/. Embryol. exp. Morph. Vol. 36, l,pp. 133-144, 1976
Printed in Great Britain
133
Comparison of growth in vitro and in vivo of
post-implantation rat embryos
By D. A. T. NEW1, P. T. COPPOLA 1 AND D. L. COCKROFT 1
From the Physiological Laboratory, Cambridge
SUMMARY
Rat embryos explanted at early head-fold stage and grown in vitro by improved culture
methods were compared with littermates in vivo. Very similar rates of growth and differentiation were obtained over a period of 48 h, while the embryos developed to around the
25-somite stage.
INTRODUCTION
Methods for studying the development of mammalian embryos in vitro are of
value only to the extent that the growth and differentiation of the embryos
resemble that in vivo. In recent years, techniques have been devised for growing
post-implantation rat and mouse embryos in vitro throughout the period of
organogenesis (reviews by New, 1973; Kochhar, 1975; Steele, 1975). The
embryos are explanted with their membranes and grown in serum, or in mixtures
of serum and synthetic culture media, with carefully regulated levels of oxygenation. Under these conditions the explanted embryos differentiate well but it has
usually been found that growth, as determined by rate of protein synthesis, is
slower than in vivo (Berry, 1968; Robkin, Shepard & Tanimura, 1972; New,
1973; Cockroft, 1973, 1976).
A likely explanation for the retarded growth of the older embryos is that they
lack a functional allantoic placenta. But the allantoic blood circulation does not
become established in normal development until about the 17-somite stage (11
days of gestation in the rat). It seems possible that at the earlier stages of
organogenesis, normal rates of growth might be supported simply by an improved nutrient medium combined with appropriate concentrations of O2 and
CO2 in the gas phase. It has recently been found that the nutrient serum can be
improved by preparing it from blood centrifuged immediately after withdrawal
from the donor rat and by heat-inactivating it before use (Steele, 1972; Steele &
New, 1974; New, Coppola & Cockroft, 1976), and that young embryos benefit
from a reduction of oxygen in the gas phase to 5 % (M. H. Jacobs, unpublished;
New et ah 1976). In this paper, rat embryos explanted at early head-fold stage
(9| days of gestation) and grown under the improved culture conditions are
compared with similar embryos grown in vivo.
1
Authors' address: Physiological Laboratory, Downing Street, Cambridge CB2 3EG, U.K.
134
D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT
4 embryos
grown
Fig. 1. Selection of embryos from the two uterine horns for comparison of growth
in vivo and in vitro.
MATERIALS AND METHODS
Embryos were obtained from rats of CFHB strain during the morning of the
10th day of gestation (9£ days post coiturri). In the embryos at this stage, neural
folds were just beginning to appear but no other organs had yet formed (Fig.
3 A). An allantoic rudiment was present as a small bud at the hind end of the
embryo. The length of the whole conceptus (embryo + membranes), excluding
the ectoplacental cone, was about 1-5 mm.
Comparisons of growth in vitro and in vivo were made between embryos from
the two uterine horns of each rat. The rat was anaesthetized with ether and one
horn of the uterus drawn out through a small incision in the abdominal wall.
This horn and its blood vessels were ligated at the cervical end between the
first and second implantation sites (Fig. 1). The blood vessels were also ligated
at the ovarian end. The ovary and uterine horn were then cut free except for the
implantation site next to the vagina, which was left in place. The whole operation took about 20 min and within half an hour the animal had recovered from
the anaesthetic and was actively moving round the cage and licking itself. Care
was taken at all stages of the operation to avoid disturbing the uterine horn and
blood vessels of the opposite side. To check whether the operation had any
effect on the growth of the embryos in vivo, we (i) compared the embryos at 32
and 48 h after operating with those of similar age in unoperated animals, and
(ii) allowed development in a few operated rats to proceed to term and compared the newborn young with those of unoperated animals.
It is known that in mice, foetal weight can be affected by both number and
position of the foetuses in the uterine horn (Healy, McLaren & Michie, 1961;
McLaren, 1965). We therefore used in this study only rats that had 5-10
embryos in each horn with a difference of not more than three embryos between
the two horns, and the embryos to be compared were taken from similar positions in the two horns (Fig. 1). The mean and standard error of the number of
Rat embryos in vitro and in vivo
135
Table 1. Embryo growth in operated and unoperated rats
Mean somites
per embryo
Mean protein (jig)
per embryo
15-7
14-5
55
51
24-6
24-7
23-5
219
223
190
Embryos at 9£ days+ 32 h from:
Operated rats
Unoperated rats (1973)
Embryos at 9\ days+ 48 h from:
Operated rats
Unoperated rats
Unoperated rats (1973)
implantation sites in the horns used to provide embryos for growth in vitro was
7-7 ±0-3, in those for growth in vivo 7-3 ±0-3.
