/. Embryol. exp. Morph. Vol. 59, pp. 131-143, 1980
Printed in Great Britain © Company of Biologists Limited 1980
The in vitro culture of
primitive-streak-stage mouse embryos
By P. P. L. TAM 1 AND M. H. L. SNOW1
From the MRC Mammalian Development Unit,
University College, London
SUMMARY
The in vitro growth and morphogenesis of mouse embryos from early primitive-streak
stage to early-somite stage is described. The embryo culture method employs a static culture
system, a conventional chemically defined medium (Dulbecco's modified Eagle's medium),
supplemented with additional glucose, glutamine and suitably prepared serum. The method
of serum preparation is important for successful culture. Both mouse serum and rat serum
support good development of primitive-streak-stage mouse embryos. Over 60% of earlystreak stage and about 90 % of late-streak stage grow and develop for 48 h in vitro. During the
first 24 h in culture, total growth of the embryos, as reflected by protein content, size and
morphology is the same as in vivo.
INTRODUCTION
The development of mammalian embryos is conveniently divided into four
phases: preimplantation, implantation and gastrulation, organogenesis, and
growth.
Embryo size militates against the in vitro culture of whole embryos during the
growth phase, but the other phases have been studied with some success. Preimplantation stages of many species are routinely grown in vitro with
minimal disturbance to either timing, form of development or subsequent
viability (Whittingham, 1975), but successful culture of the later stages is largely
confined to rats and mice (New, 1978). In the rat, using the techniques developed
by D. A. T. New, it is possible to achieve normal development of late-streak and
head-fold stages (0-5 somites) for nearly 4 days in culture and later stages, up to
134- days of gestation, for somewhat shorter periods (Buckley, Steele & New,
1979; Cockcroft, 1976). For the mouse, Clarkson, Doering & Runner (1969),
Pienkowski, Solter & Koprowski (1974) and Kochhar (1975) reported limited
success with late organogenesis stages, and Sadler (1979) describes good growth
and development of early-somite embryos in rat serum for periods up to 48 h.
The intervening phase, from implantation to head-fold formation, includes
the process of gastrulation which seems to represent a hiatus for the culture
1
Authors' address: MRC Mammalian Development Unit, Wolfson House, 4 Stephenson
Way, London, NW1 2HE, U.K.
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132
P. P. L. TAM AND M. H. L. SNOW
techniques. Many methods are available for the mouse which mimic the process
of implantation and some of these allow the development to pre-primitivestreak egg cylinders, albeit rather slowly in comparison with development in
vivo (Jenkinson & Wilson, 1970; Hsu, 1973; Hsu, Baskar, Stevens & Rash,
1974; Pienkowski et al 1974; McLaren & Hensleigh, 1975). But the major hurdle
during this phase of development seems to be primitive-streak formation - the
onset of gastrulation. At this transition point morphology of the cultured
embryos usually becomes abnormal and only limited numbers of embryos
proceed to somite stages (Hsu et al. 1974; Wiley & Pedersen, 1977). Recently a
marked improvement in the yield of somite-stage embryos has been reported by
Hsu (1979), using a system requiring careful choice of several different media
formulations and a stringent schedule of media changing. We have focused
attention on the egg cylinder to early-somite stages and below describe a
simplified method for the culture of embryos at the primitive-streak stages.
MATERIALS AND METHOD
Embryos were obtained from the random bred Q and MFI strains of mice.
Early-primitive-streak stage (6-5 days p.c.) and late-primitive-streak stage
(7-5 days p.c) embryos were studied. The embryos were dissected from the
decidua. Reichert's membrane and ectoplacental cone trophectoderm were
removed. It is important not to damage the primitive endoderm surface of the
egg cylinder. Embryos were cultured in groups of four to six in 30 mm Falcon
plastic Petri dishes containing 2-0-2-5 ml culture medium. These small dishes
were then placed in groups in larger plastic containers (large Petri dishes or
conveniently sized boxes) in which wet filter paper had been placed to ensure a
humidified atmosphere. The whole assembly was placed in an incubator at
37 °C with a humidified atmosphere of 5 % CO2 in air.
