Stemmacs media

A previously reported autofluorescence microspectroscopy protocol for fibroblasts^{25 (link)} was adopted for hMSCs based on their similar adherent nature. Selected passages of hMSCs were seeded at a concentration of at least 3.0 $\times$ 10 ${}^4$ cells/ml on glass coverslips (Schott) within square silicone wells fabricated from medical grade silicone (Wacker Chemie AG). These coverslips with cells in 1 ml of StemMACS media (Miltenyi Biotec) were then incubated in a CO ${}_2$ incubator for 24 h before autofluorescence microspectroscopy. Prior to measurements, the culture media was extracted and the remaining contents in the wells were washed twice with 1 ml of PBS. After washing, any excess PBS was remove and 100 $\mathrm{\mu }\text{l}$ of imaging solution (Thermo Fisher Scientific) was added. Five phase contrast images were obtained per coverslip at random locations through a 4 $\times$ objective. Autofluorescence images and spectra were then taken through a 60 $\times$ oil-immersion super apochromat objective (Olympus). At least 100 cells per early and senescent passage per donor were involved in each autofluorescence run. Autofluorescence output of at least 10 cells were recorded with measurements made at different locations within the silicon well (experimental repeats n = 10). This was followed by five background spectral measurements of a 100 $\mathrm{\mu }\text{l}$ volume of imaging solution placed on a clean region of the same coverslip.

Fibrotic NU tissue was collected directly from the NU site. At the time of surgery, following exposure of the fracture site, the fibrotic tissue lying directly between the fractured bone fragments was excised and collected for study.
Samples for the IP were collected at a mean time of 7.6 weeks (range 6–9) from the first stage of the technique (insertion of cement spacer). Approximately 1 cm of membrane tissue was harvested from the center of the bone defect area. Tissue was either digested by incubation in collagenase solution to generate a single cell suspension for culture initiation or flow cytometry analysis, or processed for histology [38 (link)]. A subset of samples tissue was bisected for both enzymatic digestion and processing for histology. BM aspirates were seeded directly in culture in StemMACS media (Miltenyi Biotec, Bisley, Surrey, UK) with penicillin and streptomycin (Thermo Fisher Scientific, Paisley, UK) to generate BM MSC cultures, as previously described [38 (link)].

Human fibroblasts (HS-27), adipose derived stem cells (ASC), and TNBC cells (MDA-MB-231) were all purchased as cell lines from Lonza. Cultures of individual cell types were carried out under normal conditions (37 °C, 5% CO₂). HS-27 and MDA-MB-231 were cultured in Dulbecco’s modified Eagle’s medium, 10% fetal bovine serum, 1% PenStrep (Gibco). ASCs were cultured in StemMACS media (Miltenyi Biotech), with 1% PenStrep. Co-cultures were arranged per Figure 1A. Group (i) (cancer juxtacrine group) featured all three cell types combined, in equal number (9.6 cm² = 50,000 total cells). Group (ii) (cancer paracrine group) had 25,000 HS-27 and 25,000 ASCs per 9.6 cm² and an overlying boyden chamber containing 20,000 MDA-MB-231. Group (iii) (healthy group) had 25,000 HS-27 and 25,000 ASCs per 9.6 cm².