Human endothelial sfm

The present study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki with the approval of the Institutional Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. Both U937 monocytes and HUVECs are obtained from American Type Culture Collection (Manassas, VA, USA). HUVECs are cultured with human endothelial SFM (Invitrogen, Carlsbad, CA, USA), containing 20% fetal bovine serum (Invitrogen), 100 U/ml penicillin-streptomycin (Invitrogen), 100 μg/ml heparin (Sigma-Aldrich, St. Louis, MO, USA), and 150 μg/ml endothelial cell growth supplement (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). U937 monocytes are cultured in RPMI1640 medium (Invitrogen), containing 20% fetal bovine serum (Invitrogen) and 100 U/ml penicillin-streptomycin (Invitrogen). These two cell lines are both cultured in a 5% CO₂ incubator (37°C and 90% humidity) (Thermo Fisher Scientific, Waltham, MA, USA).

The T84 human colonic cell line was obtained from the European Collection of Animal Cell Cultures (Salisbury, England) and grown in a 1:1 mixture of Dulbecco-Vogt modified Eagle’s medium and Ham’s-F12 medium (DMEM/F12) supplemented with 50 μg/mL penicillin, 50 μg/mL streptomycin (Sigma, St Louis, MO, USA) and 4% foetal bovine serum (Invitrogen, Carlsbad, CA, USA) as previously described [21 (link)]. Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and maintained in human endothelial SFM (Invitrogen) with 50 μg/mL penicillin, 50 μg/mL streptomycin, 20% foetal bovine serum, 20 ng/mL FGF (Invitrogen), 10 ng/mL EGF (Invitrogen) and 1 μg/mL heparin (Sigma) as previously described [25 (link)].

Brain microvascular endothelial-like cells (iBMECs) were prepared as previously described [8 (link)]. In Brief, human iPS cells were cultured in iPSC medium (Dulbecco’s Modified Eagle’s Medium/Ham’s F12 (Wako Pure Chemical Industries (Wako), Osaka, Japan) containing 20% knockout serum replacement (Invitrogen, Carlsbad, CA, USA), 2 mM l-glutamine (Wako), 1% minimal essential medium with non-essential amino acids (Invitrogen), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA)) at 37℃ in 5% CO₂ for 6 days. Then the culture medium was changed to EC medium (Human Endothelial-SFM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% platelet-poor plasma derived bovine serum (PDS) (Alfa Aesar, Haverhill, MA, USA), 20 ng/mL FGF2, and 10 µM all-trans retinoic acid (RA) (Tocris Bioscience, Bristol, UK)). After 2 days, the cell were detached by Accutase (Nacalai Tesque, Kyoto, Japan) and re-prated on 0.3-cm² Transwelll-Clear permeable inserts (0.4 µm pore size, Sabeu GmbH & CO. KG, Northeim, Germany) coated with a mixture of fibronectin (100 µg/mL; Wako) and collagen IV (400 µg/mL; Nitta gelatin, Osaka, Japan). These cells were then used to construct the in vitro BBB model.

Primary human umbilical vein endothelial cells (HUVECs) were prepared from fresh human umbilical veins as previously described (Barbieri et al., 1981 (link)) and maintained in Human Endothelial SFM (Gibco, Life Technologies Corporation, Grand Island, NY, USA, #11111044) supplemented with 20% Fetal Bovine Serum (FBS) (Dutsher, Bernolsheim, France, #500105 A1A), epidermal growth factor (EGF, 10 ng/mL, Invitrogen), bFGF (10 ng/ml, prepared in the protein purification facility of our institute), heparin (10 ng/ml, Sigma, Saint Quentin Fallavier, France, #H3149), and antibiotics (Gibco, #15140–122). HUVECs were used up to the 6th passage for all experiments. For assessment of FN protein production and deposition, cells were trypsinized (Gibco, #15400–054) and grown in 2% FN-depleted serum for 48 hr before protein extraction. The HEK293FT cell line from Life Technologies (Saint Aubin, France) was maintained in DMEM (Gibco, #31966–021) supplemented with 10% FBS and 1 X Non-essential Amino Acid supplement (Sigma, #M7145). Absence of Mycoplasma sp. contamination was routinely verified by PCR as described elsewhere (Kong et al., 2001 (link)).