Feeder layers cell culture

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. 

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. 

In fact the transition from bench to market of combination products is often hindered by a series of scientific and economic issues. Both the determination of the ideal cell type and the ideal biomaterial for the specific application can be problematic. Often, the use of autologous differentiated cells would be the best solution, but their use may not be feasible because of limitation on isolation and expansion. Moreover, the use of autologous cells also can introduce infections risks deriving from the use of xenogenic factors or animal feeder layers in culture. Hopefully, adult and embryonic stem cells may provide alternative solutions. Also the determination of the most suitable biomaterial for the specific application is a challenge. Most of the currently available synthetic materials are subjected to a foreign body reaction that can lead to serious complications when implanted in the human body. The use of natural scaffolds circumvents this problem, but it introduces other drawbacks such as inappropriate mechanical properties. Smart combinations of synthetic scaffolds modified with natural... 

Murakami, D., Yamato, M., Nishida, K., Ohki, T., Takagi, R., Yang, J., et al. (2006b). Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells. Joumai of Artificial Organs, 9,185-191. 

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). 

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... 

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). 

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. 

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). 

Feeder layer A layer of cells (usually lethally irradiated for animal cell culture) upon which are cultured a fastidious cell type. 

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