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1 Introduction

Adult somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) through the expression of exogenous transcription factors [1]. Human (h) iPSCs are an attractive candidates for use in cellular-based therapies, as they circumvent ethical and immunological barriers associated with human embryonic stem cell (hESC) [2]. hiPSCs have the capacity for self-renewal and can differentiate into any cell type within the body. iPSCs can drug screening, drug discovery, and toxicology assays. Also, hiPSCs offer a unique platform for in vitro human disease modeling. Patient-specific iPSCs and their differentiated derivatives can provide valuable information about disease pathogenesis [3, 4]. In fact, hiPSCs may revolutionize personalized medicine as it allows the creation of genetically customized cell lines to each patient and through the mode of responders versus non-responder “trial in a dish” models [5, 6].

One of the for front challenges that impedes the implementation of hiPSCs is its production at a relevant clinical number. Conventionally, hiPSCs are expanded in two-dimensional static culture [7]. They are typically grown under feeder or feeder-free conditions in the presence of basic fibroblast growth factor (bFGF) to maintain pluripotency [8, 9]. Most of these methods use two dimensional adherent culture, which present difficulties for large-scale and production. Other disadvantage with static culture systems are such as time consumption in feeding and passaging, culture-to-culture variability, and nonhomogeneous culture conditions. Consequently, these culture systems are not good candidates for clinical application.

Scale-up production of hiPSCs in relevant clinical quantities can be achieved with the use of stirred suspension bioreactors, which offer several advantages over conventional adherent culture systems. Suspension bioreactors facilitate the large-scale expansion of hiPSCs required for clinical studies with lower cost [10–12]. They provide more homogeneous culture environment and offer a flexible platform for various modes for culturing cells and/or derivative tissues as aggregates, on microcarriers, or on scaffolds [13]. Suspension bioreactors maintain allow for online monitoring and control of culture parameters [12] while maintaining cell pluripotency, which can be determined by marker analysis or functional tests such as differentiation potential or energy metabolism [14, 15], found in suspension bioreactors seems to induce pluripotency in cells grown in this vessel [16, 17]. Here, shear stress seems to cell pluripotency [18, 19]. Thus, suspension bioreactors is ideal for the development of standardized, fully controlled, and scalable culture processes to produce a clinically relevant quantity of hiPSCs. A detailed protocol for bioreactor expansion of hiPSCs is described in the following sections.

2 Materials

All medium and reagents should be cell culture-tested reagents and prepared in sterile conditions. Filtering non-sterile reagents through 0.2 mm filters is highly recommended. It is also strongly recommended to test each batch of hiPSC culture reagents to prevent adverse effects of batch-to-batch variation.

2.1 hiPSC Culture Reagents

  1. 1.

    hiPSC medium: mTeSR™ feeder-free medium (Stem Cell Technologies, Vancouver, Canada) is recommended. This medium comes in 500 mL bottles with separate base medium and growth factor components. 200 mL of mTeSR™ will be required for culture of hiPSCs in each 100 mL bioreactor.

  2. 2.

    Extracellular matrix: Matrigel™ ECM (BD Biosciences, Franklin Lakes, NJ) is recommended for static culture using mTeSR™ medium. Resuspend Matrigel™ and coat the culture dishes according to the manufacturer’s instructions.

  3. 3.

    Dissociation enzymes: Accutase™ (Stem Cell Technologies) is recommended for single-cell dissociation of hiPSCs in both static and suspension culture systems. Other enzymes, such as Collagenase and trypsin, are not recommended for dissociation of hiPSC aggregates derived in suspension culture system (see Note 4). Accutase™ should be stored at −20 °C. It is highly recommended to aliquot Accutase™ into smaller volumes since repeated freeze-thaw cycles or extended storage at 4 °C will reduce Accutase™ activity.

  4. 4.

    ROCK inhibitor: 10 μM Y-27632 (Stemgent, Cambridge, MA) treatments are recommended for single-cell dissociation steps.

2.2 Cell Counting and Viability Assessment

  1. 1.

