Accumax

Bone marrow cells were collected by flushing the femurs and tibias using PBS. After washing the collected cells in PBS, cells were differentiated in DMEM containing 10% FBS, 1/100 dilution of antibiotic-antimycotic, and 40 ng/mL macrophage colony-stimulating factor (416-ML; R&D Systems, Minneapolis, MN, United States), and the medium was changed every 3 days. After 5–7 days from the start of differentiation, the attached BMDMs were collected using Accumax™ (17087-54; Nacalai Tesque). To induce innate immune memory, BMDMs were treated with 50 ng/mL LPS (tlrl-peklps; InvivoGen, San Diego, CA, United States) for 24 h before use.

The iPSC lines with doxycycline-inducible MYOD1 expression vector (B7-M, B7-MSMNKD, SMA-M, and SMA-MOE) were converted into myogenic cells as previously described (Tanaka, 2013 (link)). In brief, 4.0 × 10⁵ iPSCs were seeded onto Matrigel (Corning)-coated 24-well plates in Primate ES Cell Medium (ReproCELL). At day 0, the medium was exchanged with Primate ES Cell Medium containing doxycycline (TaKaRa) (1.0 µg/ml). From day 1, the medium was exchanged everyday with Minimum Essential Medium Α (Gibco) containing 10% KnockOut Serum Replacement (Gibco) and doxycycline (1.0 µg/ml). Cells were collected with Accumax (Nacalai Tesque) on days 0, 3, and 6 after the myogenic conversion and analyzed thereafter.

HeLa, 293FT, iPSCs, and their modified cell lines expressing fluorescent proteins were seeded into a multi-well plate the day before transfection. Transcribed RNAs were transfected into the cells using Lipofectamine MessengerMAX (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cell clumps after at least 13 days of cardiomyocyte differentiation were transferred to conical tubes and harvested after the cell clumps naturally fell down. The harvested cell clumps were resuspended in collagenase I solution (2 mg/ml) supplemented with DNase I (10 μg/ml) and rotated for more than 2 hours at 37°C. After the incubation, the supernatant was removed, and the pellet of cell clumps was resuspended in Accumax (Nacalai Tesque) and incubated for about 30 min at 37°C. Then, the cell suspension was pipetted to dissociate the clumps to single cells and diluted by fresh differentiation medium 4. The dissociated cell suspension was centrifuged for 5 min at room temperature, and the supernatant was aspirated. The cells were resuspended in differentiation medium 4 and counted using Countess II (Thermo Fisher Scientific). The transfection conditions are listed in table S4.

Polarized M1 and M2 macrophages were detached from tissue culture flask using Accumax™ (Nacalai Tesque, Japan). 2 × 10⁵/ml M1 and M2 macrophages were washed with PBS and stained using antibodies for CD11b-PE, CD14-FITC, CD86-FITC, *CD80-APC/H7, CD206-APC and CD200R-PE (Miltenyi Biotec, Germany) (*BD Biosciences, United States) for 10 min at 4 °C in the dark. Antibodies with similar conjugates were stained in separate tubes. The cells were washed with MACS buffer to remove excess antibodies. Fluorescence was measured via flow cytometry using a FACSCanto™ II flow cytometer and FACSDiva software (BD Biosciences, United States). Histograms were plotted using FlowJo™ version 10 software (BD Biosciences, United States).

The Colon‐26 tumor‐bearing mice (female; about 8 weeks; n = 3; average weight = 18 g; average tumor size = 400 mm³; BALB/cCrSIc; Japan SLC) were euthanized the next day after administration of PBS (200 µL)/A‐gyo (200 µL, 1 × 10⁸ CFU)/AUN (200 µL, 1 × 10⁹ CFU) injection intravenously. To analyze the immune cells in tumors, tumors were collected from mice in different groups after i.v. injection of sample for 3 or 24 h, homogenized into single‐cell suspensions with Accumax (Nacalai Tesque), and 1 × 10⁶ cells were stained with fluorescein isothiocyanate (FITC)‐ or Alexa Fluor 488‐labeled antibodies (listed in Table S8, Supporting Information) according to the manufacturer's protocols, and then classified by flow cytometry (CyFlow Cube 6, Sysmex, Kobe, Japan) by analyzing 4 × 10⁴ cells for each sample.

Primary neurons from wild-type (WT) mice (ICR) brains were obtained from the cerebral cortices of fetal mice (14–16 days of gestation). The cerebral cortices were washed thrice using the Hanks’ Balanced Salt Solution (HBSS; Nacalai Tesque, Japan) and incubated in Accumax (Nacalai Tesque) and DNase I (Sigma-Aldrich, MO) for 5 mins at 37 °C, followed by the dissociation by gentle pipetting for the flowthrough into the 100 μm cell strainer (Greiner). Obtained cells were suspended in DMEM (Nacalai Tesque) containing 10% fetal bovine horse serum and 10% horse serum (Gibco, MA) and plated on dishes coated with 0.1% poly-ethyleneimine (Sigma) in 0.05 M boric acid buffer. After 1 h, cells were resuspended in the neurobasal medium (Gibco) containing GlutaMAX™-I(Gibco), B-27 supplement (Gibco), and 1% penicillin/streptomycin (Nacalai Tesque). After two days, the medium of the culture was replaced by 10 μM Cytarabine (Ara C, Tokyo chemical industry, Japan) in a neurobasal medium and cultured for an additional seven days.

For long-term expansion, hiPSCs were grafted to prepared substrates with ON2 and seeding densities were adjusted to meet the subculture frequency of once a week on iMatrix-511, FN, and VN or twice on GNF. The most used serum-free medium mTeSR1 and xeno-free media E8 and AK02N were used as positive controls in our study. hiPSCs were maintained at 37 °C with 5% CO₂ and 5% O₂, and the culture media were changed daily.
Passages were carried out once or twice a week according to matrix conditions, when the cell density reached > 80% of the coating surface. For the subculture, hiPSCs on iMatrix-511, FN, or VN were rinsed once with D-PBS, treated with enzymatic cell dissociation buffer Accumax (17087-54, NACALAI TESQUE, Kyoto, Japan) at 37 °C for 4 min, detached either as clusters for FN and VN, or as single cells for iMatrix-511. In case of cells on GNF, they were treated with enzyme-free PBS-based EDTA cell dissociation buffer (13151014, Gibco, USA) at 37 °C for 4 min after being rinsed once with D-PBS. Collected cells were engrafted on prepared plates with new media. For the first 24 h, 10 µM ROCKi was used to improve the survival of single hiPSCs. This procedure was repeated for at least 10 passages over 2 months before further characterization assessment.