Ion Concentration ,Recombinant Proteins ,030104 developmental biology ,Research Design ,Recombinant DNA ,Tyrosine ,Biochemical engineering ,Critical quality attributes ,Design space ,Biotechnology - Abstract
A Quality by Design (QbD) concept was applied to characterize a cell culture process for production of the recombinant Factor VIII (rFVIII). We characterized the production bioreactor process and defined the design space by applying risk assessment to determine potential critical process parameters (CPPs) impacting critical quality attributes (CQAs). Characterization studies were subsequently performed using a qualified scaled-down model (SDM) and a multi-factorial design of experiment (DOE) approach to determine both the individual and combined impacts of the potential CPPs on CQAs. Among the operating parameters characterized, production temperature, production pH and a shift in the timing of production affected rFVIII activity and tyrosine sulfation level. Finally, we identified CPPs and established a design space for the cell culture process to identify appropriate conditions for routine manufacturing.
- Published
- 2019
186. Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture
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Duygu Dikicioglu, Christopher Spencer, Richard Turner, William Holmes, Rahul Pradhan, David J. Sewell, Nigel K.H. Slater, Stephen G. Oliver, Ray Field, Oliver, Stephen [0000-0001-6330-7526], Slater, Nigel [0000-0002-0207-9440], Dikicioglu, Duygu [0000-0002-3018-4790], and Apollo - University of Cambridge Repository
- Subjects
0106 biological sciences ,0301 basic medicine ,Cell Survival ,Process development ,Low resource ,gravity cell settling ,Bioengineering ,CHO Cells ,01 natural sciences ,Applied Microbiology and Biotechnology ,Article ,ARTICLES ,03 medical and health sciences ,Bioreactors ,Cricetulus ,Perfusion Culture ,Cricetinae ,010608 biotechnology ,By-product ,Bioreactor ,Animals ,Microscale chemistry ,microscale process development ,Bioprocess Engineering and Supporting Technologies ,perfusion reactors ,Equipment Design ,Replicate ,Hydrogen-Ion Concentration ,Chinese hamster ovary ,Oxygen ,030104 developmental biology ,Batch Cell Culture Techniques ,upstream processing ,Environmental science ,Perfusion ,Biotechnology ,Biomedical engineering - Abstract
Without a scale‐down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high‐capacity microscale system that is typically used in fed‐batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 106 mg/cell/day and 7 × 10 7 cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.
- Published
- 2019
187. Substrate-source flexibility of an exponential-fed perfusion process to produce plasmid DNA for use as leishmaniasis vaccine
- Author
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Armando Tejeda-Mansir, Roberto Guzman, Angelica García-Rendón, and Aurora García-Rendón
- Subjects
process flexibility ,0106 biological sciences ,0303 health sciences ,lcsh:Biotechnology ,Tropical disease ,Leishmaniasis ,macromolecular substances ,Biology ,medicine.disease ,01 natural sciences ,Virology ,03 medical and health sciences ,plasmid dna ,Plasmid dna ,vaccine ,lcsh:TP248.13-248.65 ,medicine ,growth modelling ,perfusion culture ,030304 developmental biology ,010606 plant biology & botany ,Biotechnology - Abstract
The use of plasmid DNA (pDNA) for human vaccines is a novel approach against leishmaniasis, a neglected tropical disease with severe clinical manifestations. The development of feasible bioprocesses to obtain such vaccines is a public-health priority. The aim of this work was to investigate the substrate-source flexibility of an exponential-fed perfusion (EFP) system to produce the plasmid pVAX1-NH36 for use as a leishmaniasis vaccine. Batch and EFP cultures were conducted using Escherichia coli DH5α as a host and glucose or glycerol as a carbon source. The culture kinetics of the cell, substrate and plasmid concentrations were measured. Mathematical kinetics models were fitted to experimental data and used to describe the system comportment (r2 > 0.95). Plasmid productivities of 13.3 mg/(L h) using glucose and 19.4 mg/(L h) using glycerol were obtained. These levels represent a 1–3-fold increase in performance index compared with previously reported cultures using E. coli DH5α. The novel aspect of this work is the demonstration of the flexibility of EFP cultures for production of pDNA vaccines. Our data suggest that E. coli engineering to increase pDNA production using glucose can be circumvented with an EFP culture, reducing the host strain development costs. In addition, the greater productivity of EFP cultures entails a reduction in manufacturing costs.
