hiPSCs
Two hiPSC lines, 1231A335 and Ff-I14s04, were obtained from the Center for iPS Cell Research and Application of Kyoto University (Kyoto, Japan). Ff-I14s04, an HLA homozygous iPSC line, was established as previously described35. The use of the iPSC lines was approved by the ethical review committee of Shonan Health Innovation Park (Fujisawa, Kanagawa, Japan) and Kyoto University. For the 1231A3 line, cells were maintained according to the protocol issued by the Center for iPS Cell Research and Application (CiRA, Kyoto University). Briefly, we cultured the cells on an iMatrix511™ (Nippi, Tokyo, Japan)-coated culture surface using Stem Fit® AK02N (Ajinomoto, Tokyo, Japan). We incubated the cells at 37 °C, 95% humidity, and 5% CO2 atmosphere unless stated otherwise. Every 7 days, the cells were passaged by treatment with TrypLE™ (Thermo Fisher Scientific, Waltham, MA, USA). For the Ff-I14s04 line, we maintained the cells in the StemFit AK03N medium (Ajinomoto) and passaged them using EDTA (0.5 mM) twice weekly.
Design of microwell bag and jig
We developed the membranes for the cell culturing bag using our original films. The gas-permeable membrane was made of a 110 μm-thick linear low-density polyethylene (LLDPE) film. The gas-impermeable membrane was made of a multilayer film consisting of three layers: LLPDE, gas-impermeable ethylene–vinyl alcohol copolymer (EVOH), and adhesive resin (AD). The thickness of the gas-impermeable membrane was 118 μm, and the ratio of the film thickness was 57:15:23 (LLDPE:AD:EVOH) (Supplementary Fig. S6). We measured gas permeability using a gas/water vapor permeation analysis system (GTR-10X; GTR TEC, Kyoto, Japan) (Supplementary Fig. S6).
For the lower membrane, hemispherical microwells were thermoformed in a staggered pattern in a 50 cm2 hexagonal cell culturing area of the film. The diameter and number of the microwells are shown in Supplementary Fig. S3. For an upper membrane as a top cover, a vertical wall was thermoformed along the edge of the culture area to provide space for the medium. The height of the vertical wall, defined as the bag height, was designed as 2, 4, 6, and 8 mm for medium volumes of 10, 20, 30, and 40 mL, respectively. A port for injecting or draining the culture solution into/from the bag was placed in the center of the short side of the rectangular bag. The culture area is hexagonal in consideration of the ease of draining the culture solution from the port and future expandability, such as the installation of multiple ports. The upper and lower membranes and port components were heat-welded around the hexagonal culture area to form a bag. At the tip of the port, a Needleless Injection Site (#2901280167; Qosina, Ronkonkoma, NY, USA) to connect a syringe was attached via a short tube. When using the gas-impermeable membranes, the inner surface side in contact with the cells was designated as the LLDPE layer. We coated the inner culture surface with 2-methacryloyloxyethyl phosphorylcholine polymer (LIPIDURE®; NOF, Tokyo, Japan) to inhibit cell adhesion.
To increase the usability of the culture bag, we developed a jig composed of three parts: a base plate on which the culture bag was placed, a pressing plate that pressed the culture bag from above, and a cover that supported the pressing plate (Supplementary Fig. S1). The pressure applied to the bag by the pressing plate was set to 200–400 kgf/m2. A part of the base plate and the pressing plate was transparent, through which the culture bag contents could be observed without detachment from the jig. In addition, the external dimensions of the jig are of the SBS standard size of 128 mm × 86 mm, allowing operation similar to that of commercially available culture plates.