Culture media
Four chemically defined media have been tested, supplemented with various
sera. The media, NCTC-109, Waymouth's, RPM1 1640, and Dulbecco's
modified Eagle's medium (DMEM) foetal calf serum (FCS) and newborn calf
serum (NCS) were obtained from Gibco Bio-Cult Ltd. Mouse serum (MS) was
collected in the laboratory from MFI, Q, GW, C3H, C57BL and a number of
hybrid strains. The serum was obtained by immediately centrifuging the blood
collected from etherized mice, in a way similar to the collection of rat serum
(Steele, 1972). Rat serum (RS) was collected from albino rats of unknown
strain which were kindly supplied by Dr C. Hetherington, Clinical Research
Centre, Harrow.
Benzyl penicillin (100 i.u./ml) and streptomycin sulphate (50 /^g/ml) (Glaxo)
were added to all media.
In vitro culture of primiiive-streak-stage mouse embryos
133
Assessment criteria
The development of the embryos in culture was assessed by comparison of
morphology with the embryos of equivalent post-coital age in utero. Special
emphasis was placed on the development of head fold, heart, neural tube,
somites and gut formation. The size of the embryos was measured from cameralucida drawings done at x 60 magnification. After macroscopic examination,
some embryos were fixed in Sanfelice's fluid, wax embedded and examined
histologically. The remaining embryos were used for the determination of
protein content by the colorimetric method of Lowry, Rosebrough, Farr &
Randall (1951).
RESULTS
In pilot experiments late-streak-stage embryos were grown in each of the
synthetic media supplemented with 20 % FCS (after McLaren & Hensleigh,
1975). After mixing but before gassing with 5 % CO2 in air these media have
different osmolarities and pH values at 20 °C (Table 1). Development in all
media was somewhat slower than in vivo but two important observations were
made during these pilot studies: (1) the best yolk-sac and head-fold morphology
seem to be correlated and were seen in Waymouth's and DMEM, media with a
high glucose concentration; (2) after 24 h embryos in DMEM were growing
more vigorously, estimated on the basis of yield of metaphase plates from a
conventional air drying chromosome spreading technique. The second observation is supported by the evidence of further culture. Comparatively few embryos
grew well in Waymouth's or RPMl 1640 during the second 24 h period, whereas
in DMEM 50 % proceeded through somitogenesis and developed beating hearts.
Despite inferior head-fold morphology nearly 38 % of embryos in NCTC 109
progressed into somitogenesis. A more detailed comparison between DMEM and
NCTC 109 confirmed that the high glucose medium is superior (Table 2).
Further study therefore focused on DMEM and variations based on that
medium.
A series of experiments was carried out to investigate optimal concentrations
of serum (FCS, NCS, MS and RS) and the importance or otherwise of adding
pyruvate, glutamine and bicarbonate to the basic medium just prior to use (to
compensate for decomposition during storage in solution in the case of pyruvate
and glutamine, or to correct for pH variations introduced with serum in the
case of bicarbonate). These experiments produced the following results.
(1) For late-primitive-streak-stage embryos, 30 % FCS or NCS supports
normal development of some embryos but 50-100 % mouse serum or rat serum
is optimal for embryonic development;
(2) Additional L-glutamine is beneficial when added to the medium at a final
concentration of 2 mM;
134
P. P. L. TAM AND M. H. L. SNOW
Table 1. A comparison between the four media used in pilot studies. Each was
supplemented with 20 % FCS. pH and molarity were measured on complete
medium but prior to gassing with 5 % CO2 in air
Medium
Glucose
content
(mg/1)
PH
20 °C in air
Osmolarity
(mOsm)
NCTC-109
DMEM
Waymouth
RPMI-1640
800
3600
4000
1600
7-96
7-86
7-66
7-83
285
345
312
287
Table 2. Development of late-streak-stage embryos in NCTC-109 or
DMEM supplemented with 20 % FCS
% showing normal development of:
Medium
No. of
embryos
NCTC-109
DMEM
170
114
Yolk sac Head fold Endoderm*
49
83
49
65
Somites,
heart
51
51
48
66
* Development of gut invaginations was taken as evidence for definitive endoderm
formation.