- Published
- 2019
188. Development of a multi-scale model to simulate mesenchymal stem cell osteogenic differentiation within hydrogels in a rotating wall bioreactor.
- Author
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Győrgy, Romuald, Kostoglou, Margaritis, Mantalaris, Athanasios, and Georgiadis, Michael C.
- Subjects
- *
MESENCHYMAL stem cell differentiation , *MULTISCALE modeling , *GELATIN , *HYDROGELS , *MESENCHYMAL stem cells , *GENETIC regulation - Abstract
Demand for mesenchymal stem cell (MSC)-based therapies for various clinical applications has been steadily increasing. Suitable implants can be obtained by in-vitro osteogenic differentiation of stem cells to meet this demand, at least in part. This paper proposes a multiscale population balance model for the osteogenic differentiation of mesenchymal stem cells encapsulated within alginate-gelatin hydrogels in a rotating wall bioreactor. The mathematical model incorporates key genes and metabolic pathways linked to proliferation and osteogenesis and captures spatial as well as cell cycle heterogeneity within the hydrogels inside the bioreactor. The discretized formulation of the model consists of 12,563 simultaneous ordinary differential equations. Concentrations of key metabolites (e.g. , glucose, pyruvate, glutamine, lactate) were compared with experimental data from static well plate cultures, rendering the model predictions as adequate. Gene activation (Runx2 and osteonectin) in alginate-gelatin-bead-encapsulated cells was slightly delayed compared with well-plate cultures. Global sensitivity analysis revealed that parameters related to gene expression (decay rates and gene transcription activation constants) carry the most significance and should be the focus of future parameter estimation. Hydrogel bead size does not meaningfully impact simulation outcomes for bead diameters in the 2–3 mm range. The mathematical model proposed in this work can function as a foundation for model-based bioreactor and bioprocess optimization when coupled with a high-performance computing system. • A multiscale model of a rotating wall bioreactor. • Multiscale approach connects reactor scale effects with intracellular activity. • Concentrations of key metabolites were compared with experimental data. • Parameters related to gene expression carry the most significance. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
189. The effect of hyperosmolality application time on production, quality, and biopotency of monoclonal antibodies produced in CHO cell fed-batch and perfusion cultures
- Author
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Xiang Wu, Ying Zhang, Chen Zheng, Qiang Fu, Jinyan Qin, Zheng Huang, Yanchao Wang, and Zhigang Xia
- Subjects
medicine.drug_class ,Sodium ,Cell ,chemistry.chemical_element ,CHO Cells ,Sodium Chloride ,Monoclonal antibody ,Applied Microbiology and Biotechnology ,Cell Line ,Andrology ,03 medical and health sciences ,Bioreactors ,Cricetulus ,Perfusion Culture ,Cricetinae ,medicine ,Animals ,Cytotoxicity ,Cell Proliferation ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Chemistry ,Chinese hamster ovary cell ,Osmolar Concentration ,Antibodies, Monoclonal ,General Medicine ,Immunoglobulin E ,medicine.anatomical_structure ,CD52 Antigen ,Batch Cell Culture Techniques ,Renal physiology ,Perfusion ,Biotechnology - Abstract
Hyperosmolality has been commonly investigated due to its effects on the production and quality characteristics of monoclonal antibodies (mAbs) produced in CHO cell fed-batch cultures. However, the application of hyperosmolality at different times and its effect on biopotency have seldom been researched, especially in perfusion culture. In our study, different degrees of hyperosmolality induced by sodium chloride were investigated in anti-IgE rCHO cell fed-batch cultures and anti-CD52 rCHO cell perfusion cultures during the initial and stable phases. The results showed that the initial hyperosmolality group (IHG) in fed-batch and early phase of perfusion cultures exhibited significant suppression of the viable cell density yet an enhancement in specific productivity, whereas the stable hyperosmolality group (SHG) achieved higher mAb production in both fed-batch and perfusion cultures. Additionally, the SHG produced less aggregates and acidic charge variants than IHG in fed-batch culture, which differed from perfusion cultures. However, the contents of non-glycosylation heavy chain (NGHC) and man5 were higher in SHG than in IHG in fed-batch cultures at plus 60 and 120 mOsm/kg, which was similar to perfusion cultures. Furthermore, the biopotency in the IHG was higher than in the SHG at plus 60 and 120 mOsm/kg in fed-batch cultures, which is similar to complement-dependent cytotoxicity (CDC) efficacy in perfusion cultures. The biopotency of all group was acceptable, except FI3. Thus, the study shows that hyperosmolality at a certain level could be beneficial for both mAb production, quality and biopotency, which could play an important role in process development for commercial production.
