Automated induced pluripotent stem cell culture system

In the development of a mass production system for iPS cells, it is desirable that a uniform quality of cultured cells is maintained for a long-term. Stem cell culture is dependent on manual processes performed by skilled technicians at all stages [12]. Therefore, quality and safety is limited by the technique and skill of the worker [14]. In particular, iPS cells are very difficult to handle, as they have a tendency to change state easily upon each passage or operation because of which it is difficult to obtain consistent results with iPS cells. Therefore, it is necessary to automate the operations for a series of cultures. We developed a culture system capable of providing a stable supply of normal mouse iPS cells using ACCS (Figure 1A). This device automates stem cell culture, allows optimization, and enhances safety. ACSS automatically performed injection/aspiration of cell and liquid by the rotation of peristaltic pumps and the switching of the flow paths. Detachment system could dissociate the adherent cells by giving vibration to the culture chamber. Parameters such as fluid flow rate, volume, dilution ratio, enzymatic reaction time, and detachment time, were optimized and can be controlled through external PC.

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)

Mouse iPS cell cultivation was carried out in chamber uniquely designed for ACCS (Figure 2A). A disposable cell chamber (DCC) was a closed chamber with a total volume of 4.65 ml. We dispensed 1 × 10⁵ iPS cells into each DCC. Oxygen demand is a critical factor in stem cell culture; therefore, tight control of the culture environment was necessary [15]. In DCC, air exchange was performed through an aerated filter on top face. Dissolved oxygen levels in the medium in the chamber were maintained between 50–80% of the saturation of air after 2 days of cell culture (Figure 2B), comparable to conventional cell culture using a dish (data not shown).

Passaging of induced pluripotent stem cells in the automated culture device

In iPS cell culture, cell dissociation is the most important step. Usually, stem cell dispersion during manual passage is carried out using enzymatic and mechanical methods that are least disruptive to pluripotency [16]. iPS cells should remain as single cells from suspension to passage because a majority of the cells would not persist in a pluripotent state if they are not dissociated into single cells [17]. We optimized the method using both enzyme treatment and mechanical dissociation to achieve single cell automatic passage (Figure 3A). To monitor iPS cell pluripotency during cell culture, we utilized the fact that iPS cells are derived from transgenic mice line in which green fluorescent protein (GFP) is under the control of the Nanog promoter [18]. The Nanog-GFP transgenic mice express GFP in a pattern that is identical to that of Nanog, a known pluripotent marker.

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

The growth characteristics of iPS cells propagated in ACCS were comparable to those propagated manually. Repeated medium changes and passaging every 2 days by the automated method so that did not compromise the integrity of the iPS cell culture. No microbial contamination was observed, even after 4 weeks. Throughout the long-term culture, there were no morphological changes in mouse iPS cells cultured by the automated culture system (Figure 4A). No significant change was observed in Nanog-GFP positive areas and frequency during culture, suggesting that the undifferentiated state, growth rate and viability of iPS cells was maintained for over 17 passages, i.e., about 4 weeks of culture (Figure 4B, C).

Expression of pluripotency-associated markers over multiple passages in automated culture device

Next we examined the expression of pluripotent markers in iPS cells in automated culture over a period of 4 weeks using immunohistochemistry, alkaline phosphatase assay, and quantitative Reverse Transcription Polymerase Chain Reaction (qPCR). Stage-specific embryonic antigen (SSEA)-3, which is expressed in human embryonal carcinoma cells, is widely used for the identification of stem cells [19, 20], whereas SSEA-1 is expressed in undifferentiated mouse iPS cells [21, 22]. We showed that during 4 weeks of automated culture, the expression of SSEA-1 and SSEA-3 was maintained in mouse iPS cell cultures (Figure 5A). Undifferentiated iPS cells are known to exhibit tissue nonspecific alkaline phosphatase activity [22]. Using alkaline phosphatase assay we observed that the alkaline phosphatase activity was maintained for over 4 weeks (Figure 5B). Furthermore, we examined the expression of pluripotency markers by quantitative qPCR. Nanog, Oct3/4, and Sox2 mRNAs have all been identified as undifferentiated cell marker genes in iPS and embryonic stem (ES) cells [23], while CD13 is expressed in fibroblasts [24]. After 4 weeks of automated culture, expression of these marker mRNAs was equivalent to the pre-culture levels (Figure 5C). These results indicate that the automated cell culture device did not alter gene expression levels for pluripotency-associated transcription factors, indicating that maintenance culture of mouse iPS cells is possible for long-term.

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.

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 [18] 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.

Immunocytochemical analysis

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)

Statistics

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.