Mem non essential amino acids solution

HeLa cells were maintained in DMEM High Glucose medium (Nacalai Tesque) supplemented with 10% FBS (Biosera), 1 × MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific) and 1 mM sodium pyruvate (Sigma). 293FT cells were maintained in DMEM High Glucose medium supplemented with 10% FBS, 1 × MEM Non-Essential Amino Acids Solution, 1 mM sodium pyruvate and 2 mM (1 ×) l-Glutamine (Thermo Fisher Scientific).

Mouse embryonic fibroblasts (MEFs), bovine fetal fibroblasts (bFFs) and 293FT cells were cultured in complete Iscove′s Modified Dulbecco′s Medium (IMDM; 12200-036, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; SH30071.03, Global Life Sciences Solutions USA LLC, Marlborough, MA, USA), 1% GlutaMAX (3505006, Thermo Fisher Scientific, Waltham, MA, USA), 1% MEM Non-Essential Amino Acids Solution (11140050, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (15140122, Thermo Fisher Scientific, Waltham, MA, USA). The biPSCs were cultured in KnockOut DMEM-F12 (12660-012, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% KnockOut Replacement Serum (10828028, Thermo Fisher Scientific, Waltham, MA, USA), 1% GlutaMAX, 1% MEM Non-Essential Amino Acids Solution, 1% penicillin/streptomycin and 3.85 μM β-mercaptoethanol (21985-023,Thermo Fisher Scientific, Waltham, MA, USA) and were either supplemented or not with 10 ng/mL recombinant basic FGF (bFGF, 100-18B, Peprotech, Rocky Hill, NJ, USA), 1000 IU/mL recombinant mouse LIF protein (ESGRO, ESG110, EMD Millipore Corporation, Billerica, MA, USA) and 2i (1 mM PD0325901 and 3 mM CHIR99021, Sigma-Aldrich, St. Louis, MO, USA).

The A549 (CRM-CCL-185) and HEK293 (CRL-1573) cell lines were purchased from ATCC (Manassas, VA, USA). A549 cells were cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA, 21870076) supplemented with 10% fetal bovine serum (FBS), GlutaMAX Supplement (Thermo Fisher Scientific, 35050061), Sodium Pyruvate (Thermo Fisher Scientific, 11360070), MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific, 11140050), and Antibiotic-Antimycotic (Thermo Fisher Scientific, 15240062) at 37 °C in a 5% CO₂ atmosphere. HEK293 cells and mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, 11995073) supplemented with 10% FBS, MEM Non-Essential Amino Acids Solution, and Antibiotic-Antimycotic at 37 °C in a 5% CO₂ atmosphere.

Human Dermal Fibroblast (HDF) (Lonza, Switzerland) cells were cultured on cultureware coated with 0.1% gelatin (PAN Biotech, Germany) in Dulbecco’s Modified Eagle’s Medium (DMEM) – low glucose (PAN Biotech, Germany) with 10% fetal bovine serum (FBS) (Biowest, France), 1% penicillin–streptomycin (10,000 U/mL) (Gibco, USA), 1% GlutaMAX™ (100 ×) (Gibco, USA), 1% MEM non-essential amino acids solution (100 ×) (Gibco, USA) and 50 mM 2-Phospho-L-ascorbic acid trisodium salt (MilliporeSigma, USA).
HaCaT cells (AddexBio, USA) were cultured on cultureware coated with 0.1% gelatin (PAN Biotech, Germany) in DMEM (Gibco, USA) with 10% FBS (Biowest, France), 1% penicillin–streptomycin (10,000 U/mL) (Gibco, USA), 1% GlutaMAX™ (100 ×) (Gibco, USA) and 1% MEM non-essential amino acids solution (100 ×) (Gibco, USA).

For co-culture experiments, hydrogels containing MDA-MB-231 breast cancer cells (ATCC, Manassas, VA, USA) were printed on top of the ASC-laden constructs after 21 days of adipogenic culture. MDA-MB-231 breast cancer cells were expanded in growth medium composed of DMEM (1 g/L glucose, Life Technologies) supplemented with 10% FBS, 1% L-glutamine, 1% MEM non-essential amino acids solution, 1% sodium pyruvate, 0.6% HEPES, and 1% penicillin/streptomycin (all Life Technologies). After harvesting, MDA-MB-231 cells at passage 6 were suspended in hyaluronic acid-based hydrogel precursor solution and printed at 1 bar as described above. Before transferring the suspension into the glass rings for UV irradiation (see above), differentiated ASC spheroid-laden hydrogels were put at the bottom inside the glass ring. The hydrogel–cancer cell suspension was then dispensed on top and UV-crosslinked at 365 nm for 10 min. The resulting co-culture hydrogels (bottom: adipogenically differentiated ASC spheroids, top: MDA-MB-231 cells) were cultured for 9 days in maintenance medium consisting of DMEM/F12, 10% fetal bovine serum, 1.7 µM insulin, and 1% penicillin/streptomycin. Samples were harvested at d0 and d9. Double gels with either empty top or bottom compartment (only ASC spheroids at the bottom or only MDA-MB-231 at the top) served as monoculture controls.

Cell preparation tubes containing sodium citrate (Becton-Dickinson, Franklin Lakes, NJ, USA) were used to isolate PBMCs from whole blood. For storage, liquid nitrogen was used to freeze PBMCs in 10% RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA), 10% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA), and 80% fetal bovine serum (FBS) (Lonza, Cologne, Germany). PBMCs were cultured in RPMI 1640 medium containing 2 mM glutamine (Life Technologies), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 50 mg/mL gentamicin sulfate (Lonza), and 10% FBS. After two washes in phosphate buffered saline (Welgene, Seoul, Korea), the PBMCs were resuspended at 1 × 10⁶ cells/mL in RPMI 1640 medium supplemented with 1% sodium pyruvate (Life Technologies), 1% MEM Nonessential Amino Acids Solution (Life Technologies), and 10% FBS, followed by incubation overnight. Assays for NK cells were always conducted following incubation for 16–20 h. To determine NK cell counts, peridinin chlorophyll protein-conjugated anti-CD3, and phycoerythrin-conjugated anti-CD56 and monoclonal antibodies (BD Biosciences, San Jose, CA, USA) were added to 200 mL of diluted blood that had been incubated on ice for 20 min, and the cells were then analyzed by flow cytometry (BD FACSCalibur, BD Biosciences). The NK cell abundance was calculated as (NK cells/mL sample) = [(CD3/CD56 cell count)/ bead count] × 100.

A total of 45 colorectal cancer cell lines were used (see Additional file 1). All lines were maintained in MEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1 × antibiotic/antimycotic, 1 × MEM Non-Essential Amino Acids Solution, and 10 mM HEPES buffer solution (all from Life Technologies, Carlsbad, CA) at 37 °C and 5% CO₂. All lines were tested to be negative for mycoplasma contamination (PCR Mycoplasma Detection Set, Takara) and possible cell line cross-contamination was ruled out by clustering analysis of genome-wide mRNA expression and promoter methylation data at the time of these experiments.