/. Embryol. exp. Morph. Vol. 17, 3, pp. 513-25, June 1967
With 1 plate
Printed in Great Britain
513
Development of explanted rat embryos
in circulating medium
By D. A. T. NEW 1
From the Strangeways Research Laboratory, Cambridge
INTRODUCTION
The inaccessibility of rat embryos in vivo has prompted several previous
attempts to grow them in culture. It has been shown that embryos explanted at
head fold or early somite stages with their membranes will develop on the
surface of plasma-embryo extract clots (New & Stein, 1964; New, 1966 a)
or in homologous liquid plasma or serum (Nicholas & Rudnick, 1934, 1938;
Jolly & Lieure, 1938; New, 1966a, b). The explants can conveniently be cultured
in watch glasses, as in the standard tissue culture method of Fell & Robison
(1929), and development is improved if they are incubated in 60-90 % O2 and
3-5 % CO2. Under these conditions the older embryos grow well for 30-40 h
developing to 25- to 30-somite stages. However, no embryos have been observed
to develop much further than this, and those explanted at stages more advanced
than 30 somites have always died fairly quickly.
By the 30-somite stage the whole conceptus (embryo+membranes) has
attained a diameter of about \ cm and in vivo grows extremely rapidly, increasing
in weight about fivefold every 24 h. A likely cause of its failure in watch-glass
cultures would seem to be insufficiently rapid diffusion of oxygen, and possibly
also of nutrients and waste products, through the static medium. If this is so,
better development should be obtained in flowing medium. The only previous
attempt to do this appears to be that of Nicholas (1938), who claimed good
results but gave no indication of the proportion of embryos developing or stage
of development attained. I have therefore investigated further the growth and
development of rat embryos in flowing medium, and the results obtained from
embryos explanted on the 1 lth, 12th and 13th days of pregnancy are recorded in
this paper.
MATERIALS AND METHODS
Nutrient medium
The culture medium used, except where stated otherwise, was homologous
serum containing 50 /^g/ml streptomycin. The serum was obtained from blood
taken with a siliconed hypodermic syringe and needle from the dorsal aorta of
1
Author's address: Strangeways Research Laboratory, Wort's Causeway, Cambridge, U.K.
514
D. A. T. NEW
rats anaesthetized with ether. No anticoagulant was added. The clotted blood
was allowed to stand overnight in centrifuge tubes, and the serum decanted after
centrifugation the following morning. The serum was stored for periods of up
to 1 week at 1-4 °C or for longer periods at —10 °C.
Apparatus
The 'circulator' drawn to scale in Text-fig. 1 was designed to allow continuous
observation of the embryos, to circulate the serum without the use of mechanical
pumps, and to meet the requirements of sterility, cleanliness and simplicity of
O2/CO2
Text-fig. 1. A 'circulator' for growing embryos in flowing medium. The triangle of
glass tube is filled with serum and circulation is maintained by O2/CO2 entering
through the filter C and flowing as a stream of bubbles up tube D. The bubbles
collapse in chamber B. The embryos are contained in the detachable chamber F.
Drawn to scale ( x | ) .
construction. It consists essentially of a triangle of thick-walled glass tubing of
3 mm bore through which serum is continuously recirculated and oxygenated
by a stream of O2/CO2 bubbles flowing up the sloping tube D. The bubbles are
discharged into the large chamber B, where serum drains from them and they
collapse; if the rate of flow is fairly rapid the chamber may become filled with
bubbles and a few may escape through tube A, but they are so thin-walled that
the loss of serum is negligible.
Rat embryos in culture
515
For steady circulation the level of the serum in B must always be above the
inlet from tube D. For a circulator of tubing of 3 mm bore designed as in
Text-fig. 1 the minimum amount of serum required is about 5 ml and the maximum rate flow obtainable is about 20 ml/min. The rate of flow can be estimated
from the rate of movement of bubbles up tube D. If a stopcock is incorporated
in the circulator at E steady circulation can be obtained at any desired rate
between 0 and 20 ml/min. Without a stopcock the minimum steady flow obtainable (by reducing the O2/CO2 supply) is about 10 ml/min; slower than this
the flow becomes irregular.
