Dulbecco phosphate buffered saline (dpbs)

Cells were obtained from participants who underwent total hip arthroplasty surgery at Boston Medical Center between 2019 and 2020. All human research was done under a Boston University School of Medicine Institutional Research Board Approved protocol: “Bone Tissues Repository,” IRB Number: H-35199, with patients’ consents per current HIPAA regulations prior to surgery and specimen collection. The femoral head and reamings, from the coring of the acetabulum, were collected during total hip arthroplasty. After multiple washes using DPBS (Hyclone Laboratories) containing an antibiotic-antimycotic mixture (Thermo Fischer Scientific) cells were suspended in DPBS. 24 million cells/well were seeded in each well of 6-well plates (Corning Inc.), treated with Animal Component-Free Cell Attachment Substrate (Stem Cell Technologies) diluted 1:150 in DPBS. Cells were cultured in an incubator at 37°C, 5% CO₂, and > 90% humidity. A half media change was performed on day 4 and a full media change on day six after plating. Cells were then grown in basal medium supplemented with osteoinductive factors (Stem Cell Technologies) for 21 days before RNA extraction.

Human embryonic stem cell lines H9 (Wicell, Madison, WI, USA), H1 (Wicell, WA01) and induced pluripotent human stem cells BJ-EOS clone 4YA (provided by the Ellis lab) [64 (link)] were cultured in mTeSR complete medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 50 units/mL penicillin and 50 mg/mL streptomycin. These cells were cultured on Matrigel (Corning, New York, NY, USA) treated 60 mm plates as per the manufacturers specifications [64 (link)].
Cells were passaged by washing the cultures with Dulbecco’s phosphate-buffered saline without calcium and magnesium (DPBS, Stem Cell Technologies) twice followed by accutase (Stem Cell Technologies) treatment. Once the cells were in a single cell suspension fresh medium was used to inhibit accutase activity. Cells were then counted and aliquoted for subsequent experiments. Cell were maintained in medium containing 10 μM Rho-associated kinase (ROCK) inhibitor (Y-27632; Stem Cell Technologies) for 24 h after passaging and then returned to their regular medium.
Human foreskin fibroblast cells (HFF) [64 (link)] were cultured on gelatin coated plates in high glucose (25 mM) DMEM medium supplemented with; 15% (v/v) embryonic stem cell FBS, 1.0 mM sodium pyruvate, 0.1 mM non-essential amino acid and 0.1 mM 2-mercaptoethanol.