TERATOLOGY 45~293-301 (1992)
In Vitro Development of Rat Embryos Undergoing Organogenesis in Heparin-Plasma GREGORY J. KESBY Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, England
ABSTRACT This study examines the use of heparin-plasma as a culture medium for mammalian postimplantation whole-embryo culture. The growth and differentiation of head-fold rat embryo explants over 48 hours in a standard serum medium was compared with development of same stage explants over 48 hours in a plasma medium prepared using sodium heparin. Heparin disrupted the morphological differentiation of embryos, in a concentration-dependent manner, from 25 pg sodium heparin/ml media (i.e., 5 IU/ml media), with overall embryo growth being adversely affected from a concentration of 200 kg sodium heparin/ml media (i.e., 40 IU/ml media). Defects of cranial neural tube development were the first apparent structural anomalies resulting from culture in heparin media. Forebrain development was grossly abnormal and associated with failure of eye development. As the heparin concentration in media increased, the cephalic neural folds remained widely open and the edges became increasingly everted, although differentiation of the heart, otic primordia, and pharyngeal arch persisted. Similar concentration-dependent dysmorphogenic effects were seen when embryos were cultured in the standard serum media with added heparin. A minimum heparin concentration of 100 pg sodium heparin/ml media (i.e., 20 IU/ml media) was required to effectively inhibit coagulation of the plasma medium over the 48 hour culture period. Although embryonic growth was not adversely affected a t this heparin concentration, morphological differentiation was severely disrupted. Therefore, heparin is not a suitable anticoagulant for the preparation of plasma for use in postimplantation whole-embryo culture. Mammalian postimplantation whole-em- coagulation; it is not a physiological fluid bryo culture is used extensively in the in- and significantly differs from plasma in convestigation of both normal and abnormal stitution (see Young and Bermes, '86). embryonic development. Whole-embryo cul- When compared with plasma, the concenture techniques will support embryonic tration of certain coagulation factors and growth and differentiation a t rates that are other enzymes are decreased in serum due equivalent to those achieved in utero over either to destruction or co-precipitation in the 48 hour period from the head-fold stage the coagulation process (Alper, '74). The seof neurulation to the differentiation of some rum concentrations of glycolytic enzymes, 26-28 somite pairs (New et al., '76a). Nev- potassium, and other intracellular compoertheless, at a cellular level, differences in nents are increased as a result of haemolyenzymatic and other biochemical activities sis (Ladenson et al., '74) and a variety of of conceptuses growing in vitro and in utero physiologically potent agents including sehave been observed (Huber and Brown, '82; rotonin, lysosomal enzymes, ATP, ADP, pBrown et al., '91). The factors responsible glucuronidase, prostaglandins, and other for these differences have not yet been determined but they may reflect, in part, the use of serum as a culture medium. Serum is the fluid end-product of plasma Received May 13, 1991; accepted October 1, 1991. 0 1992 WILEY-LISS, INC.
294
G.J. KESBY
arachidonate metabolites are released by platelets and enter the serum. Plasma can be used in the whole-embryo culture system if its coagulation is effectively inhibited. However, the calcium chelating anticoagulants (oxalate, fluoride, citrate, EGTA, and EDTA) cannot be used to prepare plasma for use in embryo culture since normal embryonic development requires a physiological concentration of free calcium ions (Ellington, '83; Smedley and Stanisstreet, '85). Heparin, on the other hand, is a naturally occurring anticoagulant that does not significantly affect the free calcium ion concentration of plasma. Heparin maintains the fluidity of plasma, both in vivo and in vitro, by greatly increasing the activity of antithrombin 111, the main plasma inhibitor of activated clotting factors. There is little information available concerning the use of heparin-plasma in postimplantation whole-embryo culture. Nicholas and Rudnick ('34), using watch glass culture dishes exposed to air and leaving the Reichert membrane of embryos intact, were the first to document that heparin-plasma was capable of supporting development (albeit limited) of both presomite and early somite rat conceptuses in vitro. Much later, Sanyal and Wiebke ('79), using an intermittently gassed, rolling-bottle culture system, reported that growth of the early-somite rat embryo was significantly retarded in heparin-plasma, with embryos cultured in plasma synthesizing considerably less protein and nucleic acid than embryos cultured in serum. However, the concentration of heparin used for the preparation of plasma by Sanyal and Wiebke (100 IU/ml blood) was approximately five times the minimum concentration usually required for the preparation of plasma from whole blood (Young and Bermes, '86). In light of the earlier report by Steele and New ('74) that the protein contents of head-fold rat embryo explants cultured for 48 hours in plasma prepared using either 0.02% heparin (i.e., approximately 35 IU heparidml blood) or 1.3%sodium oxalate did not significantly differ with the protein contents of same stage embryos cultured in serum, the possibility that heparin-plasma, prepared using minimum effective anticoagulant concentrations, may support normal embryonic growth and differentiation in vitro could not be excluded. Therefore, this study was undertaken to