Table 3. Development of late-streak embryos in DMEM supplemented
with either 30 % FCS or 30 % NCS
/o of embryos showing normal development of:
Serum
FCS1
NCS
Mouse No. of culture
strain embryos
(h)
Yolk
sac
Neural
fold
Head/
brain
Q
41
MFl
24
19
MFI
22
73
79
62
41
67
94
86
82
0
10
0
4
Q
95
86
87
79
90
90
91
91
24
48
24
48
24
48
24
48
0
42
0
36
Heart
Gut
3
43
0
16
57
94
54
82
22
57
16
41
84
90
73
73
Somites
(no.)
Not scored
Not scored
20 (40±0-4)
Not scored
78 (6-9±0-2)
Not scored
77 (6-5 ±0-4)
Not scored
(3) Sodium pyruvate should be omitted from the stock DMEM and added to
complete the medium immediately prior to use (at a concentration of 0-1 ITIM);
(4) The preparation of serum is also important. Heat inactivation of serum
should be done immediately before use. Embryotrophic properties are destroyed
by freezing and storage afcer inactivation, but active serum can be stored at
In vitro culture of primitive-streak-stage mouse embryos
135
Table 4. Development of primitive-streak-stage embryos in whole mouse
serum, DMEM+ 50 % mouse serum, whole rat serum and DMEM+50% rat
serum
Embryos cultured
Culture
medium
100% MS
MS:DMEM
(1:1)
100%RS
RS:DMEM
(1:0
No. and (%) showing
normal development at
Age
(days p.c.)
No.
24 h
48 h
6-5
7-5
6-5
7-5
6-5
7-5
6-5
7-5
7
8
92
55
24
11
64
26
6(86)
8 (100)
60 (65)
52 (95)
21 (88)
11 (100)
56 (88)
22 (84)
5(71)
7(88)
55 (60)
40 (73)
19(79)
1(9)
35 (55)
21(81)
Somite
number*
(mean ± S.E.)
Not scored
4 0 ±0-2
3-8±O-3f
5-9±O-3
6-3 ±0-7
6-2 ±0-4
6-2 ±0-6
6-5 ±0-4
* Somite number of the embryo was scored at 48 h in vitro for 6-5-day embryos and at
24 h in vitro for 7-5-day embryos. The somite number of equivalent aged (8-5-day) embryo
in vivo was 5-2±03 (23).
f Significantly different from normal, P < 005 (/-test for difference).
— 20 °C for several months with little loss of potency. Serum should not be
filter-sterilized if this can be avoided, and certainly not following heat inactivation. However gamma-irradiated, non-filtered serum is not suitable for culture.
In several replicate experiments using gamma-irradiated NCS not one embryo
made normal head folds.
Table 3 shows the respective merits of FCS and NCS for late-streak-stage
embryos. Embryos cultured in NCS-supplemented medium developed much
better than in FCS-supplemented medium for the first 24 h. More embryos
showed development of yolk sac, neural fold, beating heart, invaginating foregut
and formed more somites. Development of embryos in FCS + DMEM was very
poor in the second 24 h. Although development in NCS + DMEM was retarded,
a high percentage of the embryo showed development of neural tube, initial
closure of cephalic fold and elongation of trunk.
