Dialysis device for high-density suspension culture
The dialysis-culture system included upper and lower dialysate compartments (Fig. 1a). A 40-µm mesh bottom-cell strainer (PluriSelect, Leipzig, Germany) was used as the upper culture compartment, with this insert modified by cutting and removing the bottom mesh layer. To selectively permeate nutrition or waste products, we affixed a 12-kDa MWCO Spectra/Por 4 dialysis membrane (Spectrum Chemical, New Brunswick, NJ, USA) to the bottom side of the strainer using alkyl-α-cyanoacrylate-based surgical-grade tissue adhesive (Aron Alpha A; Daiichi Sankyo, Japan). The upper compartment was then placed in six-deep well plates (Corning, NY, USA) as dialysate-compartment inserts. As a control condition, the cell strainer was directly affixed to the bottom surface of six-well untreated plates (Iwaki, Tokyo, Japan) using Aron Alpha A tissue adhesive (Daiichi Sankyo). All devices were sterilized using an ethylene oxide gas sterilizer before use. The detailed device construction was explained in Supplementary method 1.
Permeability test in cell-free conditions
The effect of FP003 on device performance was measured by glucose and lactic acid measurements during a 12 h penetration test under cell-free conditions. Low-glucose DMEM (2 mL; Sigma-Aldrich, St. Louis, MO, USA) containing 0.2% FP003 and 0.8 g/L lactic acids (Sigma-Aldrich) was added to the upper culture compartment, and 15 mL of high-glucose DMEM (Sigma-Aldrich) was placed in the lower dialysate compartment. The dialysis-culture system was then placed in a 120-rpm rotary shaker. We collected 50-µm samples of the medium every 2 h and measured changes in glucose and lactic acid concentration using a YSI 2950 multipurpose bioanalyzer (YSI, Yellow Springs, OH, USA).
To test the ability of the device to accumulate macromolecules in the upper culture compartment and differences in permeation of differently sized molecules, 2 µg/mL FITC (Sigma-Aldrich) at different molecular weights (4, 10, and 20 kDa) was added to the upper culture compartment. This experiment was performed using DMEM basal medium in both compartments, similar to the penetration test for macromolecules. Medium samples were collected after 12 h, and FITC concentration was measured using a Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Waltham, MA, USA).
Monolayer hiPSC culture
The TkDN-4M hiPSC line was provided by the Stem Cell Bank, Centre for Stem Cell Biology and Regenerative Medicine, University of Tokyo (Tokyo, Japan)56. The 201B7 was provided by the Center of iPS Research and Applications (CiRA), Kyoto University (Kyoto, Japan), and Ff-I01s01 was provided by the Center of iPS Research and Applications Foundation (CiRA Foundation), Kyoto University (Kyoto, Japan). All cells were acclimatized and maintained in vitronectin-coated tissue-culture dishes using a complete supplemented Essential 8 (E8) culture medium (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer instructions. The hiPSCs used in this study were cultured in <30 passages.
LDH release assay
HiPSC aggregates were formed by inoculation of a 2 × 106 single-cell suspension/well in six-well plates for 24 h culture in complete medium supplemented with 0.2% free-fatty acid-bovine serum albumin (BSA) in a 90-rpm rotary shaker. The formed aggregate was transferred to dialysis culture system (D/MR(−)) with or without 0.2% FP003 and cultured with a rotary shaker at 120 rpm. The cells were harvested by sedimentation in 10-mL tubes, and the supernatant was isolated for LDH analysis using an LDH cytotoxic assay kit (Wako Pure Chemical, Osaka, Japan) according to manufacturer instructions. The culture medium containing released LDH isolated from the single-cell hiPSCs suspension that was intentionally treated with 0.2% Tween 20 was used as a positive control. The luminescence of each sample was measured by Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Massachusetts, USA)
Caspase 3/7 activity assay
HiPSC aggregates were formed by inoculating single cells at 2 × 106/well in six-well plates and culturing in a complete medium supplemented with 0.2% free-fatty acid-BSA on a rotary shaker at 120 rpm for 24 h. The formed aggregates were transferred to the dialysis culture system (D/MR(−)) with or without 0.2% FP003 and cultured on a rotary shaker at 120 rpm for 24 h. Afterward, 1:1 ratio of Caspase-Glo® 3/7 reagent (Promega, Wisconsin, USA) was mixed with the aggregates suspension in a 10 ml conical tube (Corning, USA). The mixture was then moved into six-well plates and rotated at 90 rpm at room temperature for 2 h. The serial number of single-cell hiPSCs suspension that was treated with 200 ng/ml Doxorubicin (Sigma-Aldrich) for 24 h was used as a calibrator. The luminescence of each sample was measured by Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Massachusetts, USA).