The embryos are housed in the embryo chamber F, which is held in the circulator by short lengths of silicone tubing. The chamber is made from glass tubing
square in cross-section (7x7 mm internal) so that the embryos can be observed
through the flat sides without distortion. To prevent the embryos circulating
with the serum they are anchored to a strip of rayon fabric (as in the tissue
culture method of Shaffer, 1956) attached to a piece of glass coverslip.
The O2/CO2 mixture is obtained from a cylinder of 95 % O2, 5 % CO2 equipped
with a regulator valve to maintain the pressure at about 1 lb/in2. It is first humidified by bubbling through water, then passed through the filter C which contains
a fine powder (e.g. light kaolin, fine carborundum, or jeweller's rouge) packed
tight between two plugs of glass wool. The purpose of the filter is to remove any
infective particles, and to control the flow of gas so that only a few millilitres
per minute pass into the circulator and the bubbles in tube D form steadily.
Serum can be injected into or withdrawn from the circulator through the
vertical side tube after detaching the filter C.
The bent tube A is convenient for hooking the circulator on to a rack, and
several circulators can be thus placed side by side in a small hot box or incubator.
If the same hot box contains a low-power binocular microscope (Plate 1, fig. A)
the embryos in each circulator can be examined in turn without cooling them or
interrupting the circulation of the serum.
The complete circulator, with embryo chamber and gas filter, is heat-sterilized
before use.
Procedure
All embryos described in this paper were from the 'Hooded' or 'Wistar'
strains of rats, and were explanted with their embryonic membranes by a method
similar to that described previously (New & Stein, 1964; New, 1966 a). Each
conceptus was dissected free of the surrounding decidua in Tyrode saline. The
Reichert membrane was then torn open and used to anchor the explant within
the embryo chamber. The trophoblast layer on the outside of the membrane is
very 'sticky' and if pressed against the surface of a plasma clot, or on to a piece
of rayon fabric (Text-fig. 2), it will adhere sufficiently firmly to prevent the explants circulating with the serum. Preliminary trials showed that clots and rayon
both gave good results, but rayon was found the simpler to use, particularly if
516
D. A. T. NEW
the embryos were attached to it before placing it in the embryo chamber.
Rayon was therefore employed in all the experiments described in this paper, as
follows:
Small strips of rayon (cellulose acetate) fabric 5 x 20 mm were flattened and
stuck down to pieces of glass coverslip of the same size by means of three or
four small drops of acetone. After heat-sterilization the rayon coverslips were
Yolk sac
Reichert
membrane
Text-fig. 2. Method of anchoring embryo in circulating medium. Reichert's membrane with its adherent cells is torn open and pressed down into rayon fabric attached
to a piece of glass coverslip. It adheres firmly.
Rayon coverslip with
attached embryos
Saline
Text-fig. 3. Method of inserting embryos into embryo chamber with lower part of
'circulator' immersed in saline.
immersed in a dish of Tyrode saline together with the explanted embryos. The
Reichert membranes were torn open and the explants were attached, two to each
rayon coverslip. They could then be transferred to the circulators, but to avoid
Rat embryos in culture
517
damaging them this manoeuvre was done without taking them out of the saline.
Each circulator, with the embryo chamber attached at one end only, was held in
a retort stand over the dish containing the embryos so that the lower part of the
circulator was below the level of the saline (Text-fig. 3). A rayon coverslip with
attached embryos was gently raised from the floor of the dish and slid into the
embryo chamber. The circulator was then removed from the dish, care being
taken to keep it sufficiently tilted to prevent the embryo chamber empyting of
saline. Most of this remaining saline was displaced (and ran out of the open end
of the embryo chamber) by injecting 2 ml serum into the side tube. The embryo
chamber was thenfixedin position, the rest of the serum added, and the circulator
placed in the hot box and connected to the O2/CO2 supply.
Except where stated otherwise, two embryos and 4-6 ml serum were placed in
each circulator. When making comparisons of embryonic growth under various
culture conditions, embryos from each litter were always divided among the
different circulators in such a way as to eliminate any effects of differences
between litters.
The main criteria used in estimating the duration of survival and development
of the embryos in cultures were the rate of heart beat, persistence of the blood
circulation, and the final number of somites and protein content. Protein
determinations were always made on the embryos alone after dissecting them
free of the embryonic membranes; the embryos were digested in NaOH and the
protein was estimated by comparison with standard solutions of bovine plasma
albumin, using the colorimetric method of Lowry, Rosebrough, Farr & Randall
(1951).
