HM and TS are employees of Kyokuto Pharmaceutical Industrial Co., Ltd. YY, SG, YO, and KK are employees of Tokyo Electron Limited. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: CT HM YO KK TS SK. Performed the experiments: CT HM YY HI TY. Analyzed the data: CT HM HI TY SG KK TS SK. Contributed reagents/materials/analysis tools: HM YY SG YO KK TS. Wrote the paper: CT HM YO KK TS SK.
Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed “patterned culture”), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed “non-patterned cultures”). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.
Cell therapy using human pluripotent stem cell (hPSC)-derived cells has been used for the treatment of several diseases. Applications include the use of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells for the treatment of acute spinal injury [
All experiments using human cell lines and animals were reviewed and approved by the committee for non-clinical research and the committee for animal experimentation of the Foundation for Biomedical Research and Innovation (FBRI).
In this study, we used a newly developed product, SPM cell culture medium. This is a feeder-independent, serum-free, defined medium for hPSCs and is manufactured by Kyokuto Pharmaceutical Industrial, Japan. It contains 21 amino acids (L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine) as well as 12 vitamins (L-ascorbic acid, cobalamin, biotin, folic acid, I-inositol, niacinamide, d-calcium pantothenate, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, α-tocopherol and 4-aminobenzoic acid). It also includes trace elements, fatty acids, bovine serum albumin (BSA) and growth factors including 100 ng/mL bFGF (Peprotech, London, UK) at the time of preparation. It is stored at 4°C after thawing. The BSA was derived from New Zealand in compliance with the TSE guideline [
The hESC lines KhES-1 (Riken BRC) [
hPSCs in single cell cultures were harvested with TrypLE Select and resuspended with chemically defined freezing medium (STEM-CELLBANKER; TakaraBio, Shiga, Japan) (1 x 106 cells/mL) followed by transfer to cryovials (Iwaki, Tokyo, Japan) using a standard slow-freezing method. Cryovials were placed into a freezing container (Nalgene Cryo 100B0C Freezing Container, Nalgene, NY, USA) and cooled overnight in a -80°C freezer (MDF-U32V, Panasonic). Cells were then transferred to and stored in a -150°C freezer (MDF-1155AT, Panasonic) for at least 1 week before performing thawing experiments. For thawing, cryovials were warmed in a water bath at 37°C until the icy masses disappeared, and cell suspensions were transferred to 15 mL centrifuge tubes (BD Bioscience, MA, USA,) and diluted by addition of 4 mL SPM medium containing 10 μM Rock inhibitor Y-27632 (Nacalai Tesque, Kyoto, Japan). Cells were pelleted by centrifugation (120 x g, 3 min) and resuspended in 1 mL of fresh SPM medium containing 10 μM Y-27632 and seeded in 1 well of rhVTN-N-coated 6-well dishes as single cells and cultured at 37°C, 5% CO2. Media were changed every day.
hPSCs were transferred to ultra-low attachment 6-well plates (Corning, NY, USA) to demonstrate their 3 germ layer differentiation potential by forming embryoid bodies (EBs). Cell clumps were incubated in the culture medium without bFGF and the medium was changed every three days. After 21 days of cultivation, EBs were transferred to 0.1% gelatin-coated 6-well plates. Cells were cultured for another 9 days with the same medium for differentiation, changing medium every three days. Expression of lineage-specific genes was examined by quantitative RT-PCR at the end of culture and cells were immunostained with antibodies for differentiation markers. For teratoma formation assays, 1 x 106 hPSCs that had been cultured for 20 passages in SPM on rhVNT-N-coated dishes were embedded in 100 μL of Matrigel (BD Bioscience) and were transplanted under the epidermal space of the neck of NOD.Cg-
Differentiated cells from embryoid bodies were fixed on dishes with 4% PFA overnight at 4°C, then washed 3 times with PBS(-) for 5 min. Expression of α-SMA, β-tubulin or AFP in cultured cells was detected with the indicated antibody: anti-α-SMA antibody (1:200; V6630, Sigma, Tokyo, Japan); anti-β-tubulin antibody (1:100 T4026; SIGMA); anti-AFP antibody (1:100 MAB1368; R&D Systems, MN, USA). Binding was visualized with a secondary antibody labeled with Alexa Fluor 488 (1:500; A11008, Life Technologies) or Alexa Fluor 594 (1:500; A11062, Life Technologies), and cells were counterstained with DAPI (D1306, Life Technologies). An Olympus IX71 fluorescence microscope was used for fluorescent observation.
