Reprogramming Peripheral Blood Mononuclear Cells (PBMCs) with the CytoTune-iPS Sendai Reprogramming Kit

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells that exhibit a pluripotent stem cell-like state similar to embryonic stem cells (Meissner et al., 2007; Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007). While these artificially generated cells are not known to exist in the human body, they show qualities remarkably similar to those of embryonic stem cells (ESCs); thus, they are an invaluable new source of pluripotent cells for drug discovery, cell therapy, and basic research.

There are multiple methods to generate iPSCs, including retrovirus-mediated gene transduction and chemical induction. While retroviral vectors require integration into host chromosomes to express reprogramming genes, DNA-based vectors, such as adenovirus, adeno-associated virus, and plasmid vectors exist episomally and do not require integration; however, they may still be integrated into host chromosomes at certain frequencies. Unlike these vectors, the CytoTune reprogramming vectors do not integrate into the host genome or alter the genetic information of the host cell (Fusaki et al., 2009; Li et al., 2000; Seki et al., 2010).

CytoTune-iPS Sendai Reprogramming System uses vectors based on replication in competent Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune Reprogramming System uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the functional temperature sensitivity mutations introduced into the key viral proteins.

The CytoTune-iPS Sendai Reprogramming Kit contains four SeV-based reprogramming vectors, each capable of expressing one of the four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety.

• CytoTune Sendai Reprogramming Vectors (Cat. no. A1378001)
  Note: For successful reprogramming, you need all four reprogramming vectors.
• Peripheral blood mononuclear cells (PBMCs) (AllCells, Cat. no. PB003F) to reprogram
  Note: You can also use PBMCs extracted from blood by a conventional method (i.e., Ficoll-Paque
  purification) instead of using frozen PBMCs.
• StemPro-34 SFM (1X), Liquid (Cat. no. 10639-011)
• L-Glutamine (Cat. no. 25030)
• SCF (c-kit Ligand), Recombinant Human (Cat. no. PHC2111)
• FLT-3 Ligand, Recombinant Human (Cat. no. PHC9414)
• Thrombopoietin (TPO), Recombinant Human (Cat. no. PHC9514)
• IL-6, Recombinant Human (Cat. no. PHC0065)
• Gibco Mouse Embryonic Fibroblasts (Irradiated) (Cat. no. S1520-100)
• Optional: Human Neonatal Foreskin Fibroblast Cells (strain BJ; ATCC no. CRL2522) as a positive
  reprogramming control
• DMEM with GlutaMAX-I (High Glucose) (Cat. no. 10569-010)
• KnockOut DMEM/F-12 (Cat. no. 12660-012)
• Fetal Bovine Serum (FBS), ES Cell-Qualified (Cat. no. 16141-079)
• KnockOut Serum Replacement (KSR) (Cat. no. 10828-028)
• MEM Non-Essential Amino Acids (NEAA) (Cat. no. 11140-050)
• GlutaMAX-I Supplement (Cat. no. 35050-061)
• Basic FGF, Recombinant Human (Cat. no. PHG0264)
• 2-Mercaptoethanol, 1000X (Cat. no. 21985-023)
• Penicillin-Streptomycin, Liquid (Cat. no. 15140-122)
• Polybrene Hexadimethrine Bromide (Sigma, Cat. no. H9268)
• Attachment Factor (Cat. no. S-006-100)
• TrypLE Select Cell Dissociation Reagent (Cat. no. 12563) or 0.05% Trypsin/EDTA (Cat. no. 25300)
• DPBS Without Calcium or Magnesium (Cat. no. 14190-144)
• TRIzol LS Reagent (Cat. no. 10296-010)
• SuperScript VILO cDNA Synthesis Kit (Cat. no. 11754-050)
• AccuPrime SuperMix I (Cat. no. 12342-010)
• Mouse Anti-Tra1-60 Antibody (Cat. no. 41-1000)
• Mouse Anti-Tra1-81 Antibody (Cat. no. 41-1100)
• Mouse Anti-SSEA4 Antibody (Cat. no. 41-4000)
• Rabbit Anti-SeV Antibody (MBL International Corporation, Woburn, MA; Cat. no. PD029)
• Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody (Cat. no. A11029)
• Alexa Fluor 594 Goat Anti-Mouse IgG (H+L) Antibody (Cat. no. A11032)
• Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody (Cat. no. A11034)
• Alexa Fluor 594 Goat Anti-Rabbit IgG (H+L) Antibody (Cat. no. A11037)
• Sterile cell culture hood (i.e., biosafety cabinet) equipped with a stereo microscope
• Inverted microscope
• Incubator set at 37°C, 5% CO2
• Water bath set at 37°C
• Sterile serological pipettes (5-mL, 10-mL)
• Centrifuge
• 15-mL centrifuge tubes
• 60-mm and 100-mm tissue culture-treated dishes
• 6-well tissue culture-treated plates
• 25-gauge 1½-inch needle