With early-primitive-streak stages, FCS and NCS are inadequate, and
embryonic development is abnormal. For these earlier stages, MS or RS is
essential for good cultures and is also an improvement for late-primitive-streak
stages. At the time of culture, 6-5-day embryos were either in the pre-primitivestreak or early-primitive-streak stage (Fig. 1). After 24 h in vitro, 65-88 % of the
embryos cultured in mouse serum or rat serum (whole or diluted) reached the
late-primitive-streak stage (Table 4) and showed normal development of
embryonic and extraembryonic structure, principally the yolk sac (Figs. 2 and
3). There was significant but not excessive expansion of the yolk sac and amniotic
cavity. The size and protein content of the embryos developed in vitro for 24 h
136
P. P. L. TAM AND M. H. L. SNOW
Table 5. Growth of embryos in vitro, culture medium is DMEM+50 %
mouse serum
Groups
Somite
numbers
(n = no. of
embryos)
Mean±s.E.
6-5 d m vivo
7-5 d m vivo
6-5d+24hm vitro
—
—
—
8-5dmv/vo
6-5 ±0-2 (55)8h
6-5d + 48hmv/fro 5-0±0-4(23)g
7-5d + 24hm vitro 6-7±0-2(61)h
Size of embryos
(n = no. of
embryos)
Mean±s.E.
Protein content (/*g) per embryo
(« = no. of replicate
determinations)
Mean ± S.E.
—
1-23 ±005 (8)
0177 ± 0015 (22)d*
7-20 ± 0-49 (13)a
d
0168 ±0013 (34) *
717 ±0-46 (ll) a
(retarded embryos: 410±0-25 (6))
1 -934 ± 0-078 (41)? t
18-74 ± 0-84 (31)b0
1-501 ±0-053 (26)f
939±0-73 (13)c
1-933 ±0-061 (61)et
18-40 ± 0-96 (30)b
* Size of the embryo was expressed as the product of the width and height of the embryonic
part of the egg cylinder, unit was mm.2.
t Size of the embryo was expressed as the body axis length from the head fold to the tail
bud, unit was mm.
a, b, d, e, g, h: no significant difference; c, f: significantly different at P < 001 (/-test for
difference).
was the same as those at 7-5 days in vivo (Table 5). In the embryos failing to
develop normally, expansion of the yolk sac was usually excessive and a single
large vesicle resulted. The abnormal embryos also contained less protein (Table
5). About 55-79 % of the embryos developed for another 24 h to the head-fold
and early-somite stage (Table 4). Equivalent-aged (8-5 day) embryos in vivo have
five to seven pairs of somites, well developed head fold, closing neural tube,
invaginating foregut and early formation of heart tube. The cultured embryos
showed a wide range of developmental stage: from presomite neural plate to
early-somite stage with prominent head folds and beating heart (Figs. 4 and 5).
The more advanced embryos had normal numbers of somites, but a shorter
FIGURES
1-7
Fig. 1. Early-primitive-streak stage (6-5-days p.c.) embryos at the time of explanation. Bar = 500 /*m.
Fig. 2. Late-primitive-streak-stage (7-5-days^.c.) embryos in vivo. Bar = 500/tm.
Fig. 3. Early-primitive-streak-stage embryos cultured for 24 h in vitro. Bar = 500 /on.
Fig. 4. Early-primitive-streak-stage embryos cultured for 48 h in vitro. Bar = 500 /*m.
Fig. 5. One of the best embryos developed to early-somite stage at the end of the
culture. Extraembryonic membranes were dissected away. Bar = 500 /tm.
Fig. 6. Four embryos after culturing for 48 h in vitro, starting from early-primitivestreak stage. Yolk sacs were dissected away. Bar = 500 fim.
Fig. 7. Four 8-5-days early-somite-stage embryos in vivo. Yolk sacs and allantois
were removed. Bar = 500 /tm.
In vitro culture of primitive-sireak-stage mouse embryos
1
3
137
138
8
P. P. L. TAM AND M. H. L. SNOW
9
10
11
Fig. 8. Late-primitive-streak stage (7-5 days p.c.) embryos at the time of explanation. Bar = 500/tm.
Fig. 9. Late-primitive-streak-stage embryos cultured for 24 h in vitro. Bar = 500 /.cm.
