Clonal Growth in Vitro of Human Cells With Fibroblastic Morphology

Abstract

In previous papers (1-3) we have described methods for plating single human cells so that each multiplies in isolation to form a well defined colony which constitutes a clonal population. The procedure is simple and quantitative, and completely comparable to the standard plating procedure used by microbiologists for growth of colonies from single bacterial cells. In the work published thus far we have described application of this method only to cells of epithelioid morphology. The present communication demonstrates that fibroblasfic cells, although requiring more stringent care, can also be plated by this technique. Experiments comparing growth and genetic properties of these two cell types obtained from a variety of human organs are presented and some implications of these results for the problem of differentiation are discussed.Definitions.--The terms, done, plating e fidency, and parental strain are used as previously defined (2, 4, 5). Fibroblastic morphology: Elongated form of a glass-attached cell, which is spindle-shaped or possesses multiple, needle-like extensions of the protoplasm, as shown in Fig. 1 a. Colonies of such cells display rough edges. Epithelioid morphology: Cell form consisting of polygonal bodies whose angles are roughly equal in size, as shown in Fig. 1 b. These cells usually pack together fairly tightly, display less surface area and are less migratory than fibroblastic cells (2). In this paper these two terms refer only to the morphologic appearance of the glass-attached cells, without implications concerning their origin in vivo. Genetic or kereditary character: A character, the potentiality for which is transmitted to all the asexual progeny of the original individual even after many passages in an environment which does not permit development or expression of the character. Not all genetic potentialities are genic; i.e. under exclusive control of specific nuclear genes.

Genetics of Somatic Mammalian Cells

“…Actively growing cultures have been obtained from persons of both sexes, varying in age from a few weeks to more than 40 years. Under the conditions here described, the cells obtained are almost invariably spindie-shaped (fibroblastic), probably because of the selective advantage enjoyed by these more migratory cells in nutritionally complete medium (4). When the cell layer becomes confluent, it is tryps'mized by removal of the medium, a single wash with Solution 7 of Table I, then incubation for 5 to 8 minutes at 37°C.…”

“…The presence of toxic factors in serum is insidious in that they may not make their presence obvious in the mass cultures used in routine farming until after a long lag period. Cells so intoxicated may require weeks or months for return to healthy growth as previously described (4). However, the use of single cell plating can reveal even very low levels of serum toxicity immediately because of the greater sensitivity of the cells in the isolated state.…”

“…Their constitutions have been changed in various ways over previously described formulations (4,5). The use of embryo extract has been eliminated and embryonic (and occasionally newborn) mammalian sera have been substituted in its stead.…”

“…These, however, are nutritionally more exacting than the epithelial-like variety, since they will not grow like the latter in media supplemented only with adult mammalian sera, and more readily succumb to the presence of toxic agents (4). While methods for cultiva-GENETICS O1~ SOMATIC MAMMALIAN CELLS.…”

“…While methods for cultiva-GENETICS O1~ SOMATIC MAMMALIAN CELLS. III tion of these cells have been described (4), the need for inclusion of embryo extract in the growth medium has made their culture precarious because of the frequency with which toxic batches of embryo extract occur, despite great care taken to standardize the preparative procedures.…”