- Methodology article
- Open Access
Establishment of automated culture system for murine induced pluripotent stem cells
- Hiroyuki Koike†1,
- Koji Kubota†2,
- Keisuke Sekine†1,
- Takanori Takebe1,
- Rie Ouchi1,
- Yun-Wen Zheng1,
- Yasuharu Ueno1,
- Naoki Tanigawa2 and
- Hideki Taniguchi1Email author
© Koike et al.; licensee BioMed Central Ltd. 2012
- Received: 9 April 2012
- Accepted: 10 October 2012
- Published: 5 November 2012
Induced pluripotent stem (iPS) cells can differentiate into any cell type, which makes them an attractive resource in fields such as regenerative medicine, drug screening, or in vitro toxicology. The most important prerequisite for these industrial applications is stable supply and uniform quality of iPS cells. Variation in quality largely results from differences in handling skills between operators in laboratories. To minimize these differences, establishment of an automated iPS cell culture system is necessary.
We developed a standardized mouse iPS cell maintenance culture, using an automated cell culture system housed in a CO2 incubator commonly used in many laboratories. The iPS cells propagated in a chamber uniquely designed for automated culture and showed specific colony morphology, as for manual culture. A cell detachment device in the system passaged iPS cells automatically by dispersing colonies to single cells. In addition, iPS cells were passaged without any change in colony morphology or expression of undifferentiated stem cell markers during the 4 weeks of automated culture.
Our results show that use of this compact, automated cell culture system facilitates stable iPS cell culture without obvious effects on iPS cell pluripotency or colony-forming ability. The feasibility of iPS cell culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.
- Induced pluripotent stem (iPS) cell
- Automated cell culture system (ACCS)
- CO2 incubator-scale
Since the development of induced pluripotent stem (iPS) cells, their use has been anticipated in various areas, including regenerative medicine and drug discovery [1, 2]. The advantages of iPS cells include their multipotency, and they can be established from individuals, allowing the creation of pluripotent stem cells from any donor with any genetic background [3–5]. Hepatocytes derived from iPS cells are useful in evaluating drug sensitivity and toxicity and also in understanding highly variable pathological conditions [6, 7]. Obtaining mature cells like hepatocytes for drug development is impeded by short supply, high cost, and variable quality . To solve these problems, directed differentiation of iPS cells into somatic lineages in vitro has been attempted extensively [9–11]. Once the creation of mature, functional hepatocytes from iPS cells is successful, the development of stable supply system of iPS cells will be necessary for their translation to these applications.
In this regard, it is important to establish an automated cell culture system (ACCS), which facilitates stable and standardized iPS cell culture and enables researchers to handle sufficient quantities of iPS cells. To date, such ACCS are difficult to handle in a space-limited research laboratory. Therefore, iPS cell culture is still dependent on manual techniques. Cell culture conditions, such as duration of treatment with cell detachment solution, fluid flow, and seeding cell density, are difficult to control. To preserve the pluripotency of stem cells, culture requires precise control by highly skilled operators because complicating factors cause difficulty in scaling-up the stem cell culture system [12, 13]. To establish an ACCS for iPS cells in a limited space, it is necessary to standardize cell culture operations.
In this study, we describe an ACCS that enables automated iPS cell culture in a commonly used cell culture incubator. We standardized the maintenance of iPS cell culture and demonstrated long-term subculture of iPS cells using a device that automates both the positioning of seeding cells on feeder cells and their passaging.
Automated induced pluripotent stem cell culture system
ACCS was designed to be as compact as possible for industrial use (Figure 1B). It requires no centrifugation step for cell collections. As described above, iPS cells need careful maneuver; therefore, we suggest the need for an automated system for mass production in a biomedical or pharmaceutical plant. This small system is suitable for commercial adoption because it can be incorporated flexibly into a plant.
Cell culture in a disposable cell chamber (DCC)
Passaging of induced pluripotent stem cells in the automated culture device
Detached iPS cells, suspended with shaking incubation in the cell detachment unit after a 30 min-protease treatment, dissociated into single cells. After dissociation, cells were collected and plated in new DCCs through a tube with inserted needle. Morphological analysis showed that iPS cells were successfully dissociated into single cells after the passaging process (Figure 3A). No difference was observed in morphology and size of mouse iPS cell colonies between the DCC culture and manual culture in a dish (Figure 3B,C). Moreover, the iPS cell colonies in DCC showed intense Nanog-GFP fluorescence after automated culture (Figure 3B).