The embryos for culturing were explanted with the visceral yolk-sac and
ectoplacental cone intact but with the Reichert membrane torn open (Fig. 3 A).
They were incubated at 38 °C in small cylindrical stoppered bottles rotated continuously at 40-50 rev/min for periods of 32 or 48 h (New, Coppola & Terry,
1973). The bottles were of 30 ml capacity containing 4 ml of culture serum, but
some of the embryos were transferred after 24 h of incubation to bottles of
60 ml capacity containing 8 ml of serum. Usually, four embryos were placed in
each bottle. The culture serum was obtained from CFHB rats, without regard
to the sex or age of the animals, and pooled before use. The serum was always
prepared from blood centrifuged immediately after extraction from the rat (I.C.
serum of Steele & New, 1974) and was heat-inactivated (56 °C for 30 min)
before being added to the culture bottles. The gas phase was 5 % O2/5 % CO2/
90 % N 2 for the first 22-24 h, followed by 20 % O2/5 % CO2/75 % N 2 . Some of
the cultures received a further gassing at 32 h with 20 % O2/5 %CO2/75 % N 2 or
40 % O2/5 % CO2/55 % N 2 .
Development of the embryos, in vitro and in vivo, was assessed by the number
of somites formed, the condition of the blood circulation, the closure of the
neural tube, the adoption by the embryo of the foetal (ventrally concave)
position, and the fusion of allantois and chorion. Growth was assessed by
measuring the crown-rump length and the yolk-sac diameter, and by determining the protein content of the embryo (without its membranes) by the colorimetric method of Lowry, Rosebrough, Farr & Randall (1951).
RESULTS
Growth of embryos in operated and unoperated rats
(i) Table 1 shows the mean values for somite number and protein content of
all the embryos grown in vivo in one horn of the uterus for 32 h and 48 h after
removal of the opposite horn (i.e. all the in vivo embryos listed in Tables 2 and
3). These values are compared with those of embryos of the same age in
136
D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT
Table 2. Comparison of embryos grown for 32 h in vivo and in vitro
(All values are means per embryo)
CrownNumber
Yolk sac
rump
of
embryos diam (mm) length (mm)
Rat
no.
1 In vivo
In vitro
2 In vivo
In vitro
3 In vivo
In vitro
4 In vivo
In vitro
5 In vivo
In vitro
4
4
4
4
4
4
3
3
4
4
3-4
2-8
3-4
2-9
3-4
2-9
2-2
2-5
3-7
2-9
2-2
2-3
2-2
2-2
20
2-3
Not
determined
2-4
2-5
Somite
number
Protein (/*g)
(mean ± S.E.)
170
16-2
14-8
15-8
15-5
15-5
12-7
150
170
59±7
51 ±3
46±4
52 ±5
Not
determined
38 + 6
43 ±1
76±2
74 ±6
180
unoperated rats. Data for the latter were obtained from four unoperated pregnant
rats examined as part of the present series of experiments, and from a curve of
normal embryo growth prepared previously (New, 1973). The comparison gives
no indication that growth of the embryos in the operated rats is retarded.
(ii) Embryonic development in four operated rats was allowed to continue to
term. Birth of normal young occurred at 22, 22, 24 and 24 days of gestation
respectively, with a mean litter number of 8-2 and a mean weight for each
newborn of 6-1 g. Four unoperated animals all gave birth at 21 to 22 days of
gestation, with a mean litter number of 12-0 and a mean weight per newborn of
6-0 g. The slight difference in the time of birth of the two groups makes it
uncertain how far the weights of the newborn can be used for exact comparison
of rates of embryo growth. However, the results do not suggest that either
growth or development of the embryos in the operated rats was affected
significantly.
Comparison of embryos in vitro and in vivo after 32 h
Thirty-two hours after the time of explantation, all the embryos in culture had
a good yolk-sac blood circulation. Those embryos with 17 or more somites, and
an occasional embryo with 16 somites also showed a rudimentary blood circulation in the allantois. In all the 19 embryos grown in vivo, and in 15 out of the
19 grown in vitro, the allantois was now tightly fused with the chorion; in the
remaining four it had enlarged but remained 'free' in the extra-embryonic
coelom. Apart from this, all the embryos appeared to be normal and without
malformations (Fig. 3B).