Preliminary attempts at growing the embryos in circulating serum were
encouraging and showed that they could develop better in these conditions than
in watch-glasses. Experiments were then aimed at answering the following
questions:
(1) Do the embryos require as nutrient medium serum from female rats of the
same stage of pregnancy, or are other homologous sera or even synthetic
chemically defined media equally effective?
(2) During the period in culture, do the embryos exhaust the serum of essential
nutrient materials or poison themselves with accumulated waste products, and if
so can development be prolonged by increasing the amount of serum or transferring the embryos to fresh serum?
(3) How sensitive are the embryos to differences in rate of flow of the serum?
(4) What proportion of embryos survive to different stages of development
under optimum conditions?
The results will be described in four sections corresponding to these lines of
enquiry.
518
rr,
D. A. T. NEW
r
j .
RESULTS
Type of medium
Table 1 shows the results of an experiment in which four embryos explanted
at 10-15 somites were grown in serum from male rats and four in serum from
female rats 11-16 days pregnant. The serum was circulated at 2-8 ml/min.
The embryos were still thriving after 24 h, with a good yolk-sac blood circulation, but had ceased developing after 40 h. Six of the embryos reached 30- to
32-somite stages and synthesized about 300 /ig or more of protein. There was no
difference between the embryos grown in the male serum and those in the
pregnant-female serum.
Table 1. Records of embryos explanted at 10- to 15-somite stages {about 40 fig
protein) and grown in circulating serum from male rats or from female rats
11-16 days pregnant
The yolk-sac blood circulation is recorded in four grades, from + + +, indicating rapid
circulation through the entire capillary network, to — , indicating complete absence of
circulation.
After 24 h
After 40 h
K
Serum
Male
Pregnant
Embryo
no.
Circulation
1
2
3
4
5
6
7
8
+++
+++
+++
+++
++
+++
+++
+++
Heartbeats
per min
130
100
140
110
75
105
140
120
Circulation
+
—
+
+
Protein
Heartbeats
No. of content
per min somites
Og)
130
100
135
_
160
155
170
32
28
32
30
22
32
31
32
400
235
405
240
105
295
340
420
Table 2 shows the results of a similar experiment on embryos explanted at
22-28 somites. The serum was circulated at 15-20 ml/min. The embryonic
hearts beat at a steadily increasing rate for about 18 h, but by 24 h were beginning to slow down and by 40 h had failed. The embryos all developed to stages
between 40 and 46 somites and synthesized about 1 mg protein. Again there was
no difference between the embryos grown in the two types of serum.
In other experiments the effect has been studied of adding to the serum
erythrocytes or chemically defined media. The addition of erythrocytes
proved unsatisfactory; there was much haemolysis and sedimentation and the
embryos in such cultures developed less than controls. The addition of 50 %
Waymouth's medium also gave very poor results, and the results were not
improved by the addition of dextran to raise the osmotic pressure to that of
whole serum. Results with 50 % serum+ 50 % '199' medium were better, and
Rat embryos in culture
519
Table 2. Record of embryos explanted at 22- to 28-somite stages {about 150300 fig protein) and grown in circulating serum from male rats or from female
rats 11-16 days pregnant
The yolk sac blood circulation is recorded as in Table 1. After 40 h the heartbeat had failed
in all embryos.
After 24 h
After 18 h
Serum
Embryo
no.
Circulation
1
2
3
4
5
6
7
8
9
Male
Pregnant
10
11
12
After 40 h
Heartbeats
per min
Circulation
Heartbeats
per min
No. of
somites
Protein
content
(/fg)
165
175
180
180
165
195
170
180
170
195
180
210
+ Hh +
+ -\
+ -\
-+
+-\-+
+4
+ 4-+
+ Hh +
+ -\
-+
+
+
4-4-+
+4
150
165
155
160
145
170
160
165
160
165
175
180
42
40
42
43
42
46
41
42
42
41
43
43
1,215
855
1,055
1,300
1,235
1,475
955
1,190
1,120
950
1,615
1,245
Table 3. Development after 40 h as indicated by number of somites, and growth
as indicated by protein content, of embryos explanted at 22-28 somites and grown
in different amounts of circulating serum
Serum per embryo (ml)
Mean
Mean
and S.D.