• To maintain sterile culture conditions, carry out all of the procedures using sterile laboratory practices in a laminar flow hood.
• You can use the CytoTune-iPS Sendai Reprogramming Kit to reprogram a wide range of cell types in proliferative and quiescent states. However, the reprogramming efficiency may vary among different cell types (~0.01%–1%).
• For successful reprogramming, transduce your cells using all four reprogramming vectors.
  Note: For successful reprogramming, all four Yamanaka factors (i.e., Oct4, Sox2, Klf4, and c-Myc) need to be expressed in your host cell.
• Each CytoTune-iPS Sendai Reprogramming Kit of four tubes supplies sufficient reagents to transduce cells in 2 wells of a 6-well plate (5 × 10⁵ cells/well) at an MOI of 3.
• The titer of each CytoTune Sendai reprogramming vector is lot-dependent. For the specific titer of your vectors, refer to the Certificate of Analysis (CoA) available on our website.
• Viral titers can decrease dramatically with each freeze/thaw cycle. Avoid repeated freezing and thawing of your reprogramming vectors. Viral titer is not guaranteed for kits that have been refrozen or thawed.
• Prior to starting, ensure that the media are equilibrated to 37°C and appropriately gassed.
• For positive control, we recommend performing a reprogramming experiment with human neonatal foreskin fibroblast cells (strain BJ; ATCC no. CRL2522). Note that experimental conditions may vary among target cells and need to be optimized for each cell type. The example given in the following protocol does not guarantee the generation of iPSCs for all cell types.
• For a complete list of frequently asked questions, refer to our CytoTune-iPS Sendai Reprogramming Kit FAQs.

10 μg/mL bFGF Solution (1000 μL)

1. To prepare 1 mL of 10 μg/mL bFGF solution, aseptically mix the following components:
   

   Component	Volume
   bFGF	10 μg
   DPBS without Calcium and Magnesium	980 μL
   10% KSR	10 μL

2. Aliquot and store at –20°C for up to 6 months.

SCF (c-kit Ligand), FLT-3 Ligand, Thrombopoietin (TPO), and IL-6 Stock Solutions

SCF (c-kit Ligand), FLT-3 Ligand, Thrombopoietin (TPO), and IL-6 are supplied lyophilized. Prepare stock
solutions as described in their specific product inserts.

1 mg/mL Collagenase Type IV Solution

1. Add DMEM/F-12 to Collagenase Type IV to make a 10 mg/mL stock solution. Gently vortex to suspend and filter sterilize the solution. This solution can be aliquoted and frozen at –20°C until use.
2. Make a working solution of 1 mg/mL Collagenase Type IV in DMEM/F-12. The working solution can be used for 2 weeks if properly stored at 2–8°C (store in aliquots to avoid repeated warming).

MEF/Fibroblast Medium (for 100 mL complete medium)

1. To prepare 100 mL of complete MEF/fibroblast medium, aseptically mix the following components:
   

   Component	Volume
   DMEM	89 mL
   FBS, ESC-Qualified	10 mL
   MEM Non-Essential Amino Acids Solution, 10 mM	1 mL

2. Complete MEF medium can be stored at 2–8°C for up to 1 week.

PBMC Medium (for 500 mL complete medium)

PBMC medium consists of complete StemPro-34 medium supplemented with the appropriate cytokines. Follow the procedure below to prepare 500 mL of complete PBMC medium.

