Adsc bm

Mono- or heterospheroids were generated on an in vitro 384 well hanging drop array platform as described previously [19 (link),55 (link),56 ,57 (link),58 (link)]. Briefly, to generate CSC monospheroids, single cell suspensions of CSC were diluted, in such a manner that a 20 µL drop contained 12 CSCs. For heterospheroids, CSC and MSC were diluted in a 1:3 ratio, such that a 20 µL drop contained three CSCs and nine MSCs. Spheroids were cultured in a 1:1 medium mixture of RPMI and ADSC-BM (Lonza) supplemented with 1% penicillin/streptomycin and 1% L-glutamate. Live cell imaging using phase contrast microscopy was utilized to follow the formation of CSC or CSC/MSC spheroids. Spheroids were generated from CSCs isolated from the OVCAR3 cell line, or OVCAR3 CSCs with human adipose MSC (MSC). Two matched patient samples (Patient 1 and Patient 2) were also utilized to generate CSC/CA-MSC spheroids.
Additionally, heterospheroids were manufactured from CSC and siPDGFB MSC, in order to assess changes in platinum resistance and CSC characteristics within heterospheroids. CSC^gfp/siPDGFB MSC heterospheroids were also generated, to be able to isolate the CSC^gfp compartment using fluorescent activated cell sorting using protocols described previously [20 (link)], for subsequent qPCR analysis.

Human bone marrow MSCs (BMSCs) and adipose MSCs (ADSCs) purchased from Lonza (Basel, Switzerland) were cultured in mesenchymal stem cell basal medium (MSCBM, Lonza, Basel, Switzerland) or ADSC growth medium (ADSCBM, Lonza, Basel, Switzerland), respectively. Cells were cultured for 15 days following thawing from passage one and then after every seven days for successive passages. Cells were used to isolate EVs until passage six for BMSCs and seven for ADSCs and viability after starvation was established around 90% for both cell types (90% ± 2% for BMSC and 92% ± 3% for ADSC). For in vitro experiments, dermal microvascular endothelium cells (HMEC-1) were obtained by the ATCC (Manassas, VA, United States) and cultures following manufacturer’s instruction with MCDB131 medium (Thermo Fisher Scientific, Waltham, MA, USA). Normal Human Dermal Fibroblasts (NHDF) are primary adult fibroblasts (Lonza, Basel, Switzerland). NHDF were cultured in FGM-2 Growth Media (Lonza, Basel, Switzerland) supplemented with bullet kit and 1 mL Mycozap PR (Lonza, Basel, Switzerland). Normal Human Epidermal Keratinocytes (adult skin) (NHEK) were purchased from Lonza and cultured with KGMTM Gold Keratinocyte Growth Medium (Lonza, Basel, Switzerland).