Epithelial cell isolation
All methods were carried out in accordance with relevant guidelines and regulations. And all experimental protocols were approved by the ethics committee of Shanghai Jiao Tong University affiliated Shanghai general hospital. Human foreskin keratinocytes (HFKs) were isolated from young humans undergoing circumcision. The method was described previously [1]. Briefly, most of the fibrous dermal tissue was first removed, leaving a thin dermis and epidermal layer. Then, the layer was cut into 1 mm × 1 mm cube and incubated in 20 ml 0.05% Trypsin-EDTA (Gibco, USA) with 100 μg/ml penicillin and streptomycin antibiotics (Gibco, USA) at 4 °C overnight. The next day, after trypsin-EDTA was quickly neutralized, the tissue cubes were digested with 0.1% collagenase I (Gibco, USA) at 37 °C for 1 h. Finally, primary HFKs were obtained and suspended in keratinocyte growth medium (KGM) with 10% fetal bovine serum (Sigma, USA). KGM was prepared by mixing equal volumes of DMEM and DMEM with Ham’s F-12 medium (Termo Fisher Scientific, USA). KGM supplement was containing 0.3 μM hydrocortisone (SAXIZON, Japan), 140.0 mU/mL insulin (Sigma, USA), 2.0 nM triiodothyronine (Sigma, USA), 0.2 μM epidermal growth factor (Termo Fisher Scientific, USA), 1.0 nM cholera toxin (Wako PureChemical Industries, Japan), 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B (Gibco, USA). HFKs (5 × 104 cells/cm2) were seeded in 60 mm culture dishes in 5 ml complete medium (P0) and cultured at 37 °C with 5% CO2. Second-passage cells (P1) were used for the ECS culture.
Human ECS culture
Second-passage cell suspensions were diluted and seeded in 60 mm dishes at 2 × 104 cells/cm2. ECS cultures were maintained with medium changes every day for 2 weeks. ECSs were then peeled off for preservation experiments.
ECS preservation
Fresh ECSs, not subjected to storage, served as controls (n = 3). After harvesting, each ECS was sealed and randomly selected for storage in KGM at 4 degrees (refrigerated-1, n = 3). ECSs were also preserved in KGM supplemented with 0.5 mM ascorbic acid (Sigma, USA) and 10 μM Y27632 (Selleck, USA) at 4 degrees (refrigerated-2, n = 3). Following 7 days of storage, new KGM was added to the groups and rewarmed in a 37 °C incubator for 1 h. The ECSs and medium were collected for future analysis.
The programmed cryopreservation was performed as described in our previously published studies [8, 10]. Briefly, by a two-pump perfusion system (2132 MicroPerpex, LKB BROMMA, Sweden), cryoprotectant DMSO (Sigma, USA) was introduced with a linear gradient rising from 0 to 1.5 M. Cryoprotectant agents (CPA0) not containing (Frozen-1) or containing (Frozen-2) 0.1 M fructose, 0.5 mM ascorbic acid, and 10 μM Y27632 in KGM were prepared. The ECSs were cryopreserved in triplicate. The ECS was nipped by a designed polytetrafluoroethylene porous clip and then transferred to an Aluminum freezing box (diameter × height, 59 mm × 17 mm) with 15 ml cryopreservation medium. A 5-slot ampoule cryochamber (Planer KRYO 10 Series III Freezer, UK) was run by the pre-set cooling program. The boxes were finally plunged into liquid nitrogen. After 7 days of cryopreservation, thawing was carried out rapidly by holding in air for 1 minute to boil off any liquid nitrogen and swirling in a water bath at 40 degrees. When completely thawed, the ECSs in the frozen groups were washed using the pumps at the same flow rates as before. Interstitial DMSO in the ECSs was progressively eliminated by pumping medium into the reservoir. The ECSs were replenished with new KGM and rewarmed at 37 degrees. After 1 h of incubation, the ECSs and medium were collected for future analysis.
Integrity and mechanical testing of ECSs
Gross observations were made to determine the integrity of preserved ECSs. For mechanical testing, the preserved ECSs in groups were prepared as longitudinal strips (30 mm in length, 10 mm in width). The ECS thickness was measured by dial thickness gauges. Before testing, those cell sheets were maintained in serum-free medium at 37 degrees. The mechanical properties of the ECSs were measured using an FR-108C system (FARUI, Inc. Shanghai, China) at a constant speed of 10 mm/min. According to the manufacturer’s software, the ultimate loads were recorded and analyzed. Each group was tested in triplicate.
Scanning electron microscopy (SEM)
Samples were fixed in 1% osmic acid (Sigma, USA) for 30 min at room temperature and dehydrated by an ascending series of ethanol (from 50 to 100%). After air-drying overnight, samples were coated with gold. SEM (VegaII XMU instrument Tescan, Czech-Republic) was used to observe the morphology of ECSs.