    Trypan Blue (Bio-Rad, Hercules, CA) and the Bio-Rad cell counting system are used to calculate the total and viable cells in static and suspension culture. Alternatively, manual counting using a hemocytometer or any other cell counting and viability assessment method can be used.

2.3 Pluripotency Assessment

  1. 1.

    PCR: Reverse transcriptase (RT)- and quantitative (q)-PCR are conducted to determine the expression of pluripotency-related genes including Oct-4, Sox2, Klf4, Nanog, SSEA-3, SSEA-4, and REX-1.

  2. 2.

    Flow cytometry: Fixed, permeabilized, and blocked hiPSCs are incubated with fluorescently labeled antibodies against Oct-4, Nanog, SSEA-3, SSEA-4, and REX-1 (all BD, Biosciences) according to the manufacturer’s instructions. The labeled cells are then analyzed using a flow cytometer (BD) registering at least 10,000 events.

  3. 3.

    Teratoma assay: One million dissociated hiPSCs are suspended in 1× PBS− (Invitrogen) and injected into the thigh muscle of an immunodeficient SCID mouse (Taconic Farms, Hudson, NY). Tumor cell masses are usually formed in 4–6 weeks. Sections from newly formed tissue are analyzed by regular histology procedures to determine the presence of three germ layers in obtained tissues.

2.4 Anaerobic Energy Metabolism Assessment

Manipulating glycolic over cellular respiratory pathways is a useful indicator of iPSC pluripotency. The growing condition of iPSCs in bioreactors supports glycolysis which is an ideal condition for pluripotency. Glycolytic energy metabolism can be confirmed through different tests; however, in the following we show the most common tests, which are applicable and easily adaptable to most of the laboratories.

  1. 1.

    PCR: RT- and q-PCR are conducted to determine and quantify the expression of mitochondrial biogenesis genes as indicators of mitochondrial numbers. The genes are ND1, ND5, and MT-CYB.

  2. 2.

    Flow Cytometry: Mitochondrial mass and level of reactive oxygen species (ROS) are measured by flow cytometry following treatment of hiPSCs with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes®, Life Technologies, Carlsbad, CA) and Mito-tracker green (Molecular Probes®, Life Technologies, Carlsbad, CA), respectively.

  3. 3.

    Bioluminescence Luciferase-based Assay: The level of intracellular ATP is measured using ATPLite bioluminescence luciferase-based assay (Perkin Elmer, Waltham, MA). hiPSCs are used as input cells and then luminescence is quantified with a luminometer (Berthold Technologies, Bad Wildbad, Germany), following the manufacturer’s instruction. About 100,000 cells are used as input. The results are presented as nanomoles of ATP per cell.

2.5 Suspension Bioreactor Materials

  1. 1.

    Bioreactor vessel: 100 mL suspension bioreactors (NDS Technologies, Vineland, NJ) with magnetic impellers should be siliconized prior to use with Sigmacoat (Sigma) following the manufacturer’s instructions. Siliconization of the vessel is important to prevent attachment of hiPSC aggregates to vessel’s glass surface. This process should be repeated every 6 months or as required.

  2. 2.

    Magnetic stir plate: Magnetic stir plates with variable speed are required. There are many suppliers for stir plates in the market. Stir plates from VWR (VWR International, Radnor, PA) are used in our lab. Please ensure that stir plate can maintain a speed of 100 rpm, and can be housed in a cell culture incubator. If your unit is not designed to be housed at 37 °C, the temperature in the incubator may reach 90–100 °C effectively killing all mammalian cells.

3 Methods

3.1 Preparation of hiPSCs in Static Culture

  1. 1.

    hiPSCs can either be cultured under feeder-dependent or feeder-free conditions. Under feeder-dependent conditions either inactivated (see Note 1) human or murine fibroblasts can be used. Under feeder-free conditions Matrigel™ is recommended, used according to the manufacturer’s instructions. When using mTeSR™, hiPSCs medium is privileged (see Note 2) for static culturing of hiPSC, as this medium will be used within the suspension bioreactor.

  2. 2.

    For single-cell passaging under static culture conditions, it is recommended to use Accutase™ and the ROCK inhibitor Y-27632. Normally, hiPSCs are treated with 10 μM Y-27632 1 h before dissociation and for 24 h after passaging (see Note 3).

