Hanks balanced salt solution (hbss)

We have previously reported a method to prepare OMCSs^{11 (link)}. Briefly, BMSCs obtained from primary culture were seeded at a density of 1 × 10⁴ cells/cm² onto 60-mm cell culture dishes for subculture in regular medium containing 10 nM dexamethasone (Sigma, St. Louis, MO, USA) and 0.28 mM ascorbic acid phosphate (AscP; L-ascorbic acid phosphate magnesium salt n-hydrate; Wako Pure Chemical Industries, Kyoto, Japan). OMCSs were formed by culture to confluency (approximately 2 weeks), and then rinsed twice with PBS (Gibco Life Technologies, Carlsbad, CA, USA). The sheet was lifted by a mechanical retrieval method using a cell scraper.
OMCSs were divided into seven groups according to the preservation method: fresh (control group), storage in culture medium at 37°C (37°C MEM group), storage in culture medium at room temperature (22°C MEM group), storage in culture medium at 4°C (4°C MEM group), storage in Hank’s balanced salt solution (HBSS; Gibco) at 37°C (37°C HBSS group), storage in HBSS at 22°C (22°C HBSS group), and storage in HBSS at 4°C (4°C HBSS group). For preservation, OMCSs were picked up using tweezers and placed into a 50-ml tube (Falcon) with 50 ml culture medium or HBSS (six OMCSs per tube). Tubes were then transferred to an incubator (37°C), a clean bench (room temperature: 22°C), or refrigeration chamber (4°C) and stored for 24 h.

Inguinal adipose tissues of Balb/c mice were isolated in Hank’s Balanced Salt Solution (HBSS, cat.no.14025092, Life Technologies, USA) and washed twice with HBSS at 280 g to remove blood and liquid. Clear fat’s tissues were cut into 3 mm small fragments and incubated in the solution of HBSS contained 100 units/ml collagenase type I (cat.no.17100-017, Life Technologies, USA) along with 3 mM CaCl₂ for two hours at 37°C and 5% CO₂. After neutralization with fetal bovine serum (FBS, ref.no.16000-044, Gibco Life Technologies, USA), cells were centrifuged, the pellet was washed in HBSS, and filtered through 70 μm then 40 μm nylon mesh. mAd-MSCs were cultured in Minimum Essential Medium Eagle-Alpha Modification (α-MEM, cat.no.32561-037, Life Technologies, USA) containing 100 U/ml penicillin/streptomycin and 10% FBS in 25 cm² flasks. The flasks were incubated at 37°C in the humid atmosphere at 5% CO₂. Non-adherent cells were removed by medium replacement after every 2~3 days. Upon confluence at 70% density, cells were sub-cultured through 1X TrypLE select (cat.no.12563-011, Gibco Life Technologies, USA) in 1:3 ratio. Passage 2 mAd-MSCs were used for transplantation studies in AKI model (8 (link)).