/. Embryol. exp. Morph. Vol. 29, 2, pp. 473~483, 1973
473
Printed in Great Britain
Development in culture of rat foetuses explanted
at 12 5 and 13 5 days of gestation
By D. L. COCKROFT 1
From the Physiological Laboratory, Cambridge
SUMMARY
1. As a result of the relatively simple operation of opening the yolk sac and thus exposing
the foetal capillary circulation to flowing medium, it has proved possible to grow 12-5- and
13-5-day rat foetuses for a period of 42 h in culture.
2. 25 % rat serum, 75 % Tyrode has been found to be a satisfactory and economic culture
medium, and has the added attraction that foetal survival is improved compared with that
obtained in whole rat serum.
3. For both 12-5- and 13-5-day foetuses grown with open yolk sacs, a gas mixture of
95 % O.,, 5 % CO2 in equilibrium at 1 atm with culture medium flowing at about 15 ml/min
has been found to give the best results.
4. Under these conditions, foetuses explanted at 12-5 days increased their protein content
from 1 0-1 -3 mg to 2-3-2-7 mg during culture. Their somite number was 40-44 at explantation
and reached 50-55 during culture.
5. Foetuses explanted at 13-5 days increased their protein content from 3-2-40 mg to
6-1-7-5 mg during culture. Their somite number was 51-55 at explantation and reached 60-63
during culture.
INTRODUCTION
In vitro culture techniques provide a controlled system for the study of the
effects of drugs and teratogens (e.g. Turbow & Chamberlain, 1968) and are
convenient for examining certain aspects of foetal physiology (e.g. Shepard,
Tanimura & Robkin, 1970; Netzloff, Johnson & Kaplan, 1968). During the
period from 11-14 days of gestation in the rat, significant features of foetal
development are the differentiation of the limbs and the ability to utilize an
increasing range of substrates in energy metabolism (Cox & Gunberg, 1972).
The culture of 12-5- and 13-5-day rat foetuses could therefore be useful in the
study of such problems, and since the method used here involves completely
exposing the foetus, extirpation and grafting experiments would be feasible.
Some experiments of this type have been done on the mouse embryo in vitro
(Smith, 1964) and on the rat foetus in vivo (Nicholas, 1934).
The culture of post-implantation rat embryos in homologous serum for periods
of 2 or 3 days is possible over a range of ages. The lower limit at present is
7-5 days of gestation, i.e. about 24 h after implantation (New & Daniel, 1969;
Steele, 1972). The limiting factor in the case of the older embryos is probably
1
Author''s address: Physiological Laboratory, Downing Street, Cambridge, CB2 3EG, U.K.
474
D. L. COCKROFT
inadequacy of the oxygen supply, imposed by the non-functioning of the allantoic placenta in culture (New & Coppola, 19706). After about 11 days of
gestation, this placenta becomes increasingly important in vivo in the transfer
to the foetus of oxygen and nutrients. The normal culture system used in this
laboratory uses the yolk-sac placenta as the main organ of oxygen transfer.
The yolk sac surrounds the foetus after the 10th day and if explanted intact
continues to grow with the foetus in culture. At each age there is an optimum
oxygen concentration with which the culture medium should be equilibrated
(New & Coppola, 1970a). Hitherto, 11-5-day rat embryos have been the oldest
which can be grown at atmospheric pressure, using circulating medium and a
gas mixture of 95 % O2, 5 % CO2. By raising the gas pressure to 2 atm and hence
making more oxygen available to the yolk sac, it has proved possible to extend
the circulating medium culture technique to 12-5-day foetuses (New & Coppola,
19706). During a culture period of 40-45 h, these foetuses achieved a 55 %
increase in protein content and increased their somite number from about 40
to 50-55. However, further development of the foetus was prevented by the
harmful effect of the high oxygen pressure on the yolk sac itself, which soon
lost its blood circulation and ceased to have any placental function.
