Animal component free cell attachment substrate

ADSCs cultured in FBS containing media, DMEM (HyClone) supplemented with 10% FBS (HyClone), SFM (CellCor, Xcell Therapeutics). StemPro MSC SFM XenoFree (Thermo Fisher Scientific), or MesenCult-ACF Plus Culture Kit (STEMCELL Technologies) were compared. ADSCs were seeded at 4 × 10³ cells/cm² in T25 flasks (Corning) and passaged every 4 days, except every 3 days for SFM. Flask of FBS containing media and SFM group did not need to be pre-coated. When using StemPro MSC SFM XenoFree and the MesenCult-ACF Plus Culture Kit, the flasks were pre-coated with CELLstart Substrate (Thermo Fisher Scientific) and Animal Component-Free Cell Attachment Substrate (STEMCELL Technologies), respectively. At each passage, ADSCs were detached and counted using a NucleoCounter NC-250 (ChemoMetec, Allerod, Denmark) and seeded in new T25 flasks. The population doubling time (PDT) was determined at each passage by the formula: PDT = culture period in hours / {log (number of cells T₀) – log (number of cells T₁)} / log(2), where T₀ is the seeding cell number and T₁ is the cell number at harvest. The accumulation cell number (ACN) was determined at each passage by using formula: ACN = (ACN of previous passage / T₀) x T₁.

iMPCs were generated from iPSCs using a protocol established in our lab (Diederichs and Tuan, 2014 (link)). When iPSCs reached 80% confluency, expansion medium was replaced with STEMdiff™-ACF Mesenchymal Induction Medium (Stemcell Technologies) for 3 days. Afterwards, MesenCult™-ACF Plus Medium (Stemcell Technologies) was used. On day 6, differentiated cells were detached and re-plated onto flasks that were pre-coated with Animal Component-Free Cell Attachment Substrate (Stemcell Technologies). The MesenCult™-ACF Plus Medium was used to expand these cells to 80% confluency. After being dissociated with ACF Enzymatic Dissociation Solution and ACF Enzyme Inhibition Solution (Stemcell Technologies), the iMPCs were grown on regular tissue culture flasks in expansion medium [DMEM/F-12 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco), and 1% antibiotics-antimycotics (Gibco)]. iMPCs at passage 4 were used for all experiments.

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.