Morphological analysis of induced pluripotent stem cells over multiple passages in the automated culture device
Expression of pluripotency-associated markers over multiple passages in automated culture device
To evaluate whether the iPS cells cultured with ACCS have maintained differentiation property, embryoid bodies (EB) were generated after period of culture. EB were plated on the dish and cultured for additional 2days. Expression of the differentiation markers was examined by qPCR. Gene expression of ectoderm, mesoderm, and endoderm markers were increased respectively. This result indicated that iPS cells cultured with ACCS have maintained the multi-lineage differentiation potential.
In this study, we established an automated culture that enabled multiple passages of mouse iPS cells without affecting their pluripotency. We believe that ACCS established in this study has met the requirement of standardized iPS cell quality, which was verified by morphology, proliferation, and expression of markers for undifferentiated cells. In a recent study, it was demonstrated that mouse iPS cells are closer to the ground state than human pluripotent stem cells and human iPS cells can be put back in the ground state under particular culture conditions . Unlike mouse iPS cells, human iPS cells must passage as cell aggregates, and dissociation into single cells leads to differentiation. It will be useful to propagate human iPS cells with ACCS by optimization of parameters, such as flow rate and disperse reaction time. It will also be possible that human iPS cells cultured with ACCS by simply applying mouse iPS cell condition establish in this study under the presence of ROCK inhibitors which enhance human iPS cell survival as a single cell [26, 27]. This automated culture system, which produced a steady supply of pluripotent stem cells of constant quality, will serve as a base technology for the creation of pluripotent stem cells in bulk. For the application of these stem cells in advanced medicine, the next challenge will be to develop a method to efficiently induce differentiation of pluripotent stem cells with an automated culture system.
Automatic cell culture
ACCS consisted of three main components: a computer including software, control box, and cell culture system (Figure 1). The cell culture system is operated with the computer through the control box. The cell culture system is a cell-passage machine in an incubator comprising a cell detachment device, a cell chamber stack tower, and a solution exchange system. Cells that were pre-cultured in DCC were placed in the stack tower and detached mechanically in a protease solution (TrypLE Express, Life technologies Co.) after a phosphate-buffered saline wash. After detachment, the cells were dissociated by passing through a needle inserted into the DCC cell suspension diluted with culture medium, then dispensed into new DCCs in the stack tower. All steps after pre-culture were processed automatically by ACCS. Oxygen concentration in DCCs was measured using an optical oxygen sensor (Microx TX3, PreSens Precision Sensing GmbH).
Culture of mouse induced pluripotent stem cells
The Nanog-GFP mouse iPS cells line iPS-MEF-Ng-20D-17 established as described previously  were cultured for 4 weeks using the automated culture device. Chambers coated with 0.1% gelatin were seeded automatically with mitomycin-inactivated mouse embryonic fibroblast (MEF) isolated from non-visceral tissues of day E13.5 mouse embryos 6 h prior to every iPS cell passaging from another chamber loaded MEF suspension. All animal experiments were approved by the Ethics Committee of Yokohama City University and were conducted according to the institutional guidelines. We plated iPS cells at a density of 10,000 cells/cm2. The culture medium comprised of Dulbecco’s modified Eagle’s medium (GIBCO®; Life Technologies) containing 15% Knockout™ Serum Replacement (Life technologies), GlutaMAX™ (Life technologies), non-essential amino acids, and β-mercaptoethanol. Repeated passaging was performed every 2 days by automated method. In the passage process, mouse iPS cells were dissociated with TrypLE™ Express (Life technologies) as mixture with MEFs and dispersed into single cells and diluted by fresh medium before seeding. The dissolved oxygen concentration in DCC was kept for 2 days cultivation, so that iPS cell growth would not be affected. For automated culture, centrifugation steps were performed outside the automated device. After every passage, cells were subjected to morphological evaluation. For quantification of iPS cell growth, fluorescent images were processed using IN Cell Developer Toolbox software (GE Healthcare, Fairfield, CT, USA), and the sizes of iPS cell colony were measured as GFP positive area.
