**1. Introduction**

Despite the over a century old tradition of using the human amniotic membrane (hAM) successfully for tissue regeneration in clinics [1–5], 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 [7–10]. 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 [12–15] 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 [16–19].

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 [20–22]. 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 Dulbeccos Modified Eagles 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% CO2. 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.

**Figure 1.** Cellular viability of reflected and placental amnion in fresh biopsies (day 0), biopsies mounted on CellCrown™ inserts (DIS), and biopsies kept floating (FLO) under common cell culture conditions for 21 days (**A**). Viability was measured with the EZ4U assay. Mean ± standard deviation (SD), n = 4 (donors). Samples of human amniotic membrane distended on CellCrown™ inserts (in 6 well plates) (**B**), and non-distended ("floating") (**C**) at day 0. DIS: distended biopsies; FLO: floating biopsies; OD: optical density. \*\**p* < 0.01, \*\*\**p* < 0.001.

#### *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 bu ffer (20 mM Tris, 135 mM KCl, pH 7.4). Boiling bu ffer (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 reagen<sup>t</sup> with Lumat LB 9507 (Berthold, Bad Wildbad, Germany). For details, see Supplementary Material. n = 4 (biological replicates).
