Calcein am

Gelatin from bovine (type B, ~226 g Bloom) was purchased from Sigma-Aldrich (St. Louis, MO, USA). N-hydroxysuccinimide (NHS), N,N-Dimethylformamide (DMF), 3-(4-hydroxyphenyl) propionic acid (HPPA), aqueous hydrogen peroxide (H₂O₂, 31% w/w), horseradish peroxidase (HRP, 190 U mg⁻¹), catalase (bovine liver), collagenase, and 4% w/v paraformaldehyde in PBS were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Water-soluble carbodiimide hydrochloride (WSCD·HCl) was purchased from the Peptide Institute (Osaka, Japan). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Nissui (Tokyo, Japan). Calcein-AM was purchased from Nacalai Tesque Inc., Kyoto, Japan). Propidium iodide (PI) was purchased from Dojindo, Kumamoto, Japan). Phalloidin-iFluor 647 Reagent (ab176759) was purchased from Abcam (Cambridge, UK), and -Cellstain^®- DAPI solution was obtained from Dojindo (Kumamoto, Japan).

Gelatin from porcine skin (type A) and poly(ethylene oxide) (PEO) (Mw = 900,000) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Tyramine hydrochloride (96%) was purchased from Combi-Blocks, Inc. (San Diego, CA, USA). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Peptide Laboratory Inc. (Osaka, Japan). HRP (190 U/mg), H₂O₂, and N-hydroxysuccinimide (NHS) were obtained from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). Gelatin-Ph (1.95 × 10⁻⁴ mol-Ph/g) was synthesized through conjugation with Tyramine hydrochloride using EDC and NHS as previously described [25 (link)]. Propidium iodide (PI), 2-morpholinoethanesulfonic acid, monohydrate (MES), and Cellstain-CytoRed solution (CytoRed) were obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Calcein-AM and Dulbecco’s phosphate buffered saline (PBS) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Opti-MEM and Lipofectamine 3000 transfection reagents were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

For BBB-Glioblastoma experiments, BBB-in-a-CUBE at Day 6 were incubated with 40 μM Reversan or an equal volume of DMSO as control for 4 hr before being transferred to a chip with Glioblastoma-in-a-CUBE at Day 3, and the chip was assembled as described above. Glioblastoma medium was added to the basal side and BBB medium with 5 μM Vincristine (Tocris, 1257) added to the apical side. Following a 4 hr incubation at 37 °C, the media were discarded and Glioblastoma CUBEs retrieved from the chip. The CUBEs were washed once in live cell imaging solution (LCIS; Invitrogen, A14291DJ), then incubated with 1.5 μM Calcein-AM (Nacalai Tesque, 19177-14) and 1.5 μM propidium iodide (Nacalai Tesque, 19174-31) diluted in LCIS for 20 min at 37 °C. Samples were washed once in LCIS before LIVE/DEAD imaging using a fluorescence microscope with sectioning function (BZX-700, Keyence) equipped with 10× lens (NIKON PlanFluor 10×/0.30). Projection image of 3 slices with 20 µm pitch (40 µm total thickness) was taken from the side closest to the BBB for analysis. The number of live and dead cells were counted using Imaris software (Bitplane, v9.0.2).4–5 technical samples were measured in each independent experiment, and 3 independent experiments were performed in this study.

Human healthy donor peripheral blood mononuclear cells (PBMCs) were isolated using Lymphocyte Separation Solution (Nacalai tesque, Kyoto, Japan). Human T cells and NK cells were enriched from PBMCs by cell depletion of B cells and monocytes using anti-CD19, and anti-CD14 conjugated magnetic beads with Magnisort technology (ThermoFisher). To facilitate visualization, purified T cells were stained with 2 µM Calcein-AM (Nacalai tesque) in PBS for 30 min at 37 °C followed by co-culture. Isolated cells were maintained in a T cell medium; RPMI1640 supplemented with 10% FBS, 100 U/mL human IL-2 and 10 ng/mL human IL-7 (both from Biolegend, CA, USA).