Adherent cultures were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and rinsed with phosphate-buffered saline. For SSEA-1 and SSEA-3 staining, cells were pre-incubated with 10% goat serum (Sigma-Aldrich, Missouri, USA) for 1 h at RT. Cells were incubated overnight at 4°C with the appropriate concentration of primary antibodies in 1% goat serum solution (mouse-anti-SSEA-1 1:200, Santa Cruz Biotechnology; rat-anti-SSEA-3 1:200, Millipore). Antigens were visualized using the appropriate fluorophore-conjugated secondary antibodies (Alexa Fluor® 555 goat anti-mouse IgG (H+L), 1:500, Life Technologies; Alexa Fluor® 488 goat anti-rat IgG, 1:500, Life Technologies). Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (1:2,000 in Apathy’s mounting medium, Sigma-Aldrich) for 5 min. Fluorescence images were captured using a fluorescence microscope (IX-71, Olympus, Tokyo, Japan). For evaluation of cell dissociation, cells were fixed with 4% PFA for 10 min at RT and then washed with phosphate-buffered saline. Phase contrast images and fluorescence images of GFP-expressing cells were captured using a wide-field fluorescence microscope (DMI6000B, Leica Microsystems). We checked that Nanog-GFP fluorescence in iPS cells was diminished during the fixation and immunocytochemical staining operations and was negligible compared to the positive signal.
Alkaline phosphatase assay
For alkaline phosphatase staining, cells were fixed with citrate solution with added acetone and formaldehyde for 30 s at RT, rinsed with deionized water, and treated with alkaline dye mixture (Sigma-Aldrich).
EB formation and differentiation
For EB formation, hanging drop method was performed. Hanging drops (one droplet [30μl] contains 1000 iPS cells cultured with ACCS) were placed on the lid of a 100 mm dish filled with phosphate-buffered saline (PBS) and cultured for 7days. EBs were transferred to attachment cultures for further differentiation.
Quantitative analysis using real-time PCR (qPCR)
qPCR Primers Used in the Present Study
Data were presented as the mean ± SD. Statistical differences were analyzed using the Mann–Whitney U test. P values less than 0.05 were considered significant.
We are grateful to Dr. Yuka Suzuki and Ms. Atsuko Kamohara for the technical assistance. This work was supported in part by grants from the Strategic Promotion of Innovative Research and Development (S-innovation, 62890004) of the Japan Science and Technology Agency (JST). This work was also supported in part by the Grants-in-Aid (21249071, 20591532) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan; by the Specified Research Grant from the Takeda Science Foundation; and by a grant from the non-profit organization, Japan Insulin Dependent Diabetes Mellitus (IDDM) network.
- Takahashi K, Yamanaka S: Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006, 126: 663-676. 10.1016/j.cell.2006.07.024.View ArticleGoogle Scholar
- Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S: Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007, 131: 861-872. 10.1016/j.cell.2007.11.019.View ArticleGoogle Scholar
- Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ: Disease-specific induced pluripotent stem cells. Cell. 2008, 134: 877-886. 10.1016/j.cell.2008.07.041.View ArticleGoogle Scholar
- Pick M, Stelzer Y, Bar-Nur O, Mayshar Y, Eden A, Benvenisty N: Clone- and gene-specific aberrations of parental imprinting in human induced pluripotent stem cells. Stem Cells. 2009, 27: 2686-2690. 10.1002/stem.205.View ArticleGoogle Scholar
- Jin ZB, Okamoto S, Osakada F, Homma K, Assawachananont J, Hirami Y, Iwata T, Takahashi M: Modeling retinal degeneration using patient-specific induced pluripotent stem cells. PLoS One. 2011, 6: e17084-10.1371/journal.pone.0017084.View ArticleGoogle Scholar
- Xie HG, Kim RB, Wood AJ, Stein CM: Molecular basis of ethnic differences in drug disposition and response. Annu Rev Pharmacol Toxicol. 2001, 41: 815-850. 10.1146/annurev.pharmtox.41.1.815.View ArticleGoogle Scholar
- Burridge PW, Thompson S, Millrod MA, Weinberg S, Yuan X, Peters A, Mahairaki V, Koliatsos VE, Tung L, Zambidis ET: A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS One. 2011, 6: e18293-10.1371/journal.pone.0018293.View ArticleGoogle Scholar