  3. 3.

    One million viable hiPSCs are required to seed each 100 mL suspension bioreactor vessel. Therefore, it is recommended to have at least double that amount in static culture per bioreactor, since a number of cells will undergo apoptosis during single-cell dissociation even in the presence of Y-27632.

3.2 Transferring hiPSCs from Static to Suspension Culture

  1. 1.

    Add Y-27632 (10 μM) to the culture medium of static-cultured hiPSCs 1 h prior to single-cell dissociation (see Note 4).

  2. 2.

    Discard the culture medium and add a sufficient amount of Accutase™ to cover the whole dish/es. Incubate the hiPSCs at 37 °C, and check cell dissociation every 1–2 min (see Note 5).

  3. 3.

    Stop enzymatic reaction by adding fresh medium, and break up any cell clumps with gentle pipetting. Pellet the hiPSCs by centrifugation at 800 × g for 5 min.

  4. 4.

    Discard the supernatant and resuspend the cells at a density of one million/milliliter in a sterile 15 mL conical tube containing fresh mTeSR™ medium with 10 μM Y-27632. Incubate the cells in suspension culture (in low-adherent tissue culture plates) for 1 h at 37 °C in 5 % CO2 (see Note 6).

  5. 5.

    Prepare a 100 mL suspension bioreactor with 100 mL of fresh, pre-warmed (see Note 7) mTeSR™ medium with 10 μM Y-27632 and 0.1 nM Rapamycin.

  6. 6.

    Add one million hiPSCs to each 100 mL suspension bioreactor and place the bioreactors containing hiPSCs at 37 °C, 5 % CO2, and 100 rpm (see Note 8).

3.3 Culturing hiPSCs in Suspension

  1. 1.

    It is highly recommended to change the medium 24 h after initial seeding (see Note 9).

  2. 2.

    Collect all 100 mL of medium with hiPSCs in two sterile 50 mL conical. Spin the conicals at 800 × g for 5 min, and carefully remove as much of the supernatant as possible with aspiration. Do not pour off the old medium as the small hiPSC aggregates will not adhere to the bottom of the conical and will be lost. We recommend leaving the last 1–5 mL of old medium per conical so as not to accidently remove the hiPSCs.

  3. 3.

    Prepare the bioreactor with 100 mL of new pre-warmed mTeSR™ medium and 0.1 nM Rapamycin. It is not recommended to add Y-27632.

  4. 4.

    Place the bioreactor vessel at 37 °C, 5 % CO2, and 100 rpm. The medium usually does not need replacing until day 6 of culture; however, it can be replaced as often as desired.

3.4 Passaging of hiPSCs in Suspension

  1. 1.

    On day 6 of bioreactor culture, medium and cells are collected from the bioreactor into two 50 mL sterile conical tubes. Centrifuge 50 mL tubes at 800 × g for 5 min. Discard (without pouring) old medium leaving 1–2 mL. Add 10 μM Y-27632 to the medium and cells and incubate at 37 °C, in 5 % CO2 for 1 h.

  2. 2.

    Transfer hiPSCs to a 15 mL sterile conical tube and spin down at 800 × g for 5 min. Discard as much of the old medium as possible, and add 1 mL of Accutase™. Incubate the cells at 37 °C, 5 % CO2 checking occasionally under a dissecting microscope to observe dissociation of aggregates. Following aggregate dissociation into single cells, break up any small clumps by gentle agitation.

  3. 3.

    Once the majority of the aggregates have been dissociated into single cells, break up any small clumps by gentle pipetting.

  4. 4.

    Repeat steps 3–6 under Section 4.2, and steps 1–4 under Section 4.3.

3.5 Expansion Rate Calculation

It is recommended to count hiPSCs and assess the viability at each day of bioreactor culture by the following steps:

  1. 1.

    Take only 5 mL sample from the bioreactor and repeat steps 1–3 of Section 4.4 (minding only to take 5 mL and not the entire volume).

  2. 2.

    Add new 5 mL of fresh medium to the bioreactor.

