α mem

HEK-293 cells were cultured up to 80%–90% confluence in T75 flasks (TPP) containing HEK-293 medium [α-MEM (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), 1x glutagro (Corning)] and appropriate antibiotics for selection (vide infra) at 37°C and 5% CO₂. Cells were passaged every 3–4 days using TrypLE (Gibco) up to a maximum of 20 passages.

Following digestion, LN cells were resuspended in 10 mL cell media and plated on 10 cm dishes coated with 0.2% gelatin (Sigma). Complete mouse endothelial Cell Medium (EC, Cell Biologics) was used to grow heterogenous cultures containing FRCs, LECs, and BECs. Minimum Essential Media Alpha (αMEM, Corning) supplemented with 10% FBS and 1% P/S was used to grow homogenous cultures of FRCs. Dishes were placed in a 37°C cell incubator and after 24 h, washed with PBS and EC media to remove non‐adherent cells. EC media was replaced every 3 days. After 7 days, LN cell cultures primarily contained adherent stromal cells with clear fibroblast and endothelial cell morphologies. Stromal cells were incubated with 0.25% trypsin for 2–3 min at 37°C and gently harvested with a cell scrapper. The presence of nonhematopoietic LNSCs was enriched using CD45 MicroBeads and QuadroMACS™ Separator (Miltenyi Biotec). Up to 10⁸ cells were incubated with CD45 MicroBeads in MACS Buffer (PBS, 0.5% FBS) for 15 min at 4°C. The labeled suspension was applied to a LD column on the Separator and the column was rinsed with MACs buffer, according to the manufacturer's instructions. The effluent containing the CD45⁻ cells was collected. Following depletion, 95%–99% of the collected cells were CD45⁻. LNSCs were resuspended in EC or αMEM media, counted, and plated on 0.2% gelatin for later experiments.

KPC689 cells were cultured in RPMI (Corning) supplemented with 10% FBS (Gemini) and 1% penicillin-streptomycin (Corning). The KPC689 cancer cell line was isolated from an autochthonous pancreatic tumor of Pdx1^cre/+; LSL-Kras^G12D/+; LSL-Trp53^R172H/+ (KPC) mice as previously described (17 (link)). Bone marrow-derived MSCs were obtained from the Cell Therapy Laboratory at the University of Texas MD Anderson Cancer Center and cultured in αMEM (Corning) supplemented with 20% FBS, 1% nonessential amino acids (Corning), 1% L-glutamine (Corning), and 1% penicillin-streptomycin. HEK293T/17 (293T) cells were cultured in DMEM (Corning) supplemented with 10% FBS and 1% penicillin–streptomycin. HEK293T cells were obtained from ATCC and short tandem repeats (STR) fingerprinting performed to confirm their identity. The cells were screened and tested negative for mycoplasma. All cells were cultured in 37°C and 5% CO₂.

To estimate the number of colony-forming units (CFU-Fs) in the obtained marrow suspensions, nucleated cells were plated in triplicate at a density of 8, 0 × 10³/cm² (Corning Incorporated, New York, NY, USA) in 2 mL of α-MEM supplemented with 20% FBS. After 3 days of incubation at 37°C in 5% CO₂, the nonadherent hematopoietic cells were removed, and the medium was changed. At day 14, the colonies were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with 1% crystal violet (Sigma-Aldrich). Colonies with more than 50 cells were counted [48 (link)]. The efficiency of colony formation was expressed as the mean colony number relative to the 100000 bone marrow nucleated cells plated.

TSPCs were seeded at 1 × 10⁴ per well in a 12-well plate and cultured in α-MEM (Corning) supplemented with 1% Glutagro^TM (Corning), 10% fetal bovine serum (FBS) (Gibco^TM), and 1% penicillin/streptomycin (Corning). After 48 h, cells were exposed to T3 (10⁻⁶ M) and L-ascorbic acid (10⁻⁷ M), as previously optimized [18 (link)]. Treatment was repeated every 3 days, concurrently with medium change. At days 1, 7, and 14, cells were collected for qRT-PCR and immunofluorescence (IF) assays.