Expansion and Culture of UC-MSCs

Human umbilical cord mesenchymal stromal cells (UC-MSCs) were obtained from previous work conducted by Mennan et al. [42 (link)]. Briefly, umbilical cords were sourced from the Robert Jones and Agnes Hunt Orthopaedic Hospital, with informed patient consent (10/H10130/62). Isolation occurred by mincing the tissue before enzymatic digestion with 1 mg/mL collagenase I for 1 h at 37 °C [43 (link)]. Hereafter, UC-MSCs were expanded on tissue culture plastic before further expansion using the Quantum hollow-fibre bioreactor cell expansion system (Terumo UK Ltd., Surrey, UK).
For this study, passage 5 UC-MSCs were cultured on plastic in DMEM-F12 (Fisher Scientific UK Ltd., Loughborough, UK), supplemented with 10% foetal bovine serum (FBS, Fisher Scientific UK Ltd., Loughborough, UK) and 1% penicillin-streptomycin (P/S, Fisher Scientific UK Ltd., Loughborough, UK) in a 37 °C incubator with 5% CO₂. After 48 h, UC-MSCs were washed with PBS (Fisher Scientific UK Ltd., Loughborough, UK) thrice, before exchanging for the four different media compositions (see Table 2).