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The transparent chamber adapted for cell culture and permitting access to the contained living tissue.

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THE TRANSPAREXT CHADlBER ADAPTED FOR CELL
CULTURE AND PERNITTING ACCESS TO THE
CONTAINED LIVIKG TISSUE
R. G. WILLIAMS
Department of A n a t o m y , Uwiversity of Pennsytvania
ONE FIGURE
The original procedure for introducing cells into a transparent chamber inserted in a rabbit’s ear was described by
Clark ct al. in 1930. This consisted in using a ‘round table
type’ chamber provided with an oblique hole in the table opening on the growing region which, when not in use, was
occluded by a glass plug sealed in with paraffin. Small pieces
of tissue could be introduced from below through this hole
and deposited either above, in, o r below the thin layer of
growing tissue. This was technically a difficult method and
in many ways unsatisfactory. To simplify the handling and
introduction of minute transplants Dr. H. T. Kirby-Smith
developed a form of micromanipulator. With such an instrument very small bits of tissue could be handled. However,
the proportion of unsuccessful implantations was very great
and it was thought that the technique should be simplified.
Accordingly, a chamber was constructed in such a manner
that when inserted in the ear the central growing region could
be exposed from above without disturbing the assembly in
the ear. The tissue could then he covered again and the
chamber allowed to continue its normal course. This process
could be repeated many times 011 the same specimen.
’The work in this laboratory on the development of methods for studying living
cells and tissues in transparent chambers installed in the living mammal is being
aided by a grant f r o m the Rockefeller E’oundation.
4s7
488
It. G . WILLIAMS
The ij-pe of chamber here clescribed is the ‘pre-formed’
tissue type (Clark et a]., ’30). It is used for stiidics x-here it
is desirable to deal with tlie origiiial tissues of the ear. The
‘round table. t j p e ’ may be used when it is essential to d ~ a l
with iiemly formed tissues.
The chamber is coiistructed of celluloicl. This material may
be obtained from the Eastman Kodak Company in tliicknesses
up to 0.5 mm. under the trade name Kodaloid. The thicker
pieces a r e sold as ‘Viscoloid’ by the DixPont Viscoloid
Company. The various parts of the chamlwr are shown in
figure 1. To make the top, a ring of viscoloid is prepared
having an outside diameter of 3.5 cm. and an inside diameter
of 2 em. The inside edge is beveled on a grinding wheel. Over
the hole in this ring a piece of clear mica 0.004 inch thick and
2.4 cm. in diameter is glued. The glue used is that developed
by Varian (’X). Three holes a r e drilled iii tlie riscoloid
ring a s shown in figure 1.
The bottom or observation side of the chamber consists of
two parts. Part 1 is R ring c a t from viscoloid 1.2 mm. thick
having tlie dimensions given in figure 1. To the inner margin
of this piece another ring of riscoloid 2 mm. high and 2 mm.
thick is fused with a mixture of equal parts of absolute alco1101 and ether. On top of this ring a circular pi
0.5 mm. thick, with an ontsicle diameter of 3.3 em. and a n inside
diameter of 1.6 em., is fused in a similar may. The thickness
of the piece is then rednced t o 0.36 mm. by rubbing it against
a piece of 00 sand paper. Three holes are bored in the outer
ring t o correspond t o the three lioles in tlie top. Three equally
spaced holcs m e tlicii drilled through the thick part of this
piece (fig.1)’using a no. 55 twist drill in a higli speed drill
press. The inner ends of these holes are countersniik t o
receive the heads of three brass bolts wliicli h a r e been ground
square and reduced to less than 0.5 mm. in thickness. The
bolts are forced in place and the alcohol and ether mixture
applied around them. Pieces of thin celluloid, 0.075 mm., a r e
then fiisecl over the heads so that they will not come i n direct
coiitact with the tissue ~-1iei1the chamher is installed.
TRA4NSPAREA-T CIXAMBER, FOR CELL CULTUBE
489
Part 2 of the base is formed of celluloid rings having an
outside diameter of 2.3 em. a n d an inside diameter of 1.7 cm.
stacked one on another until a height of 3 mm. is reached. On
top of this a ring of celluloid, 1.2 mm. thick, wit11 a n outside
diameter of 3 em. and a n inside diameter of 1.7 cm. is placed
TOP
BASE
PART 2
PART 1
S P A C E F O R T I 5 S U E-----------
--BOLT
SPACE FOR C A R T I LAGE--
- X U 8B ER W A S H E R
--N
REMOVABLE
c o v E R------------’
HEAD
UT
C R O S S SECTION
Fig. 1 Diagram giving the materials and dimensions for constructing the
ehambcr. Cel. refers t o cellnloid. Figures givcn in milhmeters represent the
thickness of the various pieces. Natural size.
