2 . M E T H O D S
49
extract and, after a time, the cells isolated in this way began to multiply
within the confined space of the tube, thus forming colonies. Eventually
small colonies of cells emerged from the tubes into the medium (Fig.
3a). Even so, there was great mortality among the isolated cells and
only a very small percentage of the tubes with single cells produced
colonies. Moreover, the cells no longer looked quite like fibroblasts,
as these cells normally appear when they emerge from fresh tissues,
and it is probably better to regard them as "cells adapted to tissue
culture conditions", i.e. "tissue culture cells" with many of their normal
potentialities modified or actually in abeyance, rather than to consider
them as normal and typical fibroblasts or mechanocytes.
It seems probable, therefore, that the normal mechanocyte cannot
survive alone in the usual plasma and extract media, unless the medium
is to some extent preconditioned, as by other cells, or alternatively
unless the cell undergoes some sort of adaptation or mutation, so that
it can then tolerate this environment without assistance from neighbouring cells. This point is discussed more fully elsewhere (p. 167), but
meanwhile it is important to remember that all types of cell are not
readily amenable to culture in isolation and, indeed, the latter may in
some cases only be possible after the cell has altered its normal activities
in some way. Other classes of cell, e.g. the malignant HeLa cell, the chick
macrophage and one or two other types do not seem to have any such
difficulties and can be cultured relatively easily under a wide variety
of conditions; some may even be grown in protein-free media of known
composition (Fig. 4).
B. P R E P A R A T I O N OF CELL SUSPENSIONS
If tissues are finely minced and the ensuing suspension of cells
allowed to settle on the bottom of a flask, many of the cells can survive
and grow, eventually producing viable strains of cells which may be
suitable for some purposes, but the nature of the cells in these strains
remains obscure and better methods are now available for obtaining
cells in suspension with less damage. These latter methods have become
of great importance for the study of cellular interactions, cell aggregation, cellular differentiation and for the culture of viruses. They will,
therefore, be discussed in some detail.
Procedures for the preparation of cell suspensions were extensively
reviewed by Rinaldini (1958) and only certain aspects of the matter
will be emphasized here with particular stress on those relevant to the
use of dissociated cells in cell aggregation and differentiation studies
(Chapter 14). Single cells in suspension can be obtained either by
dissociation of fresh tissues, or from established cell cultures.
49
extract and, after a time, the cells isolated in this way began to multiply
within the confined space of the tube, thus forming colonies. Eventually
small colonies of cells emerged from the tubes into the medium (Fig.
3a). Even so, there was great mortality among the isolated cells and
only a very small percentage of the tubes with single cells produced
colonies. Moreover, the cells no longer looked quite like fibroblasts,
as these cells normally appear when they emerge from fresh tissues,
and it is probably better to regard them as "cells adapted to tissue
culture conditions", i.e. "tissue culture cells" with many of their normal
potentialities modified or actually in abeyance, rather than to consider
them as normal and typical fibroblasts or mechanocytes.
It seems probable, therefore, that the normal mechanocyte cannot
survive alone in the usual plasma and extract media, unless the medium
is to some extent preconditioned, as by other cells, or alternatively
unless the cell undergoes some sort of adaptation or mutation, so that
it can then tolerate this environment without assistance from neighbouring cells. This point is discussed more fully elsewhere (p. 167), but
meanwhile it is important to remember that all types of cell are not
readily amenable to culture in isolation and, indeed, the latter may in
some cases only be possible after the cell has altered its normal activities
in some way. Other classes of cell, e.g. the malignant HeLa cell, the chick
macrophage and one or two other types do not seem to have any such
difficulties and can be cultured relatively easily under a wide variety
of conditions; some may even be grown in protein-free media of known
composition (Fig. 4).
B. P R E P A R A T I O N OF CELL SUSPENSIONS
If tissues are finely minced and the ensuing suspension of cells
allowed to settle on the bottom of a flask, many of the cells can survive
and grow, eventually producing viable strains of cells which may be
suitable for some purposes, but the nature of the cells in these strains
remains obscure and better methods are now available for obtaining
cells in suspension with less damage. These latter methods have become
of great importance for the study of cellular interactions, cell aggregation, cellular differentiation and for the culture of viruses. They will,
therefore, be discussed in some detail.
Procedures for the preparation of cell suspensions were extensively
reviewed by Rinaldini (1958) and only certain aspects of the matter
will be emphasized here with particular stress on those relevant to the
use of dissociated cells in cell aggregation and differentiation studies
(Chapter 14). Single cells in suspension can be obtained either by
dissociation of fresh tissues, or from established cell cultures.