Secretory Characteristics and Viability of Human Term Placental Tissue After Overnight Cold Preservation

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 supernatants delivery and sampling (within 35 min; data not shown), with duration were determined using homologous radioimmunoassays performed of cold preservation (17–24 h; data not shown), or with drug as described previously (Robyn et al., 1971; Polliotti et al., 1990). administration during delivery (Meuris et al., 1996). Sensitivities of the assays were 0.6 µg HPL/ml and 1.5 mIU HCG/ml (2nd International Standard distributed by the World Health Experimental design Organization) respectively. All samples obtained from the same Freshly delivered and preserved explants were incubated in vials placenta were systematically measured within the same assay. Total (three per vial) containing the HEPES incubation medium, and placed hormone amounts released during incubations corresponded to the in a shaking water bath (35 cycles/min) heated at 37°C under a 100% sum of individual amounts assayed at each time interval. The large O2 atmosphere. Incubation started with a 3ϫ60 min equilibration placenta-related variations in hormone release led to the expression, period in order to reach a steady basal HCG and HPL release, as for each individual experiment, of changes in hormone release evoked described previously (Polliotti et al., 1990). Further experimental by Ca2ϩ,Co2ϩ or temperature modifications, with reference to a periods were conducted according to two designs. baseline value (100%) defined as the amount of hormone released by First, in order to assess the secretory capacity and the reactivity of the explants during the first 30 min of the experimental period. the placental tissue, the initial 180 min equilibration period was The CPP32-like activity was measured spectrophotochemically followed by an 18ϫ5 min experimental period. Explants were using the Caspase-3 Cellular Activity Assay Kit Plus (Biomol, transferred, at each time interval, to glass vials containing 1 ml of Plymouth, PA, USA). The release of p-nitroaniline (pNA), cleaved incubation medium. The increase in Ca2ϩ or Co2ϩ concentration and by the tissue extract CPP32, from the DEVD tetrapeptide (Asp-Glu- the temperature lowering were performed from the 30th min until Val-Asp)-pNA substrate was measured at 405 nm after a 60 min the 60th min of this experimental period. When the concentration of incubation at 37°C, using a pNA calibration curve. Results were divalent cations, Ca2ϩ 10 mmol/l or Co2ϩ 0.5 mmol/l, was modified expressed as pmol pNA released/min/µg DNA. Three control reactions in the incubation medium, the concentration of NaCl was adjusted were performed: a blank control using all reagents except substrate accordingly to keep osmolarity constant. The media collected at each DEVD–pNA, a positive control obtained with the addition of a known time interval were stored separately at –20°C until assayed for HCG amount of human caspase-3, and a negative control obtained after and HPL. At the end of the incubation, explants from each vial were preincubation with DEVD–formaldehyde, a CPP32 inhibitor. weighed and stored at –20°C for further determinations of final LDH activity was measured spectrophotometrically according to a protein, DNA, HCG and HPL contents. Experiments were repeated previously published method (Bergmeyer et al., 1974), modified as with five placentae. follows. The assay was conducted in 1 ml of HEPES–NaOH buffer The second experimental design was conducted in order to assess pH 7.6 (HEPES 50 mmol/l, EDTA 1 mmol/l, L-lactic acid 50 mmol/ the viability of the placental tissue after longer time incubations. The l and NADϩ 2 mmol/l). The kinetics of NADH formation was initial equilibration period was followed by a 3ϫ60 min experimental monitored at 30°C by following absorbance changes at 340 nm during 757 N.Cirelli et al.

