1. Introduction
Despite the over a century old tradition of using the human amniotic membrane (hAM) successfully for tissue regeneration in clinics [
1,
2,
3,
4,
5], properties of the hAM are still subject of research.
The hAM starts to develop around day 7.5 of human gestation, far earlier than the formation of the three germ layers [
6]. Forming the amniotic cavity, the hAM expands during the course of pregnancy and is supposed to rupture at term. It is usually discarded after childbirth, and, although of embryonic origin, the use and application of hAMs does not raise ethical issues. The hAM can be classified into different sub-regions. While both amniotic membranes are attached to the chorion, placental amnion (and chorion) covers the placenta, and reflected amnion (and chorion) is located opposite it. After preparation, the hAM is a thin, flexible and almost translucent membrane, harboring two vital cell populations. Human amniotic epithelial cells (hAECs) form a layer that, in vivo, faces the fetus, and is in direct contact with the amniotic fluid. Underneath, human amniotic mesenchymal stromal cells (hAMSCs) are embedded in a layer of extracellular matrix. Both cell populations have been proven to have stem cell characteristics, such as the ability to differentiate into lineages of all three germ layers in vivo and in vitro [
7,
8,
9,
10]. Furthermore, the cells express markers of pluripotency, an otherwise solely embryonic feature [
11]. Properties of the hAM have been extensively described, as it is known to be anti-inflammatory [
12,
13,
14,
15] and immune-modulatory [
14,
15]. Moreover, remarkably, no substantial immune reactions upon application have been reported. We and others, furthermore, showed significantly different properties of cells of placental and reflected amnion in previous studies [
16,
17,
18,
19].
Up until the turn of the century, the hAM has normally been used in a denuded or decellularized form, making use of the composition of its extracellular matrix (reviewed in [
5]). With increasing evidence of the stem cell properties of hAECs and hAMSCs, using hAMs with their original vital cell populations for tissue regeneration has come more and more into focus (reviewed in [
5]). In other tissues and cell types, in recent years, researchers have concentrated on mitochondria in particular, as it has been shown that functional mitochondria are required to support tissue regeneration processes [
20,
21,
22]. While beneficial properties of amniotic cells have been known for more than two decades, sustaining cellular viability of the hAM remains a challenge. For example, cryopreservation of hAMs under conditions reported does not result in any viable cells [
23]. However, storage under common cell culture conditions is also not applicable, since several studies have shown decreasing cellular viability of the hAM [
9,
10,
24]. To our knowledge, reasons for this rapid decrease of viability in vitro are not known so far.
However, in vivo, net loss of extracellular matrix [
25] and apoptosis of hAECs are involved in the mechanisms leading to rupture of the membranes at term (reviewed in [
26]). Parry therefore suggested that apoptosis of amniotic cells in vivo is probably a consequence of loss of tissue tension [
26]. Of note, in vivo, the hAM is distended by a factor of 1.75 [
27], a tensile strength that, ex vivo, is no longer existent. We therefore hypothesized that for in vitro culture of hAM, tensile strength plays an important role in the maintenance of cellular viability and that mitochondria play a critical role in this process.
The aim of this study was to clarify whether tissue distention controls apoptosis in a mitochondria-dependent manner and whether it can impact the viability of hAMs. To achieve this aim, we examined cellular viability, mitochondrial activity and activation of apoptotic pathways in distended compared to non-distended (floating) hAM samples in culture.
2. Materials and Methods
2.1. Preparation of the Human Amniotic Membrane (hAM)
Human placentae from caesarean sections were collected with informed consent of the patients and approval of the local ethical commission (Ethikkommission des Landes Oberösterreich, 21 May 2014), in accordance to the Declaration of Helsinki. Placentae were transported within 4 h in 500 mL Ringer solution. Placentae from caesarean sections of premature deliveries, emergency caesarean sections and placentae with detached amniotic membranes were excluded from the study. Placental (P) and reflected (RA) regions were separated, the hAM was peeled off the chorion and washed with cold phosphate-buffered saline (PBS).
2.2. Cultivation of hAM Samples
For tissue distention, fresh hAM was mounted on CellCrown™ inserts (Scaffdex, Tampere, Finland) (
Figure 1B) and incubated in Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1%
l-glutamine and 1% penicillin/streptomycin (“culture medium”) at 37 °C, a humidified atmosphere and 5% CO
2. On the day of measurement or sample freezing, biopsies of 8 mm diameter of distended samples were punched. Non-distended (floating) samples (
Figure 1C) were kept under the same conditions. All samples were measured at day 0 and incubated for 7 days (B-cell lymphoma 2-associated X protein (BAX), B-cell lymphoma (BCL)-2), 14 days (mitochondrial morphology, caspase 3), or 21 days (mitochondrial membrane potential, mitochondrial respiration, ATP concentration). The culture medium was changed twice weekly.
2.3. Cell Viability Assay
Cell viability of hAM biopsies (8 mm diameter) was quantified with the EZ4U—Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria). The assay was performed according to the manufacturer’s protocol. Briefly, the substrate solution was diluted 1:10 in DMEM without phenol red supplemented with 1% l-glutamine (Sigma-Aldrich, St. Louis, MO, USA). Biopsies were added to the solution and incubated for 3 h 45 min at 37 °C and 5% CO2. Plates were shaken for 15 min and the optical density (OD) was measured with a microplate reader (BMG Labtech, Polarstar Omega, Ortenberg, Germany) at 450 nm with 620 nm as reference. n = 4 (biological replicates).