assess the ability of a heparin-plasma culture medium, prepared using low concentrations of sodium heparin, to support growth and differentiation of head-fold rat embryo explants undergoing organogenesis in vitro. MATERIALS AND METHODS
Animals Outbred Wistar rats (CFHB; Anglia Laboratories, Huntingdon, UK) were used in this study. One male was housed with either two or three females overnight; the presence of spermatozoa on microscopic examination of vaginal smears taken on the following morning was taken as evidence of mating and that day was designated as day 1 of pregnancy. Plasma and serum preparation Plasma and serum were prepared from male and female rats anaesthetised with diethyl ether and bled from the abdominal aorta. For the preparation of plasma, measured volumes of blood were mixed with known volumes of aqueous sodium heparin solution (from porcine or bovine lung mucosa; 195 IU/mg; Leo Laboratories, UK) to yield concentrations of 5, 10, 20, 40, and 80 IU/ml whole blood. The anticoagulated plasma fraction was separated by centrifugation at 2,OOOg for 5 min and pooled for each heparin concentration. Pooled serum was prepared from the cell-free plasma clots of immediately centrifuged blood as described by New et al. ('76b). Both the pooled serum and pooled plasma were heated for 30 min at 56°C before use in culture (New et al., '76b). Culture media preparation In all experiments, the culture medium was an equal volume's mixture of either serum or heparin-plasma, with Dulbecco's modification of Eagle's medium (DMEM; 4.5 mg glucose/ml;Flow Laboratories, UK) that had first been diluted to 300 mOsm/litre with filter sterilised, double-distilled and deionised water (dilution
In Vitro Development of Rat Embryos Undergoing Organogenesis in Heparin-Plasma GREGORY J. KESBY Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, England
ABSTRACT This study examines the use of heparin-plasma as a culture medium for mammalian postimplantation whole-embryo culture. The growth and differentiation of head-fold rat embryo explants over 48 hours in a standard serum medium was compared with development of same stage explants over 48 hours in a plasma medium prepared using sodium heparin. Heparin disrupted the morphological differentiation of embryos, in a concentration-dependent manner, from 25 pg sodium heparin/ml media (i.e., 5 IU/ml media), with overall embryo growth being adversely affected from a concentration of 200 kg sodium heparin/ml media (i.e., 40 IU/ml media). Defects of cranial neural tube development were the first apparent structural anomalies resulting from culture in heparin media. Forebrain development was grossly abnormal and associated with failure of eye development. As the heparin concentration in media increased, the cephalic neural folds remained widely open and the edges became increasingly everted, although differentiation of the heart, otic primordia, and pharyngeal arch persisted. Similar concentration-dependent dysmorphogenic effects were seen when embryos were cultured in the standard serum media with added heparin. A minimum heparin concentration of 100 pg sodium heparin/ml media (i.e., 20 IU/ml media) was required to effectively inhibit coagulation of the plasma medium over the 48 hour culture period. Although embryonic growth was not adversely affected a t this heparin concentration, morphological differentiation was severely disrupted. Therefore, heparin is not a suitable anticoagulant for the preparation of plasma for use in postimplantation whole-embryo culture. Mammalian postimplantation whole-em- coagulation; it is not a physiological fluid bryo culture is used extensively in the in- and significantly differs from plasma in convestigation of both normal and abnormal stitution (see Young and Bermes, '86). embryonic development. Whole-embryo cul- When compared with plasma, the concenture techniques will support embryonic tration of certain coagulation factors and growth and differentiation a t rates that are other enzymes are decreased in serum due equivalent to those achieved in utero over either to destruction or co-precipitation in the 48 hour period from the head-fold stage the coagulation process (Alper, '74). The seof neurulation to the differentiation of some rum concentrations of glycolytic enzymes, 26-28 somite pairs (New et al., '76a). Nev- potassium, and other intracellular compoertheless, at a cellular level, differences in nents are increased as a result of haemolyenzymatic and other biochemical activities sis (Ladenson et al., '74) and a variety of of conceptuses growing in vitro and in utero physiologically potent agents including sehave been observed (Huber and Brown, '82; rotonin, lysosomal enzymes, ATP, ADP, pBrown et al., '91). The factors responsible glucuronidase, prostaglandins, and other for these differences have not yet been determined but they may reflect, in part, the use of serum as a culture medium. Serum is the fluid end-product of plasma Received May 13, 1991; accepted October 1, 1991. 0 1992 WILEY-LISS, INC.
