Stemmacs msc expansion media

The AML-MSC isolation and culture, 3D scaffold specifications, and 3D culturing setup were previously described (Borella et al., 2021 (link)). Briefly, AML-MSCs were seeded in the scaffold and cultured in StemMACSTM MSC Expansion Media (Miltenyi Biotec) for MSC expansion for 5 days. Then, AML cells were added to the scaffold in proper medium and exposed to the best drug combinations that resulted from the monoculture setting. We used the highest synergistic doses found by ZIP synergy analysis, tested to be safe in MSCs, that are I-BET151 (2 µM), sunitinib (5 µM), and thioridazine (10 µM). Venetoclax was used (1 µM). Treatment was conducted for 48 h.

Umbilical cords (UC) were collected from pregnant women after baby delivery at the surgery of Vinmec International Hospital. The UCs were washed three times with ethanol 70% and then 3–5 times with PBS to sterilize and remove blood. The UC was cut into small pieces and transferred into a 50 mL conical tube following by an incubation with 500 U collagenase Type I solution (Gibco, Massachusetts, USA) for 1 h at 37°C with shaking. After digestion, solution was diluted in chilled PBS and centrifuge at 1,400 × g/10 min at 4°C to collect cell pellet. The cell pellet was resuspended in a MSC culture media (StemMACS^TM MSC Expansion Media, Miltenyi Biotec, Bergisch Gladbach, Germany) and transferred into cell culture flasks (Nunc, Thermo Scientific, Massachusetts, United States) coated with solution CTS^TM CELLstart^TM substrate (diluted in PBS at ratio 1: 300) (Gibco, Massachusetts, USA) and incubated at 37°C and 5% CO₂ for cell expansion. With each 5 mg of UC, cell pellets were seed into 1 T25 cell culture flask. Culture media were replaced by every 2 days of culture. When the cells reached 80% confluency, the cells would be split by 0.05% trypsin for the next passage.

All BMA samples (5 mL) were processed as previously described [21 (link)] Following the lysis of RBCs using ammonium chloride, and after washing the cell pellet twice with 10 mL of PBS, the nucleated cells were re-suspended in complete MSC media (StemMACS™ MSC Expansion media, Miltenyi Biotec, Bisley, UK) in a volume equivalent to the original BMA, then seeded in a 6-well plate (Corning, New York, NY, USA ) at 750 µL/well containing 1 mL of StemMACS™ media to enable MSC attachment. After 48 h, the media were changed to low glucose DMEM supplemented with antibiotics and 10% human serum (Sigma-Aldrich, Dramstadt, Germany) for MSC expansion to passage 1 (p1). We have previously shown that the expansion of MSCs, particularly in FCS-containing media, affects their colony characteristics and gene expression profiles, while the use of human serum leads to a better preservation of their native characteristics [22 (link),23 (link)]. Half media changes were performed twice weekly, and the cells were trypsinised and expanded when approximately 60–70% confluent. P1 MSCs were then frozen at −80 °C in aliquots and used for further analysis, as described below.

Fourteen primary brain tumor samples were obtained from the neurosurgery department at Skåne University Hospital in Lund, Sweden. Ethical permit H15 642/2008. The tissue was washed in phosphate buffered saline (PBS, Life Technologies, Carlsbad, CA, USA), placed in a Petri dish with StemMACS MSC Expansion Media (Miltenyi Biotec, Bergisch Gladbach, Germany) and minced with a scalpel. Afterwards, cells were washed in PBS and then incubated in Accutase (Sigma-Aldrich, Stockholm, Sweden) at 37 °C for 20 min. They were rotated every 5 min to prevent sedimentation. Finally, cells were passed 2–3 times through an 18 G needle before being filtered through a 100 µm nylon mesh. The single cell suspension was put in cell culture flasks with MSC Expansion Media.

Procured hBM MSCs were expanded with StemMACS MSC Expansion Media (MiltenyBiotec) supplemented with Primocin (1:1000, InvivoGen), which we refer to as expansion or basal medium (BM) interchangeably, in T75 plastic flasks (Thermo Fisher Scientific). When they reached confluence, cells were trypsinized using trypsin‐ (0.25%), phenol red (Thermo Fisher Scientific).