10 min. The blank reading consisted of the HEPES–NaOH buffer without NAD. The results were expressed as mU/µg DNA Table I. Measurements of DNA, protein, human chorionic gonadotrophin (1 U ϭ 1 µmol NADH produced/min at 30°C). (HCG) and human placental lactogen (HPL) before, after or during incubation of freshly delivered and preserved placental tissue Microscopy Pa For histological observations, 5 µm sections of placental tissue, by Placental sample Freshly delivered Preserved groups of three explants, embedded in paraffin were placed onto Tissue content silanated slides and rehydrated. At least three sections were stained DNA (µg/mg explant) with haematoxylin and eosin for morphological assessment. For three Initial 3.77 Ϯ 0.21 3.87 Ϯ 0.18 NS additional tissue sections, nuclei containing fragmented DNA were Final 4.20 Ϯ 0.58 4.00 Ϯ 0.45 NS identified using the TdT-mediated biotinylated dUTP nick end- Protein (µg/µg DNA) Ϯ Ϯ labelling (TUNEL) method, as previously described (Cirelli et al., Initial 7.84 0.83 8.32 0.67 NS Final 9.12 Ϯ 1.62 8.55 Ϯ 1.09 NS 1999). Endogenous peroxidase was inactivated by covering the HCG (mIU/µg DNA) sections for 10 min with 0.6% (v/v) H2O2 containing 0.1% Initial 4.60 Ϯ 0.99 4.77 Ϯ 1.52 NS (w/v) NaN . Peroxidase activity was evidenced by the DAB Final 4.49 Ϯ 1.18 4.20 Ϯ 1.17 NS 3 µ (3,3Ј-diaminobenzidine; Fluka Chemica, Buchs, Switzerland) staining HPL (ng/ g DNA) Initial 138 Ϯ 17.4 123 Ϯ 14.9 NS (Vacca et al., 1980). Slides were counterstained with methyl green, Final 85.0 Ϯ 23.1 102 Ϯ 23.0 0.017 dehydrated, then mounted with a coverslip for histological examina- Secretory release tion. All tissue sections from the same placenta were processed in HCG (mIU/µg DNA) the same TUNEL experiment. For each set of experiments, a positive Total 5.62 Ϯ 1.22 2.56 Ϯ 0.66 0.039 Ϯ Ϯ control (testicular tissue from hamster, provided by Dr Nonclercq, Equilibration period 3.81 1.28 1.24 0.34 0.043 Experimental period 1.80 Ϯ 0.49 1.32 Ϯ 0.43 NS Department of Histology, Universite´ de Mons-Hainaut, Mons, HPL (ng/µg DNA) Belgium) was systematically included. In the absence of the TdT Total 111 Ϯ 9.89 53.1 Ϯ 6.13 0.006 enzyme, positive nuclei were never observed within testicular and Equilibration period 83.2 Ϯ 9.21 30.9 Ϯ 3.27 0.005 Ϯ Ϯ placental tissue. Experimental period 27.6 2.69 22.2 3.19 NS The incidence of the peroxidase activity staining was microscopic- Ϯ ally evaluated by two independent observers using a micrometer reticle Mean values ( SE) refer to 270 min incubations repeated with five groups of three explants from five placentae. (Omnilabo, Brussels, Belgium). The observations were processed on Cold preservation was conducted overnight in the HEPES-buffered medium. five slides for each experimental condition. For each slide, 10 fields aPaired t-test. (at magnification ϫ200) were taken into account and the number of NS ϭ not significant (P Ͼ 0.05). TUNEL-positive nuclei was expressed as a percentage of the total nuclei counted (approximately 10 000). Only transverse villous sec- Replacement of the HEPES-buffered medium by the RPMI tions ranging from 125 to 625 µm2 were considered for this assessment. 