2 . M E T H O D S
41
In the chambers using agar or serum-agar gels for supporting the
cells, there is no evidence that the cells make use of the agar itself,
other than as a mechanical support. Plasma gels are, on the other hand
often digested, or by some other means liquefied, by the tissues placed
upon them, and for this reason they are both more and less satisfactory
as bases upon which to observe cellular behaviour; more, because they
supply satisfactory nutriment and are biologically more appropriate;
less, because the base upon which the culture has to spread is unstable,
and of indeterminate composition.
C. FLASK C U L T U R E S
With the realization that the useful life of a hanging-drop culture
was not more than a few days, and that renewal of the medium without
subculturing was a difficult and uncertain procedure, the flask technique was developed at an early stage by Carrel (1923). In this technique several pieces of tissue are placed on the bottom of a small
(3-5 cm diameter) flattened flask fitted with an oblique side arm (a
Carrel flask) (Fig. l b ) . In the method as originally devised, the tissues
are planted in a thin layer of plasma which is then usually clotted
in situ by the addition of a little embryo extract. When the clot is firm
enough, a supernatant fluid medium is provided which can be changed
from time to time. The air in the flask can be replaced by a known gas
mixture and the p H of the medium may be adjusted in this way. In
such flasks, cultures of somewhat larger quantities of tissue than those
possible in hanging-drops can be kept alive and relatively undisturbed
for peroids of days, or even weeks or months, if the plasma clot is
adequately patched. This patching is a procedure which is always
made necessary sooner or later, because the tissues tend to liberate
proteolytic enzymes or other agents which cause dissolution of the clot.
The depth and consistency of the clot are important factors in determining the nature and extent of the outgrowth of the tissues. The thinner
and shallower the coagulum, the more loosely does the tissue extend,
and thus the colony spreads further in a given time and may give a spurious effect of more rapid growth.
When this technique was first developed, it appeared likely that it
might be very useful in the analysis of growth and of the factors which
accelerate and retard it. It has certainly served its purpose in such
studies both with respect to the study of mitosis in the outgrowing cells
and in determining some of the factors which increase or decrease the
migratory powers of these cells. For example, the effects of embryo and
various tissue extracts, of proteoses, amino acids and other growthstimulants and the effects of cyanides, aldehydes and various other
c*
41
In the chambers using agar or serum-agar gels for supporting the
cells, there is no evidence that the cells make use of the agar itself,
other than as a mechanical support. Plasma gels are, on the other hand
often digested, or by some other means liquefied, by the tissues placed
upon them, and for this reason they are both more and less satisfactory
as bases upon which to observe cellular behaviour; more, because they
supply satisfactory nutriment and are biologically more appropriate;
less, because the base upon which the culture has to spread is unstable,
and of indeterminate composition.
C. FLASK C U L T U R E S
With the realization that the useful life of a hanging-drop culture
was not more than a few days, and that renewal of the medium without
subculturing was a difficult and uncertain procedure, the flask technique was developed at an early stage by Carrel (1923). In this technique several pieces of tissue are placed on the bottom of a small
(3-5 cm diameter) flattened flask fitted with an oblique side arm (a
Carrel flask) (Fig. l b ) . In the method as originally devised, the tissues
are planted in a thin layer of plasma which is then usually clotted
in situ by the addition of a little embryo extract. When the clot is firm
enough, a supernatant fluid medium is provided which can be changed
from time to time. The air in the flask can be replaced by a known gas
mixture and the p H of the medium may be adjusted in this way. In
such flasks, cultures of somewhat larger quantities of tissue than those
possible in hanging-drops can be kept alive and relatively undisturbed
for peroids of days, or even weeks or months, if the plasma clot is
adequately patched. This patching is a procedure which is always
made necessary sooner or later, because the tissues tend to liberate
proteolytic enzymes or other agents which cause dissolution of the clot.
The depth and consistency of the clot are important factors in determining the nature and extent of the outgrowth of the tissues. The thinner
and shallower the coagulum, the more loosely does the tissue extend,
and thus the colony spreads further in a given time and may give a spurious effect of more rapid growth.
When this technique was first developed, it appeared likely that it
might be very useful in the analysis of growth and of the factors which
accelerate and retard it. It has certainly served its purpose in such
studies both with respect to the study of mitosis in the outgrowing cells
and in determining some of the factors which increase or decrease the
migratory powers of these cells. For example, the effects of embryo and
various tissue extracts, of proteoses, amino acids and other growthstimulants and the effects of cyanides, aldehydes and various other
c*