Feeder layers

The first batch of cells consisted of AC 133+ cells cultivated in the diffusion chambers submerged on top of the feeder (feeder -AC 133 cells /Fl-C). The second batch consisted of human embryonic liver cell suspension directly cocultured with AC133+ cells at equal initial quantities (5x10 ) in the diffusion chambers submerged in the 6-well plates without additional feeder layer (FC-C). Third batch of experiments represented AC133+ cells cultured in the DC surrounded by FL condition media (condition media-AC133+ cells/CM-C). In the control group cells were cultivated in the same condition without any additions and without feeder layers. 

We currently established cultural system (amphycultural diffusion capsules) that allowed for conditions favorable for stem cell expansion in vitro. Many cell types and culture protocols and their combination with cytokines, growth factors, feeder layers can be implemented with ADC. Capsules are characterized by high perfusion rates that ensure that allow dilution of inhibitory autocrine factors and support long-term cell expansion. We have shown that ADC in vitro provides optimal cellular microenvironment that supports long term hematopoiesis (Bilko et al. 2005). 

Our data suggests that hematopoiesis can be sustained for prolong cultivation periods in the presence of feeder layer cells or condition media supported culture models. Prolonged support of primitive hematopoietic cells (undifferentiated cells such as promyelocytes, myelocytes and metamyelocytes) and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient and that direct cell-to-cell contacts may not be exclusively required for successful long term in K/fro hematopoiesis. 

Obtained results suggest that stromal cells play important role in long term maintenance of hematopoiesis ex vivo. They induce proliferation and partially inhibit terminal differentiation and prolong hematopoiesis in long term in vitro cultures. Feeder layer derived from human embryos was adequate model for AC133+ cultures and had stimulating effect on the proliferation of the progenitor cells in vitro. 

Detailed analysis of the cultured cells indicated, that direct cell-to-cell contact of the stromal and hematopoietic cells are not necessary for hematopoiesis, and that diffusible factors produced by feeder layers are sufficient. Prevailing proliferation in AC133+ cultures with feeder layers, the ability of cells to form... 

Mitomycin C can be used as an alternative to y radiation for blocking cell proliferation of the feeder layer of CD 154 expressing cells (21, 28). However, to our knowledge, this methodology has not been used for N1H3T3 cells. 

Before the discovery and development of recombinant human cytokines, a feeder layer of stromal cells was essential for the cultivation of hematopoietic cells. The first system for the successful expansion of hematopoietic cells from murine origin was described by Dexter more than 20 years ago [76,77]. Some years later this principle was transferred to human cells [78]. Stromal culture of hematopoietic cells is a generic term that covers a variety of cultivation concepts, as indicated in Fig. 3. 

Liquid cultures of purified CD34+ (HPC) with a preestablished MSC feeder layer result in the expansion of CD34+/38+ HPC and CD34 738 hematopoietic SCs and support the growth of mature hematopoietic total nucleated cell (TNC) progeny (Figure 9). 

Fetal hamster fibroblast cultures (lethally irradiated feeder layer) ++ No... 

A serum-free medium supplemented with insulin, transferrin, ethanolamine and selenium (ITES) allows growth of certain hy-bridomas at 17-74% the rate found with 15% FBS (Wolpe, 1984) and Cleveland et al. (1983) devised a protein-free medium for growth of myeloma cells which, with addition of BSA at 2.5 mg/ml, forms the basis of Costar s SF-1 and SF-X supplemented media. Cloning is still very difficult in serum-free media, but feeder layers can be replaced by culture supernatants from human endothelial cells (HECS Astaldi, 1983) or Ewing s sarcoma cells (ESG Ley et al., 1980) — see 5.8.5. 

Suspend in growth medium and plate out onto feeder layers ( 8.1.4). 

Cells can be obtained from biopsy samples after extensive washing in PBS containing antibiotics and antimycotics. 1 cm squares of tissue can be incubated with dispase as described for endothelial cultures. ( 6.9) or with PBS-A supplemented with 1 mM EDTA. Cells are plated out onto feeder layers ( 8.1.4). 

An alternative method of producing a feeder layer is to incubate the monolayer with mitomycin C (10-6M) for 16 h which causes crosslinking of the DNA (Iyer and Szybalski, 1964). The monolayer is then washed three times with BSS to remove the mitomycin C and may be used directly or after subculture into smaller vessels. 

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. 

The dishes should be left undisturbed as epidermal cells take 2-3 days to attach firmly. Thereafter the medium should be replaced twice weekly. The growth of fibroblasts is largely suppressed but the epidermal cells form colonies of keratinocytes which push back the feeder layer at the periphery. 

Then disaggregate the keratinocyte colonies with 0.02% EDTA 0.05% trypsin (1 1). The keratinocytes may be replated with a fresh feeder layer if required. 

If the teratocarcinoma cells become confluent they will begin to differentiate into many different cells types, but this process is not so dramatic as the differentiation which occurs if cells isolated by method 2 are transferred to a vessel without a feeder layer. Then the cells attach very poorly and form clumps in suspension. These clumps remain healthy and quickly differentiate (Evans, 1972) to form an outer layer of endoderm cells. The presence of endoderm can be shown by assaying for the serine protease plasminogen activator which is a marker typical of endoderm cells (Strickland et al., 1976). 

Approximately 105 transfected embryonic stem cells are selected. Transfected embryonic stem cells are grown on layers of y-irradiated embryonic fibroblast cells. Transfected cells are plated on G-418 resistant embryonic fibroblasts feeder layers and selected in G-418 and after 36 h they are selected in Fialuridine (FIAU) or gancyclovir. 

Keeping ES cells undifferentiated is a key to success in achieving germ line transmission of the targeted allele. To prevent differentiation, ES cells must grow either on monolayers of mitotically inactivated fibroblast cells (embryonic feeder [EF] cells) or in the presence of leukemia inhibitory factor (LIF). In our experiments, we use both feeder layer cells and LIF-supplemented media for ES cell culture and maintenance. 

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