Human Wharton’s jelly stem cells (hWJSC) isolation, culture and cryopreservation

Human Wharton’s jelly stem cells were isolated from umbilical cords obtained from healthy neonates after at least 36 weeks gestation. Informed consent was obtained from the parents prior to umbilical cord collection which was facilitated by Celligenics Pte Ltd − a registered tissue bank in Singapore. Umbilical cords were first washed in Hanks’ balanced salt solution (HBSS) to remove the blood. Umbilical cords were then cut into pieces which were subsequently cut open lengthwise. The Wharton’s jelly was then carefully excised from each piece and cut into small pieces. These small pieces of Wharton’s jelly were then transferred into a tissue culture dish containing hWJSC culture medium (Celligenics proprietary formulation). The explanted tissue was incubated in a 37 °C incubator with 5% carbon dioxide (CO2). Culture medium was changed every 2–3 days while cells migrated out from the tissue. When there was substantial outgrowth from the explants, explants were removed and the cells were dissociated from the dish by incubating with TrypLE (recombinant trypsin) at 37 °C in an incubator with 5% CO2 for 15 min after washing with HBSS. After TrypLE incubation, the TrypLE was diluted with culture medium and the dissociated cells were collected into a centrifuge tube. Collected cells were centrifuged at 300g for 5 min, and resuspended in culture medium for counting after removing the supernatant. Cells were counted by trypan blue, cryopreserved in CryoStor10 and placed in liquid nitrogen for long term storage until use.

Human WJSC characterisation

Cryopreserved hWJSCs were removed from liquid nitrogen storage and rapidly thawed in a 37 °C water bath. Thawed cells were diluted with culture medium, centrifuged at 300g for 5 min and resuspended in culture medium for counting by trypan blue after removing the supernatant. hWJSCs were seeded in six-well culture plates in duplicate wells. Culture medium was changed every 2–3 days, and cells were sub-cultured every 4–5 days. For trilineage differentiation, hWJSCs at passage 4 were seeded in 24-well culture plates and differentiated using StemPro™ chondrogenesis/osteogenesis differentiation kit and MesenCult™ adipogenic differentiation kit according to manufacturers’ instructions. After differentiation, cells were stained with alizarin red for osteogenesis, oil red O for adipogenesis and alcian blue for chondrogenesis, and then imaged under bright field using an inverted microscope at 4× objective.

For cell surface marker expression, passage 3 hWJSCs were dissociated by incubation with TrypLE (recombinant trypsin) at 37 °C in an incubator with 5% CO2 for 5 min, collected and centrifuged, and fixed in 2% paraformaldehyde (one million cells per 100 µL) at 4 °C for 15 min. After fixation, hWJSCs were washed twice by adding FACS buffer (2% FBS in PBS) to the cells, centrifuging the cell suspension and removing the supernatant for each wash. Thereafter, hWJSCs were incubated with antibodies to human CD34, CD73, CD166 (APC conjugated), CD14, CD45, CD90, and CD105 (FITC conjugated) at 4 °C for 15 min. For controls, hWJSCs were incubated with isotype (IgG) control antibodies (APC or FITC conjugated) at 4 °C for 15 min. Unbound antibody was removed by repeating the above wash step three times. The stained hWJSCs were resuspended in FACS buffer and data was acquired with FACSCelesta™ Cell Analyzer (Becton Dickinson, USA) and analysed using Facsdiva v8.0.2 (Becton Dickinson, USA).

Secretome production

Secretome from human WJSCs was produced according to methods described in patent application WO 2023/101603 A1. Briefly, hWJSCs were seeded in Corning® HYPERFlasks®. At 95–100% confluency, culture medium was removed and hWJSCs were washed twice with HBSS. Cells were then incubated in serum-free medium. Secretome was periodically collected by removing the spent culture medium (the secretome) from the cell culture vessel and replacing with fresh medium every 2–3 days. Collected secretome was concentrated via ultrafiltration and stored at −80 °C for downstream applications.

