Feeder cells, preparation

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). 

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. 

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

Day 0 Split ES cells on 8.5-cm feeder plate as described in Section C. Inactivate fresh feeder and prepare eight 8.5-cm feeder plates. The culture of the feeder for inactivation should be started 1 week before electroporation. The fibroblasts should be prepared from embryos carrying the Neo gene, so that they... 

Prepare feeder cells one to three days before fusion. 

In traditional experiments with the hamster cell clonal assay, primary cultures were prepared from 12- to 14-day-old embryos as needed for each experiment. To enhance plating efficiency, small numbers of cells varying from about 300 to 1000 per dish were seeded onto a sparse lawn of previously X-irradiated feeder cells. Initially, feeder cells were usually from rats, although hamster cells have been used more recently. The cultures were then treated with chemicals for periods of time that varied from a few hours to 8 days, and were then examined for toxicity and transformation. 

In addition to MEFs, several human cell lines have been shown to be suitable feeders for hPSC culture. It is rational to assume that adhesion-based cultures of hPSCs rely on the natural ECM polymers deposited by the feeder cells for attachment and proliferation. On the basis of this idea, researchers began to use the ECM components secreted by feeder cells. There are two types of ECMs that are employed for hPSC culture decellu-larized ECMs prepared from human fibroblasts from foreskin, dermal, or other human cell lines (which are chemically undefined but not xenogenic) [28,43-46] and single or mixed components of chemically defined ECMs, such as fibronectin, laminin, and vitronectin [47]. These ECMs are used as coating materials on cell culture dishes or scalfolds [48]. 

Stacey, G. N., Cobo, E, Nieto, A. et al. 2006. The development of feeder cells for the preparation of clinical grade hES ceU Unes ChaUenges and solutions. / Biotechnol 125 583-8. 

Trypsinization is performed as described above. Cells in the suspension are counted and prepared in a suspension containing 4.2-6.7 x 104 cells/cm2. To each apical well of the 24-well plate 400 til of the cell suspension are added apically. The Feeder Tray contains 40 ml of complete medium (feeding cells from the basolateral side). 

Primary mouse embryonic fibroblast (EMFI) cells (37) or the STO fibroblast cell line (38) is the most commonly used feeder layers. Details for the preparation of a stock of EMFI or STO cells are described elsewhere (39). A brief protocol for the preparation of EMFI feeder layers will be given here. 

Prepare the appropriate-sized feeder layer containing plates for the cells that are to be recovered (24-well plates for 96-well frozen plates or 6 cm plates for cryovials containing approx 5-10 x 10 cells). 

Once prepared, feeders can be kept in culture for up to 2 wk prior to use, but must remain as an intact monolayer. Most researchers prepare feeders on gelatin-coated plates. Although this is not essential for the propagation of the fibroblasts themselves, it promotes tight adherence to the substratum, and is also essential for the adherent growth of ES cells to any regions devoid of fibroblasts. 

When ready for passage, aspirate the medium, wash wells with PBS and add 25 pL trypsin. Incubate for 10 min at 37°C, then add 175 pL of complete medium Using a multichannel pipet, disperse cells, and passage 100 pL to a fresh 96-well plate containing feeders, this plate will serve to prepare frozen stocks Passage the second 100-pL aliquot to 24-well plates (with or without feeders) containing 500 pL complete medium, these cultures will serve for DNA analysis see Note 16). 

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