2.4. Laser Scanning Confocal Microscopy
hAM samples were placed in 2-well chambered cover glass (Nunc™ Lab-Tek™, St. Louis, MO, USA) and stained with mitochondrial membrane potential sensitive fluorescent dye (500 nM tetramethylrhodamin-methylester (TMRM; VWR, Radnor, PA, USA (excitation/emission: 543 nm/585 nm)) for 45 min at 37 °C and 5% CO2. Imaging was performed with an inverted confocal microscope (LSM510, Carl Zeiss, Oberkochen, Germany). Image analysis (mean fluorescence) was performed with ZEN2009 Software (release version 6.0 SP2; Carl Zeiss). n = 2–3 (biological replicates).
2.5. High Resolution Respirometry
Mitochondrial respiratory parameters were monitored using high resolution respirometry (Oxygraph-2k, Oroboros Instruments, Innsbruck, Austria). Mitochondrial ROUTINE respiration, reflecting total mitochondrial oxygen consumption, was measured by incubating 14 hAM biopsies (8 mm diameter) in DMEM at pH 7.2 and 37 °C. For details, see
Supplementary Material. Mitochondrial states were calculated as the negative time derivative of oxygen concentration (rate of oxygen uptake), and corrected for non-mitochondrial respiration (myxothiazol, 1 µM). Data were calculated in µM O/min/14 biopsies and are displayed in percent of placental amnion at day 0. n = 4 (biological replicates).
2.6. ATP Measurement
Liquid nitrogen frozen hAM biopsies (8 mm diameter) were homogenized in Precellys tubes with ceramic beads (Keramik-Kit 1.4 mm Peqlab VWR, USA) in a ball mill (CryoMill MM301, Retsch, Haan, Germany) with 500 µL of Tris-HCl buffer (20 mM Tris, 135 mM KCl, pH 7.4). Boiling buffer (400 µL of 100 mM Tris/4 mM EDTA, pH 7.75) was added to 100 µL hAM homogenate, incubated for 2 min at 100 °C and centrifuged at 1000×
g for 2 min. ATP measurements were performed with the ATP Bioluminescence Assay Kit CLS II (Roche, Basel, Switzerland) in accordance with the manufacturer’s protocol using luciferase reagent with Lumat LB 9507 (Berthold, Bad Wildbad, Germany). For details, see
Supplementary Material. n = 4 (biological replicates).
2.7. Histology
Amnion biopsies were fixed for 24 h in 4% formalin and samples were embedded in paraffin. Immunohistochemistry against caspase 3 was performed with an anti-cleaved caspase 3 antibody 1:100 (Cell Signaling Technology, Danvers, MA, USA). Immunohistochemical negative controls were performed by replacing the primary antibody with buffer. Immunohistochemical sections were quantified with ImageJ software (National Institutes of Health, version 1.51j8, Bethesda, MD, USA). n = 3 (biological replicates).
2.8. Transmission Electron Microscopy
Biopsies were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde for 2–3 h at room temperature and post-fixed with 1% OsO4 in 0.1 M cacodylate buffer. Dehydration and embedding in Epon resin were carried out according to standard protocols. Sections (70 nm) were contrasted with 2% uranyl acetate. Images were acquired with an electron microscope (Tecnai20, FEI Europe, Eindhoven, Netherlands) equipped with a 4K EagleCCD camera and processed with Adobe Photoshop. n = 2 (biological replicates).
2.9. Reverse-Transcription Quantitative PCR Analysis
Samples of hAM biopsies (8 mm diameter) were snap-frozen in liquid nitrogen and kept at −80 °C until further analysis. Total RNA extraction, mRNA reverse transcription and qPCR were performed by TAmiRNA GmbH (Vienna, city, Austria). n = 3 (biological replicates).
Total RNA extraction: total RNA was extracted from 10 amnion biopsies (8 mm diameter) using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Tissue was homogenized with 700 µL Qiazol; following incubation at room temperature for 5 min, 140 µL chloroform was added to the lysates, which were incubated for 3 min at room temperature and centrifuged at 12,000× g for 15 min at 4 °C. Precisely 350 µL of the upper aqueous phase was transferred to a miRNeasy mini column, and RNA was precipitated with 525 µL ethanol followed by automated washing with RPE and RWT buffer in a Qiacube liquid handling robot. Finally, total RNA was eluted in 30 µL nuclease free water and stored at −80 °C until further analysis.
mRNA reverse transcription and qPCR (RT-qPCR): messenger RNA quantification was performed using the TATAA Grandscript cDNA synthesis and SYBR Grandmaster mix kit (TATAA Biocenter, Göteborg, Sweden). Total RNA (500 ng) was used for reverse transcription and all steps were carried out according to recommendations by the manufacturer. PCR amplification was performed in a 96 well format in a Roche LC480 II instrument (Roche, Mannheim, Germany) with the following settings: 95 °C for 30 s followed by 45 cycles of 95 °C for 5 s, 63 °C for 15 s and 72 °C for 10 s and subsequent melting curve analysis. To calculate the cycle of quantification values (Cq-values), the second derivative method was used. Cq-values were subsequently normalized to the geometric mean of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), hypoxanthine phosphoribosyltransferase (HPRT1) and ubiquitin C (UBC) mRNA levels, by subtracting the gene of interest Cq-value from the respective geometric mean of the four references. Primer sequences of BAX and BCL-2 used for mRNA reverse-transcription quantitative PCR analysis are shown in
Table S1.
2.10. Statistical Analysis
Data were analyzed using GraphPad Prism software (GraphPad Software 5.01, San Diego, CA, USA) by one-way ANOVA followed by Bonferroni post hoc test. In all tests, n (sample size) represents biological replicates (donors). Results are presented as mean ± SD. Level of significance was set at 0.05 and is indicated as *p < 0.05, **p < 0.01, or ***p < 0.001.