1640 culture medium during the 4°C preservation period did not modify protein and hormone explant contents, whereas Statistical analysis hormone amounts released during incubation were lower than The statistical significance of differences between mean amounts per from freshly delivered explants. The total hormone release placenta of two experimental groups was assessed using paired was reduced by 62.4 Ϯ 3.42% for HCG and by 53.9 Ϯ 2.87% Student’s t-test. A two-tail P-value of Ͻ 0.05 was considered for HPL. These decreases did not differ from those observed significant. when tissue was preserved in the HEPES-buffered medium. In order to verify the integrity of the stimulus–secretion Results coupling cascade, extracellular Ca2ϩ concentration was raised The DNA contents measured before or after incubation were from 1 to 10 mmol/l during 30 min. The Ca2ϩ increase always similar in freshly delivered and preserved tissues (Table I). elicited a marked stimulation of the HCG and HPL releases The initial protein, HCG and HPL contents in relation to (P Ͻ 0.001; Figure 1). Whether incubations were performed DNA were similar after delivery or after cold overnight immediately after placenta collection or after an overnight preservation (Table I). The contents were not significantly preservation period, the secretory responses were of similar modified after 37°C incubation. The final protein and HCG amplitude for both hormones. The amplitude of the HCG contents were similar whichever explants were incubated and HPL secretory responses to a second rise in calcium immediately after placenta delivery or after the overnight concentration to 10 mmol/l (from the 270 to 300th min) was preservation. By contrast, the final HPL content in preserved not significantly different from that following the first Ca2ϩ explants was significantly higher (18.3 Ϯ 4.61%) than in elevation (data not shown). Moreover, the addition of 0.5 explants incubated immediately after placenta delivery mmol/l Co2ϩ during 30 min resulted in a marked inhibition (Table I). of HCG and HPL release (P Ͻ 0.001; Figure 1). The inhibitory The total amounts of HCG and HPL secreted into the effect of Co2ϩ observed on freshly delivered and preserved medium during the entire incubation time were lower when explants was of similar magnitude. Lastly, temperature experiments were conducted following overnight preservation lowering from 37°Cto4°C during 30 min also resulted in a (Table I). These differences resulted from a larger release decrease in hormone release (P Ͻ 0.001; Figure 1). This during the equilibration period from freshly delivered explants inhibition was similar in freshly delivered and preserved than from preserved explants (Table I). Hormone release during explants. The effects of Ca2ϩ,Co2ϩ and temperature variations the experimental period was not statistically different between on HCG and HPL releases were reversible phenomena (data freshly delivered and preserved tissues (Table I). not shown). 758 Characteristics of cold-preserved placental tissue

Table II. TUNEL, caspase-3 (CPP32) and lactate dehydrogenase (LDH) measurements before, after or during incubation of freshly delivered and preserved placental tissue

Placental sample Freshly delivered Preserved Pa

TUNEL-positive nuclei (% total number) Initial 0.460 Ϯ 0.111 0.590 Ϯ 0.145 NS Final 0.610 Ϯ 0.125 0.753 Ϯ 0.063 NS CPP32 content (pmol pNA released/min/µg DNA) Initial 0.103 Ϯ 0.103 0.224 Ϯ 0.204 NS Final 0.227 Ϯ 0.199 0.263 Ϯ 0.237 NS LDH content (mU/µg DNA) Initial 1.16 Ϯ 0.170 0.766 Ϯ 0.069 NS Final 0.759 Ϯ 0.132 0.731 Ϯ 0.094 NS LDH release (mU/µg DNA) Total 0.114 Ϯ 0.032 0.056 Ϯ 0.017 0.048 Equilibration period 0.093 Ϯ 0.016 0.052 Ϯ 0.008 0.043 Experimental period 0.020 Ϯ 0.014 0.005 Ϯ 0.005 NS

Mean values (Ϯ SE) refer to 360 min incubations repeated with five groups of three explants from three placentae. Cold preservation was conducted overnight in the HEPES-buffered medium. aPaired t-test. NS ϭ not significant (P Ͼ0.05); pNA ϭ p-nitroaniline; TUNEL ϭ TdT- mediated biotinylated dUTP nick-end labelling.