Total RNA was extracted using an RNeasy micro kit (74004, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. One μg of total RNA was used to synthesize cDNA with the QuantiTect Reverse Transcription Kit (205311, QIAGEN). Quantitative PCR (qPCR) was performed with TaqMan hPSC Scorecard Panel (A15870, Life Technologies)[
hPSCs were harvested and dissociated to single cells with TrypLE Select. The cells were washed once in a final concentration of 2% human serum albumin (Tanabe-Mitsubishi Pharma, Osaka Japan) in PBS(-). A total of 5 × 105 cells was incubated with the same buffer containing 1/100 volume of Alexa Fluor 647-conjugated anti-SSEA-3 (BD, 561145) or BV421-conjugated anti-TRA-1-60 (562711, BD Bioscience) for 30 min at 4°C. The dead cells were distinguished with 7-amino-actinomycin D (7AAD, 559925, BD Bioscience). The cells were analyzed with a FACS Aria II cell sorter (BD Bioscience) and the data were analyzed using FlowJo software (FlowJo LLC, OR, USA).
G band karyotyping service was provided by Nihon Gene Research Laboratories, Inc., (Sendai, Japan). Briefly, hiPSC were treated with colcemid (Sigma) and were harvested by treatment with 0.25% trypsin/EDTA. Cells were fixed on slides with Carnoy’s solution and soaked in Giemsa stain solution (Merck Millipore). After washing with water, 50 metaphase spreads were screened and 20 of them were evaluated for chromosomal rearrangements by microscopy (Eclipse E600, Nikon, Tokyo, Japan) at 1000x magnification for G-band analysis. For multi-color fluorescein
Micropatterning of culture dishes was achieved by photolithographic technology as follows. The entire bottom of each polystyrene 35-mm dish (BD Bioscience) was coated with a water-soluble PVA-based, UV-sensitive polymer (BIOSURFINE-AWP, Toyo Gosei Co., Ltd., Tokyo Japan) that became insoluble after crosslinking with UV irradiation at 30 mW/cm2 for 1 sec with an MA6 mask aligner (SUSS MicroTec AG, Garching, Germany). The spots for cell culture were masked whereas the area not used for cell culture was not masked, permitting UV exposure. The masked area remained soluble and could be washed with Milli Q water. rhVTN-N was directly coated on the bottom of the polystyrene dish on the masked area by incubating 0.5 μg/cm2 of rhVTN-N for 1 h at room temperature just prior to use. rhVTN-N did not coat the unmasked insoluble PVA after UV irradiation. In this way, we were able to readily prepare rhVTN-N-coated micropatterned dishes.
hPSCs (hESCs/hiPSCs) cultured on SNL feeder cells were harvested either with CTK (clumps) or CTK followed by TrypLE Select (single cells). They were subsequently seeded on rhVNT-N-coated dishes. The first passage of cell clumps was split at ratios of 1:1 to 1:2 and cultured for 4 or 5 days. hPSCs must adapt to their new feeder-free environment and recover from the perturbation of cell growth after the first passage. Then, they were passaged at 1:4 or 1:5 with GCDR for subsequent passages (from the second passage onward). Cells (3–5 x 105) in a single cell suspension were seeded either on 35-mm dishes (the entire surface was coated with rhVNT-N: non-patterned culture) or on 35-mm dishes micropatterned with 199 discrete spots (1 mm in diameter) coated with rhVNT-N (patterned culture) for the first passage and cultured for 4 days. Cells (2 x 105) in a single cell suspension were seeded on either non-patterned dishes or patterned dishes and cultured for 4 days from the second passage onward as their proliferation potential stabilized during their first cultivation on rhVNT-N-coated dishes. TrypLE Select was used for dissociation. The culture protocol is shown in
(Top) hPSCs cultured in cell clumps on feeder cells (SNL) were harvested with CTK to culture in cell clumps on recombinant rhVTN-N-coated dishes with SPM medium by splitting at 1:1 or 1:2 ratios (clump culture). (Middle, Bottom) Alternatively, hPSCs were harvested with CTK followed by TrypLE Select to seed 3 or 4 x 105 single cells on either rhVNT-N fully coated 35-mm dishes (single cell, non-patterned culture) or on rhVNT-N spot-coated 35-mm dishes (single cell patterned culture). hPSCs were cultured for 5 to 7 days and passaged at ratios of 1:4 or 1:5 splits with GCDR in cell clumps. hPSCs (2 x 105 single cells) were seeded on each 35-mm dish for both non-patterned and patterned dishes, cultured for 4 days and dissociated with TrypLE Select.