The experiments described here were performed in order to find the effect
on growth and differentiation of explanting 12-5- and 13-5-day rat foetuses with
the yolk sac opened and the amnion removed so that the foetal surface was
directly exposed to the flowing medium, and to discover the optimum oxygen
concentration at each age. Comparisons were made with foetuses of the same
age grown totally enclosed in their yolk sacs in the normal way, using a range
of oxygen pressures at each age. In addition, since the size of these foetuses
demands a large volume of culture medium per foetus, it was decided to try
diluting the rat serum used as a culture medium with Tyrode saline.
MATERIALS AND METHODS
Source of foetuses
Male and female rats of the CFHB strain were caged together in the late
afternoon, and the following morning vaginal smears were made. If sperm were
found in a smear, that rat was considered to be half a day pregnant. Females
12-5 or 13-5 days pregnant were killed in the early afternoon by cervical
dislocation. In experiments in which more than one rat was used, the foetuses
from each rat were shared equally between treatments.
Explantation
Foetuses were explanted using techniques similar to those described by New
(1967). The uterus of a 12-5- or 13-5-day pregnant rat was removed and placed
in a dish of sterile Tyrode saline. It was opened with fine forceps and each
conceptus was cut away with cataract knives. The decidua was removed
with watchmaker's forceps and Reichert's membrane opened, exposing the
Rat foetuses in culture
475
Viteiline
blood vessels
Yolk sac
L -shaped
slit cut in
yolk sac
Alluntoic
placenta
I cm
Fig. 1. Diagram of 13-5-day rat foetus in yolk sac. To open the yolk sac, a slit is made
at the site shown on the surface of the yolk sac behind the foetus. The foetus is
pulled through the hole head first and the amnion (not shown) is removed. The
result of this operation is shown in Fig. 2.
yolk sac. Some foetuses were cultured with the yolk sac intact but in others the
yolk sac was opened as follows. Using a pair of watchmaker's forceps ground
to give a cutting action, an L-shaped slit was cut in the yolk sac, carefully
avoiding any major blood vessels (Fig. 1). The foetus was pulled head first
through this slit. The amnion was then removed and the yolk sac and the blood
vessels leading to it were tucked under the tail.
The foetus thus prepared with open or closed yolk sac was attached by means
of flaps of Reichert's membrane to a strip of collagen-coated terylene fabric
stuck to a piece of glass coverslip, 5 x 20 mm (Fig. 2). One 13-5-day foetus or
two 12-5-day foetuses were attached to each piece of terylene coverslip. The
coverslip with its attached foetus(es) was then placed inside the culture chamber
of a glass circulator - a device for circulating oxygenated medium past the
foetus (New, 1967). Ten ml of medium was put inside each circulator; i.e.
5ml/l2-5-day foetus, 10 ml/13-5-day foetus.
The circulator was mounted in a Perspex-walled incubator, or, if hyperbaric
oxygen was required, in a pressure chamber (New & Coppola, 19706). The
temperature was maintained at 37£ °C. The rate of flow of the medium through
the (10 mm)2 section culture chambers was 15 ml/min (Fig. 3).
Gas mixtures
In experiments with different serum/Tyrode concentrations, the gas mixture
used was 95 % O2, 5 % CO2 at 1 atm, and the foetuses were cultured with open
yolk sacs.
D. L. COCKROFT
Fig. 2. Rat foetus of 13-5 days with opened yolk sac attached to a piece of terylenecovered glass coverslip. The preparation is ready to be placed inside the culture
chamber of a circulator.
Fig. 3. Rat foetus explanted at 13-5 days with open yolk sac inside (10 mm)2 section
culture chamber. Oxygenated medium isflowingpast the foetus. The photograph was
taken 20 h after explanation.
The proportion of carbon dioxide in hyperbaric gas mixtures was reduced
from the 5 % in mixtures used at 1 atm so that its partial pressure remained
the same (about 38 mmHg). Hence the gas mixture used at 1-5 atm (gauge
reading O5kgf/cm2) was 96-6% O2, 3 4 % CO 2 ; at 2 atm (1-Okgf/cm2),
97-5% O2, 2-5% CO 2 ; and at 3 atm (2-0 kgf/cm2), 98-3% O2, 1-7% CO2.
(Note: 1 atm = 101-3 kNirr 2 .)