Figure 1. Effect of raising calcium or cobalt concentrations and of TUNEL-stained nuclei was similar in the periphery and in the decreasing temperature to 4°C, on human chorionic gonadotrophin central part of tissue explants. (HCG; upper panel) and human placental lactogen (HPL; lower No difference in CPP32-like activity was found between panel) releases from the 210th to the 240th min of incubation, by samples collected either just after the delivery or after the freshly delivered (open bars) and preserved (stippled bars) tissues. Results are expressed as the percentage of mean hormone release overnight preservation period, or after the incubation (Table II). during the previous 30 min period. Values (mean Ϯ SE) refer to The LDH contents of explants measured after the delivery 270 min incubations repeated with five groups of three explants were similar to those found following overnight preservation from five placentae. All relative releases are significantly different (Table II). Concurrently, LDH content after incubation did not Ͻ from those observed during the previous 30 min period with P differ from that measured before incubation. The percentage 0.001 (*). Differences between relative releases observed on freshly delivered and preserved tissues were not significant using the paired of LDH released into the incubation media, compared with Student’s t-test (NS). the corresponding LDH final content, was lower for preserved explants than for explants incubated after the delivery (7.66 Ϯ 2.33% and 15.0 Ϯ 4.22% respectively; P ϭ 0.034). In order to examine further the viability of placental explants, Interestingly, Ͼ80% of total LDH release was observed during longer incubations (360 min rather than 270 min) were the equilibration period in freshly delivered and preserved conducted immediately after placenta collection, or after the tissues (81.6 Ϯ 14.0% and 92.9 Ϯ 15.4% respectively; Table II). overnight preservation period at 4°C. In haematoxylin and Replacement of the HEPES-buffered medium by the RPMI eosin-stained sections, no histological difference was identified 1640 culture medium during the 4°C preservation period did before and after incubation between explants from freshly not modify the incidence of TUNEL-positive nuclei and the delivered and preserved tissues. The large majority of nuclei CPP32 and LDH explant contents. The total amount of LDH were considered to be morphologically normal, though some released during incubations was less than that from freshly (which were randomly distributed in the section) presented delivered explants, but similar to that released from HEPES- evidence of shrinkage and condensation of chromatin. No preserved tissue. The average percentage of LDH released necrotic area was detected. Morphologically normal red blood from RPMI 1640-preserved tissue into the incubation media cells could be seen in intervillous spaces as well as in fetal was 7.18 Ϯ 0.11% of the corresponding LDH final content. vessels, even after incubation. The amount of LDH released during the first 3 h amounted to Ͻ The incidence of TUNEL-positive nuclei remained 1% in 84.8 Ϯ 2.30% of the total release. both preserved and freshly delivered tissues (Table II). Follow- ing a 360 min incubation period, the incidence of positive nuclei did not differ from that observed in the same placenta Discussion before incubation (Table II). These nuclei were distributed Although the easy accessibility of human placenta is of great randomly within tissue, and could be observed in trophoblastic benefit in overcoming a number of restrictions related to cells, in Hofbauer cells from the villous stroma, and in experimental research on human tissue, the limited availability endothelial cells from fetal capillaries. The incidence of of placental tissue during working hours may lead to difficulties 759 N.Cirelli et al. in this respect. Thus, in order to overcome such technical A low percentage of TUNEL-positive nuclei (Ͻ1%) was problems, a rapid, simple and efficient experimental model of found in explants fixed just after delivery or after cold placental tissue preservation was developed. preservation. This low incidence of DNA fragmentation was Following the cold overnight preservation procedure, total not increased after6hofincubation. Moreover, the CPP32- protein, HCG, HPL and LDH contents were similar to those like activity, previously immunolocalized in placental sections found in samples collected from the same placenta just (Huppertz et al., 1998) and quantified in tissue homogenates after the delivery. Moreover, on completion of a subsequent for the first time in the present study, was not modified in incubation period, those contents remained unchanged. Con- preserved explants. All these data confirm a low incidence of tents remaining in preserved explants after incubation were apoptosis within human term placentae obtained after delivery equal to (for HCG, total protein and LDH) or even higher (Qiao et al., 1998; Axt et al., 1999) and after a subsequent than (for HPL) those observed in explants incubated just after incubation at 4°C (Cirelli et al., 1999). Further incubations of delivery. These data indicate that intracellular protein levels placental tissue at 37°C during 6 h were not associated with were maintained in the placental tissue preserved overnight at higher apoptotic death. 