Somites
Protein
42
39
40
40
42
41
42
36
41
40
37
36
39-7
—
1005
615
1000
905
1115
885
1230
835
990
745
740
665
—
894 ±176
2-2-5 renewed
4-5
2-2-5
r
Somites
44
35
44
41
43
43
44
41
39
43
42
39
41-5
—
Protein
1525
570
1890
960
965
1075
985
965
815
905
870
1125
—
1054 ±330
Somites Protein
46
44
37
39
37
42
46
39
41
43
39
37
40-8
—
1900
1290
650
845
520
1040
1355
915
810
1225
900
610
—
1005 ±371
520
D. A. T. NEW
the few embryos that have been grown in this mixture developed nearly as well
as controls, but until further trials have been made it is uncertain whether it is
generally as good a medium as whole serum.
Amount of medium
Table 3 summarizes the results from 36 embryos explanted at 22-28 somites,
which were divided into three equal groups and grown in different amounts of
serum. Twelve of the embryos (group 1) were grown in 2-2-5 ml serum per
embryo, 12 (group 2) in 4-5 ml per embryo, and 12 (group 3) in 2-2-5 ml per
embryo of which about two-thirds was twice replaced by fresh serum during the
period of the culture. In all the cultures the serum was circulated at 10-20 ml/min.
The table shows that average values for both the final number of somites and
final protein content are a little higher in the two groups of embryos grown with
extra serum than in the group with minimum serum. However, the differences
in protein content are not statistically significant; comparison of groups 1 and
2 gives P = 0-17, and of groups 1 and 3 gives P = 0-38.
Table 4. Development after 40 h as indicated by number of somites, and growth as
indicated by protein content, of embryos explanted at 22-28 somites and grown in
different amounts of circulating serum
Serum per embryo (ml)
3
3 initially+ 9 after 17 h
Somites
Protein
Somites
Protein
39
43
43
42
39
36
37
35
Mean
39-2
735
1015
875
1035
890
780
830
705
Mean and s .D.
858 ± 117
43
46
45
38
Mean
430
1250
1510
920
930
Mean and S.D.
1152 ±245
The results shown in Table 4 are from two groups of embryos, one grown in
3 ml serum per embryo throughout the period of the culture, the other with an
initial 3 ml per embryo to which was added a further 9 ml after 17 h. Again the
embryos with extra serum developed a little better, and here the difference in
mean protein between the two groups is just significant (P = 003).
From Tables 3 and 4 taken together it appears that increasing the amount of
serum above the usual 2-3 ml per embryo may have slightly improved growth,
but that any improvement was small compared with the amount of extra serum
added; 2-4 times the usual amount of serum increased growth by only about
10-35 %
Rat embryos in culture
521
In another experiment two 25-somite embryos were grown as long as possible
in a circulator containing 6 ml serum, and this serum was then used as nutrient
medium for young (15-somite) embryos cultured in watch-glasses. These young
embryos developed as well as controls in fresh serum, suggesting that embryonic
growth of the embryos in the circulator had not been limited by nutritional
deficiencies or accumulation of excretory products in the serum, although the
circulator contained only 3 ml serum per embryo.
Explanted at Explanted at
7-9 somites 10-15 somites
1000 -
Explanted at
22-28 somites
Extra protein synthesized (tig)
200 -
W 1 10
W 1 10
W 1 4-815-20
Flow (ml/min)
Text-fig. 4. Development as indicated by extra somites formed, and growth as
indicated by extra protein synthesized, of embryos grown in watch-glasses of serum
(W) and in serum circulating at different rates.
Rate of flow of medium
Text-fig. 4 shows the effect of different rates of flow of the serum in the circulators on embryonic growth and development. Each rectangle in the histogram
is the mean of determinations made on 5-7 embryos.
For the younger embryos there appears to be an optimum rate of flow. They
grow better in serum flowing at 1 ml/min than in static serum in watch-glasses
(W) or serum flowing at 10 ml/min. Particularly striking is the difference between
the effects of the slower and faster rates of flow on the embryos explanted at
7-9 somites, and the difference in amount of protein synthesized by these is
highly significant (P = < 0001).
