Bovine serum: An alternative to rat serum as a culture medium for the rat whole embryo culture

Toxic. in Vitro Vol. 4, No. 4/5, pp. 598~01, 1990 Printed in Great Britain. All rights reserved

0887-2333/90 $3.00+ 0.00 Copyright © 1990 Pergamon Press pie

BOVINE SERUM: AN ALTERNATIVE TO RAT SERUM AS A CULTURE MEDIUM FOR THE RAT WHOLE EMBRYO CULTURE S. KLUG, C. LEWANDOWSKI,L. WILDI and D. NEUBERT Institute for Toxicology and Embryopharmacology, Garystr. 5, 1000 Berlin 33, FU Berlin, FRG Abstract--The results reported here demonstrate the ability of supplemented bovine serum to serve as a culture medium for rat whole embryos. After 48 hours' culture in bovine serum supplemented with Tyrode's buffer and methionine, 9.5-day-old rat embryos were at a stage of development comparable with that of embryos cultivated in homologous serum, although some deficiency in the formation of haemoglobin in embryonic blood cells could be observed. However, supplementation of the culture medium with haemoglobin overcame this deficiency. The procedure used for preparing the culture medium is described in detail and some advantages of bovine serum are discussed.

Introduction

Culturing of whole early post-implantation embryos has become an important tool in reproductive biology and toxicology. Meanwhile this technique, with its well known advantages, is an established method in several laboratories (e.g. Beck et al., 1987; Brown and Fabro, 1982; Clode et al., 1987; Fantel, 1982; Langenfeld et al., 1988; Neubert, 1984; Neubert et al., 1985; Tesh, 1988). Since the establishment of this system homologous serum has been the main culture medium used until now. F o r large-scale testing with this method it became necessary to use a medium that could be easily obtained while providing conditions comparable with homologous serum. Attempts to replace rat serum with heterologous serum were performed with human serum (Chatot et al., 1980; Gupta and Beck, 1983), monkey serum (Klein et aL, 1982) and serum from cows (Dealy et al., 1986) and from dogs (Flynn et al., 1987). Except for those studies using human serum, all other experiments dealing with the growth and development of rat embryos in the sera of other species could be regarded as rather isolated studies. Until now, no group of investigators has decided to work exclusively with serum of another species over a period of several years. The use of human serum might be discussed for specific experiments dealing with special problems (e.g. diabetes). However, studies using human serum as a culture medium over an extended time period has led to variable growth and development, and the incidence of abnormalities in control groups (up to 58%) has been rather high (Chatot et al., 1984). For these reasons we decided in 1983 to perform the whole embryo culture using the serum of cows, a large species that provides large quantities of blood without the need to kill the donor. Some of the data and experiences obtained with this culture medium over the last six years are presented here. Materials and Methods

9.5-day-old embryos (Wistar rats) were cultured for 48 hr (first 24 hr following the mating period were

called "day 0" of pregnancy if sperm were detected in the vaginal smears) according to the method described by Cockroft (1977) and New (1978). In 7 ml medium 3-4 embryos were cultured in rotating bottles (25 rpm) at 38.5°C. Initially the culture bottles were gassed with 10% 02, 5% CO2 and 85% N2. After 36 hr of culture the bottles were regassed with 50% 02, 5% CO2 and 45% N2. Supplemented bovine serum (6ml bovine serum + 1 ml Tyrode's buffer supplemented with 75/zg methionine/ml culture medium and 3 mg D-glucose/ml) was routinely used as a culture medium. For some experiments the medium was additionally supplemented with 2.5 mg bovine haemoglobin/ml. Preparation o f serum. The blood was drawn from the jugular vein of non-pregnant, non-medicated, metabolically stable cows and was immediately centrifuged at 3000rpm for 10rain. The fibrin clot was removed from the wall of the tube and two more centrifugations (3000rpm/10min) followed. The sapapled serum was then heat-inactivated (30 min at 56°C) and pre-filtered. Subsequently the serum was sterile filtered through Millipore filters (0.2/~m) in portions of 6 ml into the culture bottles and sealed. The serum was stored frozen at - 1 8 ° C until needed. The rat serum used in the present experiments was gained by puncture of the abdominal aorta of anaesthetized rats and handled according to the method described by Steele and New (1974). At the end of the culture period the embryos were evaluated with respect to growth and development using a scoring system similar to that of Brown and Fabro (1981). The data were stored, described and evaluated by using a computer. The complete method has been described previously (Klug et aL, 1985a). Histological examination of a representative number of embryos was performed routinely. Results