Small-scale bag culture of undifferentiated iPSCs
A day before cell seeding, the bag was half-filled with medium, attached to the jig, and incubated at 37 °C, 95% humidity, and 5% CO2 atmosphere for > 18 h to remove the microbubbles caught in each microwell. When using the gas-impermeable membranes, the microbubbles were floated by gently tapping outside the bag, and the floated bubbles were sucked out through the port with a syringe. The next day, cells for seeding were dissociated into single cells and centrifuged at 150×g for 2 min; thereafter, the supernatant was removed to obtain a cell pellet. The cells were suspended at a ratio of 250 cells per microwell in a medium at 37 °C. Before cell seeding, the bag was detached from the jig, which remained at 37 °C, and cell slurry was injected into the bag, followed by removal of air with a syringe. After mixing by gentle inversion, the bag was placed on a hot plate (HP-4530 N; AS ONE Corporation, Osaka, Japan) set at the target temperature. The culture solution in the bag was kept warm while mixing by applying palm pressure onto the bag from above. The bag containing the cells was reattached to the jig in the incubator and incubated at 37 °C for 2 days.
Differentiation of iPSCs into iPICs
The hiPSCs (Ff-I14-s04) were differentiated into iPICs using a previously reported method36, with some modifications, as shown in Supplementary Table S1. Dissociated undifferentiated iPSCs were resuspended at a density of 2.0 × 105 cells/mL cells using AK03N containing 10 μM Y-27632 (FUJIFILM Wako) and seeded into a 100 mL bioreactor (ABLE Corporation, Tokyo, Japan). The agitation rate was set to 55 rpm using Bio Jr.8 (ABLE). The cells were cultured for 24 h to induce aggregation. The culture medium was changed according to the following schedule.
Stage 1 (3 days): DMEM (#10569010; Thermo Fisher) containing 1 × B27® supplement (#17504001 or A1895601; Thermo Fisher), 1% DMSO (FUJIFILM Wako), 0.1% Pluronic® F68 (Sigma-Aldrich, Saint Louis, MO, USA), activin A (10 ng/mL; PeproTech, Cranbury, NJ, USA), and CHIR99021 (3 μM; Axon Medchem, Reston, VA, USA). CHIR99021 was added to the culture medium only on the first day.
Stage 2 (4 days): MCDB131 medium (#10372019; Thermo Fisher) containing 0.5 × B27, glucose (final concentration 10 mM; FUJIFILM Wako), NaHCO3 (1.5 g/L; FUJIFILM Wako), GlutaMAX™ (2 mM; Thermo Fisher), ascorbic acid phosphate magnesium salt (PMS) (58 mg/L; FUJIFILM Wako) and keratinocyte growth factor (KGF) (50 ng/mL; R&D Systems, Minneapolis, MN, USA), and 0.1% Pluronic® F68.
Stage 3 (3 days): improved MEM (#10373017, Thermo Fisher) containing 0.5 × B27, ascorbic acid PMS (58 mg/L), KGF (50 ng/mL), LDN-193189 (100 nM; MedChemExpress, Monmouth Junction, NJ, USA), 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)cyclopamine (0.5 μM; Toronto Research Chemicals, Toronto, Canada), 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB) (10 nM; Santa Cruz Biotechnology, Dallas, TX, USA), and 0.1% Pluronic® F68.
Stage 4 (4 days): improved MEM containing 0.5 × B27, ascorbic acid PMS (58 mg/L), KGF (100 ng/mL), epidermal growth factor (50 ng/mL; R&D Systems), nicotinamide (10 mM; Stemcell Technologies, Vancouver, Canada), phorbol 12,13-dibutyrate (0.5 μM; Sigma-Aldrich), activin A (5 ng/mL), and 0.1% Pluronic® F68.
Stage 5 (2 days): MCDB131 medium containing 0.5 × B27, glucose (final concentration 20 mM), NaHCO3 (1.5 g/L), 1:100 GlutaMAX™, SANT-1 (0.25 μM; Sigma-Aldrich), TTNPB (10 nM), activin receptor-like kinase 5 inhibitor II (ALK5i II) (10 μM; Santa Cruz), LDN-193189 (100 nM), triiodothyronine (T3) (1 μM; Sigma-Aldrich), basic fibroblast growth factor (50 ng/mL; PeproTech), XAV939 (1 μM; Sigma-Aldrich), Y-27632 (10 μM), and 0.1% Pluronic® F68.