- Published
- 2018
190. Influence of Culture Conditions on Cell Proliferation in a Microfluidic Channel
- Author
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Kae Sato, Miwa Sato, Aya Furuta, Mizuho Yokoyama, and Mai Hirai
- Subjects
Cell Survival ,Microfluidics ,Cell ,Cell Culture Techniques ,02 engineering and technology ,01 natural sciences ,Analytical Chemistry ,HeLa ,Perfusion Culture ,Lab-On-A-Chip Devices ,medicine ,Humans ,Lactic Acid ,Cell Proliferation ,Microchannel ,biology ,Chemistry ,Cell growth ,Cell Cycle ,010401 analytical chemistry ,Endothelial Cells ,Equipment Design ,Microfluidic Analytical Techniques ,021001 nanoscience & nanotechnology ,biology.organism_classification ,Culture Media ,0104 chemical sciences ,Endothelial stem cell ,Glucose ,medicine.anatomical_structure ,Cell culture ,Biophysics ,0210 nano-technology ,HeLa Cells - Abstract
Microfluidic devices have emerged as a new cell culture tool, which can mimic the structure and physiology of living human organs. However, no standardized culture method for a microfluidic device has yet been established. Here, we describe the effects of various conditions on cell proliferation in a microchannel with a depth smaller than 100 μm. Primary endothelial cell proliferation was suppressed with a decrease in the culture medium volume per cell culture area. Moreover, cell growth was compared with or without medium flow, and the optimum culture condition was determined to be 1 μL/h flow in a 65-μm-deep microchannel. In addition, glucose consumption was greater under fluidic conditions than under static conditions, and the ability of tumor (HeLa) cells to convert glucose into lactate appeared to be higher in a static culture than that in a fluidic culture. Overall, our results will serve as a useful guide for designing a microfluidic cell culture platform in a channel smaller than 100 μm.
- Published
- 2018
191. The effect of macropore size of hydroxyapatite scaffold on the osteogenic differentiation of bone mesenchymal stem cells under perfusion culture
- Author
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Dongqin Xiao, Yumei Liu, Jie Weng, Wei Zhi, Cheng-Dong Zhang, and Feng Shi
- Subjects
biology ,Cell growth ,Chemistry ,Cellular differentiation ,Mesenchymal stem cell ,osteogenic differentiation ,Cell biology ,Biomaterials ,Perfusion Culture ,stomatognathic system ,HAp scaffolds ,Osteocalcin ,biology.protein ,macropore size ,Alkaline phosphatase ,AcademicSubjects/SCI01410 ,Osteopontin ,Stem cell ,AcademicSubjects/MED00010 ,perfusion culture ,Research Article - Abstract
Previous studies have proved that dynamic culture could facilitate nutrients transport and apply mechanical stimulation to the cells within three-dimensional scaffolds, thus enhancing the differentiation of stem cells towards the osteogenic phenotype. However, the effects of macropore size on osteogenic differentiation of stem cells under dynamic condition are still unclear. Therefore, the objective of this study was to investigate the effects of macropore size of hydroxyapatite (HAp) scaffolds on osteogenic differentiation of bone mesenchymal stem cells under static and perfusion culture conditions. In vitro cell culture results showed that cell proliferation, alkaline phosphate (ALP) activity, mRNA expression of ALP, collagen-I (Col-I), osteocalcin (OCN) and osteopontin (OPN) were enhanced when cultured under perfusion condition in comparison to static culture. Under perfusion culture condition, the ALP activity and the gene expression of ALP, Col-I, OCN and OPN were enhanced with the macropore size decreasing from 1300 to 800 µm. However, with the further decrease in macropore size from 800 to 500 µm, the osteogenic related gene expression and protein secretion were reduced. Computational fluid dynamics analysis showed that the distribution areas of medium- and high-speed flow increased with the decrease in macropore size, accompanied by the increase of the fluid shear stress within the scaffolds. These results confirm the effects of macropore size on fluid flow stimuli and cell differentiation, and also help optimize the macropore size of HAp scaffolds for bone tissue engineering.
