/ . Embryol. exp. Morph. Vol. 49, pp. 17-25, 1979
Printed in Great Britain © Company of Biologists Limited 1979
Culture of early somite mouse embryos
during organogenesis
ByT. W. SADLER 1
From the Department of Anatomy, School of Medicine, University of
Virginia
SUMMARY
Early somite (2-4) mouse embryos were explanted and then maintained in culture for
24 or 48 h intervals. Various types of media were tested and it was determined that rat serum
supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal
calf serum + Waymouth's (1:1) supported some growth, but did not equal the success of
using rat serum alone. During the 48 h culture period in rat serum, embryos developed to
stages indistinguishable from embryos maintained for a similar time in vivo.
INTRODUCTION
The organogenic period of mammalian embryological development has been
a focal point of investigations by embryologists, teratologists and others
interested in determining mechanisms of normal and abnormal organ differentiation. However, due to the inaccessibility of the fetus in vivo, problems have
arisen in examining and manipulating specific developmental events during
critical organogenic stages. Thus, much attention has been directed toward
developing life support systems capable of maintaining mammalian embryos
in vitro (Clarkson, Doering & Runner, 1969; Robkin, Shepard & Tanimura,
1972; New, Coppola & Terry, 1973; New, Coppola & Cockroft, 1976a, b;
Gunberg, 1976). One of the most successful of these techniques has been developed by Denis New at Cambridge and it is now possible to culture early
stage rat embryos, ranging from 1\ to 13^ days of gestation, throughout much
of the organogenic period, using New's procedure (New et ah, 1976a, b; New,
1978). Thus rat embryos explanted at the primitive streak or early head-fold
stage can be maintained for approximately 4 days in culture with a rate of
organogenesis similar to that observed in vivo (New, 1978; Buckley, Steele &
New, 1978).
In addition to rat embryos, later stage (3CM10 somite) mouse embryos have
been cultured successfully in whole embryo systems for shortened (24-36 h
1
Author's address: Department of Anatomy, Box 439, School of Medicine, University of
Virginia, Charlottesville, Virginia 22901, U.S.A.
18
T. W. SADLER
culture periods (Kochhar, 1975). Culture of later stage embryos permits some
fundamental investigations into embryological events, but rarely affords the
opportunity to observe development of an organ system from primordial stages
to a definitive form. Culture of early stage mouse embryos has also been attempted, but without the success of using rat or later staged mouse embryos (Clarkson et al. 1969; Hernandez-Verdun & Legrand, 1975). Fcr example, in these
investigations a large percentage of embryos failed to establish a normal
circulation, rotation, or growth rate compared to similar stage embryos maintained in vivo (Clarkson et al. 1969; Hernandez-Verdun & Legrand, 1975).
Due to the lack of success with mouse embryo cultures and because many
laboratories employ mice for embryological and teratological investigation, it
was determined that development of a system, capable of supporting early stage
mouse embryos for extended times in culture, would be beneficial. Therefore,
the following study was undertaken.
MATERIALS AND METHODS
Ninth day (plug day = 1st day) ICR mouse embryos were extracted from the
uterus and placed on a watch glass containing sterile Tyrcde's solution. Under
a dissecting microscope, the decidua and Reichert's membrane were removed,
leaving the visceral yolk sac and a small ectoplacental cone intact (Fig. 2,
insert). Somite counts were made and each embryo was placed in a separate
sterile 10 ml culture flask containing 1-5 ml of rat serum, fetal calf serum
(Flow Laboratories, Dublin, Virginia) or a combination of 50% fetal calf
serum, 50% Waymouth's media MB 752/1 (K. C. Biological, Lenexa, Kansas).
Each flask was then gassed with 5 % O2, 5 % CO2, 90 % N2, placed on a rotator
wheel, and rotated at 30 rev./min at 38 °C. At 12 h cultures were re-gassed with
the same gas mixture. After 24 h cultures were either terminated or embryos
were removed and placed separately in either 10 or 25 ml flasks containing
1-5 ml of fresh media and a new atmosphere of 20% O2, 5 % CO2, 75% N 2 .
This same gas mixture was replenished at 36 h and cultures were terminated
at 48 h. Upon termination at 24 h or 48 h, embryos were examined for gross
abnormalities and evaluated for heart beat and embryonic and visceral yolk
sac circulation. Heart beat and circulation were measured on a scale of 0 to
3+ with 3+ heartbeat 3+ circulation being optimum (rapid uninterrupted
heart beat and circulation). Embryos were then prepared for total protein
analysis, by the Lowry method (Lowry, Rosenbrough, Farr & Randall, 1951),
or for histological observation by fixation in modified Karnovsky's fixative
(2% glutaraldehyde, 2 % paraformaldehyde) followed by embedding and sectioning (1 fim) in araldite.