Table 2 gives further details. In four out of the five rats used for the experiment, the mean yolk-sac diameter of the embryos in vitro was less than in vivo.
Rat embryos in vitro and in vivo
D
Scries
65'
250
137
82"
89 ",
20",; O,
20",;O 2
112
-h
200
o
c. 150
100
50
Treatment
/'// vitro
At 24 h
At 32 h
-0",'. O,
20",; o 2
20 " , ; o ,
60 ml bottle
8 ml new serum
60 ml bottle
8 ml new serum
20% O,
40 •;,; o .
40';,, O2
Fig. 2. Comparison of final embryo protein in vivo and in vitro, after five different
culture treatments. Each rectangle in the histogram shows the mean and standard
error of 12 embryos. Further details of thefiveseries of embryos (A-E) are given in
Table 3.
But there was no significant difference in crown-rump length, somite number, or
protein content.
It appears that, for the first 32 h after explantation, the rate of growth and
development of the embryo itself is the same in vitro as in vivo. But in some of
the explants there may be slight abnormalities of the embryonic membranes.
Comparison of embryos in vitro and in vivo after 48 h
Fifteen rats were used in this experiment, providing a comparison between 60
embryos grown for 48 h in vitro with 60 left to continue growth for the same
duration in vivo. Nearly all the explanted embryos still had a good yolk-sac
blood circulation at the end of the culture period and many also had a circulation in the allantois. In about 20 % of the cultured embryos, the allantois had
failed to fuse with the chorion. All the embryos had turned into the ventrallyconcave 'foetal' position, but in a few (< 10 %) the posterior tip of the trunk
was bent sideways as though the final stages of turning had not been completed.
These two abnormalities appeared to be unrelated. In view of the sensitivity of
neural tube development to variations in the culture conditions (New et al.
1976), all the cultured embryos were examined particularly carefully for
abnormalities in this region, but no failures of closure of the tube or other
malformation of brain or spinal cord were observed.
138
D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT
Table 3. Comparison of embryos grown for 48 h in vivo and in vitro
(All values are means per embryo)
Series
B
D
Number
Crownof
Yolk sac
rump
embryos diam (mm) length (mm)
Rat
no.
10
11
12
13
14
15
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
In vivo
In vitro
3-7
3-4
3-3
2-7
4-4
41
4-5
4-3
41
3-4
3-9
3-6
40
3-8
3-8
3-6
40
3-3
3-5
30
40
3-9
3-4
3-3
4-3
4-2
3-6
3-7
4-2
3-6
4-6
41
3-7
3-4
4-4
4-3
3-8
3-8
4-2
4-2
3-7
3-8
4-3
4-3
2-5
3-8
3-9
40
4-2
3-9
3-4
3-7
4-3
3-8
4-4
3-6
3-8
3-9
3-4
3-4
Somite
number
Protein (/*g)
(mean ± S.E.)
230
22-5
26-7
26-7
26-5
25-5
24-8
240
24-3
23-5
23-3
220
23-8
23-5
24-8
24-5
24-8
22-3
25-0
25-3
25-8
25-3
24-8
25-3
250
26-8
23-3
25-3
23-3
23-3
178 ±7
101 ±12
292+13
190±3
255 ±12
179±3
226+19
173 ±7
252 ±12
163 ±13
195 ±21
136 ± 6
177 ± 6
144±9
182± 13
184± 13
191 ± 3
122 ±9
233 ± 14
201 ± 6
274 ±8
209 ±13
223 ±40
240 ±11
224 ±15
268 ±17
201 ± 17
233 ±10
182±5
177 ±5
Five variants of the culture procedure were tried, each using three of the
fifteen rats, as follows:
Series A. The culture bottles were regassed with 20 % O2 (i.e. 20%O 2 /
5%CO 2 /75%N 2 ) a t 2 4 h but not subsequently; the serum was not renewed.
Series B. Regassed with 20 % O2 at 24 h and transferred to 8 ml fresh serum
in a larger (60 ml) bottle.
Series C. Regassed with 20 % O2 at 24 h and at 32 h; serum not renewed.
Series D. Regassed with 20 % O2 at 24 h, and with 40 % O2 at 32 h; serum not
renewed.