- Published
- 2021
192. A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion
- Author
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Biju Parekkadan, Tony Houwayek, Patrick Erickson, Alexandra Burr, and Matthew Teryek
- Subjects
Cell Survival ,Microfluidics ,Circadian clock ,Biophysics ,Cell Culture Techniques ,Residence time (fluid dynamics) ,01 natural sciences ,Biochemistry ,Article ,Cell Physiological Phenomena ,03 medical and health sciences ,Perfusion Culture ,Bioreactors ,Bioreactor ,Transient (computer programming) ,Secretion ,Molecular Biology ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Chemistry ,010401 analytical chemistry ,Cell Biology ,0104 chemical sciences ,Culture Media ,Cell culture ,Biological system - Abstract
Cells exchange substances with their surroundings during metabolism, signaling, and other functions. These fluxes are dynamic, changing in response to external cues and internal programs. Static cultures are inadequate for measuring these dynamics because the environments of the cells change as substances accumulate or deplete from medium, unintentionally affecting cell behavior. Static cultures offer limited time resolution due to the impracticality of frequent or prolonged manual sampling, and cannot expose cells to smooth, transient changes in stimulus concentrations. In contrast, perfusion cultures constantly maintain cellular environments and continuously sample the effluent stream. Existing perfusion culture systems are either microfluidic, which are difficult to make and use, or macrofluidic devices built from custom parts that neglect solute dispersion. In this study, a multiplexed macrofluidic perfusion culture platform was developed to measure secretion and absorption rates of substances by cells in a temporally controlled environment. The modular platform handles up to 31 streams with automated fraction collection. This paper presents the assembly of this dynamic bioreactor from commercially available parts, and a method for quantitatively handling the effects of dispersion using residence time distributions. The system is then applied to monitor the secretion of a circadian clock gene-driven reporter from engineered cells.
- Published
- 2021
193. Developments and opportunities in continuous biopharmaceutical manufacturing
- Author
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Abraham M. Lenhoff and Ohnmar Khanal
- Subjects
Time Factors ,Drug Industry ,Cost-Benefit Analysis ,Immunology ,Review ,Drug Costs ,Unit (housing) ,Biopharmaceuticals ,Workflow ,03 medical and health sciences ,0302 clinical medicine ,Immunology and Allergy ,Production (economics) ,Humans ,Technology, Pharmaceutical ,Biomanufacturing ,biosimilars ,continuous manufacturing ,perfusion culture ,Biosimilar Pharmaceuticals ,030304 developmental biology ,Pace ,filtration ,0303 health sciences ,viral inactivation ,Biological Products ,bioprocessing ,Biosimilar ,Investment (macroeconomics) ,process analytical technology ,Biopharmaceutical manufacturing ,Risk analysis (engineering) ,030220 oncology & carcinogenesis ,chromatography ,Lower cost ,Business ,monoclonal antibodies ,Biotechnology - Abstract
Today’s biologics manufacturing practices incur high costs to the drug makers, which can contribute to high prices for patients. Timely investment in the development and implementation of continuous biomanufacturing can increase the production of consistent-quality drugs at a lower cost and a faster pace, to meet growing demand. Efficient use of equipment, manufacturing footprint, and labor also offer the potential to improve drug accessibility. Although technological efforts enabling continuous biomanufacturing have commenced, challenges remain in the integration, monitoring, and control of traditionally segmented unit operations. Here, we discuss recent developments supporting the implementation of continuous biomanufacturing, along with their benefits.
