Liberase mnp s

In the SSCF facilities, AT was washed twice with Dulbecco’s phosphate-buffered saline with calcium and magnesium solution (DPBS +/+, Gibco, Life Technologies, Carlsbad, CA, USA) and enzymatically digested for 45 min at 37 °C under constant agitation in a cell culture incubator. For the latter, Liberase® MNP-S (Roche, Basel, Switzerland), i.e., a mix of collagenase I and II, and neutral proteases, was used. Enzymatic digestion was then stopped with sterile, clinical grade 1% human serum albumin (HSA, CSL Behring, King of Prussia, PA, USA) in DPBS without calcium and magnesium (DPBS −/−, Life Technologies); the hydrophilic phase was collected and subsequently filtered with 100-μm and 40-μm sieves. At the final step of SVF manufacturing, the hydrophilic phase was centrifuged and the resulting cell pellet, i.e., the autologous SVF, was resuspended in 5% HSA.
In the AP-HM facilities, the autologous SVF was obtained using the Celution 800/CRS automated processing system (Lorem Cytori, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, the collected lipoaspirate was washed with Ringer’s lactate (RL; Baxter, Deerfield, IL, USA) and enzymatically digested with Celase, a GMP cocktail of enzymes provided with consumables (Worthington Biochemical Corp., Lakewood, NJ, USA). The cells were concentrated, washed, aseptically recovered, and resuspended in RL.

Adipose tissue was harvested from the inguinal fat pads of five male BALB/c mice (Jackson Laboratory) at the age of 8–12 weeks and pooled for further processing. The tissue was washed twice with prewarmed PBS prior to mincing. Two units of Liberase MNP-S (Roche) were mixed in 1 mL prewarmed Dulbecco's modified eagle medium (DMEM) (Invitrogen) per gram fat and incubated at 37°C 225 rpm shaking for 30 minutes followed by mixing with a pipette to release cells. Afterwards, mixture was centrifuged at 500 g for 10 min and cell pellet was washed three times with PBS. The resulting cell pellet was resuspended and propagated in complete ASCs culture medium (DMEM) supplemented with 20% fetal bovine serum (FBS, PAN-Biotech), 100 U/mL Penicillin (Sigma), and 100 μg/mL streptomycin (Sigma) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. C2C12 cells (ATCC) were cultured in growth medium consisting of DMEM, 10% FBS, 100 U/mL Penicillin, and 100 μg/mL streptomycin. Myoblasts were subcultured before reaching 80% confluency and used for all experiments before passage 7.
For lentiviral production, human embryonic kidney 293T cells (HEK293T, ATCC) were grown in DMEM containing 10% FBS and used in passages below 20.