1. Thaw the frozen StemPro-34 Nutrient Supplement at 4°C overnight.
2. After thawing, mix the supplement well by gently inverting the vial a couple of times, and then aseptically transfer the entire contents of the vial to the bottle of StemPro-34 SFM. Swirl the bottle to mix and to obtain a homogenous complete medium.
3. Aseptically add L-Glutamine to a final concentration of 2 mM (5 mL of 200 mM L-Glutamine to 500 mL of medium).
4. Add the following growth factors to the indicated final concentration:
   

   Cytokine	Final concentration
   SCF	100 ng/mL
   FLT-3	100 ng/mL
   TPO	20 ng/mL
   IL6	10 ng/mL

5. The complete medium has a shelf life of 30 days when stored at 2–8°C, in the dark.

Human iPSC Medium (for 100 mL complete medium)

1. To prepare 100 mL of complete human iPSC medium, aseptically mix the following components:
   

   Component	Volume
   KnockOut DMEM/F-12	78 mL
   KnockOut Serum Replacement	20 mL
   MEM Non-Essential Amino Acids Solution, 10 mM	1 mL
   GlutaMAX-I Supplement	1 mL
   2-mercaptoethanol, 1000X	100 μL
   Penicillin-Streptomycin (optional)	1 mL
   bFGF (10 μg/mL)*	40 μL

   * Prepare the iPSC medium without bFGF, and then supplement with fresh bFGF when the medium is used.
2. Complete human iPSC medium can be stored at 2–8°C for up to 4 weeks.

TOP

Gelatin coating culture vessels

1. Cover the whole surface of each new culture vessel with Attachment Factor (AF) solution and incubate the vessels for 30 minutes at 37°C or for 1 hour at room temperature.
2. Using sterile technique in a laminar flow culture hood, completely remove the AF solution from the culture vessel by aspiration just prior to use. Coated vessels may be used immediately or stored at room temperature wrapped in Parafilm sealing film for up to 24 hours. It is not necessary to wash the culture surface before adding cells or medium.

Thawing Gibco MEFs (Irradiated)

1. Remove the cryovial containing inactivated MEFs from the liquid nitrogen storage tank.
2. Briefly roll the vial between hands to remove frost, and swirl it gently in a 37°C water bath.
3. When only a small ice crystal remains in the vial, remove it from water bath. Spray the outside of the vial with 70% ethanol before placing it in the cell culture hood.
4. Pipet the thawed cells gently into a 15-mL conical tube.
5. Rinse the cryovial with 1 mL of pre-warmed MEF medium. Transfer the medium to the same 15-mL tube containing the cells.
6. Add 4 mL of pre-warmed MEF medium dropwise to the cells. Gently mix by pipetting up and down.
   Note: Adding the medium slowly helps the cells to avoid osmotic shock.
7. Centrifuge the cells at 200 × g for 5 minutes.
8. Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre-warmed MEF medium.
9. Remove 20 μL of the cell suspension and determine the viable cell count using your method of choice (e.g., Countess Automated Cell Counter).

Plating MEFs

1. Centrifuge the remaining cell suspension (step 9, Thawing Gibco MEFs) at 200 × g for 5 minutes at room temperature.
2. Aspirate the supernatant. Resuspend the cell pellet in MEF medium to a density of 2.5 × 10⁶ cells/mL.
3. Aspirate the gelatin solution from the gelatin coated culture vessel.
4. Add the appropriate amount of MEF medium into each culture vessel (refer to Table 1, below).
5. Into each of these culture vessels, add the appropriate amount of MEF suspension (refer to Table 1, below).
   Note: The recommended plating density for Gibco Mouse Embryonic Fibroblasts (Irradiated) is 2.5 × 10⁴ cells/cm².
6. Move the culture vessels in several quick back-and-forth and side-to-side motions to disperse the cells
   across the surface of the vessels.
7. Incubate the cells in a 37°C incubator with a humidified atmosphere of 5% CO₂.
8. Use the MEF culture vessels within 3–4 days after plating.

Table 1. Amount of Inactivated MEFs Needed

The following protocol has been optimized for peripheral blood mononuclear cells (PBMCs) (AllCells, Cat. no. PB003F). We recommend that you optimize the protocol for your cell type.

        Day -4: Seed PBMCs

1. 4 days before transduction, thaw PBMCs and gently transfer the cells into one well of a 24-well culture plate. Add 1 mL of StemPro-34 Medium to the cells while gently agitating to mix. Note: We recommend using the wells in the middle section of the 24-well plate to prevent excessive evaporation of the medium during incubation.
   Note: You can also use a conventional method to extract PBMCs from blood (i.e., Ficoll-Paque purification) instead of using frozen PBMCs.
2. Centrifuge the cell suspension at 200 × g for 10 minutes, discard the supernatant, and resuspend the cells in 1 mL of complete PBMC medium (complete StemPro-34 medium containing the appropriate cytokines; aliquot the cytokines and add fresh daily) to 500,000 cells/mL.
3. Place the cells in a 37°C, 5% CO₂ incubator and incubate overnight.
   