294
G.J. KESBY
arachidonate metabolites are released by platelets and enter the serum. Plasma can be used in the whole-embryo culture system if its coagulation is effectively inhibited. However, the calcium chelating anticoagulants (oxalate, fluoride, citrate, EGTA, and EDTA) cannot be used to prepare plasma for use in embryo culture since normal embryonic development requires a physiological concentration of free calcium ions (Ellington, '83; Smedley and Stanisstreet, '85). Heparin, on the other hand, is a naturally occurring anticoagulant that does not significantly affect the free calcium ion concentration of plasma. Heparin maintains the fluidity of plasma, both in vivo and in vitro, by greatly increasing the activity of antithrombin 111, the main plasma inhibitor of activated clotting factors. There is little information available concerning the use of heparin-plasma in postimplantation whole-embryo culture. Nicholas and Rudnick ('34), using watch glass culture dishes exposed to air and leaving the Reichert membrane of embryos intact, were the first to document that heparin-plasma was capable of supporting development (albeit limited) of both presomite and early somite rat conceptuses in vitro. Much later, Sanyal and Wiebke ('79), using an intermittently gassed, rolling-bottle culture system, reported that growth of the early-somite rat embryo was significantly retarded in heparin-plasma, with embryos cultured in plasma synthesizing considerably less protein and nucleic acid than embryos cultured in serum. However, the concentration of heparin used for the preparation of plasma by Sanyal and Wiebke (100 IU/ml blood) was approximately five times the minimum concentration usually required for the preparation of plasma from whole blood (Young and Bermes, '86). In light of the earlier report by Steele and New ('74) that the protein contents of head-fold rat embryo explants cultured for 48 hours in plasma prepared using either 0.02% heparin (i.e., approximately 35 IU heparidml blood) or 1.3%sodium oxalate did not significantly differ with the protein contents of same stage embryos cultured in serum, the possibility that heparin-plasma, prepared using minimum effective anticoagulant concentrations, may support normal embryonic growth and differentiation in vitro could not be excluded. Therefore, this study was undertaken to
assess the ability of a heparin-plasma culture medium, prepared using low concentrations of sodium heparin, to support growth and differentiation of head-fold rat embryo explants undergoing organogenesis in vitro. MATERIALS AND METHODS
Animals Outbred Wistar rats (CFHB; Anglia Laboratories, Huntingdon, UK) were used in this study. One male was housed with either two or three females overnight; the presence of spermatozoa on microscopic examination of vaginal smears taken on the following morning was taken as evidence of mating and that day was designated as day 1 of pregnancy. Plasma and serum preparation Plasma and serum were prepared from male and female rats anaesthetised with diethyl ether and bled from the abdominal aorta. For the preparation of plasma, measured volumes of blood were mixed with known volumes of aqueous sodium heparin solution (from porcine or bovine lung mucosa; 195 IU/mg; Leo Laboratories, UK) to yield concentrations of 5, 10, 20, 40, and 80 IU/ml whole blood. The anticoagulated plasma fraction was separated by centrifugation at 2,OOOg for 5 min and pooled for each heparin concentration. Pooled serum was prepared from the cell-free plasma clots of immediately centrifuged blood as described by New et al. ('76b). Both the pooled serum and pooled plasma were heated for 30 min at 56°C before use in culture (New et al., '76b). Culture media preparation In all experiments, the culture medium was an equal volume's mixture of either serum or heparin-plasma, with Dulbecco's modification of Eagle's medium (DMEM; 4.5 mg glucose/ml;Flow Laboratories, UK) that had first been diluted to 300 mOsm/litre with filter sterilised, double-distilled and deionised water (dilution