33
JEEM
17
522
D. A. T. NEW
The embryos explanted at 22-28 somites benefit from increased rates of
flow up to the maximum (15-20 ml/min) that has been tried. The difference in
protein synthesis between the embryos growing in serum flowing at 15-20 ml/min
and those in serum flowing at 1 ml/min is highly significant (P = 0-001).
Growth of embryos of this age explanted into watch-glasses (W) containing
similar amounts of serum per embryo is very limited.
Survival of embryos
Text-fig. 5 shows the percentage surviving to different stages of development,
as indicated by number of somites, of 34 embryos explanted at 7-15 somites and
grown in serum circulated at 0-5-3-0 ml/min, and of 89 embryos explanted at
22-28 somites and grown in serum circulated at 8-20 ml/min.
16
20
24
28
32
36
40
44
48
Somites
Text-fig. 5. Percentage surviving to different stages of development as indicated by
number of somites, of embryos explanted at 7-15 somites and 22-28 somites and
grown in circulating homologous serum.
About half the younger group of embryos developed to 30-35 somite stages,
but none survived longer. Over half the embryos explanted at 22-28 somites
developed to 40-46 somite stages (Plate 1, fig. B).
A characteristic feature of the development of the younger embryos was the
abnormal formation of a large blood vessel bypassing the umbilical vessels to
the allantoic placenta (Text-fig. 6). With its formation blood stopped or failed
to begin flowing through the vessels of the allantoic placenta, and also gradually
ceased flowing through the capillaries of the yolk sac, although it often continued
to circulate for some time round the embryo. No such' allantoic bypass' was ever
observed in embryos explanted at stages older then 22 somites.
Seven embryos were explanted at 38-40 somites from rats 12-13 days
/ . Embryo/, exp. Morph., Vol. 17, Part 3
PLATE 1
Fig. A. Hot box (base 45 x 45 cm) containing six 'circulators' as shown in Text-fig. 1 and a
binocular dissecting microscope.
Fig. B. Two embryos taken from the same rat on the 12th day of pregnancy, the one on the
left (27 somites) fixed immediately and the other grown to 40 somites in circulating serum.
The 27-somite embryo is about the size and stage of development of the largest embryos that
can be grown in static medium, x 17.
D. A.T. NEW
facing p. 522
Rat embryos in culture
523
pregnant, and grown in serum flowing at 15-20 ml/min. Blood continued to
circulate through the yolk-sac capillaries for over 8 h, and in one embryo for 24 h,
but the embryos developed only to 41- to 46-somite stages. Three embryos explanted (1 embryo per circulator, with a larger embryo chamber) at about
50 somites did not develop, and the blood circulation began to fail within 2 h.
It appears therefore that the 45-somite stage is about the oldest that can be
maintained by these conditions of culture.
Aorta
Allantoic
placenta
I
'
/
""Heart
Text-fig. 6. Formation of a large blood vessel bypassing the umbilical vessels in an
embryo explanted at the 12-somite stage and grown in circulating serum to 32
somites. Drawn from life, x 12.
DISCUSSION
The results show that rat embryos can be grown in circulating homologous
serum from 5- to 10-somite stages to 30-35 somites, and from about 25 somites
to 40-45 somites.
Within these two periods of development the explanted embryos grow well,
and are very similar to controls in vivo both in rate of growth and in degree of
differentiation. They do not begin to degenerate until they have reached a certain
stage of development, which for the younger group is about the 30-somite stage
and for the older group the 40-somite stage; they then die fairly rapidly.
The question arises why the embryos explanted at 5- to 10-somite stages do
not develop to 40 somites. The main difference between embryos that have
been grown in culture from stages younger than 15 somites and those grown from
stages older than 22 somites is that the younger embryos develop a large blood
vessel bypassing the allantoic placenta (Text-fig. 6) and the older ones do not.
It seems very likely therefore that failure to establish or maintain a blood vascular
connexion with the allantoic placenta is the cause of death at the 30-somite
stage. This is surprising because the allantoic placenta of explanted embryos is
held against the rayon coverslip on the floor of the embryo chamber (Textfig. 2), and even with a blood circulation is unlikely to make a significant contribution to the nutritional or respiratory needs of the embryo. Perhaps the cells
of the allantoic placenta synthesize essential substances with a hormonal
function, and the formation of the bypass vessel prevents these reaching the
33-2
524
D. A. T. NEW
embryo; or the bypass might cause a general lowering of blood pressure, resulting in the failure of the yolk-sac circulation which is always one of the first signs
of degeneration. Further experiments will be necessary to determine the correct
explanation.