Rat serum was prepared from male Sprague-Dawley retired breeder rats by
withdrawing blood from the abdominal aorta following ether anesthetization.
Twelve-15 ml of blood was obtained from each rat and was immediately centri-
Culture of early somite mouse embryos
19
fuged at 2000 g for 5 min (Steele & New, 1974). Following centrifugation, the
fibrin clot was removed and the sample re-centrifuged as before. Serum was then
decanted (3-4 ml/rat) and heat inactivated in a water-bath at 56 °C for 30 min.
Streptomycin sulphate (50/tg/ml) was added and the serum was either used
immediately or stored up to 2 weeks at - 1 0 °C. Excess ether was removed
prior to culture by gassing the media with 5 % O2, 5 % CO2, 90 % N 2 .
RESULTS
At the time of culture, 9th day embryos had 2-4 somites, open neural folds
and no heartbeat or circulation (Fig. 2, insert). After 24 h in vitro all embryos
cultured in rat serum rotated normally, had 16-19 somites, closed neural
folds, and a 3 + heartbeat 3 + circulation. The visceral yolk sac and amnion
expanded and a vascular supply to the visceral yolk sac and ectoplacental cone
(via umbilical vessels) was established. Macroscopically, these embryos did not
differ from embryos maintained in vitro (Figs. 1 and 2) and histologically no
abnormal sites of necrosis were observed. Developmental events such as optic
vesicle formation, forelimb-bud initiation, and heart growth appeared normal
(Figs. 1 and 2). Total growth as measured by total protein analysis also appeared
normal and no significant differences in protein content were observed between
similar staged embryos maintained in vivo or in vitro (Table 1).
Embryos cultured 48 h (24 h in 10 ml flasks and 24 h in 25 ml flasks containing rat serum) also appeared grossly normal when compared to 11th day
embryos maintained in vivo (Figs. 3 and 4). Extended development of the
craniofacial area, somite numbers, and forelimb buds occurred normally and
on schedule. Furthermore, 22 of 23 embryos cultured had normal heartbeats
and circulation (3 + , 3+). Histologically these embryos showed no abnormal
necrotic areas (Silver & Hughes, 1973) and events such as optic vesicle formation
appeared to occur in a manner comparable to in vivo specimens having similar
numbers of somites (Figs. 5 and 6). Total protein analysis also showed no
significant difference between these embryos and 11th day embryos grown in
vivo (Table 1).
Embryos maintained for 48 h in 10 ml flasks containing rat serum did not
grow as well as embryos which were transferred to 25 ml flasks at 24 h. Somite
counts were lower (Table 1), limb buds were smaller, and heart rates and circulation were decreased (2 + , 2 + ) in 14 of 22 embryos. Developmental abnormalities were also observed, including four embryos with forelimb defects
(one limb smaller than the other), six with enlarged pericardial cavities, and five
which failed to rotate. Total proteins were also significantly decreased from
values obtained from embryos maintained in 25 ml flasks (P < 0 01) and in
vivo (P < 0-01) (Table 1).
Use of fetal calf serum alone severely retarded embryonic growth and embryos
grown in this medium were grossly malformed. After 24 h these embryos
Figs. 1, 2. Macroscopic comparison of 17-somite (S) mouse embryos maintained in vivo (10th day, Fig. 1) and in vitro (24 h
culture, Fig. 2) showing similar stages of development. Heart (H), optic vesicle (OV), otic placode (OP), x 100. Insert:
Early somite (2-4 S) mouse embryo as prepared for culture showing head folds (HF), visceral yolk sac (YS) and ectoplacental
cone, x 50.
d
a
on
>
Culture of early somite mouse embryos
21
Table 1. Comparison of somite numbers and total proteins between embryos
maintained in vivo and in various preparations of culture media
Media
Rat serum
In vivo, 10th day
FCS
FCS + Waymouth's
Rat serum
(10 ml flasks)
Rat serum
(25 ml flasks)
In vivo, 11 th day
Duration
Number
of
of
culture
embryos
(h)
17
24
13
8
48
48
5
Total
somites
(mean ± S.D.)
Total
proteins (/tg)
(mean ± S.D.)
17-8 ±0-9
17-8 ±0-8
Not determined
13±0-9
71 ± 7-9
80 ±15-8
22 ±10-7
142 + 231
21
48
Not determined
242 ±23-8
13
14
48
—
28-2 ±0-9-5
28-1 ±0-6
299 ± 46
305 ±26-7
developed a heartbeat (1 +) but no visceral yolk sac or embryonic circulation
and also failed to rotate. By 48 h heartbeats had ceased, no circulation existed
and no rotation had occurred. Somite counts were not possible due to the
great degree of deformity, and total protein analysis confirmed the severely
depressed amount of growth exhibited by these embryos (Table 1).