Morphological observations and flow cytometric analyses showed that hiPSC cell lines PXF#9 and 201B7 and the hESC cell lines KhES-1 and H9 could be cultured in an undifferentiated state in three ways: as cell clumps or as single cells with SPM on rhVTN-N-coated dishes (non-patterned) or on rhVTN-N-coated spots in micropatterned dishes (patterned culture) (
(A) Phase contrast microscopic observation of iPSC cell line PFX#9 at passage 15. (B) Expression of SSEA-3 and TRA-1-60 by flow cytometric analysis of PFX#9 in indicated culture conditions at passage 15. (C) Time course of cell proliferation in patterned dish from days 1 to 4 (left to right). (D) Cell proliferation area that was occupied inrhVTN-N-coated spot area (spot Φ = 1 mm, 0.79 mm2/spot, X axis). Plot also shows the number of spots and their areas (out of 199 rhVNT-N-coated spots, Y axis) on days 1 to 4 (left to right). (E) Microscopic observations of clump cultures, single cell non-patterned or single cell patterned cultures with the higher magnified area in red rectangles at passage 20. A representative undifferentiated clump colony is shown in the upper left photo. Scale bars are appended. (F) Time course (0–100 h) of the area occupied by PFX#9 cells (in mm2) in 5 randomly selected spots (0.79 mm2/spot) measured by captured image analysis software (ImageJ 1.450, National Institutes of Health, Bethesda, MD, USA) every hour. Average of cell occupation area at every hour is plotted as a dot. The dot graph shows representative results from 3 independent trials. (G) Cell density (cells/mm2) of PFX#9 in single cell non-patterned or in single cell patterned culture was calculated by dividing harvested cell number by 962 mm2 (35-mm non-patterned culture dish) or dividing harvested cell number by 156 mm2 (total 199 spots of 1 mm diameter in 35-mm patterned culture dish). The results were obtained by scoring harvested cell numbers from 18 passages of indicated cultures and are shown as a bar (mean) with error bar (standard deviation). The significance of difference between 2 groups, p = 1.45 x 10−9. Representative results of 3 independent trials are shown. (H) Growth curve of PFX#9 in non-patterned culture, patterned culture or clump culture are shown in logarithmic graphs. PFX#9 cells in patterned culture or non-patterned culture were passaged every 4 days and in clump culture on feeder-free every 6 days and on feeder (SNL) every 5 days respectively.
(A) Gene expression profiles of PFX#9 cells in the indicated culture condition (clump culture, single cell non-patterned culture or single cell patterned culture) before (undifferentiated state) and after induction of differentiation via embryoid body (EB) formation. Average of gene expression values for self-renewal (undifferentiated state), ectoderm-, mesoderm- or endoderm-related genes is shown in comparison with reference standards of TaqMan hPSC Scorecard Panel (Life Technology). (B) EB formation at day 14 from PFX#9 cells (top left). EB attached to culture dish and continued to differentiate (top right). Cells were then stained with antibodies against β-tubulin (ectoderm), α-SMA (mesoderm), AFP (endoderm) and DAPI. (C) Tissue section of teratoma in NOG mouse generated by inoculating PFX#9 cells maintained in cell clumps is shown after staining with HE. Three germ layers of tissue consisting of neural rosette (ectoderm), muscle/cartilage (mesoderm) and gut-like epithelium (endoderm) are observed. Scores in Tables are visualized in bar graph below.
A greater yield of hPSCs was harvested from non-patterned cultures than from patterned cultures (
It is possible that uneven cell growth on non-patterned cultures as evidenced by distinctively different cell morphologies in the rim and in the center of the colony may not be favorable for the maintenance of the self-renewal potential of hPSCs as a population in long-term culture (
hPSCs that were cultured with SPM on VTN-N-coated dishes as single cells and clumps were examined for their differentiation potential by forming embryoid bodies (EB). The three germ layer differentiation potentials of PFX#9 cells (
Karyotype of PFX#9 maintained with SPM on rhVNT-N-coated dish (single cell patterning culture) was analyzed by mFISH at passage 5 (left). PFX#9 from single cell non-patterned culture underwent G-Band analysis at passage 15 (right).
