Extracellular matrix cell culture surfaces

Figure 6.1 Culture methods for human pluripotent stem cells (hPSCs). hPSCs can be cultured on mouse embryonic fibroblasts (MEFs) or human feeder cells. Feeder-free culturing of hPSCs is possible on xeno-containing Matrigel. Several types of feeder-free and xeno-free cultures of hPSCs can be developed on extracellular matrix (ECM)-coated surfaces and ohgopeptide-immobilLzed surfaces. hPSCs can also be cultured on polysaccharide hydrogels or on synthetic polymer surfaces by selecting a specific GAG or a specific polymer with optimal water content. Reproduced with permission from [2] Copyright 2014 Elsevier Inc.

Bader et al. [35] and De Bartolo et al. [36] developed the flat membrane bioreactor which consists of a multitude of stackable flat membrane modules as shown in Fig. 5. Each module has an oxygenating surface area of 1150 cm. Up to 50 modules can presently be run in parallel mode. Isolated hepatocytes are co-cultured with non-parenchymal cells. Liver cells are located of a distance of 20 pm of extracellular matrix from a supported polytetrafluorethylene (PTFE) film. Medium and cells in the modules are oxygenated in the incubator by molecular diffusion of air across the non-porous PTFE membrane. The design of the bioreactor is also the basis for its proven potential for cryostorage with fully differentiated adult primary human liver cells. 

The use of plastic materials for routine cell culture on the laboratory scale was introduced at the end of the 1960s, and some characteristics of glass surface, such as hydrophobicity and negative charge, were maintained in these materials. Polystyrene is the most widely used plastic material for animal cell adhesion at present, because of its surface characteristics, its low cost, and also its transparency. For more demanding cell lines, the surface has to be submitted to a treatment that involves coating with proteins such as poly-lysine, poly-ornithine, or extracellular matrix-derived proteins such as fibronectin, laminin, and collagens. 

Fibroblasts live in the spaces between other cells and secrete the proteins of the extracellular matrix, e.g. collagen. They do not associate tightly with one another or with other cell types, but they do readily attach themselves to a substratum. In dilute culture they are observed as individual spindle shaped cells which move around the surface of the culture vessel avoiding each other. As their density increases they tend to align themselves in parallel assays (Fig. 2.1). However, fibroblasts can become chondrocytes or adipocytes in the appropriate environment (Taylor and Jones, 1979) and on transformation ( 2.2) they readily lose their contact inhibition and pile up on top of one another (Fig. 2.1)... 

Some primary and fastidious cell types will not attach and grow on regular tissue culture surfaces and require a protein coating to divide and become fully differentiated. In addition to polylysine and polyomithine, a variety of proteins that are derived from extracellular matrix are commercially available. Fibronectin, laminin and collagens are available as reagents and also as precoated plasticware. 

As sketched in Fig. 10 and mentioned before, the cells anchor to adhesive proteins that are immobilized on the surface. When cells are cultured for a certain time, they even produce their own adhesive proteins and secrete it into the space between membrane and substratum. Thus, we tried to address whether or not these adhesive proteins underneath the cell body may contribute to the total QCM readout of a confluent cell layer. Instead of limiting the analysis to preadsorbed layers of one or two purified proteins, we tried to study the complex extracellular material underneath the cells (extracellular matrix or ECM) by removing the cell bodies but leaving the macromolec-ular network of proteins and sugars behind on the substrate. The protocol required a combination of hypotonic stress and detergent extraction [16]. Microscopic inspection of reference substrates revealed that this procedure hfted the cell bodies effectively off the substrate. The surface was, however, still decorated with proteins as revealed by immunocytochemical staining. 

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