4°C, as well as in placental explants further incubated during Taken as a whole, the results demonstrate that the preserved periods of up to 360 min. Amounts of total protein, hormone tissue remains physiologically and morphologically intact as and LDH content, observed here under different experimental compared with the freshly delivered tissue. Consequently, conditions, were consistent with those reported previously for the above-described preservation procedure may be used to normal human term placenta obtained after delivery (Gustke increase placental tissue availability for research purposes. The and Kowalewski, 1975; Lee et al., 1979; Nolan et al., 1994). incubation of tissue explants allows maintenance of cellular The amplitude of HCG and HPL responses to extracellular elements in their normal morphological relationships and Ca2ϩ indicates that the preserved tissue maintains its Ca2ϩ preserves cell-to-cell communication which is particularly sensitivity at levels similar to those reported previously for important for endocrine secretory processes (Meda, 1996). freshly delivered tissue (Polliotti et al., 1990, 1992; Meuris Indeed, gap junction-mediated cell-to-cell communication has et al., 1994; Petit and Belisle, 1995). Moreover, the respons- been proposed to be a process whereby cells synchronize iveness of the overnight-preserved tissue to the addition of calcium-dependent events (Cao et al., 1997) and modulate cobalt—which is a potent competitive inhibitor of ion permea- their responsiveness to physiological agents (Munari-Silem tion through calcium channels (Hurwitz, 1986)—also supports et al., 1995). The present technical approach is likely to be of the integrity of the tissue’s plasma membrane protein equip- major advantage when compared with cell cultures, because ment. The reversibility of the secretory responses to these cell isolation may damage the plasma membrane and cause divalent cations, as well as the similar amounts of hormone degradation of cell surface proteins (Ringler and Strauss, released during the experimental period from both freshly 1990). Moreover, our incubation system with short intervals delivered and preserved explants, strongly suggest that the between medium changes makes it possible to observe secret- overnight treatment did not impair the secretory machinery. ory dynamics and, perhaps, to unmask short-term effects of Interestingly, lesser amounts of HCG and HPL were released physiological or pharmacological factors on hormone release. from preserved explants during the equilibration period Coupled to the in-vitro preservation model developed here, it (0–180 min). Similar decreases were also observed when the might facilitate the study of the physiological control of HEPES-buffered solution was replaced by a conventional hormone release while trophoblastic cells are maintained in their histological environment. culture medium such as RPMI 1640 during cold preservation. In summary, an original and feasible approach to preserve These features may be related to the lower percentage of LDH human placental tissue has been developed. The potential use tissue content released by explants incubated after an overnight of preserved tissue for experimental purposes has been valid- preservation. Besides that, more than 80% of the total LDH ated by the assessment of its secretory capacity, its physio- released during 360 min was observed during the first3h, logical responsiveness to stimuli, and its morphological integ- whether incubated tissue was freshly delivered or preserved. rity. This preservation method may be useful in increasing These results indicate that some cellular leakage occurs early the availability of limited pathological material and early during incubation, immediately after preparation of placental pregnancy trophoblastic tissue. fragments into explants. This also confirms the importance of an equilibration period in order to reach a steady state of hormone release before any experiment (Polliotti et al., 1990). Acknowledgements Moreover, and as described previously (Atwater et al., 1984; We are very grateful to Professor G.Graff for his helpful comments, as well as to Mrs L.Lammers and the nursing staff from the Erasme Sooranna et al., 1999), it should be noted that a low percentage Hospital (Brussels, Belgium) for providing placentae. This work was of the LDH tissue content released into the incubation media granted by the National Fund for Scientific Research (Belgium, and a reversible decrease in hormone release caused by FNRS) from which S.Meuris and P.Lebrun are Research Directors. temperature lowering, further substantiate membrane integrity N.Cirelli was supported by a grant in aid from the ‘Universite´ Libre within placental tissue maintained overnight at 4°C. de Bruxelles’. 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