   

   Day -3 to -1: Observe cells and add fresh medium.

4. Count the cells gently, remove 0.5 mL of the medium, and add 0.5 mL of fresh complete PBMC medium without disturbing the cells.
   Note: Cells will not proliferate, but should maintain stable cell number for first few days (PBMCs contain a variety of cells, and current media system is only targeting a small population).
   
   

   Day 0: Count cells and perform transduction

5. Count the number of cells using a hemocytometer or the Countess Automated Cell Counter to determine the viability and total number of cells.
   Note: Cells may have decreased in number. Proceed with transduction using the live cell count.
6. Harvest the cells and seed the wells of a 24-well plate with 2.5 × 10⁵–5 × 10⁵ cells/well for transduction.
7. Remove one set of CytoTune Sendai tubes from the –80°C storage. Thaw each tube one at a time by first immersing the bottom of the tube in a 37°C water bath for 5–10 seconds, and then removing the tube from the water bath and allowing it to thaw at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice.
8. Transduce the cells by adding each of the four CytoTune Sendai viruses at an MOI of 6 with 4 μg/mL of Polybrene in a volume of <0.3 mL and incubating them overnight at 37°C.
   Note: We recommend initially performing the transductions with an MOI of 6. If you obtain high transductions efficiency, you can reduce the MOI to 3.
   
   

   Day 1: Remove CytoTune Sendai virus and culture cells.

9. The next day, remove the cells and medium from the culture late and transfer to a 15-mL centrifuge tube.
10. Remove the CytoTune Sendai viruses by centrifuging the cells at 400 × g for 10 minutes, aspirating the supernatant, and resuspending the cells in 0.5 mL of complete PBMC medium in the 24-well plate.
    Note: The cells may have drastic cell death (>60%); continue with the protocol using live cell count. For the first 48 hours, view the cells under the microscope for changes in cell morphology as a validation of transduction. Expect large, aggregated cells.
11. Place the cells in a 37°C, 5% CO₂ incubator and culture them for 2 days.
    Note: While the cells are incubating (1–2 days before passaging the transduced cells), prepare two 60-mm MEF culture dishes for each well of transduced cells.
    
    

    Day 3: Plate cells on MEF culture dishes.

12. Count the cells using the desired method (e.g., Countess Automated Cell Counter) and seed the 60-mm MEF culture dishes with 20,000 and 100,000 live cells per dish in 5 mL of complete StemPro-34 medium without the cytokines. Incubate the cells at 37°C. Plate any excess cells in an additional MEF culture dish.
    
    

    Day 3–6: Replace spent medium.

13. Every other day, gently remove 2.5 mL (half) of the spent medium from the cells and replace it with 2.5 mL of fresh complete StemPro-34 medium without cytokines and without disturbing cells.
    
    

    Day 7: Start transition to human iPSC medium.

14. Prepare 100 mL of complete human iPSC medium as described in Preparing Media.
15. Remove 2.5 mL (half) of StemPro-34 medium from the cells and replace it with 2.5 mL of human iPSC medium to start the transition of the cells to the new culture medium.
    
    

    Day 8 to 28: Feed and monitor the cells.

16. 24 hours later (day 8), change the full volume of medium to iPSC medium, and replace the spent medium everyday thereafter.
17. Starting on day 8, observe the plates every other day under a microscope for the emergence of cell clumps indicative of transformed cells (see Figure 1).
    Note: For BJ fibroblasts, we normally observe colony formation on Day 12 post-transduction. However, depending on your cell type, you may need to culture for up to 4 weeks before seeing colonies.
18. Day 15 to 20 after transduction, colonies should have grown to an appropriate size for transfer. The day before transferring the colonies, prepare MEF culture plates using Attachment Factor-coated 12- or 24-well plates.
    Note: We typically harvest colonies closer to 3 weeks to avoid differentiation.
19. When colonies are ready for transfer, perform live staining using Tra1-60 or Tra1-81 for selecting reprogrammed colonies. 20. Manually pick colonies and transfer them onto prepared MEF plates.
20. Manually pick colonies and transfer them onto prepared MEF plates.