Through the work of several laboratories it is well established that homologous serum supports the growth and development of whole early postimplantation embryos of rats 'in culture. Table 1 598

B o v i n e s e r u m as a c u l t u r e m e d i u m

599

Table 1. Culture data for rat whole embryos cultured for 48 hr in either rat serum or supplemented bovine serumt

Culture medium Rat serum n ~ 17 Bovine s e r u m + Tyrode's buffer n=41 Bovine s e r u m + T y r o d e ' s buffer +methionine n = 52 Bovine serum + Tyrode's buffer + methionine + haemoglobin n = 20

Yolk sac (mm) 4.44 4.02 3.78 4.80 4.44** 4.20 4.68 4.44** 4.27 4.60 4.44* 4.27

Crownrump length (ram)

Somites (n-)

3.51 3.42 3.18 3.51 3.18 3.12 3.78 3.63 3.48

40

27

288

40

26 27 26** 25 28 27 26

216 237 182"* 144 251 218"* 194

39 37 36** 35 38 37** 36

28

251 212" 185

40

27 3.30

Score

320

27

3.58 3.42

Protein ~g/E)

27

Abn

6% --

--

40 39

A b n = frequency of the abnormalities in %. n = number of cultured embryos. tThe numbers give the median values and the third (upper number) and the first quartile (down number). *Significant difference (0.05 > P > 0.01) from rat serum (Mann-Whitney test). **Significant difference (P < 0.01) from rat serum (Mann-Whitney test).

gives the results of the experiment comparing the growth of 9.5-day-old embryos in (a) rat serum, (b) bovine serum+Tyrode's buffer, (c) bovine serum + Tyrode's buffer + methionine and (d) bovine serum + Tyrode's buffer + methionine + haemoglobin. Embryos cultivated in bovine serum supplemented with Tyrode's buffer showed a slight reduction in growth (crown-rump length, protein content) and development (somites, score). The reduced score is mainly due to anaemia but sometimes smaller fore-limb buds and retardation of the ear anlage (absence of recessus dorsalis at the otic vesicle) were seen as well. When 75/zg methionine/ml was added to the culture medium, yolk-sac diameters, crown-rump length and number of somite pairs were developed as well as in those embryos growing in rat serum; only the protein content was somewhat lower ( - 2 5 % ) . Further supplementation with 2.5mg bovine haemoglobin/ml also improved the formation of haemoglobin in the embryonic pre-erythrocytes. Embryos cultivated in bovine serum with these supplements showed no abnormalities (el. Table 1). One embryo in the rat serum group presented here showed an abnormal flexure, possibly' resulting from an incomplete embryonic rotation. This abnormality occurs sometimes spontaneously (at a very low

rate of < 3 % ) irrespective of which serum is used (cf. Table 2). Table 2 shows the data of three representative 'historic' control groups: (1) negative controls: bovine serum + Tyrode's buffer + methionine; (2) and (3) solubilizer controls: supplemented bovine serum + 5 #1 dimethylsulphoxide, and 5 #1 ethanol per 7 ml culture medium, respectively. In the presence of ethanol an abnormality rate of 3% is still consistent with the spontaneous rate (flexure of embryos) mentioned above. The relatively large number of cultured embryos and the homogeneity of the data presented in Tables 1 and 2 demonstrate that supplemented bovine serum is able to support sufficient growth and development of the cultured rat embryos. Since we did not find it necessary to supplement the medium routinely with haemoglobin the embryos were anaemic, which in turn explains the reduced score (mean.= 37 instead of 39).

