Table 3 Protocol for isolating different types of MSCs

MenSCs

Menstrual blood (5 mL) from the second day of normal menstrual period was collected with a menstrual cup and transferred into PBS containing supplementations which are listed in Table 1. The menstrual blood was diluted using the same volume of aseptic PBS. MenSCs were separated by lymphocyte separation medium with a density gradient centrifugation. The cells were inoculated in growth medium as shown in Table 2 and cultured at 37 °C with 5% CO2 and saturated humidity. The medium was changed every 3 to 4 days according to the growth of the cells.

[49]

AFMSCs

Two or 3 mL of amniotic fluid was obtained from pregnant women during routine amniocentesis at 16–18 weeks of gestation, and cells were immediately isolated by centrifugation. The supernatant was discarded, and the cell pellet was suspended in standard medium composed of low-glucose DMEM and other supplementations and incubated at 37 °C with 5% CO2 in a humidified atmosphere. The medium was changed every 3 to 4 days according to the growth of the cells.

[76]

BMSCs

The bone marrow cells were diluted in an equal volume of PBS, isolated with Ficoll, and then centrifuged. The cells were then rinsed with PBS and cultured in DMEM containing 10% FBS at 37 °C in a humidified incubator infused with 5% CO2. Adherent cells were collected after 5 days.

[77]

UCMSCs

The umbilical cords were rinsed with PBS in penicillin and streptomycin, and then the umbilical arteries and veins were removed. The remaining tissue was cut into 1 to 2 mm pieces and floated in DMEM/F12. The pieces were subsequently digested in an enzyme cocktail for 3 h at 37 °C. After this tissue was crushed with forceps and large pieces were removed, human umbilical cord MSCs were harvested and plated into a culture flask. The cells were incubated at 37 °C in an incubator with 5% CO2 at saturating humidity. The medium was changed every 3 to 4 days according to the growth of the cells.

[39]

ADSCs

ADSCs were obtained by adipose tissue (2–3 mL) digestion with collagenase A (Sigma Aldrich, Milan, Italy) and then seeded onto a T25 flask at 37 °C, 5% CO2 in DMEM containing 10% FBS and antibiotics to select adherent cells. ADSC cell lines at passages 3–6 were used for the experiments.

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DPMSCs

Human dental pulp was extracted from third molar or permanent teeth of adult subjects (18 and 35 years of age) after informed consent of patients undergoing routine extractions. Dental pulp was removed from the teeth and then immersed in a digestive solution (3 mg/mL type I collagenase plus 4 mg/mL dispase in α-MEM) for 1 h at 37 °C. Once digested, the pulp was dissociated and then filtered onto Falcon Cell Strainers to obtain a cell suspension. Cells were then plated in 25 cm² flasks and cultured in culture medium at 37 °C and 5% CO2. Cells obtained from a single dental pulp were plated at clonal density (1.6 cells/cm²). After 6 days of culture, eight cell populations were isolated from nodules originated by single cells.

[79]