Rat embryos in vitro and in vivo
139
Table 4. Comparison of different culture treatments on embryos
grown for 48 h in vitro
Treatment at
A
32 h
24 h
20%O 2
20%O 2
20%O 2
[20 %o,
60 ml bottle
8 ml new serum
40%O 2
20 % O2
20%O 2
40%O 2
60 ml bottle
8 ml new serum
J
J
No. of
embryos
Yolk sac
diam
(mm)
Crownrump
length (mm)
Somite
number
Protein (/*g)
(mean ± S.E)
12
3-8
3-6
23-9
162 ± 11
12
3-8
3-6
23-6
180± 11
12
3-7
3-4
23-9
171 ±12
12
3-7
3-5
23-9
181 ±11
Series E. Regassed with 20 % O2 at 24 h, and transferred to 8 ml fresh serum
in a larger (60 ml) bottle. Gassed with 40 % O2 at 32 h.
Figure 2 and Table 3 give further details. In Series A, the average number of
somites in the cultured embryos was similar to that of the comparable embryos
in vivo but the relative protein content was only 65 %. In Series B to E the
protein of the cultured embryos progressively increased until in Series E it was
112 % of that of the controls. In each of Series A, B and C the protein contents
of the embryos in vitro were significantly different from those in vivo (P < 0-01);
in Series D and E the differences were insignificant (P > 0-1).
Comparison of different treatments for embryos cultured for 48h
To obtain further information about the variants of culture method used in
the previous experiment, 48 head-fold embryos were explanted and grown
under four different culture treatments, care being taken that embryos from the
same litter were always divided equally between the four treatments.
The culture details and the results are summarized in Table 4. All the cultures
were regassed at 24 h and at 32 h. The results show slightly increased embryo
protein when the O2 level was raised at 32 h to 40 %. Embryo protein was also
higher in those cultures that were transferred at 24 h to 60 ml bottles with 8 ml
new serum. This may have been an effect of the fresh serum, or an effect of the
increased gas space providing more oxygen, or a lower CO2 concentration
resulting in a serum pH closer to normal values; at the end of the culture, the
pH of the serum in the 60 ml bottles was 7-4-7-5, in the 30ml bottles 7-1-7-3.
But in general the results of the four treatments were similar, and the differences of embryo protein content are not statistically significant (P > 0-1).
It should be noted that three of these treatments resemble those given to the
cultured embryos of Series C, D and E of Fig. 2. None is comparable with
Series A or B.
140
D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT
Fig. 3. (A) Head-fold stage embryos as explanted at 9\ days of gestation. (B)
Embryos grown for 32 h in vivo (upper row) and in vitro (lower row). The embryos
are all from rat 1 of Table 2. Photographed after removal of the embryonic membranes. (C) Embryos grown for 48 h in vivo (upper row) and in vitro (lower row).
The embryos are all from rat 14 of Table 3. Photographed after removal of the
embryonic membranes. All the photographs are at the same magnification.
Rat embryos in vitro and in vivo
*
4 ** -
Fig. 4. (A) Optic vesicle of embryo after 48 h in culture. The retinal area and lens
epidermis are thickening, and invagination of the optic cup is beginning. (B) Optic
vesicle of a littermate of the embryo shown in (A), which was allowed to develop for
the corresponding period in vivo. The vesicle is rounded and no retinal or lens
thickening has taken place. (C) Auditory vesicle of the embryo shown in (A).
Closure is just being completed. (D) Auditory vesicle of the embryo shown in (B),
which is at a similar stage of development to that shown at (C). All the photographs
are at the same magnification.
141
142
D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT
Comparative histology of embryos grown in vitro and in vivo
A histological examination was made of 12 embryos (obtained from three
rats) that had been grown in vitro for 48 h, and of a further 12 embryos, littermates of the cultured embryos, that had been allowed to develop in vivo for the
corresponding period. The culture schedule was the same as for the embryos of
Series E, Fig. 2. No abnormal tissue degeneration was found in any of the
embryos examined, though small differences in degree of development of corresponding in vitro and in vivo embryos were noted.
In the cultured embryos from one rat, one embryo was retarded, but the other
three were more advanced than the corresponding in vivo embryos with respect
to eye development (Fig. 4 A, B) and closure of the posterior neuropore. In
other respects, such as closure of the auditory vesicles (Fig. 4C, D), development in vitro was similar to that in vivo. The cultured embryos from the second
rat were slightly less well developed in all respects than the in vivo embryos,
though the differences were within the range of variation often found in embryos
from the same rat. For the third rat, optic vesicle development and closure of
the posterior neuropore were again slightly more advanced in vitro than in vivo,
though in other respects the embryos were equivalent.