The embryos are very sensitive to the rate of flow of the nutrient serum in the
circulators. The older embryos develop best at maximum, and the younger at
minimum, rates of flow. There are many possible ways in which varying the rate
might affect the embryos, but probably the most critical is the supply of oxygen.
Experiments are now being made to determine whether further increases in flow
rate will make possible the growth of larger embryos, and whether very slow
flow combined with a reduction in the proportion of oxygen in the equilibrating
gas will support the development of pre-somite stages.
SUMMARY
1. A method is described for growing post-implantation rat embryos,
explanted with their embryonic membranes, in circulating medium. Embryos
cultured by this method grow to about five times the maximum weight attainable
in static medium in watch-glasses.
2. Homologous serum, equilibrated with 95 % O2/5 % CO2, has proved to be
a satisfactory culture medium, and it makes no difference whether the serum is
from pregnant or non-pregnant animals. Good development is obtained in
2-3 ml serum per embryo, and little or no improvement is gained by using more
than this.
3. About 50 % of embryos explanted at 7-15 somites develop to 30- to 35somite stages and synthesize 0-3-0-5 mg protein during the period in culture.
Further development of these embryos appears to be prevented by the abnormal
formation of a large blood vessel bypassing the allantoic placenta.
4. About 50 % of embryos explanted at 22-28 somites develop to 40-46
somite stages and synthesize about 1-1-5 mg protein. Embryos explanted at
more than 45 somites do not develop.
5. The rate of flow of the circulating medium is critical. Embryos explanted at
7-15 somites develop much better in serum flowing at 1 ml/min ( = 2 cm/min
through the embryo chamber) than at 10 ml/min. Embryos explanted at 22-28
somites develop much better in serum flowing at > 10 ml/min than at 1 ml/min.
RESUME
Developpement d'embryons de rat explantes dans un milieu circulant
1. On decrit une methode assurant la croissance d'embryons de rat aux
stades posterieurs a l'implantation, explantes avec leurs annexes embryonnaires
dans un milieu circulant. Des embryons cultives a l'aide de cette technique
croissent jusqu'a environ cinq fois le poids maximum qu'ils peuvent atteindre
dans un milieu statique, dans des verres de montre.
Rat embryos in culture
525
2. Du serum homologue, equilibre avec un melange 95 % O2/5 % CO2, s'est
revele etre un milieu de culture satisfaisant, et il n'y a pas de difference selon
qu'il s'agit de serum de femelles gestantes ou non gestantes. On obtient un bon
developpement dans 2-3 ml de serum par embryon, et on n'a que peu ou pas
d'amelioration en utilisant davantage de serum.
3. Environ 50 % des embryons explantes aux stades 7-15 somites se developpent jusqu'aux stades 30-35 somites et synthetisent 0,3-0,5 mg de proteines
au cours de la periode de culture. Le developpement ulterieur de ces embryons
est inhibe par la formation anormale d'un gross vaisseau sanguin traversant le
placenta allantoidien.
4. Environ 50 % des embryons explantes a 22-28 somites se developpent
jusqu'aux stades 40-46 somites et synthetisent environ 1 a 1,5 mg de proteines. Des embryons explantes a un stade depassant les 45 somites ne se
developpent pas.
5. Le rythme d'ecoulement de milieu circulant est critique. Des embryons
explantes a 7-15 somites se developpent beaucoup mieux dans du serum
s'ecoulant au rythme de 1 ml/min ( = 2 cm/min a travers le chambre embryonnaire) que a 10 ml/minute. Des embryons explantes a 22-28 somites se developpent beaucoup mieux dans du serum d'ecoulent au rythme de plus de 10 ml/min
qu'a 1 ml/min.
I am very grateful to Mrs J. R. Wallage for technical assistance, and to Mr M. F. Applin
for the photography. Text-figs. 2 and 4 and Plate 1, fig. A have previously appeared in a
Symposium of the British Society for Parasitology, and I thank Blackwell Scientific Publications Ltd for permission to reproduce them.
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(Manuscript received 18 January 1967)