Human Reproduction vol.15 no.4 pp.756–761, 2000 Secretory characteristics and viability of human term placental tissue after overnight cold preservation N.Cirelli1,4, P.Lebrun2, C.Gueuning1, A.Moens3, (Chard, 1986; Talamantes and Ogren, 1988). The releases of J.Delogne-Desnoeck1, C.Dictus-Vermeulen1, both HCG and HPL from term placental explants are stimulated A.-M.Vanbellinghen1 and S.Meuris1 by the influx of calcium ion (Polliotti et al., 1990, 1992; Meuris et al., 1994; Petit and Belisle, 1995) in a manner 1Research Laboratory on Reproduction and 2Laboratory of Pharmacology, Universite´ Libre de Bruxelles (ULB), Brussels, concordant with the ‘stimulus–secretion coupling’ concept Belgium and 3Unite´ Ve´te´rinaire, Universite´ Catholique de Louvain (Douglas, 1968). However, in contrast to their pituitary homo- (UCL), Louvain-La-Neuve, Belgium logues whose secretory responses are modulated by defined 4To whom correspondence should be addressed at: Research external stimuli, little is known about the physiological control Laboratory on Reproduction, CPi 626, Faculty of Medicine, of the placental HCG and HPL releases (Ringler and Strauss, Universite´ Libre de Bruxelles, 808, Route de Lennik, B-1070 1990; Handwerger, 1991). Brussels, Belgium ‘In-vitro’ systems represent useful experimental approaches Collection of human term placentae for research purposes to characterize the influence of physiological and/or pharmaco- is generally limited during working hours. Preserving logical agents upon the endocrine function. Among the numer- placental tissue overnight might help to postpone experi- ous ‘in-vitro’ methods for studying secretion, incubation of ments and, by extent, to increase material availability. In placental explants has the potential advantage of maintaining this study, fragments from normal placentae were incubated the placental cells in their normal histological environment, at 37°C either immediately after delivery or after preserva- thus allowing paracrine and/or autocrine interactions among tion at 4°C in a HEPES-buffered solution or in a Roswell different cell types (Ringler and Strauss, 1990). However, Park Memorial Institute (RPMI) 1640 culture medium. tissue explant incubations often reveal declining hormone Protein, human chorionic gonadotrophin (HCG), human secretion, morphological alterations and short-term cell sur- placental lactogen (HPL) and lactate dehydrogenase (LDH) vival (Chung et al., 1969; Taylor and Hancock, 1973; Hall contents within preserved explants were similar to those et al., 1977). within freshly delivered ones. In contrast, HCG and HPL Although research on human placenta involves no particular amounts released during incubation of preserved tissue ethical problem, laboratories have to face the limited availabil- were lower than with freshly delivered tissue. Differences ity of normal and, more particularly, pathological placentae were significant only during the first 3 h of incubation. during working hours. Preserving placentae overnight might Hormone releases were similarly Ca2⍣-stimulated, and help to postpone experiments and, by extent, to increase Co2⍣- and low temperature-inhibited in preserved and material availability for research purposes. freshly delivered tissues. After preservation, LDH leakage In this study, an ‘in-vitro’ preservation methodology was was also reduced. Furthermore, before and after 37°C tested in order to increase availability of placental tissue usable incubation during 6 h, preserved tissue was morphologically for further physiological investigations. Explants from normal- indistinguishable from freshly delivered tissue and showed term placentae were incubated either directly after delivery or neither higher incidence of DNA fragmentation, nor elev- after a 4°C overnight preservation period, and then compared ated caspase-3 activity, both of which are markers of on the basis of their secretory characteristics and tissue viability. apoptosis. This study validates an original, useful and rapid method to preserve placental tissue. Consequently, this Materials and methods preservation model may facilitate the study of physiological processes regulating placental hormone secretion in normal Tissue collection and preservation and pathological conditions. Term human placentae were obtained after vaginal delivery following Key words: apoptosis/explant/placenta/release/viability normal pregnancy (37–41 weeks gestation) and immediately trans- ferred from the Obstetric Department of Erasme Hospital (three to four deliveries/day) to the laboratory located on the same campus. As previously shown (Jauniaux et al., 1991; Denison et al., 1998), Introduction for further in-vitro investigations placental tissue needs to be processed as rapidly as possible after delivery. In