Culture media
Streptomycin was added to all media to a final concentration of 50 /^g/ml
100 % rat serum and two dilutions of rat serum in Tyrode were used - 50 %
serum, 50% Tyrode; and 2 5 % serum, 7 5 % Tyrode. When oxygen concen-
Rat foetuses in culture
All
trations and the effect of open or closed yolk sacs were compared, the culture
medium used was 25 % rat serum, 75 % Tyrode.
Serum was prepared as follows, using sterile precautions. Blood was withdrawn by syringe from the dorsal aortae of rats anaesthetized with ether,
allowed to coagulate at 4 °C for 18-24 h, and centrifuged at 2500 rev/min for
10 min. The supernatant was recentrifuged and stored at —10 °C until required.
Tyrode was made up in triple-distilled water and its osmolarity adjusted to that
of rat serum (304 m-osmoles).
Assessment of growth and differentiation
The cultures were terminated after 42 h, and the foetuses were dissected free
of their membranes and placentae. Somite counts were made, counting the
somite caudal to the hind limb bud as number 32 (Witschi, 1962), and the protein
content of each foetus was measured using the colorimetric method of Lowry,
Rosebrough, Farr & Randall (1951). Foetuses cultured in vitro were compared
with zero controls (litter-mates which had been stored at 4 °C since explantation).
RESULTS
Serum ITyrode dilutions
As shown in Table 1, there are no significant differences in the protein
contents of foetuses of the same age grown in whole serum or serum diluted
with 50 % or 75 % Tyrode saline. However, Table 2 shows that foetal survival,
as indicated by persistence of the heart beat, is improved by diluting the serum
with 75 % Tyrode.
Oxygen requirements - 12-5-day foetuses
Table 3 shows that the protein content of foetuses grown with open yolk sacs
in 95 % O2, 5 % CO2 at 1 atm is more than doubled during the culture period.
This is significantly better growth (P < 001) than is obtained with closed yolk
sacs and 97-5 % O2, 2-5 % CO2 at 2 atm, when the protein increase is about
55 %. The latter figure is consistent with the results obtained by New & Coppola
(1970/?). Reducing the oxygen concentration from 95 % to 40 % at 1 atm resulted
in very poor growth of open yolk-sac foetuses (P < 0-001).
Oxygen requirements - 13-5-day foetuses
Table 4 summarizes the results of growing 13-5-day foetuses in a variety of
oxygen concentrations and pressures with open and closed yolk sacs. Of the
foetuses cultured with open yolk sacs the best results were obtained in 95 % O2,
5 % CO2 at 1 atm. In this group the protein increase during the culture period
was 60-100 %. This growth was better than that in oxygen at higher pressures
and there was little or no growth in 40 % oxygen. None of the foetuses grown
with closed yolk sacs synthesized much protein (P < 0-01 when compared with
open yolk-sac foetuses in 95 % O2, 5 % CO2 at 1 atm).
31
EMP
29
478
D. L. COCKROFT
Table 1. Protein contents of rat foetuses explanted at 12-5 and 13-5 days
of gestation and cultured for 42 h in different serum \Tyrode dilutions
The foetuses were grown with open yolk sacs in medium equilibrated with 95 % O2,
5 % CO2 at 1 atm. There are no significant differences between the mean protein
contents of groups of foetuses of the same age grown in different serum/Tyrode
dilutions.
Mean foetus protein ± S.E. G«g)*
13-5 day
Medium
12-5-day
100% serum
50 % serum \
50 % Tyrode I
25 % serum \
75% Tyrode j
Zero, controls
1966± 192 (14)
5581 ±279(15)
—
2268 ±150 (14)
5891 ±249 (15)
5813 ±194 (13)
2260±120(14)
—
6374 ±208 (13)
6099 ±179 (14)
1005 ± 60(14)
3589±166(15)
3411 ±150 (13)
3216+143(13)
5800 ±232 (14)
* No. of foetuses in parentheses.
Table 2. Survival as indicated by persistence of heart beat of
12-5- and 13-5-day rat foetuses in culture
The foetuses were grown with open yolk sacs in medium equilibrated with 95 % O2,
5 % CO2 at 1 atm.