Cultures usingfetal calf serum+Waymouth's media (1:1) also failed to develop
normally and all embryos showed a variety of defects including exencephaly
and limb malformations. However, all embryos did establish a heartbeat (2 + )
and embryonic and visceral yolk sac circulation (2+). Somite counts and total
proteins after 48 h in culture revealed that these embryos were significantly
retarded in their amount of growth compared to embryos grown in rat serum
and in vivo (P < 0-01), but also that they did significantly better (P < 001)
in culture than embryos grown in fetal calf serum alone.
DISCUSSION
The results indicate that early-somite-stage mouse embryos can be maintained
in whole embryo culture throughout much of the organogenic period. Embryos
grown in rat serum and transferred from 10 ml flasks to 25 ml flasks after 24 h
develop normally for 48 h showing no abnormal regions of necrosis and no
decrease in total protein. During this time in culture embryological events
such as cardiovascular development, forelimb-bud initiation, and craniofacial
formation appear to occur on schedule and without abnormalities. Thus, the
success of this system using mouse embryos grown in rat serum approximates
New's success using rat embryos (New et al. 1976a). Furthermore, 96 % of all embryos placed into culture survive for 48 h, thereby making it possible to employ
the system for observation and manipulation of these developing structures.
Figs. 3, 4. Macroscopic comparison of 28-somite (S) mouse embryos maintained in vivo (11th day, Fig. 3) and in vitro (48 h
culture, Fig. 4) showing similar stages of development. Heart (H), optic vesicle (OV), and otic vesicle (OP), x 60.
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on
Culture of early somite mouse embryos
Figs. 5, 6. Photomicrograph of optic vesicle formation in an 11th day mouse
embryo (Fig. 5) and in an approximately similar-stage embryo grown 48 h in
culture (Fig. 6). The outlined area demonstrates an area of physiological necrosis
(Silver & Hughes, 1973) (pyknotic debris) occurring in both embryos. Mesenchymal
tissue (M) in the cultured embryo appears to have slightly fewer cells than the in
vivo specimen. Neuroepithelium (NE). x 200.
23
24
T. W. SADLER
The results also indicate that the choice of medium is critical and medium
which has been used successfully to culture later-stage mouse embryos is
not effective for maintaining earlier stages. For example, Kochhar (1975)
has employed a combination of fetal calf serum and Waymouth's medium (1:1)
to maintain 11th and 12th day (30-45 somite) mouse embryos in culture for
periods of 24-36 h. However, this combination would not support normal
growth of 2- to 4-somite mouse embryos, nor would fetal calf serum alone.
Reasons for these results are not clear and cannot be determined until more
information is known about nutrient requirements of embryos at different
developmental stages.
The gassing protocol of increasing O2 tensions was similar to that devised by
New and co-workers and appears to approximate the oxygen tension in the
blood surrounding the rodent conceptus (New et al. 1976&). One difference from
New's procedure is that during the entire 48 h period of culture in New's
system, embryos are exposed to 6-5 ml of atmosphere per embryo. In our
system each embryo was exposed to 8-5 ml of atmosphere per embryo for the
1st 24 h (comparable to New's values) and growth proceeded normally. However,
embryos maintained at this level, i.e. those cultured for an additional 24 h
in 10 ml flasks did not grow as well as embryos grown for an additional 24 h
in 25 ml flasks in which the gas volume was 23-5 ml per embryo. These differences in gas volume requirements may be due to the larger size of mouse embryos
which were started in culture at early somite stages versus head-fold stages in
rat embryos and which had approximately 100/tg more protein than rat
embryos after 48 h in culture (New et al. 1916a).
The point at which mouse embryonic growth ceases in culture using these
techniques has not been determined and it is not clear as to how far beyond
48 h embryos can be maintained. Several preliminary experiments (unpublished
data) indicate that cultures vary in their deterioration rates, based on heartbeat
and circulation, and that only occasional cultures have 3+ heartbeat 3 +
circulation after 72 h. New has also noticed this decline in older cultures and
suggests that it might be due to a competition for nutrients between the allantoic
placenta and embryo and to the problem of supplying enough nutrients (including O2) to match the greater rate of growth increase (total protein) experienced by older embryos.
This work was supported by NIH Grant 5T32 DE07037-02 for craniofacial development
and by Biomedical Research Support Grant 5S07RR5431-15.
Culture of early somite mouse embryos
25
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BUCKLEY,
{Received 30 May 1978, revised 29 August 1978)