Human tissue sample collection and primary cell isolation

Freshly collected human fibroid and adjacent myometrial samples were obtained via the Augusta University Biorepository under an approved IRB protocol (IRB No. 644354-6) from AA women of reproductive age (22–55 years old) undergoing hysterectomy or myomectomy for symptomatic UFs. These patients had not received any hormonal supplements, including vitamin D, for 3 months prior to the day of surgery (i.e., the day of sample collection). The 8-cm³ UF tissue samples were collected. Myometrial tissue samples, of at least 2 cm from adjacent UF, were collected to exclude any mechanical or hormonal effects of the fibroids on the adjacent myometrial tissue. For preparation of the primary cell population, the collected samples were washed with calcium- and magnesium-containing Hanks’ balanced salt solution (HBSS) to remove blood and chopped into small pieces. The tissues were then digested for 3.5 h at 37 °C with shaking in an enzyme buffer of calcium- and magnesium-free HBSS containing 1% antibiotics-antimycotics, 2.5% N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [HEPES], 660 µg/mL collagenase Type IV (Worthington, NJ, USA), and 4.76 µg/mL DNase I (Sigma-Aldrich, St. Louis, MO, USA). The suspension was filtered through a 100-µm sterile nylon mesh cell strainer to remove undigested tissues and then through a 70-µm cell strainer to obtain a single cell suspension. The remaining undigested tissue was suspended in a fresh enzyme buffer and incubated for 14 h at 37 °C and filtered again to obtain a single cell suspension. Cells were plated out and incubated at 37 °C to allow them to attach to a sterile tissue culture-treated plate containing smooth muscle basal medium (SmBM), as described below.