12-5-day foetuses
Medium
100% serum
50 % serum
50 % Tyrode
25 % serum
75 % Tyrode
13-5-day foetuses
No. with No. with
No. with No. with
No.
heart beat heart beat
No.
heart beat heart beat
explanted at 24 h
at 42 h explanted at 24 h
at 42 h
14
13
1
19
15
1
14
14
7
18
15
3
14
14
17
17
Condition offoetuses after culture
At the end of the culture period, the best foetuses (grown with open yolk sacs
in 95 % O2, 5 % CO2 at 1 atm) had excellent heart beats (140-180/min explanted
at 13-5 days; 180-200 explanted at 12-5 days) and good foetal circulations. The
circulation in the open yolk sac often persisted for more than 24 h, but only in
the main yolk-sac blood vessels.
Foetuses explanted at 13-5 days and grown with open yolk sacs at 1-5 atm
also had good heart beats at the end of culture, but in much of the vascular
system the blood was static and limb differentiation was poor. When 13-5-day
Rat foetuses in culture
479
Table 3. Protein contents of rat foetuses explanted at 12-5 days of gestation
with open and closed yolk sacs and cultured for 42 h in different oxygen
concentrations
The medium used was 25 % rat serum, 75 % Tyrode. The figures in italics indicate
probability levels (P) of differences between means using the Student / test.
Mean foetus protein ± S.E. (fig)
Treatment
Open yolk sacs
40 % O a , 55 % N 2 , 5 % CO 2)
1 atm
1488 ± 83(10)
95 % O 2 , 5 % CO 2 , 1 atm
2480±184(10)
Closed yolk sacs
97-5 % O2, 2-5 % CO 2 , 2 atm
Zero, controls
1179± 48(10)
< 0001 2692 ±202 (12) ]
P < 001
2047 ± 92 (12) J
1311+ 38 (12)
Fig. 4. Two foetuses with their placentae taken from the same rat at 12-5 days of
gestation. The one on the left was stored at 4 °C at explantation. The foetus on the
right was cultured with open yolk sac for 42 h in circulating medium. The photograph was taken immediately after culture.
foetuses were grown with open yolk sacs at 2 atm, severe microcephaly and a
stunted appearance resulted. 12-5-day foetuses grown with closed yolk sacs at
2 atm suffered from static blood areas and microcephaly. All the 13-5-day
foetuses grown with closed yolk sacs were in very poor condition at the end of
31-2
97-5 % O2, 2-5 % CO2, 2 atm
Closed yolk sacs
95 % O2, 5 % CO2, 1 atm
97-5 % O2, 2-5 % CO2, 2 atm
98-3 %O 2 , 1-7%CO2, 3 atm
Zero, controls
96-6 % O2, 3-4 % CO2, 1-5 atm
95 % O2, 5 % CO2, 1 atm
Open yolk sacs
40 % O2, 55 % N2, 5 % CO2, 1 atm
Treatment
0001
3492 ± 84(9)
6086 ± 284 (9)
6500+327(9)
I
P <
3842 ±155 (9)
3856+ 133 (5)
P < o-oi
5209 ±397 (5)
I
7517 ±359 (5)
4647±185(6)
—
_
4012± 165 (6)
P < ooi
6519 ±481 (6)
4554±176(6)
_
3755±79(6)
P < o-oi
6672±543(6)
Mean foetus protein ± S.E. Og). No. of foetuses in parentheses
The medium used was 25 % rat serum, 75 % Tyrode. The figures in italics indicate probability levels
(P) of differences between means using the Student / test.
001
4460±154(6)
3978 ± 112 (6)
P <
6617±554(6)
Table 4. Protein contents of rat foetuses explanted at 13-5 days of gestation with open and closed yolk
sacs and cultured for 42 h in different oxygen concentrations
H
o
p
r
o
o
o
o
oo
Rat foetuses in culture
481
Fig. 5. Two foetuses with their placentae taken from the same rat at 13-5 days of
gestation. The one on the left was stored at 4 °C at explantation. The foetus on
the right was cultured with open yolk sac for 42 h in circulating medium. The
photograph was taken immediately after culture.
culture. Foetuses grown with open yolk sacs in 40 % O2 at 1 atm were microcephalic and severe oedema was common.