Fig. 10. Four embryos after culturing for 24 h in vitro. Yolk sacs and allantois were
removed. Bar = 500 fim.
Fig. 11. Four 8-5-day early-somite-stage embryos in vivo. Yolk sacs and allantois
were removed. Bar = 500 /tm.
body axis and significantly less protein when compared to 8-day embryos in vivo
(Table 5, Figs. 6 and 7). Histologically, the development of neural fold, heart,
gut and mesenchyme were apparently normal (Figs. 12 and 13).
Between 84 and 100 % of the late-primitive-streak-stage (7-5-day) embryos
(Fig. 8) developed normally in culture during the first 24 h (Table 4). The
embryos had six to seven pairs of somites, well-formed head folds, closing
neural tube, invaginating foregut (Figs. 14-17) and some had a beating heart.
Gross morphology was similar to the 8-5-day embryos in vivo (Fig. 9,10 and 11).
Total growth as measured by total protein content and body axis length also
In vitro culture of primitive-streak-stage mouse embryos
139
12
Figs. 12-13. Early-primitive-streak-stage embryos cultured for 48 h in vitro. NF,
neural fold. HF, head fold. HT, heart. FG, invaginating fore gut. Bar = 200 /*m.
Figs. 14-17. Late-primitive-streak-stage embryos cultured for 24 h in vitro. Sections
were through head region (Fig. 14), heart (Fig. 15), posterior trunk region (Fig. 16),
and mid trunk region (Fig. 17). Bar = 100/tm. DA, dorsal aorta; HM, head
mesendyme; HT, heart tube; NE, neuroepithelium; NF, neural fold; NT, neural tube;
PC, pericardial cavity; SM, somite; TM, trunk mesoderm.
appeared normal and no significant differences in protein content and size were
observed between similar-staged embryos maintained in vitro or in vivo (Table 5).
The few late-streak embryos failing to develop normally have invariably
140
P. P. L. TAM AND M. H. L. SNOW
Table 6. Protocol for preparation of serum supplemented DMEM
for mouse embryo culture
To make 10 vol. culture medium
Serum
Immediately centrifuge fresh blood (3000 rev./min, 5 min)
Squeeze fibrin clot, re-spin (3000 rev./min, 5 min)
Collect serum in sterile tubes
Add penicillin (100 units/ml) and streptomycin (50/ig/ml)
Store below - 20 °C in aliquots
Slowly thaw required amount, heat inactivate at 56 °C for 30 min
Denned medium
DMEM (without pyruvate but with glutamine, bicarbonate and
glucose 4000 mg/1)
L-glutamine (200 mM)
Na-pyruvate (100mM)
Sterilize by millipore filtration (0-22 or 0-45 /im pore size)
4-9 parts
01 part
001 part
Mix 5 parts of serum with defined medium, equilibrate with 5 % CO2 in air at 37 °C
for 60 min before use.
attached to the dish where the embryonic tissues grew out as a monolayer disrupting normal morphogenesis in the process. These embryos were most probably damaged during explantation from the decidua. During the second 24 h in
culture, the growth of the embryos was depressed and overall development was
retarded. However, there was further development of blood circulation (vigorous
heart beat and yolk-sac circulation) and formation of craniofacial (cephalic fold,
mandibular arch, optic vesicles) trunk (neural tube, somites) and tail-bud
structures, The best embryos had about 15-20 somites and axial rotation had
begun. Although some embryonic structures like the head and heart were of
abnormal size, histologically the tissue appeared very healthy. No extensive
necrosis was found and many cell divisions were seen especially in the neuroepithelium.
Cultures in whole serum are not significantly superior to those using mouse
serum or rat serum diluted 50 % with DMEM. There are differences between
batches of rat serum which are not associated with degree of haemolysis in preparations. Since mouse serum is pooled from many mice, variation is not seen in
these preparations. As observed by Steele (1972) the mode of serum preparation
is important, immediately centrifuged serum being better than serum centrifuged
after blood clotting. Table 6 describes the protocol of serum and medium
preparation we found best.
