Ezsphere

E0771/mCherry-luc2 cells (2 × 10⁴ cells / well) were seeded in 96-well plates optimized for tumor spheroid formation (IWAKI EZSPHERE, AGC Techno Glass Co. Ltd., Shizuoka, Japan) with 10% FBS-RPMI with murine galectin-3 (Biolegend, San Diego, CA, USA), and incubated for 72 h. The mCherry fluorescence was detected to visualize tumor spheroids (Keyence, Osaka, Japan). The size of tumor spheroids was analyzed in three independent images for each concentration of galectin-3 with Hybrid Cell Count software (Keyence).

Exactly 14 days after sorting, CD73⁺ cells were cultured for 2 days in EZSPHERE^® (AGC Techno Glass, Shizuoka, Japan), in order to generate spheroids. Spheroids were collected and suspended in PBS. Cells (1.0 × 10⁶ cells in 200 μL of PBS) were transplanted via enteral administration 0 and 5 days after DSS treatment. Trans-anal transplantation was performed as previously described, with slight modifications [22 (link)]. The cell suspension was infused into the murine colonic lumen by using a thin, flexible catheter (diameter: 2.1 mm) or a stainless steel needle (diameter: 1.9 mm). After transplantation, the mice were housed as usual, and colon tissues were collected and analyzed by flow cytometry. Control mice received an equal volume of PBS, or a solution of 0.01 mg/mL 5-aminosalicylic acid (5-ASA; Zeria Pharmaceutical Co., Ltd., Tokyo, Japan). The concentration of 5-ASA (100 mg/kg) was based on a previous paper, and 5-ASA was administered enterally in the same manner as the spheroids [23 (link)].

CD31+ cells were prepared from differentiated hiPSCs (Ff-I14). To generate embryoid bodies (Ebs), hiPSCs were seeded onto EZSPHERE (AGC TECHNO GLASS Co., Ltd., Shizuoka, Japan). Approximately 3 × 10⁵ hiPSC cells/mL were cultured in the StemFit AK03N medium containing 2 ng/mL BMP4 and 10 µM Y27632 in the absence of component C (Ajinomoto, Co., Ltd., Tokyo, Japan) (day 0). On day 1, 9 ng/mL BMP4, 10 ng/mL bFGF, and 6 ng/mL Activin A (R&D Systems, Inc., Minneapolis, MN, USA) were added to the medium. From days 2 to 7, Ebs were cultured in a single-use bioreactor and magnetic stirrer (ABLE Corporation & Biott Corporation, Tokyo, Japan). On day 2, the medium was supplemented with 9 ng/mL BMP4, 10 ng/mL bFGF, and 6 ng/mL Activin A (R&D Systems, Inc., Minneapolis, MN, USA), and removed on day 4. On day 4, the medium was supplemented with 25 ng/mL vascular endothelial growth factor (VEGF) (R&D Systems, Inc., Minneapolis, MN, USA) and 8 ng/mL bFGF, and removed on day 7. On day 7, Ebs were enzymatically dissociated and subjected to MACS (Miltenyi Biotec Inc., Tokyo, Japan) to separate CD31+ cells. CD31+ cells were maintained in EGM2 (Lonza, Inc., Basel, Switzerland) on collagen-type IV-coated tissue culture dishes. They were passaged every two days until being harvested for cryopreservation on day 13.