Cell viability assay
CCK-8 testing and trypan blue staining were employed to investigate the viability of the preserved ECSs. For CCK-8 testing (Dojindo, Japan), the ECSs were punched out with a biopsy punch (8 mm in diameter) and transferred into a 96-well plate with 100 μl CCK-8 solution (10 μl CCK-8 in 90 μl medium) in each well. The plate was then incubated at 37 °C with 5% CO2. After 1 h of incubation, the optical density was read on a spectrophotometer (Cytation Hybrid, BioTek, USA) at 450 nm. After incubated in PBS containing 0.05% trypsin-EDTA at 37 °C for 5–10 min, the ECSs were gently pipetted to single cell suspension. Cell viability was determined by staining viable cells with trypan blue (0.4% solution) (Sigma, USA) staining. Viability (%) = Live cells/Live and Dead cells × 100.
Flow cytometry analysis of human leukocyte antigen class I
The preserved ECSs were first dissociated using the methods above. The cells were then incubated with a recombinant anti-human leukocyte antigen class I (HLA-I) B8-PE IgG antibody (Miltenyi Biotec, Germany) (dilution: 1:50) for 30 min at room temperature. The expression profiles of HLA-I in groups were determined with a FACScan flow cytometer (BD, USA) using Cell Quest software. Fresh ECSs were used as controls.
Apoptosis analyzed by the TUNEL method
Preserved ECSs were fixed in 4% paraformaldehyde solution for 30 min, and then 5 μm serial frozen sections were prepared. The TUNEL Apoptosis Detection Kit (Alexa Fluor 488) (YEASON, China) was used according to the manufacturer’s instructions. After straining with 2 μg/mL DAPI solution (Servicebio, China) at room temperature for 5 min, the slides were observed immediately under a fluorescence microscope. Green fluorescence was observed at 520 ± 20 nm with a standard fluorescent filter, and DAPI fluorescence was observed at 460 nm. Green fluorescent signals localized by Alexa Fluor 488–12-dUTP were only present in the apoptotic nuclei, while DAPI stained both apoptotic and nonapoptotic cells blue.
Dihydroxyphenylacetic acid (DOPA) staining
To detect melanocyte viability in preserved ECSs, DOPA staining was performed. When exogenous levodopa is added and taken up, melanin particles were synthesized under the specific tyrosinase in melanocytes to make the cells appear black under the microscope [29]. The preserved ECS was reattached to a 60 mm dish and cultured for another 2 days. After fixation in 4% paraformaldehyde solution, fresh and preserved ECSs were used for this experiment. Following washing with PBS, the ECSs were incubated with 0.1% L-DOPA (Aladdin, China) (dissolved in PBS) at 37 °C for 5 h and counterstained with hematoxylin.
Transplantation in nude mice
Twenty-four female C57BL/6 mice (six weeks old) were divided into four groups (n = 6 per group): 1) control group: mice with skin wounds and received no treatment; 2) fresh group: mice received fresh ECS transplantation; 3) refrigerated group: mice received ECSs stored in KGM supplemented with 0.5 mM ascorbic acid and 10 μM Y27632 at 4 degrees; and 4) frozen group: mice received ECSs cryopreserved in CP0 containing 0.1 M fructose, 0.5 mM ascorbic acid, and 10 μM Y27632. After the mice were anesthetized with 2% inhaled isoflurane, 10-mm-diameter full-thickness excisional skin wounds were created under sterile conditions. Then, the wounds were closed with transplantation of the ECSs by interrupted suturing. The mice were sacrificed at day 7 and day 14 postwounding. The wound tissues were harvested and then processed for analysis.
Histological and immunohistochemical staining
Frozen sections or paraffin sections in groups were prepared as described above. Re-epithelialization and scar formation were assessed by H&E staining (Servicebio, China). Collagen deposition was detected by Masson’s trichrome staining (Servicebio, China). For immunohistochemical staining, the sections were blocked with 1% bovine serum albumin (Servicebio, China) and 0.5% Triton-X100 (Servicebio, China) and then separately treated with AE1/AE3 (Abcam, USA, prediluted), CD31(Abcam, USA, 1:500), CD68 (Abcam, USA, 1:500) and P63 (Abcam, USA, 1:500) mouse anti-human IgG antibody at 4 °C overnight. After washing three times with PBS for 5 min, the sections were incubated for 1 h with an HRP goat anti-mouse IgG antibody (Invitrogen, USA, 1:1000) at room temperature. Finally, the sections were stained with 3,3 N-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. The slides were covered with coverslips and examined under a microscope.
Statistical analysis
Data analyses were performed using IBM SPSS Statistics 19.0 software (IBM Corporation, NY, USA). The data are presented as the mean ± standard deviation. Multiple comparisons were evaluated by one-way analysis of variance. A two-tailed t-test was adopted to evaluate the significance of the differences between two comparisons. The level of statistical significance was defined as P values < 0.05.
