Collection of bovine milk samples

Bovine milk samples were obtained during routine milking from dairy farms in the Bafra region of Samsun. Colostrum samples (n = 3) were collected within 24 h postpartum, whereas mature milk samples (n = 3) were collected from bovines 30–35 weeks postpartum (Cao et al. 2019; Fadlalla et al. 2020). The number of animals included in the study was determined using a power analysis (Tang et al. 2019). After discarding the foremilk, 100 mL of milk was aseptically collected directly into sterile containers containing 100 mL of phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) presupplemented with 5% penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and 5% amphotericin B (Sigma-Aldrich, St. Louis, MO, USA). This procedure ensured a 1:1 (vol/vol) ratio between the milk and the protective buffer at the time of sampling. All samples were transported to the laboratory under aseptic conditions.

Isolation and culture of bovine milk-derived cells

Bovine milk was centrifuged at 228g for 15 min at room temperature (Nuve, Ankara, Turkey). The fat layer and supernatant were discarded, and the cell pellet was washed with sterile PBS containing 1% penicillin–streptomycin and 1% amphotericin B (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA). The cells were then re-centrifuged at 400g for 5 min to obtain a clean pellet. Finally, the cells were resuspended in a standard culture medium composed of minimum essential medium alpha (α-MEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Grand Island, NY, USA), 1% penicillin–streptomycin, and 1% l-glutamine (Capricorn Scientific GmbH, Germany). The cells were then cultured in an incubator at 37 °C and 5% CO2 (Heracell 150i, Thermo Scientific, Grand Island, NY, USA). The culture medium was replaced with fresh medium every 2 days. For subculturing and expansion, cells were detached using 0.25% trypsin–EDTA (Sigma-Aldrich, St. Louis, MO, USA) upon reaching 80–90% confluence. All experimental assays were performed using cells between passages 3 and 4 (Pipino et al. 2018).

Multipotency assays

To assess osteogenic differentiation, passage 3 cells were cultured in an osteogenic medium after they reached approximately 80% confluence and the previous culture medium was removed. The osteogenic medium consisted of low-glucose Dulbecco’s modified Eagle medium (DMEM-LG) supplemented with 0.05 mM ascorbic acid-2-phosphate (Sigma-Aldrich, St. Louis, MO, USA), 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), and 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). Additionally, the medium included 10% FBS, 1% penicillin–streptomycin, and 1% l-glutamine (Pipino et al. 2018). The medium was refreshed every 2 days, and the cells were cultured for 21 days. At the end of the culture period, cells were fixed in 4% paraformaldehyde. After fixation, cells were stained with 2% Alizarin Red S solution (pH 4.2; Sigma-Aldrich, St. Louis, MO, USA) for 1 h (Im et al. 2005).

To induce chondrogenic differentiation, milk-derived cells were incubated for 21 days in high-glucose Dulbecco’s modified Eagle medium (DMEM-HG) supplemented with 1% FBS, 1% penicillin–streptomycin, 1% l-glutamine, 100 nM dexamethasone, 1% insulin–transferrin–selenium–ethanolamine (ITS-X; Invitrogen, Carlsbad, CA, USA), 50 μg/mL ascorbate-2-phosphate, 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), and 10 ng/mL TGF-β1 (Sigma-Aldrich, St. Louis, MO, USA) (Pipino et al. 2018). Following incubation, cells were fixed in 4% paraformaldehyde and stained with 1% Alcian blue solution (Isolab, Eschau, Germany) (Murata et al. 2014).

For adipogenic differentiation, cells were cultured for 21 days in DMEM-HG-based adipogenic medium supplemented with 10% FBS, 1% penicillin–streptomycin, 1% l-glutamine, 1 mM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 100 μM indomethacin (Sigma-Aldrich, St. Louis, MO, USA), and 10 μg/mL insulin (Gibco, Thermo Fisher Scientific, Shah Alam, Malaysia) (Ghorbani et al. 2014). The medium was refreshed every 2 days. Following differentiation, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with Oil Red O (Grimstone and Skaer 1972). Histological images were acquired using a Nikon Eclipse E600 research microscope equipped with a Digital Sight DS-L1 camera system (Nikon Corporation, Tokyo, Japan).

Flow cytometry

Passage-3 cells were characterized for specific cell-surface antigens by flow cytometry (BD FACSCanto™ II Cell Analyzer, San Jose, CA, USA). A total of 1 × 105 cells were suspended in 100 μL of PBS supplemented with 2% FBS and incubated at room temperature for 30 min with FITC-conjugated anti-CD90 (BioLegend, San Diego, California, USA, cat. no. 328108), PerCP/Cyanine5.5-conjugated anti-CD105 (BD Pharmingen, San Diego, CA, USA, cat. no. 560819), and APC-conjugated anti-CD73 (BioLegend, San Diego, California, USA, cat. no. 344006) monoclonal antibodies. Antibodies were used according to the manufacturer’s instructions (5 µL antibody per 106 cells in 100 µL staining volume). Antibodies were purchased from BioLegend and BD and were validated by the manufacturers for flow cytometry applications. Specificity was confirmed on the basis of the manufacturers’ technical data sheets. After incubation, cells were centrifuged at 400g for 5 min; the supernatant was discarded, and the cells were washed with PBS. The cells were then resuspended in 200 μL of PBS, and at least 2 × 104 cells were analyzed by flow cytometry (Teshima et al. 2020; Teunissen et al. 2021).

Determination of growth curve and population doubling time (PDT)

To compare the growth kinetics of cells from different sources, growth curves based on cell counts were plotted, and the PDT was determined. Cells were seeded in 6-well culture plates (TPP, Trasadingen, Switzerland) at an initial density of 5 × 104 cells per well. The cells were harvested by trypsinization on seven consecutive days. After trypan blue staining, cells were counted manually using a hemocytometer. The PDT was calculated using the following formula: PDT = (t − t0) × log2 × (log < N/N0 >)−1, where t is time, t0 is the initial time, N is the number of cells, and N0 is the initial number of cells. All experiments were performed in triplicate for each group, and the mean values were calculated (Yang et al. 2018).

MTT assay

To further evaluate cellular proliferation and metabolic activity, an MTT assay was performed. Cells were seeded in 96-well plates at a density of 2 × 103 cells per well. Cell proliferation was monitored over a 7-day period, with measurements taken at daily intervals. For each time point, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/mL MTT, Serva, Heidelberg, Germany) was added to each well, which was then incubated at 37 °C for 4 h. Following incubation, 100 μL of solubilization solution (10% SDS prepared in 0.01 M HCl) was added to each well, and the plates were incubated for an additional 16 h. The absorbance was then measured at 570 nm using a microplate reader (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer, Vantaa, Finland). All experiments were performed in triplicate for each group (Wu et al. 2018).

Colony-forming unit assay

A clonogenic assay was performed to assess the colony-forming potential of the cells. Passage 3 cells were seeded into 6-well plates at a low density of 100 cells per well in triplicate. Following a 2-week incubation period, the resulting colonies were fixed with 4% paraformaldehyde for 10 min and stained with 0.3% (vol/vol) crystal violet for 5 min. Only colonies consisting of more than 50 cells were counted. The colony-forming efficiency (CFE) was then calculated as follows: CFE (%) = (number of colonies counted/number of cells seeded) × 100 (Pereira et al. 2012).

Statistical analyses

All data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 22 for Windows. Group comparisons were performed using Student’s t-test and the Mann–Whitney U test. A p-value of less than 0.05 was considered statistically significant.