High-density hiPSC suspension culture
To increase simplicity and reduce costs, the culture medium was divided into two main components. Basal medium included DMEM/F12 (Life Technologies, Carlsbad, CA, USA) supplemented with complex macromolecule nutrition and macromolecule growth factors (Table 1). hiPSC aggregates were formed by inoculation of a 2 × 106 single-cell suspension/well in six-well plates for 24-h culture in complete medium supplemented with 0.2% free-fatty acid-bovine serum albumin in a 90-rpm rotary shaker. On the day of culture, the dialysis-culture insert was placed in six-deep well plates, and the dialysis membrane was activated by pre-wetting the dialysis layer using 2 mL of sterile H2O 15 min to 30 min before starting the culture. After removal of the H2O, 15 mL of DMEM/F12 basal medium was added to the lower dialysate compartments. The hiPSC aggregates were harvested and transferred to the upper culture compartment using 2 mL of DMEM/F12 basal medium along with 0.2 % BSA and 0.2% FP003, which were added according to manufacturer instructions to the upper culture compartments on the first day of culture (Fig. 4b). The hiPSCs expansion was performed on a 120-rpm rotary shaker for 4 days with daily replacement of basal medium in the lower dialysate compartment and supplementation with 2% growth factors in groups not undergoing medium replacement in the upper culture compartment. To simplify the operation in D/MR(−), growth-factor supplementation consisting 100 ng/mL FGF2, 19.4 µg/mL Insulin, 10.7 µg/mL Transferrin, and 2 ng/mL TGFβ-1 was added only in the upper culture compartment every day, and the culture medium was not replaced to preserve the growth factors accumulation. (Fig. 1). As a control group, suspension culture was performed using the same volume in a blocked culture insert with (C/MR(+)) or without (C/MR(–)) manual daily complete medium replacement.
Morphological analysis
Each of the aggregate groups was moved to 12-well plates, and macroscopic and microscopic images were obtained by light microscope (Olympus, Japan), and aggregate diameter was analyzed using ImageJ 1.53 software (National Institutes of Health, Bethesda, MD, USA).
Cell counting
After morphological analysis by light microscopy (Olympus, Tokyo, Japan), the aggregates were collected, centrifuged at 1000 rpm for 3 min, and the supernatant was removed from the tube. To obtain single cells, 1 mL of TrypLE dissociation reagent (Thermo Fisher Scientific) was added to the aggregates and incubated for 10 min to 15 min at 37 °C, followed by homogenization by gentle pipetting. The cells were then diluted 100-fold in phosphate-buffered saline (PBS) and counted using a haemocytometer (Tatai-type; Japan).
Measurement of glucose and lactate concentrations
Glucose and lactate concentrations during the 4-day culture in different high-density configurations were measured by collecting 50-µL samples every 24 hand assessment using a YSI 2950 multipurpose bioanalyzer (YSI, Ohio, USA).
