Insulin

Human primary preadipocytes were obtained from subcutaneous adipose tissue of a female Caucasian subject (body mass index 21 kg/m², age 44 years) and purchased from PromoCell Product No. C-12730; Heidelberg, Germany). The preadipocytes with passage number less than 10 were used and cultured in a preadipocytes growth medium at 37°C in a 5% CO2/95% air as previously described [9 (link),10 (link)]. Upon confluence, the cells were incubated for 3 days in differentiation medium (Dulbecco’s modified Eagle’s medium-Ham’s F-12 (1:1) medium (Gibco BRL, GFigrand Island, NY) containing 32 μM biotin (Sigma), 1 μM dexamethasone (Sigma), 200 μM 3-isobutyl-1-methylxanthine (Sigma),100 nM insulin (Sigma), 11 nM L-thyroxine 8 μM rosiglitazone (Sigma), 100 U/mL penicillin, and 100 μg/mL streptomycin) and followed by maintenance medium (DMEM/f12 supplementted with 3% foetal bovine serum (Biological Industries, Israel), 100 nM insulin, 32 μM biotin, and 1 μM dexamethasone) until full differentiation into adipocytes. The maintenance medium were changed every 2 days. Culture media were collected at various time points (D0-D30) throughout the differentiation process.

The human breast cancer cell lines MCF-7 (ATCC-HTB-22), T-47D (ATCC-CRL2865), and MDA-MB-231 (ATCC-HTB-26) were purchased from ATCC (Manassas, VA, USA). MCF-7 and T-47D were cultured in DMEM-GlutaMAX (Life technologies, Carlsbad, CA, USA) plus 10% fetal bovine serum (FBS 10082-147, R&D systems) and 10 µg/mL insulin (I5500 Sigma Aldrich, St. Louis, MO, USA). MDA-MB-231 was grown in DMEM-GlutaMAX + 10% FBS at 370C in a 10% CO₂ incubator. The culture medium was obtained from Gibco by Life Technologies.

For in vitro differentiation into adipoblast, PD-MSCs were plated at a density of 2.5 × 10⁴ cells/30 mm dish and cultured in adipogenic induction medium containing 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine (IBMX), 0.2 mM indomethacin, 1.7 μM insulin (Sigma-Aldrich), 10% FBS (GIBCO-BRL), and 1% penicillin/streptomycin (GIBCO-BRL) with medium changes three times a week. After 21 days, PD-MSCs were fixed with 4% paraformaldehyde (PFA) and were analyzed by Oil-Red O (Sigma-Aldrich) staining to induce osteogenic differentiation, and PD-MSCs were plated at a density of 2.5 × 10⁴ cells/30 mm dish and cultured in osteogenic induction medium containing 1 μM dexamethasone, 10 mM glycerol-2-phosphate (Sigma-Aldrich), 50 μM L-ascorbic acid 2-phosphate (Sigma-Aldrich) 10% FBS, and 1% penicillin/ streptomycin with medium changes three times a week. After 21 days, calcium deposits in PD-MSCs were evaluated by von Kossa staining using 5% silver nitrate (Sigma-Aldrich) under light for 1 h. The differentiated cells for osteogenic and adiogenic were marked by arrowheads.

Myobundles were formed by modifying our previously published methods for engineered rodent muscle tissues (Hinds et al., 2011 (link); Juhas et al., 2014 (link)) (Figure 1—figure supplement 2). Expanded myogenic cells were dissociated in 0.025% trypsin-EDTA to a single cell suspension and encapsulated in a fibrinogen (Akron, Boca Raton, FL) and matrigel solution on laser cut Cerex frames (9.2 × 9.5 mm outer dimensions, 6.8 × 8.3 mm inner dimensions) within PDMS molds (cast from Teflon masters and pretreated with pluronic) at 15 × 10⁶ cells/ml (7.5 × 10⁵ cells per myobundle). Specifically, a cell solution (7.5 × 10⁵ cells in 17.2 µl media per bundle + 2 µl of 50 unit/ml thrombin in 0.1% BSA in PBS [Sigma, St. Louis, MO]) and a gelling solution (11 µl media + 10 µl Matrigel + 10 µl of 20 mg/ml fibrinogen in DMEM) were prepared in separate vials on ice for up to six myobundles per vial. Gelling solution was added to the cell solution and mixed thoroughly then each bundle was individual pipetted within the PDMS mold and onto the frame. The cell/hydrogel mixture was polymerized for 30 min at 37°C followed by incubation in growth media containing 1.5 mg/ml 6-aminocaproic acid (ACA, Sigma). Myobundles were kept in growth media during gel compaction (3–5 days) and then switched to low glucose DMEM with 2% horse serum (Hyclone, Logan, UT), 2 mg/ml ACA and 10 µg/ml insulin (Sigma). Frames were removed from molds at the time of switch to low serum medium and cultured dynamically in suspension for an additional 1–4 weeks. Starting from a 50 mg donor biopsy, typical cell expansion for 5 passages can allow generation of at least 1000 myobundles with a total mass of >5 g, representing a >100-fold amplification of muscle mass when going from native to engineered tissue system.
All drugs were purchased from Sigma. Clenbuterol hydrochloride, chloroquine phosphate, and cerivastatin sodium salt hydrate were prepared at 1000× stock solutions in PBS (control) and sterile-filtered for use. Lovastatin was prepared as a 10,000× stock solution in DMSO in which case DMSO was used as vehicle control. Drugs studies in myobundles or 2D cultures were initiated after 1 week of differentiation. Myobundles were replenished with fresh media and drug each day to maintain drug concentration.