Growth in serum-free medium of human colonic adenocarcinoma cell lines on microcarriers: A two-step method allowing optimal cell spreading and growth (original) (raw)
Related papers
Journal of Biological Standardization, 1985
Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occured—both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commerically-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.
Cytotherapy, 2015
Background aims. Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. Methods. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. Results. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Conclusions. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line.
Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates
Journal of Biological Standardization, 1986
Three commercially-important types of cell were grown on four different microcarrier substrates. The cells, which included normal human diploid fibroblasts (MRC-5), primary chick embryo cells and Madin-Darby bovine kidney cells (MDBK), were compared with regard to proliferation on the substrates and wirh regard ro recovery of viable cells from the same substrates. The substrates used included glass-coated microcarriers (Biosil), collagen microcarriers (Ventregel), DEAE-dextran microcarriers (Cytodex I) and collagen-linked DEAE-dextran microcarriers (Cytodex III). The established cell line (MDBK) grew well on all of the substrates and a high percentage of viable cells could be harvested from each substrate. The MRC-5 cells also grew well on all four substrates but high recovery rates were achieved only with cells grown on the glass-coated microcarriers or collagen microcarriers. In contrast, the primary chick embryo cells grew well only on the glass microcarriers and the recovery rate of cells harvested from this substrate was high. In some industrial operations, the re-utilization of cells after removal from the substrate is necessary. In these situations the appropriate choice of microcarriers for the cultivation of the cells may be critical.
Microencapsulation of human cells: Its effects on growth of normal and tumour cells in vitro
British Journal of Cancer, 1991
The growth kinetics of established human colorectal tumour cell lines (HT29, HTI 15 and COLO 320DM) and human diploid fibroblasts (Flow 2002) were studied in conventional culture and in microcapsules formed from alginatepoly(L-lysine) -alginate membranes. The tumour lines grew rapidly in microcapsules but, in the case of the substrate-adherent lines HT29 and HTl 15, only after a prolonged lag phase. This phase was reduced by serial passage in microcapsules. The anchorage-independent line COLO 320DM showed no lengthening in lag phase. Microencapsulated fibroblasts underwent negligible growth but remained viable. Some evidence for functional differentiation (microvilli, cell-cell junctions) of the tumour line HTl 15 within the microcapsules was observed. We conclude that the use of microcapsules provides an alternative system with some advantages for the study of human cancer and its metastases in vitro.
Biotechnology and Bioengineering, 1992
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 decrease 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 MRC-5 cells on Cytodex 1 microcarriers through as many as 13 microcarrier-to-microcarrier transfers, and at serum levels as low as I%, 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 inoculum build-up for production purposes. 0 1992 John Wiley & Sons, Inc.
Biotechnology Progress, 1992
Vero and HepG2 cells were cultivated on macroporous gelatin microcarriers prepared by the calcium carbonate inclusion method. Cell attachment to these microcarriers was slow. For HepG2 cells the subsequent growth was poor. Modification of the microcarriers by incorporation of (diethy1amino)ethyl-HC1 improved HepG2 attachment and subsequent growth. Optical sectioning with confocal microscopy allowed visualization of the distribution of cells within microcarriers. In most microcarriers, cells were found to preferentially populate regions close to the external surface and some cavities in the interior. Despite the incomplete occupancy of the interior of the microcarriers, high cell concentrations were achieved.
Growth of three established cell lines on glass microcarriers
Biotechnology and …, 1983
Three established cell lines were examined for growth on a newly developed microcarrier which consists of glass beads. The cells were simultaneously examined for growth on comrnercially available microcarriers made from DEAE-dextran and from plastic. Cell yields on the glass microcarriers were comparable to the cell yields on the commercially available products. Cells grown on the glass microcarriers were easily separated from the substratum by trypsinization (as were the cells grown on the plastic substratum) while the cells grown on the DEAEdextran particles were much more trypsin resistant. After removal of cells from the glass microcarriers, the cells reattached and spread out in plastic flasks as readily as cells harvested from monolayer. Scanning electron microscopy revealed dramatic differences in the appearance of the cells grown on the glass microcarriers and cells grown on the DEAE-dextran microcarriers. On the glass micmcarriers, cells attached to the substratum through long, slender filopodia while on the DEAE-dextran microcarriers, the entire edge of the cell appeared to be in contact with the substratum. This dissimilarity in attachment could underly the difference in sensitivity to trypsin-mediated detachment. Finally, the glass microcarriers were washed after being used once and retested for their ability to support cell growth a second time. Nearly identical results were obtained with the reprocessed beads as with previously unused ones.
Microcarrier culture: Applications in biological production and cell biology
Biotechnology and Bioengineering, 1981
This report reviews recent progress in microcarrier technology at the Massachusetts Institute of Technology. This progress includes new understanding of the nutritional needs of high-cell-density microcarrier cultures, the demonstration of cell growth on microcarriers in a hormone-supplemented, serum-free medium, and the continuous cell propagation of epithelial cell types in microcarrier culture by medium modification that permits efficient bead-to-bead transfer. Also, a technique for obtaining large quantities of mitotic cells by selective detachment from microcarriers is reported. Finally, recent progress in interferon production from human foreskin fibroblasts grown on microcarriers is outlined: Our improvements in the interferon "superinduction" process have increased the interferon yield per cell fivefold to tenfold.
Microparticles for Suspension Culture of Mammalian Cells
ACS Applied Bio Materials
The focus of this work is to develop a technology for the synthesis of polymer microcarriers that demonstrate mammalian cell culture adhesion on the surface of the microcarriers. Most mammalian cells are adherent in nature that requires multilayer vessels, large volume, expensive cell culture media, high manufacturing time, and high costs of cell culture supplies for the commercial-scale manufacturing of cells. The development of an efficient, scalable technology for producing large volumes of cells is a need in bioprocess industries to improve product potency. We developed a method of synthesizing soft biocompatible US FDA approved polymer based microparticle carrier system of approximately 260 ± 27 μm in diameter that serves as an adherent platform for human umbilical vein endothelial cells (HUVEC) to grow in suspension. Our preliminary experimental results showed that using the polymeric microcarrier system cell adhesion to the surface of the microcarriers was 2−3-fold higher than conventional cell culture flasks while using 10-fold lower cell culture media in a bioreactor than a tissue-culture treated flask. The survival of HUVEC on microparticles was confirmed by live cell staining (green fluorescent calcein AM), dead cell staining (ethidium homodimer-1), nuclear DAPI staining, actin cytoskeleton staining, confocal microscopy, and flow cytometry analysis. This technology will provide high cell culture productivity while reducing the costs of growing adherent cells.