Scratch wound assay

Scratch wound assays were carried out in 96-well tissue culture plates using non-immortalized human adult dermal fibroblasts (HDF) and HaCaT keratinocytes. Cells were seeded in culture medium [DMEM with 5% fetal bovine serum (FBS), 1× GlutaMAX, 1× non-essential amino acid (NEAA), 1× penicillin–streptomycin] with and without an inflammatory cocktail comprising interferon gamma (IFNg) and/or phorbol 12-myristate 13-acetate (PMA) and/or lipopolysaccharide (LPS) and allowed to grow to confluence. At confluence, cells were scratched using an in-house developed scratch assay device comprising a guide template, scratch pin and imaging template (Chen et al. 2023). After scratching, the cells were washed with HBSS to remove cell debris, imaged and subsequently incubated at 37 °C in a CO2 incubator for 2 (HaCaT) or 3 (HDF) days in fresh culture medium with and without the inflammatory cocktail. Concentrated secretome (see above under secretome production) was added together with the final selected combination of the inflammatory cocktail for studies evaluating the effect of secretome on scratch closure in the delayed scratch closure model at the time of scratch creation. Culture medium was used as a positive control for normal scratch closure response. After scratching and before incubation, scratched cells were imaged on phase contrast at 4× objective with the imaging template to enable imaging of the same area in each well. After incubation, culture medium was removed and cells were washed with HBSS before fixing in 1% paraformaldehyde (PFA) for 30 min at room temperature. After fixation, cells were stained with 0.025% safranin O and the same area was imaged microscopically at 4× objective, on bright field using the imaging template as a guide. Scratch area pre- and post-treatment was assessed by tracing and then quantifying the area without cells in pixels using FIJI Software (Schindelin et al. 2012). The percentage wound closure was calculated by dividing the scratch area after treatment by the scratch area before treatment and multiplying this value by 100.

Cytokine profile

HDF and HaCaT culture media after scratch under different treatment conditions were collected and stored at −80 °C until analysis. Cells treated with culture media only was used to obtain baseline cytokine levels. Cytokine levels in the culture media in the presence of the inflammatory cocktail with and without secretome were analysed by multiplex using the 48-Plex Bio-Plex Pro human cytokine screening panel (Bio-Rad Laboratories, USA) according to manufacturer’s instructions. Sample–antibody–bead complexes were re-suspended in sheath fluid for acquisition on the FLEXMAP® 3D (Luminex) using xPONENT® 4.0 (Luminex) software. Data analysis was done on Bio-Plex Manager™6.1.1 (Bio-Rad). Standard curves were generated with a five-parameter logistic (5-PL) algorithm, reporting values for both mean florescence intensity (MFI) and concentration data.

Cell proliferation

Cell proliferation was assessed in 96-well tissue culture plates using non-immortalized human adult dermal fibroblasts (HDF) and HaCaT keratinocytes. Cells were seeded in culture medium (DMEM with 5% FBS, 1× GlutaMAX, 1× NEAA, 1× penicillin–streptomycin) with and without an inflammatory cocktail comprising similarly of IFNg, and/or PMA, and/or LPS. Secretome was included on a subset of cells seeded with the inflammatory cocktail. Culture medium only was used as a positive control for normal proliferation response. Cells were then incubated for 3 days at 37 °C in a CO2 incubator. After incubation, the cells were washed with HBSS before fixing in 1% PFA for 30 min at room temperature. After fixation, cells were stained with 0.5% crystal violet solution for 10 min at room temperature. The crystal violet was removed and the cells washed with water until the solution was clear. Cells were air-dried and 1N acetic acid solution was added to the cells to solubilise the crystal violet. After solubilisation, the absorbance was read at 560 nm using a spectrophotometer (Victor 3, 1420, Perkin Elmer, USA) and cells in each well were quantified by comparing the absorbance with the absorbance of a cell standard which was stained and solubilised using the same method.

Equipment, reagents and chemicals

HDF was from Lonza, USA. HaCaT keratinocytes was from AddexBio, USA. DMEM, GlutaMAX, NEAA, TrypLE, PFA, and StemPro™ chondrogenesis/osteogenesis differentiation kit were from Gibco; Life Technologies, USA. FBS and HBSS were from HyClone; Cytiva, USA. Penicillin–streptomycin was from Biowest, France. PMA, LPS, and oil red O were from Sigma Aldrich, USA. Alizarin red and alcian blue were from Merck Millipore, Germany. Cryostor10 was from Biolife Solutions, USA. MesenCult™ adipogenic differentiation kit was from Stem Cell Technologies, Canada. Safranin O and crystal violet staining solutions were from Fisher Scientific, USA. PBS and acetic acid solution were from VWR; Avantor, USA. Human CD34, CD45, CD73, CD166, CD14 and CD90 antibodies, and IFNg, were from Miltenyi Biotech, Germany. Human CD105 antibody was from Biolegend, USA. The 6-well and 96-well tissue culture plates and HYPERFlasks® were from Corning, USA.

All images were acquired using an Olympus IX71 inverted microscope (Olympus, Japan).

Statistical analysis

Real Statistics Resource Pack software (Release 8.8.1) for Microsoft Excel (Office 365) was used for data analyses. Data are expressed as mean ± standard deviation (SD). Comparison between groups were performed using one way ANOVA with Tukey’s post hoc test using a two-tailed test. P values less than 0.05 were considered statistically significant.