- Published
- 2021
194. A microfluidic bubble perfusion device for brain slice culture
- Author
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Debalina Acharyya, Amirus Saleheen, Rebecca A. Prosser, and Christopher A. Baker
- Subjects
Materials science ,General Chemical Engineering ,Bubble ,Microfluidics ,02 engineering and technology ,Analytical Chemistry ,03 medical and health sciences ,Mice ,Perfusion Culture ,Slice preparation ,Carbogen ,Lab-On-A-Chip Devices ,Animals ,Droplet microfluidics ,030304 developmental biology ,0303 health sciences ,General Engineering ,Brain ,Microfluidic Analytical Techniques ,021001 nanoscience & nanotechnology ,Perfusion ,0210 nano-technology ,Ex vivo ,Biomedical engineering - Abstract
Ex vivo brain slice cultures are utilized as analytical models for studying neurophysiology. Common approaches to maintaining slice cultures include roller tube and membrane interface techniques. The rise of organ-on-chip technologies has demonstrated the value of microfluidic perfusion culture systems for sampling and analysis of complex biology under well-controlled in vitro or ex vivo conditions. A number of approaches to microfluidic brain slice culture have been developed, however these typically involve complex design, fabrication, or operational parameters in order to meet the high oxygen demands of brain slices. Here, we present proof-of-principle for a novel approach to microfluidic brain slice culture. In this system, which we term a microfluidic bubble perfusion device, principles of droplet microfluidics were employed to generate droplets of perfusion media dispersed between bubbles of carbogen gas, and brain tissue slices were perfused with the resulting monodispersed droplets and bubbles. The challenge of tissue immobilization in the flow system was addressed using a two-part cytocompatible carbohydrate-based tissue adhesive. Best practices are discussed for perfusion chamber designs that maintain segmented flow throughout the course of perfusion. Control of droplet and bubble volumes was possible across the range of ca. 4–15 μL, bubble generation frequency was well controlled in the range ca. 1–7 bubbles per min, and bubble duty cycle was well controlled across the range ca. 20–80%. Murine hypothalamic tissue slices containing the suprachiasmatic nuclei were successfully maintained for durations of 8–10 hours, with tissue remaining viable for the duration of perfusion as assessed by Ca2+ imaging and propidium iodide (PI) staining.
- Published
- 2021
195. Enhancing and stabilizing monoclonal antibody production by Chinese hamster ovary (CHO) cells with optimized perfusion culture strategies.
- Author
-
Liang K, Luo H, and Li Q
- Abstract
The perfusion medium is critical in maintaining high cell concentration in cultures for the production of monoclonal antibody by Chinese hamster ovary cells. In this study, the effects of perfusion culture strategies when using different media on the process stability, product titer, and product quality were investigated in 3-L bioreactor. The results indicated that continuous perfusion could maintain higher levels of cell density, product titer, and quality in comparison with those of the intermittent perfusion culture. Next, the perfusion culture conditions with different perfusion rates and temperature reduction methods were further optimized. When combining the high perfusion rates and delayed reduction of culture temperature at day 6, the product titer reached a higher level of 16.19 g/L with the monomer relative abundant of 97.6%. In this case, the main peak of the product reached 56.3% and the total N-glycans ratio was 95.2%. To verify the effectiveness of the optimized perfusion culture in a larger scale, a 200-L bioreactor was used to perform and the final product titer reached the highest level of 16.79 g/L at day 16. Meanwhile, the product quality (monomer abundant of 97.6%, main peak of 56.3%, and N-glycans ratio of 96.5%) could also be well maintained. This study provided some guidance for the high-efficient production of monoclonal antibody by CHO cells via optimized perfusion culture strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liang, Luo and Li.)
