Feeder cells

Yamazoe H, Iwata H (2005) Cell microarray for screening feeder cells for differentiation of embryonic stem cells. J Biosci Bioeng 100 292-296... 

Feeder cell line cDNA used for transduction SCF expression CSF-1 expression Described as... 

Not unexpectedly, addition of large quantities of recombinant SCF to TFl cocultures on parental MMCE synthesizing no SCF or transduced SI SCF did not increase the cloning efficiencies of TFl cells. Addition of recombinant SCF, even in excess of 100 ng/ml to cocultures of TFl on MMCE cells transduced to present mb SCE could not impair the growth promoting potential of these feeder cells (Eig. 2d). 

NEUTRALIZATION OF THE SCF/C-KIT INTERACTION CAN INCREASE OR INHIBIT PROLIFERATION OF TFl ON FEEDER CELLS... 

The first experiments describing the clonal growth of hemopoietic progenitor cells immobilized in a soft gel matrix in vitro were reported by Bradley and Metcalf (1966) and Pluznick and Sachs (1965). Clonogenic cells are plated in the presence of various of feeder cells, medium conditioned by the growth of different tissues or cell lines in cultures (for example, 5637 bladder carcinoma conditioned media) and colony stimulating factors such as GM-CSF, G-CSF, M-CSF, Epo, interleukins. 

Feeder cells G418 resistant murine embryonic fibroblasts. 

Last, the surgical procedures such as keratoprosthe-sis [24] surgery and comeal and limbal transplantations are on the way to be developed. Yet, the transfer of cultured stem cells is a problem due to the need of mouse-derived feeder cells these might introduce new risks of disease transfer. Until those problems are not solved, these techniques are not applicable on humans, but these problems are likely to be solved in the near future [25]. 

Feeder cells for fusion cultures (see Note 6) essential for fusions employing rat myelomas. Quickly thaw irradiated rat fibroblasts, prepared as described in Note 6, just before commencing the cell fusion. Add the cells to 10 mL of serum-free DMEM, centrifuge, and wash once in serum-free DMEM Resuspend feeders in HAT medium just before addition of the fusion mixture. Alternatively, use thymocytes from spleen donors (mouse). 

Centrifuge for 3 min at 400g, and then resuspend the cells in 200 mL HAT selection medium, and add irradiated fibroblast or thymocyte feeder cells. Plate 2-mL aliquots into four 24-well plates or, if necessary, five 96-well plates (fusions with SP2/0 myeloma) (see Note 6). 

Centrifuge cells from at least two wells of a 24-well plate that contain confluent layers of hybridoma cells Count the number of cells, and then dilute to give about 50 cells in 20 mL of HT or DMEM containing 10% FCS-containing feeder cells... 

Carefully flick off the supernatant medium from the rat feeder cells, and plate 0 2-mL aliquots of hybridoma cells into each of the 96-wells... 

Another important discovery was made when comparing MVLBisG2 and DOTAP in a number of different cell lines. As shown in Fig. 12, complexes of MVLBisG2 efficiently transfect a variety of mouse and human cells in culture [24]. Their TE reaches or surpasses that of optimized complexes prepared from commercially available DOTAP. Most importantly, complexes containing MVLBisG2 are significantly more transfectant over the entire composition range in mouse embryonic fibroblasts (MEFs). MEFs are important as feeder cells for embryonic stem cells and are a cell line that is empirically known to be hard to transfect. 

Fig. 12 Transfection efficiencies for DOTAP/DOPC and MVLBisG2/DOPC complexes in four different cell lines, plotted against the mole fraction of cationic lipid. The data points were obtained at a constant pchg (7 for HeLa cells, 4.5 for all others), corresponding to a constant amount of DNA applied to the cells for each data point in a plot. Remarkably, MVLBisG2 complexes are significantly more transfectant in mouse embryonic fibroblasts, a cell line empirically know to be hard to transfect and of large practical importance as feeder cells for embryonic stem cells. Reprinted with permission from [24]. Copyright 2006 American Chemical Society...

