α mem medium

IDG-SW3 (murine immortal osteocyte, EKC001, Kerafast, USA) cells were allowed to proliferate at 33 °C with α-MEM medium (Life Technologies, USA) containing 10 ng/mL IFN-γ at an initial seeding density of 5000 cells/cm² on type I collagen-coated plates (0.2 mg/mL in 0.2 M acetic acid). The specific cell culture medium developed for the experiment was made by mixing the conditioned medium derived from M0, M1, M2 macrophages (named Group M0-CM, M1-CM, and M2-CM), respectively, with α-MEM medium (Life Technologies, USA) at a ratio of 1:1, supplemented with 50 µg/mL ascorbic acid (Sigma, USA) and 4 mM β-glycerophosphate (Sigma, USA) for 14 days of osteogenic differentiation. The mixed medium for osteocyte culturing was replaced every 3 days.

Primary 2-month, 8-month and 22-month BMSCs were isolated by flushing the bone marrow of the femurs and tibiae as previously described [44 ]. The cells were then cultured in α-MEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin (HyClone) in a 5% CO₂ atmosphere at 37 °C. Primary BMSCs were used for the experiments at passages 2. OriCell C57/BL6 BMSCs at passage 5 were purchased from Cyagen (Cyagen, Guangdong, China) and cultured in OriCell growth medium (Cyagen) as manufacturer’s instructions. For miRNA agomimics and antagomimics transfection, lentivirus transfection and BMSCs transplantation, OriCell C57/BL6 BMSCs at passage 7–8 was used in these experiments.
For BMSCs osteogenic differentiation, cells were induced by osteogenic conditional medium when reached 70% confluence. The osteogenic culture medium containing α-MEM medium (Gibco) supplemented with 50 μM ascorbic acid, 10 mM β-glycerophosphate and 10 nM dexamethasone (Sigma-Aldrich). Culture medium was changed every two days. For BMSCs adiopogenic differentiation, cells were induced by adipogenic conditional medium when reached 90% confluence. The adipogenic medium containing DMED medium supplied with 1 mM dexamethasone, 10 nM insulin and 200 mM indomethacin (Sigma-Aldrich).

The cell lines used in this study were either purchased recently at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) or authenticated by the Multiplex human Cell Authentication test (Multiplexion). The human AML cell lines MV4-11, MOLM-13, HL60 and IMS-M2 were cultured in RPMI-1640 medium (Gibco) supplemented with 10 % fetal bovine serum (FBS, Biochrom GmbH). The human AML cell line OCI-AML3 was cultured in α-MEM medium (Gibco) supplemented with 20 % FBS. The human AML cell line OCI-AML5 was cultured in α-MEM medium (Gibco) supplemented with 20 % FBS and 10 ng/mL GM-CSF (PeproTech GmbH). Primary AML blasts and CD34⁺ progenitor cells from healthy donors were cultured in IMDM medium (Gibco) supplemented with 10 % FBS, 10 % horse serum (Gibco), and 10⁻⁶ M hydrocortisone (Sigma-Aldrich). All cells were maintained in a humidified incubator with 5 % CO₂ at 37°C.

Normal human foreskin fibroblasts (AG1522, National Institute of Aging) were grown in cultured dishes in a monolayer and the growth is contact-inhibited. Cells of less than 10 passages, routinely cultured at 37⁰ C, 95% humidity, and 5% CO₂ in α-MEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 50 mg/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA). Cells at passage 6 were frozen and shipped to Kennedy Space Center (KSC) months prior to the scheduled launch date. Two weeks prior to launch, the cells were thawed and plated in T-75 flasks. Seven days prior to the launch date, the cells were seeded in the BioCell cell culture chambers (10 flight and 10 ground control BioCells) at a density of 1x10⁶ cells per BioCell. The cells reached confluence 2 days before launch with about 92% of the cells expected in the G1 phase of the cell cycle. One day prior to the launch, media in the BioCell was replaced with fresh media, and the cell culture chambers were inserted in sealed habitats (5 BioCells per habitat) that were each flushed with CO₂ (5%) balanced air. The samples were then transported to the launch pad and kept at 20⁰ C. Ground controls were prepared following the exact same procedure used for the flight samples.

P0 female mice were culled by decapitation and ovaries dissected out into Leibovitz L-15 dissection medium (Invitrogen) supplemented with 3 mg ml⁻¹ bovine serum albumin (Sigma Aldrich); day of dissection: Day 0. Ovaries were cultured for 6 days in a 24-well culture plate (Greiner Bio-one, Stonehouse UK), on Whatman nucleopore polycarbonate membranes (Camlab Ltd, Cambridge UK, 13 mm, 8.0 μm) floating on α-MEM medium (Invitrogen) supplemented with 3 mg ml⁻¹ bovine serum albumin, incubated at 37 °C, 5 % CO₂. Culture medium was supplemented with varying doses of etoposide to produce final concentrations of 50, 100, 150 or 200 ng ml⁻¹ for the duration of the culture (Days 0–6). As with the fetal ovary cultures, etoposide was dissolved in DMSO, which was therefore also added to control medium, all media containing 0.1 % DMSO. All treatments were n = 6, with previously published work showing this to be sufficient group size [34 ].

hucMSCs were isolated and identified as previously described [4 (link)]. hucMSCs were cultured in α-MEM medium (Invitrogen) with 10% fetal bovine serum (Bioind) and were used for experiments in 3 to 5 passages. 293 T cells were purchased from ATCC and cultured in high DMEM (Gibco) containing 10% fetal bovine serum (Bovogen). All cells were maintained at 37°C with 5% CO₂.