- Published
- 2023
- Full Text
- View/download PDF
196. Extracellular matrix in the trabecular meshwork: Intraocular pressure regulation and dysregulation in glaucoma.
- Author
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Vranka, Janice A., Kelley, Mary J., Acott, Ted S., and Keller, Kate E.
- Subjects
- *
TRABECULAR meshwork (Eye) , *EXTRACELLULAR matrix , *INTRAOCULAR pressure , *GLAUCOMA , *ANTERIOR eye segment , *AQUEOUS humor - Abstract
The trabecular meshwork (TM) is located in the anterior segment of the eye and is responsible for regulating the outflow of aqueous humor. Increased resistance to aqueous outflow causes intraocular pressure to increase, which is the primary risk factor for glaucoma. TM cells reside on a series of fenestrated beams and sheets through which the aqueous humor flows to exit the anterior chamber via Schlemm's canal. The outer trabecular cells are phagocytic and are thought to function as a pre-filter. However, most of the outflow resistance is thought to be from the extracellular matrix (ECM) of the juxtacanalicular region, the deepest portion of the TM, and from the inner wall basement membrane of Schlemm's canal. It is becoming increasingly evident that the extracellular milieu is important in maintaining the integrity of the TM. In glaucoma, not only have ultrastructural changes been observed in the ECM of the TM, and a significant number of mutations in ECM genes been noted, but the stiffness of glaucomatous TM appears to be greater than that of normal tissue. Additionally, TGFβ2 has been found to be elevated in the aqueous humor of glaucoma patients and is assumed to be involved in ECM changes deep with the juxtacanalicular region of the TM. This review summarizes the current literature on trabecular ECM as well as the development and function of the TM. Animal models and organ culture models targeting specific ECM molecules to investigate the mechanisms of glaucoma are described. Finally, the growing number of mutations that have been identified in ECM genes and genes that modulate ECM in humans with glaucoma are documented. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
197. Production of recombinant human growth hormone by rCHO cells in a depth filter perfusion system.
- Author
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Kim, Jae, Kwon, Hyeong, and Oh, Duk
- Subjects
- *
RECOMBINANT protein synthesis , *SOMATOTROPIN , *CHO cell , *PERFUSION , *RECOMBINANT microorganisms , *CELL culture - Abstract
Human growth hormone is a single-chain polypeptide produced commercially from recombinant animal cells as well as recombinant microorganisms. Its increased applications have requested development of highly efficient production systems using particularly animal cells. Depth filter perfusion system (DFPS) has been developed and successfully used for production of recombinant proteins such as antibodies from recombinant Chinese hamster ovary (rCHO) cells. In this study, rCHO cells expressing recombinant human growth hormone (rhGH) were successfully cultivated in the DFPS for 2,200 h. Parameters affecting the performance of the DFPS for rhGH production were investgated. The depth filter with 40 µm pores was selected for stable operation. The shifts of culture temperature and pH were tested at 37 and 33℃, and 7.2 and 7.0, respectively. In the begining of the culture, more than 80% of the seeded cells were immobilized in 200 min by medium/cell circulation. When the culture temperature was lowered from 37 to 33℃, about 50% increase of the volumetric productivity (VP) at a perfusion rate of 2.0/day was achieved, and the VP reached 168 mg/L/d at perfusion rate of 3.0/day, showing the benefit of cultures in low temperature. In contrast, when pH was shifted from 7.2 to 7.0, maintaining rhGH concentration at about 60 mg/L and decreased perfusion rate from 2.0 to 1.0/day, the VP dropped to 50%. As the DFPS showed stable and efficient production of rhGH for long-term periods from this study, it can be an attractive choice for production of rhGH from recombinant animal cells. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