Differentiation
Foetuses explanted with open yolk sacs at 12-5 or 13-5 days and grown in
25 % serum, 75 % Tyrode equilibrated with 95 % O2, 5 % CO2 at 1 atm showed
considerable differentiation, corresponding to about 24 h in vivo (Figs. 4, 5).
The limb-buds of 12-5-day foetuses enlarged and became paddle-shaped, and
digits were formed on the forelimbs of 13-5-day foetuses. The older foetuses
also showed marked elongation of the snout and elevation of the primary
whisker papillae. When somites could be counted, 12-5-day foetuses increased
their somite number from 40-44 to 50-55 during the culture period, 13-5-day
foetuses from 51-55 to 60-63. (It was not always possible to obtain reliable
somite counts of foetuses cultured with open yolk sacs, as the tail sometimes
became covered by a flap of the opened yolk sac, resulting in its growth being
stunted, though the foetus could be in excellent condition otherwise.)
482
D. L. COCKROFT
DISCUSSION
The results presented here describe the first successful attempt to grow 13-5day rat foetuses in culture. Explanted with open yolk sacs and cultured in 25 %
rat serum 75 % Tyrode equilibrated at one atmosphere with 95 % O2, 5 % CO2
flowing at 15ml/min, the foetuses show good growth and differentiation,
corresponding to about 24 h of development in vivo. Under the same conditions,
growth of 12-5-day foetuses is also greatly improved over that obtained by
previous methods. At these stages in vivo, much of the foetuses' oxygen is
presumably supplied via the allantoic placenta. However, this placenta does not
appear to grow or function in vitro (Figs. 4, 5). Growing the foetus with the
yolk sac opened makes up for this deficiency by utilizing the capillary circulation
at the foetal surface for oxygen transfer, in addition to any contribution from
the opened yolk sac.
When 13-5-day foetuses with open yolk sacs were grown in hyperbaric oxygen
(1-5 or 2 atm) the results were inferior, presumably due to the toxic effects of
the high oxygen concentration.
The poor growth and condition after culture of all the foetuses grown with
closed yolk sacs probably results from early death of the foetuses due to oxygen
shortage. The yolk-sac circulation is unable to supply the oxygen requirements
of 12-5- or 13-5-day foetuses at atmospheric pressure, and at higher pressures
the yolk-sac circulation fails early in culture as a result of oxygen toxicity (New
& Coppola, 19706). Reducing the oxygen concentration to 40 % at 1 atm results
in very poor growth of foetuses explanted with open yolk sacs. This is perhaps
surprising in the case of 12-5-day foetuses, as the optimum oxygen concentration
for 13-5-day foetuses is 95 % at 1 atm, and it might be expected that 12-5-day
foetuses could grow with a lower oxygen concentration. It is possible, however,
that the oxygen requirements of 13-5-day foetuses cannot be fully met by the
culture method described here. When the oxygen concentration is raised above
95 % at 1 atm, the cells near the surface of the foetus may be damaged by the
high oxygen concentration, even though more centrally situated cells are being
starved of oxygen.
The results of growing foetuses with open yolk sacs in different serum/Tyrode
dilutions show that growth is at least as good in dilute serum as in whole serum.
These results are in contrast to those of New (1973), who found that diluting
serum with Tyrode resulted in poorer growth and differentiation of 10-5- and
11-5-day foetuses explanted with closed yolk sacs. This suggests that there may
be some harmful or inhibitory factor in rat serum which does not affect younger
foetuses though its dilution improved survival of older foetuses. Alternatively,
the closed yolk sac of a 10-5- or 11-5-day foetus may not allow transmission of
the harmful factor. It could be, however, that the higher viscosity of undiluted
serum is responsible for the presence around the foetus of a static layer of serum
which impedes oxygen transfer to the foetus.
Rat foetuses in culture
483
This work was supported by a Medical Research Council scholarship for training in
research methods. I am grateful to Dr D. A. T. New for advice.
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