Stage 6 (7 days): MCDB131 medium containing 0.5 × B27, glucose (final concentration 20 mM), NaHCO3 (1.5 g/L), 1:100 GlutaMAX™, ZnSO4 (10 μM; Sigma-Aldrich), heparin sodium salt (1.4 IU/mL; Nacalai Tesque, Kyoto, Japan), RO4929097 (1 μM; Selleck Chemicals, Houston, TX, USA), ALK5i II (10 μM), LDN-193189 (100 nM), T3 (1 μM), PD-166866 (1 μM; Sigma-Aldrich), and 0.1% Pluronic® F68. PD-166866 was added to the culture medium only for the last 3 days.
After 7 days of culture of stage 6, the cell clusters were collected and treated with TrypLE™ to dissociate into single cells. The cells were cryopreserved using Bambanker® hRM (GC Lymphotec, Tokyo, Japan) and stored at –150 °C until use.
iPIC cluster formation after cryopreservation in a small-scale bag and bioreactors
A day before cell seeding, 10 mL 2 × differentiation medium was added into a φ 0.5 mm and 4 mm height microwell bag and incubated at 37 °C to remove microbubbles (Supplementary Fig. S2). The 2 × differentiation medium was formulated as the Stage 7 medium: MCDB131 medium containing 2% fat-free bovine serum albumin (BSA) (FUJIFILM Wako), glucose (final concentration 20 mM), NaHCO3 (1.5 g/L), GlutaMAX™ (2 mM), 0.5 × ITS-X, ALK5i II (20 μM), T3 (2 μM), ZnSO4 (20 μM), heparin sodium salt (2.8 IU/mL), N-acetyl cysteine (2 mM; Sigma-Aldrich), Trolox (20 μM; FUJIFILM Wako), R428 (4 μM; Selleck Chemicals), TR05991851 (6 μM; Takeda original multi-kinase inhibitor), and Y-27632 (10 μM).
iPICs were thawed in a 37 °C water bath. The cells were suspended in improved MEM containing 0.5 × B27 and Y-27632 (10 μM). The cells were centrifuged at 350×g for 5 min and then resuspended in MCDB131 medium containing 2% fat-free BSA, glucose (final concentration 20 mM), NaHCO3 (1.5 g/L), GlutaMAX™ (2 mM), 0.5 × ITS-X, and Y-27632 (10 μM) to a density of 2.7 × 106 cells/mL. Cell suspension (10 mL) was added to the prepared bag. The number of iPICs seeded in the bag was 2.7 × 107 cells/mL (1500 cells/microwell), and the medium volume was 20 mL. The cells were seeded in a 30 mL bioreactor (ABLE) using a 1 × differentiation medium. The number of iPICs seeded in the bioreactor was 3.66 × 107 cells, and the medium volume was 15 mL. The agitation rate of the bioreactors was set to 30, 60, or 90 rpm. The cells were incubated at 37 °C for 4 days. Reaggregation experiment was repeated three times using the same batch of cells that were cryopreserved.
Scale-up of bag culture
A large-scale culture bag was designed at a 1000 cm2 rectangular culture area and height of 4 mm. A total of 6.5 × 105 microwells, with a diameter size of 350 μm, were thermoformed on the lower portion of the bag. The port was placed at one of the vertices of the rectangle for easy drainage. The large-scale bag was made of the same material as the small-scale bags. A jig was also designed to fit the size of the large-scale culture bag.
The iPICs were seeded into a large-scale bag using the same method as for the small-scale bag above. Briefly, a day before cell seeding, the bag was half-filled with 2 × differentiation medium and incubated at 37 °C to remove the microbubbles in the microwells (Supplementary Fig. S2). After thawing, the cells were seeded into the bag placed on a hot plate at 37 °C. The number of seeded cells was 9.75 × 108 cells (1500 cells/microwell), and 200 mL medium was added to a total volume of 400 mL. The bag was then incubated with the jig at 37 °C for 4 days.