By Day 21 post-transduction, the cell colonies on the MEF culture dishes will have become large and compact, covering the majority of the surface area of the culture dish. However, only a fraction of these colonies will consist of iPSCs, which exhibit a hESC-like morphology characterized by a flatter cobblestone-like appearance with individual cells clearly demarcated from each other in the colonies (see Figure 1). Therefore, we recommend that you perform live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated hESCs.

Note: Although colonies of “transformed” cells may emerge as early as 7 days after transduction, most of these colonies will not be correctly “reprogrammed” cells. iPSCs usually emerge a little later (around day 14 posttransduction), resemble embryonic stem cells in morphology, and express the cell surface markers Tra1-60 and Tra1-81.

One of the fastest and most reliable methods for selecting a reprogrammed colony is live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated iPSCs and enable the identification of reprogrammed cells from a variety of human cell types.
Note: Other methods of identifying iPSCs (such as alkaline phosphatase staining) are also acceptable. 1.

1. Aspirate the medium from the reprogramming dish.
2. Wash the cells once with 1X KnockOut DMEM/F-12.
3. Add the diluted primary antibody to the cells (6 mL per 100-mm dish).
4. Incubate the primary antibody and the cells at 37°C for 60 minutes.
5. Remove the primary antibody solution from the dish.
   Note: The primary antibody solution can be stored at 4°C for 1 week and re-used up to 2 times.
6. Wash cells three times with KnockOut DMEM/F-12.
7. Add the diluted secondary antibody to the cells (6 mL per 100-mm dish).
8. Incubate the secondary antibody and the cells at 37°C for 60 minutes.
9. Remove the secondary antibody solution from the dish.
   Note: The secondary antibody solution can be stored at 4°C for 1 week and re-used up to 2 times.
10. Wash cells three times with KnockOut DMEM/F-12 and add fresh KnockOut DMEM/F-12 to cover the surface of the cells (6 mL per 100-mm dish).
11. Visualize the cells under a standard fluorescent microscope and mark the successfully reprogrammed colonies for picking and expansion. Successful antibody staining can very specifically distinguish
    reprogrammed colonies from just plain transformed counterparts, and can be detected for up to 24–36 hours. This is particularly useful because it helps identifying and tracking of candidate iPS colonies before picking and the day after they are transferred into a new culture dish for expansion.

1. Place the culture dish containing the reprogrammed cells under an inverted microscope and examine the colonies under 10X magnification.
2. Mark the colony to be picked on the bottom of the culture dish.
   Note: We recommend picking at least 10 distinct colonies by the end of each reprogramming experiment and expanding them in separate 24-well MEF culture plates (see below).
3. Transfer the culture dish to a sterile cell culture hood (i.e., biosafety cabinet) equipped with a
   stereomicroscope.
4. Using a 25-gauge 1½-inch needle, cut the colony to be picked into 5–6 pieces in a grid-like pattern.
5. Using a 200 μL pipette, transfer the cut pieces to a freshly prepared 24-well MEF culture plate containing human iPSC medium.
6. Incubate the MEF culture plate containing the picked colonies in a 37°C incubator with a humidified
   atmosphere of 5% CO2.
7. Allow the colonies to attach to the culture plate for 48 hours before replacing the spent medium with fresh human iPSC medium. After that, change the medium every day.
8. Treat the reprogrammed colonies like normal human ESC colonies and passage, expand, and maintain them using standard culture procedures until you have frozen cells from two 60-mm plates.

Guidelines for generating vector-free iPSCs

• The time needed to derive vector-free iPSCs may vary depending on culture and passage conditions. In the case of human neonatal foreskin fibroblast cells (strain BJ), it takes about 2 months after gene transduction to obtain iPSCs free of CytoTune Sendai reprogramming vectors.
• To obtain virus-free clones faster, we recommend that you perform single colony subcloning for the first few passages (minimum 5) instead of bulk or pooled-clone passaging.
• To perform single colony subcloning, pick from a single colony to transfer to another 6-well plate (Passage 1). From Passage 1, pick a single colony and transfer to another 6-well plate (Passage 2) and so forth. We recommend subcloning for 5 passages and then testing for virus free iPSCs.