Haematoxylin–eosin staining of cross-sectioned aggregates
Aggregates were collected and fixed with 4% paraformaldehyde (Wako Pure Chemical) in PBS for 1 h at room temperature, washed in PBS, and cultured in 30% PBS-sucrose solution (Wako Pure Chemical) at 4 °C overnight. The sucrose solution was removed, and the aggregates were placed in a cryomold after embedding with Tissue-Tek OCT compound (Sakura, Alphen aan den Rijn, The Netherlands) at −20 °C until hardened. Thin sections (10 µm) were obtained using a cryostat and mounted onto glass slides for haematoxylin–eosin staining.
Measurement of growth-factor concentrations
The FGF2, Insulin, and TGFβ-1 concentrations were measured by Duo Set Elisa Kit (R&D Biosystems, Minessota, USA). A 100-µL solution containing diluted capture antibody was immobilized in each well of 96-well enzyme-linked immunosorbent assay (ELISA) plates and incubated overnight at 37 °C. The plates were then washed with 200 µL of wash buffer, followed by the addition of 300 µL of blocking buffer (reagent diluent) and incubation for 1 h at 25 °C. 100 µL of each medium sample or standard in reagent diluent was plated in each well and incubated for 2 h at room temperature. The plates were washed with wash buffer, and 100 µL/well of the diluted detection antibody (R&D Biosystems) was added and incubated for 2 h at 25 °C. The plates were then washed with wash buffer, and 100 µL of streptavidin-horseradish peroxidase solution (R&D Biosystems) was added and incubated at room temperature for between 20 min at 25 °C. To obtain a color reaction, the plates were washed with wash buffer, and 100 µL of a substrate solution was added to each well, followed by incubation for 20 min at 25 °C dark places. The color reaction was stopped by adding 50 µL/well stop solution. Fluorescence intensity was measured using a Wallace Arvo SX 1420 multilabel counter (PerkinElmer).
Nodal detection was performed using a human Nodal ELISA kit (LSBio, Seattle, WA, USA) according to manufacturer instructions provided with the kit. Fluorescence intensity was measured using a Wallac Arvo SX 1420 multilabel counter (PerkinElmer, MA, USA). Further information on the ELISA kit used in this analysis is listed in Supplementary Table 1.
Analysis of the genetic abnormality
Genomic DNA was isolated using Genomic DNA Purification kit (StemCell Technologies, Vancouver, Canada) followed by qPCR analysis using hPSCs Genetic Analysis kit (StemCell Technologies, Vancouver, Canada) according to manufacturer’s instruction. The genomic DNA amplification and analysis were performed by StepOnePlus kit qPCR (Thermo Fisher Scientific, MA, USA). The genetic abnormality was able to be detected when the copy number of Chr 1q-20q is <1.8 or >2.2 with a p value <0.05.
Directed differentiation assay
The aggregates were dissociated by 30-min incubation in Accutase solution at 37 °C. The cell suspension was then filtered through a 40-µm cell strainer (Corning) and counted using a hemocytometer (Tatai, Japan). The hiPSCs functional assay was performed by using STEMdiff™ Trilineage Differentiation Kit (StemCell Technologies, Vancouver, Canada) to evaluate their capability to differentiate into three germ layers. For ectoderm induction, 4 × 105 hiPSCs per well were plated in matrigel coated-24 well plates using STEMdiff™ Trilineage Ectoderm Medium (StemCell Technologies, Vancouver, Canada) supplemented with 10 µM Y-27632. The medium was replaced daily for another 6 days without Y-27632 (Wako, Japan). For mesoderm induction, 1 × 105 hiPSCs per well were plated in matrigel coated-24 well plates using mTeSR™1 medium (StemCell Technologies, Vancouver, Canada) supplemented with 10 µM Y-27632 (Wako, Japan). The medium was replaced daily by STEMdiff™ Trilineage Mesoderm Medium (StemCell Technologies, Vancouver, Canada) for another 4 days without Y-27632. For endoderm induction, 4 × 105 hiPSCs per well were plated in matrigel coated-24 well plates using mTeSR™1 medium (StemCell Technologies, Vancouver, Canada) supplemented with 10 µM Y-27632 (Wako, Japan). The medium was replaced daily by STEMdiff™ Trilineage Endoderm Medium (StemCell Technologies, Vancouver, Canada) for another 4 days without Y-27632. The gene-expression levels of the transcription markers of each lineage were analyzed by quantitative real-time PCR.