this study, the mean time Throughout pregnancy, human placenta synthesizes and between delivery and sampling was 27.5 Ϯ 3.3 min (range 15–35 secretes numerous polypeptide hormones into maternal blood. min; n ϭ 16). Villous tissue free of visible infarct, calcification or Among them, human chorionic gonadotrophin (HCG) and haematoma was sampled midway between the chorionic and basal human placental lactogen (HPL) are structurally and function- plates, from five to ten cotyledons. These central parts of cotyledons ally related to hormones produced by the pituitary gland were cut into multiple fragments (~0.5 g) which were thoroughly 756 © European Society of Human Reproduction and Embryology Characteristics of cold-preserved placental tissue rinsed in a cold Hanks’ medium (pH 7.4), gassed under a 5% CO2– period. A 1 h time interval was required to assay lactate dehydrogenase 95% O2 atmosphere and containing (in mmol/l): NaCl 137, KCl 5, (LDH) release which remained undetectable in 5 min incubation CaCl2 1, MgSO4 1, Na2HPO4 0.3, KH2PO4 0.4 and NaHCO3 4. All media. Explants were transferred, at each time interval, to glass vials reagents were of analytical grade and purchased from Sigma Chemical containing 5 ml of incubation medium. Media were collected and Co (St Louis, MO, USA), unless specified. stored at –20°C in order to further assay hormone and LDH. At the Sets of fragments (n ജ10) from the same placenta were used for end of incubations, explants were either fixed (48–96 h) in 10% immediate incubations or for postponed incubations after an overnight formaldehyde–PBS solution for histological observations or weighed preservation at 4°C. and stored at – 20°C for further determinations of final DNA, HCG, The preservation procedure consisted of bathing placental fragments HPL, LDH and caspase-3 (CPP32) contents. Experiments were overnight (17–24 h) at 4°Cinflasks containing 200 ml of medium repeated with three placentae. continuously gassed under an atmosphere of 100% O2. The preserva- tion medium was composed of the incubation medium supplemented Assays µ with penicillin 50 IU/ml and streptomycin 50 g/ml (Gibco-BRL, For cellular content determinations, tissue homogenates were prepared Gaithersburg, MD, USA). The incubation medium was composed of as following. Thawed placental samples were sonicated (15 s twice, a HEPES-buffered physiological salt solution (pH 7.4) having the 50 kHz, 50 W) either in 500 µl ice-cold PBS solution (pH 7.2) following composition (in mmol/l): HEPES 10, NaCl 139, KCl 5, containing (in mmol/l) Na2HPO4 40, KH2PO4 10 and NaCl 120 or, CaCl2 1, MgCl2 1, glucose 4.2 and 0.5% (w/v) dialysed albumin. In for CPP32 determinations, in an ice-cold cell lysis buffer (pH 7.4) ϭ comparative experiments (n 3 placentae), this cold preservation comprising (in mmol/l) HEPES 50, EDTA 0.1, dithiothreitol (DTT) medium was replaced by a Roswell Park Memorial Institute (RPMI) 1 and 0.1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-pro- 1640 culture medium (Gibco-BRL) supplemented with 5% (v/v) fetal pane sulphonate (CHAPS). Homogenates were then centrifuged for µ bovine serum, penicillin 50 IU/ml and streptomycin 50 g/ml and 10 min at 2500 g at 4°C. Pellets were used for DNA determinations maintained at 4°C under a 5% CO2–95% O2 atmosphere. whilst supernatants were assayed for protein and hormone contents. Immediately after delivery, or after the overnight preservation Protein contents were assayed in supernatants using a modification ജ period, fragments (n 10) were cut into small explants (~20 mg wet of the method of Lowry et al. (1951) (Bensadoun and Weinstein, 1976). weight) and collected in a Petri dish containing cold Hanks’ medium. The DNA was extracted from pellets using a published method Explants, randomly sampled, were either fixed (48–96 h) in 10% (Wannemacher et al., 1965), with the following modifications. First, formaldehyde–phosphate-buffered saline (PBS) solution for histo- pellets were washed twice with 0.2 mol/l perchloric acid instead of logical observations (five groups of three explants) or weighed and 10% trichloroacetic acid before the first 0.3 mol/l KOH extraction stored at –20°C for initial content determinations (five groups of three step. The final step of DNA extraction involved 0.3 mol/l KOH at explants). Remaining explants were used for incubation experiments. 37°C for 15 min instead of 0.5 mol/l perchloric acid at 96°C for Incidentally, it had been noticed previously that no significant 45 min. DNA content was estimated colorimetrically using the variation of placental hormone content was found in relation to Dische’s diphenylamine reaction (Giles and Myers, 1965). gestational age (37–41 weeks; data not shown), with time between Quantities of HCG and HPL in incubation media and