Discussion Our attempts to use human serum for large series of experilments were not very successful because of the variable development of the cultured embryos and the comparatively high occurrence of

Table 2. Culture data for representative historical control groups (negative controls and solvent controls)*

Culture medium

Yolk sac (ram) 4.56

Bovine serum + Tyrode's buffer + methionine n = 227

4.32

Bovine serum + Tyrode's buffer + methionine + D M S O t n=41

4.44

4.20

Crownrump length (ram) 3.72 3.54 3.42

4.65

Bovine serum + Tyrode's ,buffer + methionine + ethanolt n =58

4.20

27

38

214

-36 37

36 190

ffi frequency of the abnormalities in %. n = number of cultured embryos. *The numbers give the median values the third (upper number) and the first quartile (down number). tAdditive of 5/~1 dimethylsulphoxide resp. ethanol to 7 ml culture medium. Abn

38

247 210

26

0.8%

37 171

28 27

Abn

36

240 215

26

3.78

37 183

28

3.42

3.48

Score

250

26 27

3.60

Protein (pg/E)

28

3.84 3.60

4.68 4.44 4.26

Somites (n = )

3% 36

600

S. KLUG et al. Table 3. Number of laboratory animals (rats) required for the establishment of a dose-response relationship In vivo

Control Dose I Dose II Dose III Total animals:

In vitro

(Segment II study) Rats (n) 20 20 20 20

Culturemedium: rat serum embryos (n) 15-20 15-20 15-20 15-20 12 15: rats for culture +30-35: rats for serum 42-50

Culture medium: bovine serum embryos (n) 15-20 15-20 15-20 15-20

80

12 15

spontaneous abnormalities. A possible explanation for this might be found in the lack of control over the blood donors, which involves various factors (e.g. smoking, alcohol, diet, medication). Since 1983, when studies in our group revealed the ability of bovine serum to serve as a culture medium for rat whole embryos, we more or less routinely cultivated approximately 10,000 rat embryos in supplemented bovine serum. Our experiments probably represent one of the most extensive studies performed with the whole embryo culture system. The results obtained with this system were found to be identical to those achieved when using a homologous serum, and the test system tolerates sufficient concentrations of solvents such as dimethylsulphoxide and ethanol. This is of considerable importance if water-soluble test substances are to be studied. Moreover, doseresponse relationships identical to those reported in the literature on rat serum were achieved, using, for example, substances like valproic acid (Kao et al., 1981; Lewandowski et al., 1986) or acyclovir (Bechter and Taccard, 1987; Klug et al., 1985b). Aside from the p r o o f that bovine serum can replace rat serum as a culture medium for the purpose mentioned, other important advantages are worth considering: the donor animals are kept under controlled environmental conditions (standardized food, veterinarian care, etc.) allowing relatively good control over the quality of the serum. Bovine serum can easily be obtained in large quantities (up to 1 litre from one batch of blood). This means time and cost reduction in the preparation of the culture medium and furthermore, large experimental series can be performed with the same batch of serum. Another aspect becoming increasingly important concerns animal protection. In several countries obtaining blood from an anaesthetized animal is considered to be an animal experiment (irrespective of whether or not the rat is going to be sacrificed anyway). The replacement ,of rat serum by bovine serum leads to a clear-cut reduction of laboratory animals necessary for the in vitro study with this culture method. F r o m a legal point of view, obtaining the embryos from a sacrificed rat is not an animal rexperiment, while obtaining the blood by using the usual procedure (anaesthesia and puncture of a large vessel) is certainly considered an in vivo method. F o r this reason the use of bovine serum renders the whole embryo culture method to be a clear-cut in vitro technique. This means that one should not sacrifice laboratory animals for gaining the serum necessary for preparation of the culture medium, if adequate alternatives exist. The rat whole embryo culture using bovine serum as a culture medium requires less than

one-third of the laboratory animals that are required using rat serum, and even less than one-fifth of those required for an in vivo study (cf. Table 3). The fact that the embryos in bovine serum, not supplemented with haemoglobin, form little haemoglobin in their 'pre-erythrocytes' is in our experience no disadvantage for routine studies. Histological examinations (including electron microscopy) revealed no differences between 'anaemic' and 'nonanaemic' embryos. Enzyme-histological studies of the yolk-sac enzyme pattern confirmed this (R. Gossrau and G. Stein, personal communication, 1987). The data presented clearly indicate that bovine serum is well suited for the culture of rat whole embryos during early organogenesis. Especially for large-scale testing this medium provides obvious advantages, at least until a serum-free and chemically defined culture medium has been developed for routine purposes.

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