46] The culture of human tumor cells in serum-free medium

Abstract

Summary In this chapter we describe methods for growth in serum-free medium of several types of human tumors. Our laboratory and others have reported formulations of serum-free medium that will support the growth of other, additional types of human cells in culture that are not described in this chapter but are reviewed elsewhere.2,27 The serum-free medium HC described for the human colon tumors was originally devised to support the serum-free growth of a line of human colon tumor cells (HC84S) established in serum-containing medium and derived from a human colon tumor line (T84) transplantable in nude mice. Subsequently, it was found that this medium will support the serum-free growth of several (but not all) other transplantable human colon tumor lines in primary culture as well as primary culture of at least one tumor directly from a patient. The serum-free medium HA described for the human astrocytoma was devised to support the serum-free growth of a line of human astrocytoma cells (HA24) established in serum-containing medium and derived from a human astrocytoma line (T24) transplantable in nude mice. Both tumorigenic and nontumorigenic clones have been isolated from the HA24 cell line, and both will grow serum-free in the HA medium, although quantitative differences in responses of the different clones to omission of the individual components of the HA medium are seen. The serum-free medium LA described for the human lung adenocarcinoma was devised to support the serum-free growth of primary cultures of the T291 lung adenocarcinoma transplantable in nude mice. This medium will support the continuous serum-free growth of a line of lung adenocarcinoma cells originally established from cultures of the T291 tumor in serum-containing medium. The serum-free medium LE described for the human lung epidermoid carcinoma was devised to support the serum-free growth of primary cultures of the T222 lung epidermoid carcinoma transplantable in nude mice. Neither the LA nor the LE medium will support continuous, multipassage serum-free growth of cell cultures obtained from the T291 or T222 tumors. MCF-7 is an established cell line, derived in serum-containing medium from a metastatic pleural effusion of a human breast cancer.14 The medium developed for the continuous serum-free growth of the MCF-7 line will support growth in primary culture to some extent of some human breast tumors, but not all we have tested. In addition, we report in this section procedures for the partial purification of a factor from serum that stimulates the spreading of these cells, as well as others, in serum-free medium. We also describe in this chapter methods for the isolation of human tumor cells of epithelial origin for primary culture and methods for the quantitation of cells in such cultures. These methods, which are likely to be generally applicable in the isolation of most cell types for primary culture, allow us to obtain cultures with an acceptable plating efficiency while avoiding the ubiquitous problem of fibroblastic overgrowth of the epithelial population. Furthermore, the use of medium in which serum has been replaced by cell type-specific combinations of nutrients, hormones, binding proteins, and attachment factors allows us to reproduce more accurately in vitro the nutritional, hormonal and stromal influences to which the cells are subject in vivo.28 In this way we should minimize the genetic or epigenetic alteration and selection of an aberrant population of cells from those of the original culture. It is interesting to note that in several instances presented in this chapter both the growth and the differential properties of the cell types in their respective serum-free, hormone-supplemented media are closer to what we wish to attain in culture than what which we see in cultures of these cells in serum-supplemented medium. Human colon tumor cells, as well as human lung adenocarcinoma and epidermoid carcinoma, grow better in their serum-free media than in serum-containing medium. Furthermore, the keratinization of the epidermoid carcinoma cells is more marked in serum-free medium, as is the formation of villi-like, mucin-secreting structures in serum-free cultures of the colon tumor cells.3 It is our view that this is further evidence that, by the use of serum-free medium specifically designed for each cell type, we are more accurately reproducing the in vivo environment of the cells.