KhES-1 cells that were cultured with SPM in single cell suspensions were cryopreserved with chemically defined freezing medium (STEM-CELLBANKER). After thawing, they grew robustly in single cell suspension with SPM on VNT-N-coated non-patterned dishes. Gene expression profiles of cells 3 passages after thaw are shown in
(A) 1x106 KhES-1 cells in single cell suspension from single cell non-patterned culture were cryopreserved with freezing medium, STEMCELL BANKER. The cells were thawed and cultured for 3 passages as single cells before examining the gene expression profile with TaqMan hPSC Scorecard. (B) Phase contrast image of KhES-1 cells cultured with SPM as single cells on rhVNT-N-coated dishes 3 passages after thaw. (C)Expression of SSEA-3 and TRA-1-60 on KhES-1 cells before and 5 passages after thaw was evaluated by flow cytometry. (D) Growth curve of KhES-1 in single cell non-patterned flat culture after thaw (blue line) was examined in comparison with cells in single cell non-patterned culture without cryopreservation (red line). The cryopreserved KhES-1 showed the same proliferation rate as the control in the second passage after thaw. Scores in Tables are visualized in bar graph below.
In this report, we have shown that hPSCs can be cultured with a defined medium, SPM, on a very cost-effective ECM substrate (rhVNT-N) in the form of cell clumps or single cells (non-patterned or patterned). One of the features of SPM is that hPSCs cultured on feeders can be easily adapted to feeder-free conditions in the form of cell clumps or single cells without complicated sequential adaptation procedures. Such procedures may become an issue when chemically defined media and Vitronectin-N are used for cultivation and coating materials of dishes. SPM is also optimized to culture hPSCs in ECM-coated patterned culture dishes. SPM contains BSA produced in New Zealand, and it meets safety criteria as stated in the TSE guideline (EMA/410/01 rev.3) [
Photolithographic technology has been used for manufacturing patterned culture dishes. This technology can generate a patterned dish of any size and any shape with very sharp boundaries at micrometer scales. Such patterned dishes have been used for culturing mesenchymal stem cells [
A remaining concern for safety issues in clinical application of micropatterned dish is the possibility that PVA might elute into the culture medium and cause toxic effects to cells in culture. However, the growth rates of hPSCs in PVA-coated dish are comparable to those in non-coated dishes per culture area and no genetic abnormality was reported in several cell lines that have been tested for up to 20 passages. We believe that we will be able to prepare a toxicological test package for PVA prior to the application for clinical trial.
The passage of hPSCs as single cells was advantageous compared to passage via cell clumps (classical method). Some hPSC clumps include differentiated cells, and such differentiated clumps can attach to ECM after passage and proliferate. It is necessary to pick up and remove such differentiated clumps daily to maintain the culture’s undifferentiated state. We speculated that differentiated cells in single cell suspension could no longer attach to ECM with the same affinity, probably due to changes in their adhesion molecule expression profile and could be washed out at every medium change. In contrast, differentiated cells in clumps (differentiated clump) could remain attached to the ECM due to the strong affinity of the remaining undifferentiated cells for the ECM. To prove this speculation, a novel tracing method to mark cells early in differentiation was needed. Alternatively, we conducted comparison studies of gene expression profiles of cells from several culture conditions. Specifically, we used the qRT-PCR score card panel to examine the following: (a) cells from clumps that included differentiated cells (differentiated clumps), (b) cells from clumps lacking differentiated cells (undifferentiated clumps, after removal of differentiated clumps), (c) cells from single cell culture that were passaged from single cells for several passages, and (d) cells from single cell culture that were passaged from differentiated clumps (a). We found that cells from differentiated clumps most likely differentiated toward ectoderm among the three germ layers (
Patterning hPSCs in hundreds of round shaped spots provides several advantages over non-patterned single cell culture. For example, patterned culture supports controlled cell growth and the maintenance of the undifferentiated state of hPSCs by controlling the area for cell culture at around 1 mm in diameter, while cells cultured in a non-patterned dish grow unevenly and may lose the potential for pluripotency after many passages (
In conclusion, we have introduced a new hPSCs culture system, combining the use of a defined culture medium (SPM) and rhVNT-N-coated patterned dishes. This culture system provides a new method that can assure the quality of cultured hPSCs and the technological basis for the design of fully automated hPSCs mass closed culture system in the near future.
