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排序方式: 共有86条查询结果,搜索用时 15 毫秒
1.
Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers 总被引:1,自引:0,他引:1
G. Mignot T. Faure V. Ganne B. Arbeille A. Pavirani J. L. Romet-Lemonne 《Cytotechnology》1990,4(2):163-171
Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (107–2×107 cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors. 相似文献
2.
A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 107 cells per ml were obtained. The described system is scaleable. 相似文献
3.
Mortimer M. Civan Stephen R. Williams David G. Gadian Enrique Rozengurt 《The Journal of membrane biology》1986,94(1):55-64
Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional31P and19F probes of intracellular pH (pH
c
) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH
c
on external Na+ concentration (c
Na
o
). pH
c
also depended on intracellular Na+ concentration (c
Na
o
). Increasingc
Na
c
by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc
Na
o
produced a larger acid shift in pH
c
than with external K+ present. Comparison of separate preparations indicated that pH
c
was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH
c
of Swiss mouse 3T3 cells using31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. 相似文献
4.
Alexander Tschopp Augusto Cogoli Marian L. Lewis Dennis R. Morrison 《Journal of biotechnology》1984,1(5-6)
Attachment to a substrate and survival of human embryonic kidney (HEK) cells have been tested in an incubator installed in the flight-deck of the Space Shuttle ‘Challenger’ during its eighth mission.HEK cells are producing the enzyme urokinase and are presently investigated as candidates for electrophoretic separation in an apparatus developed and manufactured by McDonnell Douglas.Attachment of HEK cells to a substrate is mandatory for survival and production of urokinase after electrophoretic separation.Analysis of the samples shows that cells adhere, spread and survive in microgravity (< 10−3 ×g) conditions as well as the ground controls at 1 × g. This result represents an important step towards further bioprocessing in space. 相似文献
5.
Human diploid fibroblasts serially passaged on microcarriers exhibit a decrease in their proliferative capacity with each transfer from microcarrier-to-microcarrier. This phenomenon, which does not occur in the same time scale with cells cultured in T-flasks, has been a serious barrier to the systematic utilization of microcarriers in the scale-up of anchorage-dependent human diploid cell cultures. This decreases in cell growth with each passage is shown to be related to the serum content of the medium, with high serum concentrations resulting in a more rapid decrease in cell growth with each serial transfer. As a result, methods for reducing the serum requirement of the cells were investigated. A new medium supplement mixture, PPRF92, has been developed, which allows the serial passaging of MRC5 cells on Cytodex 1 microcarriers through as many as 13 microcarrier-to-microcarrier tranfers, and at a serum levels as low as 1%, with no decrease in the proliferative capacity of the cells until they approach their reported population doubling limit. This new supplement mixture is a significant improvement to microcarrier technology in that it enables the use of microcarriers in the early stages of inocculum build-up for the production purposes. (c) 1992 John Wiley & Sons, Inc. 相似文献
6.
Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels
Thomas J. Goodwin J. Milburn Jessup David A. Wolf 《In vitro cellular & developmental biology. Animal》1992,28(1):47-60
Summary A new low shear stress microcarrier culture system has been developed at NASA’s Johnson Space Center that permits three-dimensional
tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable,
moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs).
RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were
cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal
layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation.
In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast
population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing
themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures,
apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development,
internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected
to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining
the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords
a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue. 相似文献
7.
Ee Xien Ng Ming Wang Shu Hui Neo Ching Ann Tee Chia-Hung Chen Krystyn J. Van Vliet 《Biotechnology journal》2021,16(3):2000048
Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells. 相似文献
8.
In view of the advantages of the bulk production of clonal pancreaticbeta cells, an investigation was made of the growth and insulin secretoryfunctions of an electrofusion-derived cell line (BRIN-BD11) immobilizedon a solid microcarrier, cytodex-1 or a macroporous microcarrier,cultispher-G. For comparison, similar tests were performed usingBRIN-BD11 cells present in single cell suspensions or allowed toform pseudoislets. Similar growth profiles were recorded for eachmicrocarrier with densities of 4.4×105±0.3 cells/ml and4.2×105±0.2 cells/ml achieved using cytodex-1 andcultispher-G, respectively. Cell viability began to decline on day 5 ofculture. Insulin concentration in the culture medium reached a peak of26±2.0 ng/ml and 24±2.2 ng/ml for cells grown oncytodex-1 and cultispher-G, respectively. Cells grown on both types ofmicrocarrier showed a significant 1.5–1.8-fold acuteinsulin-secretory response to 16.7 mmol/l glucose. L-alanine (10 mmol/l) andL-arginine (10 mmol/l) also induced significant 3–4 fold increasesof insulin release. BRIN-BD11 cells immobilized on cytodex-1 or cultispher-Gout-performed single cell suspensions and pseudoislets in terms ofinsulin-secretory responses to glucose and amino acids. A 1.3-fold,2.2-fold and 1.7-fold stimulation of insulin secretion was observed forglucose, L-alanine and L-arginine respectively in single cellsuspensions. Corresponding increases for pseudoislets were1.6–1.8-fold for L-alanine and L-arginine, with no significantresponse to glucose alone. These data indicate the utility ofmicro-carriers for the production of functioning clonal beta cells. 相似文献
9.
Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster
ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated
for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion
bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 106 cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level
of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells
to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture
scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development
are highlighted.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
10.
Modulation of mesenchymal stromal cell characteristics by microcarrier culture in bioreactors
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