Quantitative real-time PCR
mRNA was isolated using Trizol reagent (Life Technologies) and reverse-transcribed using ReverTra Ace master mix (Toyobo, Osaka, Japan), followed by qPCR analysis using Thunderbird SYBR qPCR mix (Toyobo) according to manufacturer’s instructions. Gene amplification was performed using a StepOnePlus kit qPCR (Thermo Fisher Scientific) following the manufacturer’s instruction. The primer sequences used in this analysis are listed in Table 2.
Alkaline phosphatase activity assay
To detect intracellular alkaline phosphatase, aggregates were harvested and stained with an AP staining kit II (Stemgent, Cambridge, WA, USA), with several modifications for aggregate staining using gentle rotational agitation. After washing with 1× PBST, the aggregates were incubated with 2 mL fixed solution in six-well plates at room temperature for 30 min and then washed with 1× PBST and incubated with 3 mL AP substrate solution in the dark for 90 min at room temperature, followed by washing with 1× PBST. The stained aggregates were observed under a light microscope (Olympus, Japan).
FACS analysis
hiPSC aggregates were harvested and dissociated by 30-min incubation in Accutase solution at 37 °C. The cell suspension was then filtered through a 40-µm cell strainer (Corning) and counted using a hemocytometer (Tatai, Japan). In all, 1 × 106 single cells suspension was permeabilized with 0.5% Triton X for 10 min and fixed with 4% paraformaldehyde for 3 h. Afterwards, the single cells were stained with 1:1000 Alexa Fluor 488 anti-human SSEA-4 Antibody (Biolegend, San Diego, CA, USA). The single cells suspension was washed by 100 rpm centrifugation using PBS. The FACS was performed using Coulter Epics flow cytometer (Beckman Coulter, Brea, CA, USA).
Immunocytochemistry
Aggregates were collected and fixed with 4% paraformaldehyde overnight at 4 °C. To preserve the morphology during cross-sectioning, the aggregates were incubated with an 8% sucrose solution overnight following sample fixation in a cryomold using Tissue-Tek OCT compound (Sakura) and freezing for at least 6 h at −80 °C. A 20-µm thin cross-section was obtained using a Leica CM1850 cryostat (Leica Biosystems, Wetzlar, Germany) and fixed on poly-L-lysine-coated slides (Sigma-Aldrich, Missouri, USA). The sections were then stained using 1:10 dilution of Human Pluripotent Stem Cell 3-Color Immunocytochemistry Kit (R&D Biosystems, USA) in blocking buffer overnight in 4°C and counterstained with 1:1000 nuclear staining solution DAPI (Dojindo, Kumamoto, Japan) at room temperature for 10 min. The section washed with PBS and fluorescence microscopy image was performed by FV3000 Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).
To identify the cytoplasmic alkaline phosphatase, the sections were incubated with 10 µg/mL Mouse anti-Human Alkaline Phosphatase/ALPL Antibody (R&D Biosystems) overnight at 4°C. After washing with PBS, the sections were then incubated with Goat Anti-Mouse IgG Alexa Fluor 488 secondary antibody at room temperature for 3 h and counterstained with 1:1000 nuclear staining solution DAPI (Dojindo, Kumamoto, Japan) for 10 min. The section washed with PBS and fluorescence microscopy image was performed by FV3000 Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan). The detailed information about the antibody used in this study is listed in Supplementary Table 1.
Statistics and reproducibility
The data were obtained from at least three independent experiments using three different hiPSCs lines. Statistical analysis was performed using GraphPad Prism software (v.8.3.0; GraphPad Software, San Diego, CA, USA). Statistical significance was determined by one-way analysis of variance with Tukey’s multiple comparison test. A p < 0.05 was considered significant.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