Some cells, such as hybridomas, stem, and hematopoietic cells may require a class of proteins known as interleukins, especially IL-6. This compound can substitute for feeder cells (such as those of the peritoneal exudates or spleen, fibroblasts or timocytes) in the post-fusion stage of hybridoma cultures. IL-6 is secreted by monocytes, T lymphocytes, and endothelial cells and is effective in the stimulation of hybridomas of different species, including lineages that are difficult to cultivate, such as human-mouse and rat-mouse hybrids. IL-6 also presents a synergistic effect with other interleukins to increase antibody production. 

For cloning 6 cm dishes containing about 2 X 10s killed cells (i.e. irradiated or treated with mitomycin C) are used. These form the layer of feeder cells. Replace the medium in the dish of feeder cells with a suspension of the cells to be cloned. As few as 10 cells may be present but about 1000 is better. These cells settle into open spaces between the feeder cells and begin to grow and form colonies which may be isolated using cloning cylinders ( 7.1.2). 

Spleen lymphocytes ( 13.6.1) can also be used as feeder cells. These cells do not normally divide, nor do they attach to the surface of the dish and they disappear following 6-10 days in culture. 

Described below is a method for growing keratinocytes from skin biopsies using a feeder layer (Rheinwald and Green, 1975). The feeder cells may be 3T3 and BHK21 cells treated with gamma rays or with mitomycin C as described in 7.1.4. 

Mix with a suspension of feeder cells and distribute the cells into dishes. The proportions of the two cell types affect the character of the subsequent growth. 

After 4 wk, the popliteal, inguinal, and axilliary lymph nodes were removed from the mice. The thymus was also removed for use as feeder cells for the hybridomas. 

Positive hybridomas were transferred to larger wells, additional feeder cells were provided, the samples were retested, and then subcloned by limiting dilution (see Note 7). 

Irradiated embryonic fibroblast feeder cell layer to be used for growing the embryonic stem cell line G418 resistant embryonic fibroblast cells are derived from embryos that carry a targeted disruption such as the 3-2 microglobulin gene (9). 

Embryonic Feeder Cell Media (EFM) DMEM with 10% FCS and IX penicillin and streptomycin. 

Self-renewal of stem cells can be regarded as a combined phenotypic outcome of cellular proliferation and inhibition of differentiation and cell death. Consistent with this notion are the findings that self-renewal of murine embryoiuc stem (mES) cells can be achieved in the absence of feeder cells and serum in a chemically defined media condition by the combined activity of two key signaling molecules LIF/interleukin 6 (IF6)... 

Feeder cells for fusion cultures ( eNote 5. Essential for fusions employing rat myelomas) Three hours to one day before fusion, 2—4 x 10 rat fibroblasts in 10 mL of DMEM and contained in a 30 mL plastic universal are irradiated with about 30 Gy (3000 rad) of X- or T rays. Dilute with 90 mL of HT (3 h before fusion) or DMEM containing 10% FCS (1 d before fusion) and plate 1-mL aliquots into four 24-well plates or 0.2-mL aliquots into five 96-welI plates. Alternatively, use thymocytes from spleen donors (mouse). 

Cloning by the soft agar method is carried out in petri dishes 3 cm in diameter that contain a layer of normal spleen feeder cells (10 per plate) in 5% agar, over which is then layered a dilution of the hybrid cells (obtained from positive wells) suspended in a medium containing 20% fetal calf serum in 2.5% agar. A range of different dilutions of the hybrid... 

Maintenance ofhPSC Maintain hPSC (H9, H13, H14, and 19-9-11) on mouse embryonic feeder cells in hESC medium DMEM/ F12 culture medium supplemented with 20 % KOSR, 0.1 mM NEAA, 1 mM L-glutamine, 0.1 mM 3ME, and 10 ng/ml human bFGF. For feeder-free culture, maintain the hPSC on matrigel in the presence of mTeSRl medium. 

The hybridoma success rate can be increased by the inclusion of feeder cells (Fazekas de St. Groth and Scheidegger, 1980 Miner et al., 1981), e.g., mineral oil-induced peritoneal exudate cells, which enhance the survival of hybrid cells early after fusion, perhaps... 

Preparation of spleen and feeder cells The mouse killed... 

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