  3. 3.

    Using Trypan Blue and an automated cell counter or hemocytometer, record the percent viability and total cell count.

3.6 Assessment of Pluripotency and Anaerobic Energy Metabolism

At the conclusion of your experiment, it is recommended to analyze the pluripotency of the hiPSCs. It would be beneficial to check pluripotency features of hiPSCs every three to five passages following their culture in bioreactors. We usually assess the pluripotent state of hiPSCs through FACS, PCR, and teratoma analysis (see Note 10). Analysis of anaerobic energy metabolism as an indication of proliferating hiPSCs in bioreactors is also recommended. Normally, we evaluate this through PCR, flow cytometry, and bioluminescence luciferase-based assay (see Note 11).

4 Notes

  1. 1.

    Many different methods have been reported for the static culture of hiPSCs previously [20–22]. However, only culturing methods that use mTeSR™ are suitable for suspension culture. Therefore, hiPSCs should be adapted to mTeSR™ before transferring them to suspension culture.

  2. 2.

    The method which is described here to grow hiPSCs in suspension utilizes mTeSR™ hiPSCs medium. Other culture mediums may also work but culturing conditions and other growth factors may need optimization.

  3. 3.

    Y-27632 and other ROCK pathway inhibitors have been shown to allow hiPSCs dissociation into single cells without massive cell death [23, 24]. However, for the successful generation of hiPSC aggregates in suspension only Rho/ROCK pathway inhibitors that target ROCK or upstream of ROCK are effective.

  4. 4.

    It is essential to use single-cell inoculation into bioreactors, as it helps to keep the undifferentiated state of hiPSC aggregates. If partial collagenase dissociation is utilized, the risk of generating EBs is greatly increased. However, chromosomal changes can occur following prolonged enzymatic single-cell passaging of the iPSCs [25]. Hence, recently the new enzyme-free EDTA-based reagent called Versene (Versene® (EDTA) 0.02 %, Lonza, Basel, Switzerland) has been offered to the market, allowing single cell dissociation of stem cells without affecting their chromosomal status. Although we have not used Versene for single-cell dissociation of hiPSCs, it may be a useful alternative for single-cell passaging of bioreactor-cultured hiPSCs.

  5. 5.

    Normally, in static culture hiPSC colonies are tightly packed with well-defined borders. With enzymatic dissociation, individual cells will be identifiable within the colony. It is recommended not to allow single-cell dissociation to proceed until all cells are released into the medium, as this is unnecessarily may damage hiPSC. Instead, wait until most of the cells are identifiable within the hiPSC colonies, stop the reaction with fresh medium, and break up any leftover cell clumps with gentle mechanical dissociation.

  6. 6.

    The 1-h incubation time with Y-27632 promotes the formation of small “seed” aggregates (1–5 cells) and thus enhances the efficiency of aggregation and expansion of hiPSCs following transferring to the stirred bioreactor.

  7. 7.

    Pre-warm all hiPSC culture medium to 37 °C to avoid shocking the cells.

  8. 8.

    To calculate rpm of the bioreactor, do not rely on the digital/analog settings on your stir plate. Each bioreactor design will behave differently when placed on the magnetic stir plate. Verify your rpms by counting the number of impeller rotations in 10s and multiply by 6 to get your revolutions per minute. One hundred rotations per minute is optimal for hiPSC aggregate formation and expansion, if significantly slower speeds are used (60–80 rpm), aggregates will grow quickly and may develop either differentiated or necrotic centers. However, at higher speeds (120+) cells do not effectively aggregate: any resultant small aggregates will normally die.

  9. 9.

    If the Y-27632 is not removed from the bioreactor after 24 h, the aggregates will still remain viable and undifferentiated; however, the expansion rate will be significantly negatively impacted. Furthermore, the prolonged maintenance of Y-27632 in the medium might have negative effect on differentiation potential of hiPSCs for downstream studies.

  10. 10.

    The following reference describes commonly used methods to analyze pluripotency of hiPSCs [26–28].

  11. 11.

    The following references describe anaerobic energy metabolism of hiPSCs and some of the tests discussed above [29–31].