Imaging clusters, cell counting, and medium gas analysis
Clusters in microwells were observed using a phase-contrast microscope (CKX53; Olympus Corporation, Tokyo Japan) with a UPlanFL N × 4 objective lens (NA 0.13; Olympus Corporation) and imaged using a CMOS camera L-835 (Hozan Tool Industrial, Osaka, Japan) or a CCD camera DP27 (Olympus Corporation).
Before collecting the clusters, a portion of the medium was sampled from the bag, and PO2 and PCO2 in the medium were measured using i-STAT® 300F (Abbott Laboratories, Chicago, IL, USA).
To collect the clusters from the bag, the bag was detached from the jig and placed upside down to float clusters from inside the microwells. After inflating the bag by injecting air through the port with a syringe (20 mL for a small bag, 400 mL for a large bag), the entire culture solution was collected with the syringe. For culturing in the bioreactors, cells were directly sucked up from the vessel with a serological pipette. For large-scale culture bags, the bag was hung on a stand to drain all the culture solution through a tube connected to the port and collected in a centrifuge tube under gravity. At this time, large clusters over 300 μm in diameter were removed using a cell strainer (pluriSelect Life Science, Leipzig, Germany) (Supplementary Video S4).
The harvested culture solution was transferred to a 10 cm dish (from the small bags or bioreactors) or a T-225 Flask (from the large bag), and phase-contrast images were obtained for analysis of the shape and diameter of the clusters.
After taking images on a 10 cm dish, the iPIC clusters were divided into two groups using a cell strainer—those with a diameter of 300 μm or less and those with a diameter > 300 μm. Aliquots of the collected clusters were dissociated into single cells by treating with TrypLE™. The cell number was counted using a hemocytometer (One cell Counter; One Cell, Hiroshima, Japan) for undifferentiated iPSCs and by using a NucleoCounter® NC 200™ (ChemoMetec A/S, Allerod, Denmark) for iPICs. In addition, the inside of the large bag after draining the culture solution was washed twice with 50 mL PBS (FUJIFILM Wako), and the clusters remaining in the bag were collected for cell counting. After cell counting, the cells were fixed and immunochemically stained for flow cytometry analysis.
Cluster size analysis
The size of the cell clusters was measured from the phase-contrast images. For the small-scale bag, the culturing area was divided into 55 compartments with ~ 1 cm2 area to analyze the relationship between the position inside the bag and the cluster size (Supplementary Fig. S1). One image of the center of each compartment was taken. The field of view of one image was 2.88 mm × 2.16 mm. The diameters of the clusters were measured using the image processing software, WinROOF™ (MITANI Corporation, Tokyo, Japan). The outline of a cell cluster was extracted, and the diameter of the circle corresponding to the projected area was defined as the diameter of the cluster. The average diameter was calculated from all the clusters shown in an image.
To investigate the size distribution of the collected clusters, the entire harvested culture solution was transferred to a 10 cm dish or a T-225 flask, and images of the clusters dispersed and settled on the bottom surface were obtained. Multiple images were taken while repositioning so that the areas did not overlap. The field of view of one image was 2.88 mm × 2.16 mm for undifferentiated iPSCs and 3.35 mm × 2.63 mm for iPICs. The diameter of the clusters was measured using WinROOF™. The diameters of 600 clusters from each small bag and the bioreactor were measured. For the large bag, the 600-cluster measurement was repeated three times to obtain a value for 1800 clusters.
Flow cytometry analysis
Cells were fixed and permeabilized with BD Cytofix/Cytoperm™ (BD Corporation, Franklin Lakes, NJ, USA). The cells were stained for various intracellular markers for analysis on a BD LSRFortessa™ X2037. The primary antibodies used in this study are listed in Supplementary Table S2.
Data analysis and statistics
The Dunnett’s multiple-comparison test was performed at a statistical significance level of P < 0.05. All statistical analyses were performed using the Statistical Analysis System v9.3 (SAS Institute, Cary, NC, USA).