- Hay DC, Pernagallo S, Diaz-Mochon JJ, Medine CN, Greenhough S, Hannoun Z, Schrader J, Black JR, Fletcher J, Dalgetty D, et al: Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism. Stem Cell Res. 2011, 6: 92-102. 10.1016/j.scr.2010.12.002.View ArticleGoogle Scholar
- Greenhough S, Medine CN, Hay DC: Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening. Toxicology. 2010, 278: 250-255. 10.1016/j.tox.2010.07.012.View ArticleGoogle Scholar
- Iwamuro M, Komaki T, Kubota Y, Seita M, Kawamoto H, Yuasa T, Shahid JM, Hassan RA, Hassan WA, Nakaji S, et al: Hepatic differentiation of mouse iPS cells in vitro. Cell Transplant. 2010, 19: 841-847. 10.3727/096368910X508960.View ArticleGoogle Scholar
- Inamura M, Kawabata K, Takayama K, Tashiro K, Sakurai F, Katayama K, Toyoda M, Akutsu H, Miyagawa Y, Okita H, et al: Efficient generation of hepatoblasts from human ES cells and iPS cells by transient overexpression of homeobox gene HEX. Mol Ther. 2011, 19: 400-407. 10.1038/mt.2010.241.View ArticleGoogle Scholar
- Veraitch FS, Scott R, Wong JW, Lye GJ, Mason C: The impact of manual processing on the expansion and directed differentiation of embryonic stem cells. Biotechnol Bioeng. 2008, 99: 1216-1229. 10.1002/bit.21673.View ArticleGoogle Scholar
- Gobbel GT, Kondziolka D, Fellows-Mayle W, Uram M: Manual vs automated delivery of cells for transplantation: accuracy, reproducibility, and impact on viability. Neurosurgery. 2010, 67: 1662-1668. 10.1227/NEU.0b013e3181f9b1e2. discussion 1668View ArticleGoogle Scholar
- Terstegge S, Laufenberg I, Pochert J, Schenk S, Itskovitz-Eldor J, Endl E, Brustle O: Automated maintenance of embryonic stem cell cultures. Biotechnol Bioeng. 2007, 96: 195-201. 10.1002/bit.21061.View ArticleGoogle Scholar
- Serra M, Brito C, Sousa MF, Jensen J, Tostoes R, Clemente J, Strehl R, Hyllner J, Carrondo MJ, Alves PM: Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J Biotechnol. 2010, 148: 208-215. 10.1016/j.jbiotec.2010.06.015.View ArticleGoogle Scholar
- Oh SK, Kim HS, Park YB, Seol HW, Kim YY, Cho MS, Ku SY, Choi YM, Kim DW, Moon SY: Methods for expansion of human embryonic stem cells. Stem Cells. 2005, 23: 605-609. 10.1634/stemcells.2004-0297.View ArticleGoogle Scholar
- Di Stefano B, Buecker C, Ungaro F, Prigione A, Chen HH, Welling M, Eijpe M, Mostoslavsky G, Tesar P, Adjaye J, et al: An ES-like pluripotent state in FGF-dependent murine iPS cells. PLoS One. 2010, 5: e16092-10.1371/journal.pone.0016092.View ArticleGoogle Scholar
- Okita K, Ichisaka T, Yamanaka S: Generation of germline-competent induced pluripotent stem cells. Nature. 2007, 448: 313-317. 10.1038/nature05934.View ArticleGoogle Scholar
- Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science. 1998, 282: 1145-1147.View ArticleGoogle Scholar
- Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP: Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci USA. 1995, 92: 7844-7848. 10.1073/pnas.92.17.7844.View ArticleGoogle Scholar
- Stadtfeld M, Maherali N, Breault DT, Hochedlinger K: Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell. 2008, 2: 230-240. 10.1016/j.stem.2008.02.001.View ArticleGoogle Scholar
- Brambrink T, Foreman R, Welstead GG, Lengner CJ, Wernig M, Suh H, Jaenisch R: Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell. 2008, 2: 151-159. 10.1016/j.stem.2008.01.004.View ArticleGoogle Scholar
- Palmqvist L, Glover CH, Hsu L, Lu M, Bossen B, Piret JM, Humphries RK, Helgason CD: Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells. 2005, 23: 663-680. 10.1634/stemcells.2004-0157.View ArticleGoogle Scholar
- Chan EM, Ratanasirintrawoot S, Park IH, Manos PD, Loh YH, Huo H, Miller JD, Hartung O, Rho J, Ince TA, et al: Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol. 2009, 27: 1033-1037. 10.1038/nbt.1580.View ArticleGoogle Scholar
- Hanna J, Cheng AW, Saha K, Kim J, Lengner CJ, Soldner F, Cassady JP, Muffat J, Carey BW, Jaenisch R: Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci USA. 2010, 107: 9222-9227. 10.1073/pnas.1004584107.View ArticleGoogle Scholar
- Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Muguruma K, Sasai Y: A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007, 25: 681-686. 10.1038/nbt1310.View ArticleGoogle Scholar
- Xu Y, Zhu X, Hahm HS, Wei W, Hao E, Hayek A, Ding S: Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc Natl Acad Sci USA. 2010, 107: 8129-8134. 10.1073/pnas.1002024107.View ArticleGoogle Scholar
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