(A) Phase contrast microscopic observation of KhES-1 cells under the indicated culture conditions at passage 15. (B) Expression of SSEA-3 and TRA-1-60 on KhES-1 determined by flow cytometry after culture under the indicated conditions at passage 15. (C) Phase contrast microscopic observation of cell cultures (clump, single cell non-patterned and single cell patterned) for ESC H9 cells and iPSC 201B7 cells. (D) Densities (cells/mm2) of KhES-1 cells in single cell non-patterned (non-patterned) or in single cell patterned (patterned) cultures were calculated by dividing harvested cell number by 962 mm2 (35-mm non-patterned culture dish) or dividing harvested cell number by 156 mm2 (total 199 spots of 1 mm diameter in 35-mm patterned culture dish). The result was obtained by scoring harvested cell numbers from 20 passages of the indicated culture and shown as a bar (mean) with error bar (standard deviation). The significance of difference between 2 groups: p = 3.6 x 10−16. Growth curve of KhES-1 cells (E), 201B7 cells (F) or H9 cells (G) in single cell non-patterned (non-patterned), single cell patterned (patterned) cultures or clump cultures. X-axis represents days of culture. PCSs in patterned culture or non-patterned culture were passaged every 4 days and in clump culture on feeder-free every 6 days and on feeder (SNL) every 5 days.
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(A) Flow cytometric analysis of PFX#9 cells for the expression of SSEA-3 and TRA-1-60 cultured in non-patterned dishes. Gated population was sorted at passage 20 (left) and reanalyzed at passage 21 (middle) and 24 (right). (B) Patterned cell colony (spot) was removed by pipetting for cell sampling.
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Gene expression profiles of KhES-1 cells under the indicated culture conditions (clump, single cell non-patterned or single cell patterned) before (undifferentiated state) and after induction of differentiation via embryoid body (EB) formation. Average gene expression of self-renewal (undifferentiated state), ectoderm-, mesoderm- or endoderm-related genes is compared with reference standards of TaqMan hPSC Scorecard Panel (Life Technology). Scores in Table are visualized in bar graph below.
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(A) a: H9 cells cultured in a clump on a VTN-N-coated dish started to differentiate. Cell clumps having morphology of differentiation are marked as red square, undifferentiated cell clump is marked as blue square. b: undifferentiated colonies after removing differentiated colonies. c: single cell flat non-patterned culture on VTN-N. (B) Gene expression profile of H9 with TaqMan hPSC Scorecard Panel to detect trend for differentiation of hPSCs in culture conditions stated in A. Relative gene expression of two representative genes for self-renewal, ectoderm, mesoderm or endoderm differentiation are presented as bar graph. (C) a: KhES-1 cells cultured in a clump on VTN-N coated dish started differentiation (indicated by white arrows). b: Undifferentiated colonies in clump culture after removing differentiated colonies. c: Non-patterned culture after 15 passages. d: Cells passaged from “a” were dispersed into single cells and seeded on Vitronectin-N coated non-patterned dish. (D) Gene expression profile of KhES-1 cells with TaqMan hPSC Scorecard Panel to detect trend for differentiation in hPSCs in culture conditions stated in “C”. Relative gene expression of two representative genes for self-renewal, ectoderm, mesoderm or endoderm differentiation are presented as bar graph.
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(A) Karyotype of KhES-1 cells from patterned culture at passage 5 (P5) by mFISH. (B) Karyotype of KhES-1 from single cell non-patterned culture at P15 by G-Band analysis. (C) Karyotype of KhES-1 cells in clump culture at P22. (D) Karyotype of PFX#9 cells in clump culture at P25 by G-band analysis.
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Cell proliferation of PFX#9 cultured with SPM on rhVTN-N-coated patterned dish was observed in a time lapse (up to 96 h).
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We thank Naoki Nishishita and Marie Muramatsu of FBRI for technical support for cell culture.