Culture dishes

Vascular smooth muscle cells (VSMCs) were obtained by digestion of freshly isolated aortas of 10-week-old Sprague Dawley rats for 30 minutes in collagenase (1 mg/mL, Worthington Biomedical Corporation, Lakewood Township, NJ, USA) and elastase (0.5 mg/ml, Calbiochem, San Diego, CA, USA) in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Waltham, MS, USA) at 37°C. After trituration and centrifugation, the cells were seeded in culture dishes (Corning, New York, NY, USA) and cultivated in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37°C, 5% CO₂ with humidified atmosphere. The early passage cells (between 2 and 4) were used.

AGS and MKN-28 cells were purchased from the Procell Life Science&Technology Co.,Ltd. (Wuhan, China, Cat. no. CL-0022, Cat. no. CL-0291). All cells were identified by STR, and STR typing showed no cross contamination of human cells in the cell lines. No mycoplasma was detected in the two cells. Cells were cultured in RPMI 1640 (HyClone, USA, Cat. no. SH30809.01) containing 10% fetal bovine serum (FBS) (TransGen Biotech, China, Cat. no. FS301) and the culture dishes (Corning, USA, Cat. no. 150,464) were placed in an incubator containing 5% CO₂ at a constant temperature of 37℃. After the cells were overgrown in the culture dishes, the cells were digested and subcultured with 0.25% trypsin digestion solution (Beyotime Biotechnology, China, Cat. no. C0201).

Caco-2, MDCK and MDCK-MRP2 cells were donated by Prof. Yan Xie and Yueming Ma from Shanghai University of Traditional Chinese Medicine. The cells were grown in culture dishes (Corning® Costar, Cambridge, MA, USA) using DMEM supplemented with 10% FBS, 1% NEAA and 1% penicillin-streptomycin solution. The cells were cultured in 5% CO₂ and 95% O₂ with 90% relative humidity at 37°C. The medium was replaced every other day during incubation. The cells were passaged every 3–4 days between 70 and 80% confluence at a 1:5 split ratio using 0.25% trypsin-EDTA. For the transport experiments, the cells from passages between 30 and 45 were seeded at 1 × 10⁵ cells/cm² onto permeable polycarbonate inserts (0.45 μm pore size, seeding surface of 0.6 cm², Millipore, MA, USA) in 24-well plastic plates. The media in the culture plates were changed every other day for the first week after seeding and were replaced daily afterward. The integrity of the cell monolayers and tight junctions (TJ) were tested and confirmed by measuring transepithelial electrical resistance (TEER) with a Millicell® ERS-2 electrode. The cells were used for the transport experiments 21–28 (Caco-2) or 5–7 (MDCK and MDCK-MRP2) days after seeding, and only monolayers with a TEER-value above 420 Ω cm² were used during the studies.

Three-month-old New Zealand white rabbits were selected for rBMSC isolation according to an established protocol. The animals were provided by Jilin University, Changchun, China, and treated according to the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Bone marrow aspirates (5 mL) were obtained from rabbit tibia and subsequently cultured. Briefly, the isolated cell pellets were resuspended in 5.0 mL of culture medium (DMEM; Dulbecco’s Modified Eagle Medium (Gibco, Carlsbad, CA, USA) supplemented with 10% (V/V) fetal calf serum (Gibco, Carlsbad, CA, USA) and 100 IU/mL penicillin-streptomycin (Sigma, Shanghai, China)). The cells were seeded in culture dishes (Corning Costar Co., Cambridge, MA, USA) and cultured in a 37 °C and 5% carbon dioxide (CO₂) incubator. Non-adherent cells were removed when the medium was changed after 24 h. After that, the medium was replaced every three days until the cells reached 80% confluence. Then, the cells were washed twice with phosphate-buffered saline (PBS), detached by treatment with 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA, Sigma, Shanghai, China), and subcultured under the same condition until the third passage.

Human epidermoid carcinoma HEp-2 cells (HeLa-derived human carcinoma cells; ATCC, CCL23) were obtained from the Russian Cell Culture Collection (Institute of Cytology, St. Petersburg, Russia). Cells were grown in DME medium (#5648, Sigma, USA) supplemented with 10% FBS (Gibco, USA), 80 mg/L of gentamycin, and 20 mM sodium bicarbonate at 37 °C in an atmosphere of 5% CO₂. The cell line was confirmed to be free of mycoplasma infection through regular testing with Hoechst 33342 staining. Cells were seeded in culture dishes (Corning, NY, USA) at a concentration of 4–6 × 10⁴ cells/cm². Freshly prepared solutions of vitamin B_12b and filtered DDC were added 24 h after cell seeding.

Vascular smooth muscle cells were obtained, as previously described [30 (link)]. Briefly, after the rats were sacrificed, the aortas were excised, the surrounding fat and connective tissues were removed, and the lumen of the aorta was gently rubbed to remove the endothelium. The aortas were cut into small segments and transferred into a tube containing collagenase (1 mg/mL, Worthington Biomedical Corporation, Lakewood Township, NJ, USA) and elastase (0.5 mg/mL, Calbiochem, San Diego, CA, USA) in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Waltham, MS, USA) at 37 °C for 30 min. After trituration and centrifugation, the cells were seeded in culture dishes (Corning, New York, NY, USA) and cultivated in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 10,000 μg/mL streptomycin (Gibco) at 37 °C, in 5% CO₂ with a humidified atmosphere. The early passage cells (between two and four) were used.