198. Perfuse and Reuse: A Low-Cost Three-Dimensional-Printed Perfusion Bioreactor for Tissue Engineering.
- Author
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Bender RJ, Askinas C, Vernice NA, Dong X, Harris J, Shih S, and Spector JA
- Subjects
- Perfusion methods, Hydrogels, Polyesters, Printing, Three-Dimensional, Tissue Scaffolds, Tissue Engineering methods, Bioreactors
- Abstract
This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow centimeter-scale vascularized or otherwise canalized tissue constructs to be maintained in weeks long static and/or perfusion culture at an exceptionally low cost, with intermittent live imaging and media sampling capabilities. The perfusion system includes a reusable polydimethylsiloxane (PDMS) lid generated from a three-dimensional (3D)-printed poly-lactic acid (PLA) mold and several lengths of perfusion tubing. The coverslip tissue culture chamber includes PDMS components built with 3D-printed PLA molds, as well as 3D-printed PLA frames and glass coverslips that house perfusable hydrogel constructs. As proof of concept, we fabricated a vascularized hydrogel construct, which was subjected to static and perfusion tissue culture, as well as flow studies using fluorescent beads and widefield fluorescent microscopy. This system can be readily reproduced, promoting the advancement of tissue engineering and regenerative medicine research.
- Published
- 2022
- Full Text
- View/download PDF
199. Production of IgG1 monoclonal antibody 520C9 specific for human breast cancer oncoprotein c-erbB-2, using hybridoma cell line 8696 in perfusion bioreactor.
- Author
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Sen, Sucharita and Roychoudhury, P. K.
- Subjects
- *
MONOCLONAL antibodies , *HYBRIDOMAS , *MYC proteins , *BREAST cancer , *OVARIAN cancer , *CELL culture - Abstract
The monoclonal antibody 520C9 is specific for human breast oncoprotein HER-2/neu, that overexpresses in various metastatic breast and ovarian carcinomas. The production of monoclonal antibody 520C9 (mouse IgG1) from suspension hybridoma cell line HB-8696 is shown to improve by, using optimized perfusion culture strategy. The cells were grown under two different culture modes, namely, the batch culture mode and the perfusion culture mode, under constant inoculum cell density of 1.2 x 105 cells/ml. In perfusion culture, the optimum medium was introduced at different perfusion rates: 0.25, 0.5, and 0.75 wd (defined as volume of fresh medium/working volume of reactor/day). The maximum viable cell density in both culture systems has been investigated while maintaining steady state conditions. It has been observed that monoclonal antibody productivity using perfusion strategy improved significantly than that observed in batch culture. The perfusion culture system is preferred over other traditional culture techniques such as batch and fed-batch system, since it provides higher cell density for prolonged periods, higher yield of product, besides less exposure to toxic metabolites and, less exposure of hydrodynamic shear stress to the cells. In this paper, we have shown that the perfusion culture system operates in a programmed manner, and can be easily applied to other culture systems. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
200. Effect of Perfusion Culture on Localization, Intensity, and Functionality of Transporter Proteins in a Bilayer Proximal Tubule-on-a Chip
- Author
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Tatsuji Enoki, Toshikazu Araoka, Ryohei Ueno, Jun Yamashita, Minoru Takasato, Ryuji Yokokawa, and Ramin Banan Sadeghian
- Subjects
Cytosol ,Materials science ,Perfusion Culture ,medicine.anatomical_structure ,Transcytosis ,Reabsorption ,Bilayer ,medicine ,Albumin ,Biophysics ,Transmembrane protein ,Epithelium - Abstract
A biomimetic proximal tubule on-a-chip (PToC) comprised of isolated endothelial and epithelial microchannels along with a custom-made culture media perfusion system is presented. The microfluidic setup enables independently-addressable perfusion of epithelial and endothelial culture media for optimum bilayer health and function. It was shown for the first time that flow-induced shear stress not only enhances the expression level of megalin, a major proximal tubule transmembrane protein involved in albumin transcytosis, but also increases the chance of its presence/activity in the cytosol milieu. Albumin reabsorption capacity of the bilayer was improved through the epithelium subjected to shear stress, the cell height was increased, and the apical microvilli were fortified.
- Published
- 2021
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