490
a.
G. WILLIAMS
and all pieces are fused together with alcohol and ether. The
inside edge of this piece is then beveled with a small grinding
wheel or a bastard file, as shown in figure 1. The overlapping
portion of this part is drilled with a no. 54 twist drill at three
points corresponding t o the protruding bolts in part 1.
A paper washer is cut from bond paper so that it fits snugly
on the shelf z (fig. 1). The cover is made from clear mica
about 80 p thick and rests on the paper washer on the shelf a;.
These parts are held in place by the removable collar, part
2 (fig. l), which is in iurn held down by the nuts and bolts
installed in part 1 (fig. 1).
Preliminary to the installation in the ear the various parts of
the chamber are immersed in 1: 500 metaphen for 20 minutes,
except for the paper washer, which is immersed in a smoking hot, but not boiling mixture of bayberry wax and vaseline,
1to 3. Just before starting the installation the parts of the
chamber are removed from the metaphen, washed in distilled
water and dried. The paper washer is recovered from the
wax mixture and, when the wax has solidified, is placed on the
shelf of the base element. The mica cover is placed on’top
of it and pressed down. The retaining collar is then applied
and the nuts screwed down. A water- and bacteria-tight
joint is thus made between the removable cover and the rest
of the chamber. For the formula of the wax mixture I am
indebted to Dr. S. C. Williams, of this laboratory. From this
point on the installation of the chamber is the same as that
described f o r other ‘pre-formed7chambers (Clark et al., ’30).
Such a chamber when installed in an ear follows the same
course as is usual for the ‘pre-formed tissue’ chamber. After
the immediate effects of the operation have disappeared, the
cover may be removed and the chamber contents exposed.
The following precautions must be observed: The entire ear
and chamber are sterilized by immersion in metaphen, 1:5007
o r some similar antiseptic which neither injures the delicate
tissues of the car nor affects the celluloid. All subsequent
operations are carried out aseptically. The nuts over the
retaining collar are removed. The well in the chamber is then
TRANSPARENT CHAMBER FOR CELL CULTUllE
491
filled with cool Ringer’s solution, about 60°F. If the entire
procedure can be done in a 50°F. constant temperature
room, so much the better. The retaining collar is now
removed. The mica cover stays on the paper washer, and
can be raised with a needle. This should be done very
slowly, allowing the Ringer’s solution to fill the space formed
by raising the cover, which can shortly be removed with forceps. The thin tissue is then exposed under fluid and if the
operations are done slowly enough and at the proper temperature, no hemorrhage occurs. Under optimum circumstances
the tissue can be exposed f o r as much as 1 hour and no dcleterious effects occur. However, should the temperature be too
high, smelling occurs and much damage will be done the
preparation. This swelling can be decreased by a preliminary
injection of ephedrin into the base of the ear. The replacement of the cover is the reverse of taking it off. If there has
been any swelling the collar must be screwed down slowly,
taking as long as 1to 2 hours t o reach the original position of
the nuts.
For the application of the external protective splints and
shields which have added much to the success of the transparent chamber technique and f o r the routine handling and
care of the chambers, the reader is referred to the article bj7
Clark and Clark ( ’32, p. 55).
This project, which extends the possibilities for studying
living cells in vivo, was done under the stimulus of Dr. E. R.
Clark, who developed the transparent chamber methed iii
coniiectioii with his researches on living cells in the living
animal.
LITERATURE CITED
E. B., A
m E. L. CLARK 1932 Observations on the new growth of Igmphatic vessels as seen in transparent chambers introduced into thc rabbit’s
ear. Am. J. Annt., vol. 51, p. 55.
CLARK, E. R., II. T. KIRBY-SMITH,
R. 0. R E X AND R. G . WILLIAXS1930 Recent
modifications in the method of studying liping cells in the transparent
ehambws inserted i n the rahbit’s ear. Anat. Rec., vol. 47, p. 87.
VARIAN,B. B. 1931 A transparent elastic glue, used in making chambers for
insertion in the rabbit’s ear. Science, vol. 73, p. 678.
CLARK,
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