Human Cell Culture Protocols

Otto WR, Sarraf CE, William R. Otto, Catherine E. Sarraf, Otto, William R., Sarraf, Catherine E.

Mesenchymal stem cells from a variety of sites are a natural resource that using appropriate skills can be cultured in the laboratory, in scaffolds, to provide differentiated-cell replacement tissues, for clinical application. To perform such work with human cells, strict ethical integrity must be observed at all stages. Adipocytes, osteocytes and chrondrocytes are amongst the most desirable end-point cells. Hydrolytic degradable scaffolds allow implanted cells to synthesise their own extracellular matrix in situ after implantation, degeneration of the foreign scaffold to temporally match creation of the new innate one. For preliminary in vitro stem cell differentiation protocols, initial investigation is commonly performed with stem cells in commercially available porous collagen sponges or cell-free small intestinal submucosa. Differentiation of stem cells to a specific phenotype is achieved by culturing them in apposite culture media under precise conditions. Once the cells have differentiated, they are checked and characterised in a wide variety of systems. This chapter describes differentiation media for adipocytes, osteocytes, chondrocytes, myocytes and neural precursors and methods of observing their characteristics by microscopy using phase contrast microscopy, standard light microscopy and electron microscopy with tinctorial, immunocytochemical and electron dense stains, respectively. Cell sorting techniques are not dealt with here. Immunocytochemistry/microscopy staining for specific differentiated-cell antigens is an invaluable procedure, and the range of commercially available antibodies is wide. Precautions need to be considered for using actively proliferating cells in vivo, so that implanted cells remain controlled by the body's molecular signals and avoid development of malignancy.