78
A . M O S C O N A , O. A . T R O W E L L A N D E . N . W I L L M E R
the experiment, the lens paper is fixed, stained, dehydrated, cleared
and mounted whole, the "outgrowth" can be studied under the microscope, and the different cell types afford an interesting study. If the
cultures are to be examined by histological sectioning, it is best to
lift off the lens paper and place it, with the cultures attached, into the
fixative. After fixation, the cultures can usually be detached from the
paper quite easily. But in the case of some organs this may be difficult
because paper fibres have become incorporated in the base of the
culture, and even when the culture is freed a few of these fibres may
remain within it. These fibres are very tough and cause trouble during
section cutting. This difficulty can be avoided by culturing such organs
on a 1 mm thick sheet of 2 °/o agar instead of on lens paper (Trowell,
1959). The agar sheet should also be used when cultures are to be
exposed to ionizing radiation, because in this way they are spaced
1 mm from the metal and the effect of secondary electrons arising
from the metal is minimized.
Thin sheets of tissue such as skin, retina and mesentery present
a particular problem. On lens paper they stay admirably extended but
become very adherent to the paper so that subsequent histological
treatment is difficult for the reasons noted above. On agar (or on plasma
clot or free-floating) they roll up. The solution here is to use a cellulose
acetate (rayon) fabric in place of the lens paper, a device introduced
by Shaffer (1956). The tissue sheets stay well-extended on rayon; they
also become adherent to it, but this does not matter because, after
fixation, any incorporated rayon fibres can be dissolved out with acetone. Prop (1961) used nylon gauze instead of lens paper for whole
mammary gland cultures but he mounted the cultures whole and did
not section them.
Grabar and Corvazier (1960) mention a method in which the cultures
are placed directly on a dry grid and each one immediately covered
with a drop of molten (40°C) 1 °/o agar. After the agar has set, medium
is added in the ordinary way. The agar serves to attach the culture to
the grid and also to prevent cellular migration and the encapsulation
of the culture. This seems to be a promising method which should be
further investigated.
The method of supporting the cultures on a metal grid overlaid with
lens paper, rayon or agar is perhaps the most satisfactory method of
organ culture so far devised because most of the variables are under
control. It is important that the metal grids are absolutely flat and
that they stand level in the dish. The "wetness" of the cultures can
be accurately controlled by varying the amount of medium in the dish.
Normally the outer fluid level should be about 1 mm lower than the
top of the grid, otherwise the cultures will be too "wet", which means
A . M O S C O N A , O. A . T R O W E L L A N D E . N . W I L L M E R
the experiment, the lens paper is fixed, stained, dehydrated, cleared
and mounted whole, the "outgrowth" can be studied under the microscope, and the different cell types afford an interesting study. If the
cultures are to be examined by histological sectioning, it is best to
lift off the lens paper and place it, with the cultures attached, into the
fixative. After fixation, the cultures can usually be detached from the
paper quite easily. But in the case of some organs this may be difficult
because paper fibres have become incorporated in the base of the
culture, and even when the culture is freed a few of these fibres may
remain within it. These fibres are very tough and cause trouble during
section cutting. This difficulty can be avoided by culturing such organs
on a 1 mm thick sheet of 2 °/o agar instead of on lens paper (Trowell,
1959). The agar sheet should also be used when cultures are to be
exposed to ionizing radiation, because in this way they are spaced
1 mm from the metal and the effect of secondary electrons arising
from the metal is minimized.
Thin sheets of tissue such as skin, retina and mesentery present
a particular problem. On lens paper they stay admirably extended but
become very adherent to the paper so that subsequent histological
treatment is difficult for the reasons noted above. On agar (or on plasma
clot or free-floating) they roll up. The solution here is to use a cellulose
acetate (rayon) fabric in place of the lens paper, a device introduced
by Shaffer (1956). The tissue sheets stay well-extended on rayon; they
also become adherent to it, but this does not matter because, after
fixation, any incorporated rayon fibres can be dissolved out with acetone. Prop (1961) used nylon gauze instead of lens paper for whole
mammary gland cultures but he mounted the cultures whole and did
not section them.
Grabar and Corvazier (1960) mention a method in which the cultures
are placed directly on a dry grid and each one immediately covered
with a drop of molten (40°C) 1 °/o agar. After the agar has set, medium
is added in the ordinary way. The agar serves to attach the culture to
the grid and also to prevent cellular migration and the encapsulation
of the culture. This seems to be a promising method which should be
further investigated.
The method of supporting the cultures on a metal grid overlaid with
lens paper, rayon or agar is perhaps the most satisfactory method of
organ culture so far devised because most of the variables are under
control. It is important that the metal grids are absolutely flat and
that they stand level in the dish. The "wetness" of the cultures can
be accurately controlled by varying the amount of medium in the dish.
Normally the outer fluid level should be about 1 mm lower than the
top of the grid, otherwise the cultures will be too "wet", which means