It appears that histological differentiation of the embryos grown in culture is
very similar to that of the littermates in vivo.
DISCUSSION
The results show that the rates of growth and differentiation of all the
embryos grown for 32 h in culture were similar to those of littermates in vivo
(Table 2 and Fig. 3 B).
The embryos grown in culture for 48 h were more variable. This is perhaps
not surprising in view of the rapidly increasing demands of the embryo on the
culture system. At 32 h after explanation, the embryos are at about the 16somite stage. During the period 32-48 h, littermates in vivo synthesize about
three times as much protein as during the previous 32 h. A culture system that is
sufficient to support normal rates of growth up to 32 h may be inadequate for
longer periods. The inadequacies may include diminished O2, excessively high
CO2 and low pH, and insufficient nutrients or accumulating waste products.
Another reason why it becomes more difficult to maintain normal rates of
growth with increasing age of the embryo, is the failure of development of the
allantoic placenta. At 32 h, blood is just beginning to circulate in the allantois,
and from then on the embryos in vivo can be presumed to have the support of a
functional allantoic placenta. In embryos in culture, there may be an embryonic
blood circulation in the allantois but there is no maternal blood, nor any system
of spaces allowing passage of the culture medium through the placenta, and it is
very unlikely that the allantois provides any support for growth of the embryo.
Rat embryos in vitro and in vivo
143
Our results show that if incubation of the 32-h cultures is continued to 48 h,
without any other treatment, the embryo has only about 65 % of the protein
content of littermates in vivo (Fig. 2, Series A). But if the culture bottles are regassed at 32 h with 20 % O2/5 % CO2/75 % N 2 , final embryo protein in vitro
rises to over 80 % of that in vivo (Series C). Further small gains can be made by
raising the O2 level at 32 h to 40 % and by transferring the embryos during the
culture period to a larger bottle with fresh serum; the protein content of the
embryos in vitro then becomes very similar to that of the controls in vivo
(Series D, E).
The regassing at 32 h is clearly important. The fact that a gas mixture containing 40 % O2 gave slightly better results than 20 % O2 (Fig. 2, Series C, D ;
and Table 4) suggests that at least part of the effect on embryonic growth results
from a raised oxygen level. But these concentrations must give oxygen tensions
in the culture serum of around 320mmHg and 160mmHg respectively, far
higher than those found anywhere in the uterus. It seems likely that their
beneficial effect on embryos in vitro is to cause increased transport of oxygen to
the embryo by the yolk-sac, which compensates for the lack of a functional
allantoic placenta.
Regassing also removes the excess CO2 resulting from embryonic respiration,
and by restoring the CO2 level to 5 %, helps to maintain the serum pH at around
7-4. Both these effects are potentially beneficial but our data are insufficient to
establish whether they were significant. Cultures in which the serum was renewed
(and increased) at 24 h showed improved embryonic growth (Fig. 2, Series B
compared with A, and E compared with D; also Table 4) but these were always
transferred to 60 ml bottles with a larger gas volume; it is uncertain therefore
whether the improvement was primarily the result of the renewed serum or of
factors - respiration, pH, etc. - related to the gas volume.
Is embryonic differentiation at 48 h the same in vitro as in vivo ? Table 3 shows
that under optimum culture conditions (Series D and E) the number of new
somites formed by the embryos in culture is the same as in the littermates in vivo.
In overall appearance, the two groups of embryos are usually indistinguishable
(Fig. 3C). Histologically, the resemblance is also very close (Fig. 4). The only
abnormalities that we have observed were in a few embryos (< 10 %) where the
posterior tip of the trunk was bent sideways - possibly a result of incomplete
turning of the embryo (Deuchar, 1975)-and in about 20% of the embryos
where the allantois failed to fuse with the chorion.
We conclude that the available culture methods enable rat embryos explanted
at head-fold stage to be grown in vitro for 48 h at the same rate of growth and
differentiation as in vivo. We have described in another paper (New et ah 1976)
the further growth of these embryos for 72-95 h.
We would like to thank Mrs S. M. Jackson for valuable technical assistance and the
Medical Research Council forfinancialsupport.
144
D. A. T. NEW, P. T. COPPOLA AND D. L. COCKROFT
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{Received 13 January 1976; revised 12 March 1976)