In vitro culture of primilive-streak-stage mouse embryos
141
DISCUSSION
The results of this study indicate that primitive-streak-stage mouse embryos
can be cultured through gastrulation to early-somite stage in a simple static
culture system. The embryos develop best in whole mouse or rat serum, and in
medium supplemented with 50 % mouse or rat serum. Over 60 % of the earlyprimitive-streak-stage embryos develop to the early-somite stage. Developmental events such as gastrulation, head-fold and neural-tube formation, gut
invagination, heart growth and formation of the first six to eight somites all
occurred on schedule and appeared normal. Total growth of embryos, as
measured by protein content and size, is essentially the same as in vivo during
the first 24 h in culture. Growth of embryos is depressed in the second 24 h and
development of embryos beyond the early-somite stage (equivalent to 8-5 days
in vivo) was retarded. It seems, therefore, that this culture system is most suitable
for the observation and experimental manipulation of embryos between
primitive-streak stages and early-somite stage for periods of morphogenesis of
about 24 h. The inadequacy of the present method for culturing embryos beyond
the somite stages could be twofold. First, the essential nutrient and energy substrate in the culture medium may be depleted during the initial culture period
and would not support normal growth of older embryos. Rat embryos at and
beyond the head-fold stage are known to have a stringent requirement of
nutrients and energy substrates for normal development in vitro (Cockroft,
1979). Secondly, it has been shown that early stages of rat and mouse embryos
require initially a low oxygen tension in the culture system and as development
progresses a higher oxygen tension is necessary (New, Coppola & Cockroft,
1976; Buckley et al. 1978; Sadler, 1979; Morriss & New, 1979). The use of
5 % CO2 in air (i.e. about 20 % O2) may be beneficial for early embryonic
development in this study, because in the static culture described, gaseous
diffusion may be less efficient and a low oxygen tension is therefore maintained
in the medium. The same gas phase would be insufficient for older embryos
when there is a more demanding oxygen requirement. The transfer of cultured
late-streak-stage embryos from a static culture to a rolling-bottle culture during
the second 24 h of culture may perhaps improve the success rate and allow
normal development of embryos beyond the early-somite stage (Sadler, 1979).
During the development of early-primitive-streak-stage embryos, the primitive endoderm occasionally becomes raised into fluid-filled blisters over the
surface of the embryonic part of the egg cylinder. Such embryos do not develop
normally. Similarly, overexpansion of the yolk sac and amniotic cavity are
often accompanied by degeneration of embryonic structures. All these phenomena suggest that the normal osmoregulation between embryonic compartments and the environment has failed to operate. Whether this is due to intrinsic
errors in embryonic physiology during culture or because media/culture conditions overwhelm the embryo's regulatory capacities is not known. It may not
10
EMB
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142
P. P. L. TAM AND M. H. L. SNOW
be coincidence that the most successful culture medium also has the highest
osmolarity.
The differences seen in sera prepared in different ways may suggest a way to
identify the embryotrophic factor(s). Heat inactivation does not remove these
factors but does alter them so that they can be destroyed at least partially by
freezing and removed by Millipore filtration. The observation that nitration of
fresh serum will lower its potency may indicate that the factors are already
partly denatured before heat inactivation. Taken in conjunction with the fact
that immediately centrifuged serum, i.e. serum collected before blood clotting
occurs, is superior in embryo cultures, it is possible that the differences in serum
types with respect to their embryotrophic properties are created entirely by
artifacts of preparation. Commercially available serum is generally collected
after whole blood has clotted and is sterilized before dispatch by membrane
filtration. These various preparative steps may have depleted the serum of
identifiable factors such as, but in addition to, the proteins found in immediately
centrifuged serum, but which are apparently absent in serum collected after
whole blood clotting (Klein, Minghetti, Jackson & Vogler, 1978).
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