Protocol for generating vector-free iPSCs

1. When passaging iPSC colonies, prepare duplicate plates; one for immunostaining and one for further
   passaging.
2. Perform immunostaining on one plate using anti-SeV antibodies (see below).
3. If any colonies stain positive, perform cell cloning on the other duplicate plate.
4. Repeat immunostaining with anti-SeV antibodies on the cloned colonies until all colonies in a plate are negative.
5. If all colonies are negative for anti-SeV antibodies, passage the cells and confirm the absence of the
   CytoTune Sendai reprogramming vectors by RT-PCR.

Immunocytochemistry with Anti-SeV Antibodies

1. Wash cells once with DPBS
2. Fix the cells in 4% paraformaldehyde for 5 minutes at room temperature.
3. Wash cells twice with DPBS.
4. Add the anti-SeV antibody (MBL, Cat. no PD029) diluted in 0.1% Triton X-100 in DPBS to the cells and
   incubate for 1 hour at 37°C.
5. Remove the antibody solution. Wash the cells 3 times with DPBS.
6. Add the secondary antibody diluted in 0.1% Triton X-100 in DPBS and incubate for 1 hour at 37°C.
7. Remove the secondary antibody solution from the dish. Wash the cells 3 times with DPBS.
8. Visualize the cells under a fluorescence microscope.

RT-PCR Protocol for detecting the SeV genome and transgenes

1. Extract the total RNA from 5 × 10⁶ iPSCs using the TRIzol Reagent following the instructions provided with the reagent. As a positive control, use cells set aside at the last step of the reprogramming procedure.
2. Carry out a reverse transcription reaction using 1 μg of RNA (from step 1, above) and the SuperScript® VILO™ cDNA Synthesis Kit following the instructions provided with the kit.
   Note: Because the CytoTune Sendai reprogramming vectors are based on SeV, which is an RNA virus, reverse transcription is required for detecting the presence of the SeV genome in your reprogrammed cells.
3. Carry out the PCR using 10 μL of cDNA from the reverse transcription reaction (step 2, above) and
   AccuPrime™ SuperMix I with the parameters below. For the RT-PCR primer sequences and the expected product size, refer to the Table 2.
   

   Step	Temperature	Time	Cycles
   Denaturation	95°C	30 seconds	30–35
   Annealing	55°C	30 seconds	30–35
   Elongation	72°C	30 seconds	30–35

4. Analyze the PCR products using 2% agarose gel electrophoresis.
   Note: If you still detect CytoTune Sendai virus in your iPSC lines after more than 10 passages, and have performed RT-PCR to show that Oct4, Sox2, and Klf4 (these vectors do not have the temperature sensitive mutations) are absent from your cells, then you can perform temperature shift to remove the cMyc gene. CytoTune Sendai hc-Myc tends to persist in the cells longer than the other CytoTune Sendai reprogramming vectors. However, because this vector contains a temperature sensitivity mutation, you can enhance its removal and obtain complete absence of Sendai virus by incubating your cells at 38–39°C for 5 days.

Table 2. RT-PCR primer set used for detecting the SeV genome and transgenes in cells reprogrammed using the CytoTune Sendai reprogramming vectors

 * Primer contains SeV genome sequences. Pairing of these primers with transgene-specific primers allows specific detection of transgenes carried by the CytoTune Sendai reprogramming vectors. Note that the same reverse primer is used for detecting Sox2, Klf2, and Oct3/4.

iPSC freezing medium

1. Prepare the Freezing Media A and B immediately before use.
2. In a sterile 15-mL tube, mix together the following reagents for every 1 mL of freezing medium A needed:
   

   Human iPSC medium	0.5 mL
   KnockOut Serum Replacement	0.5 mL

3. In another sterile 15-mL tube, mix together the following reagents for every 1 mL of freezing medium B
   needed:
   

   Human iPSC medium	0.8 mL
   DMSO	0.2 mL

4. Place the tube with freezing medium B on ice until use (you can keep freezing medium A at room
   temperature). Discard any remaining freezing medium after use.

Freezing iPSCs

1. Prepare the required volume of fresh freezing medium and place it on ice.
2. Aspirate the culture medium and rinse the dishes twice with DPBS without Ca2+ and Mg2+ (2 mL per 35-mm  or 4 mL per 60-mm dish).
3. Gently add Collagenase IV solution to the culture dish (1 mL per 35-mm or 2 mL per 60-mm dish).
4. Incubate the dish with cells for 5–20 minutes in a 37°C incubator with a humidified atmosphere of 5% CO2.
   Note: Incubation times may vary among different batches of collagenase. Therefore, the appropriate incubation
   time should be optimized by examining the colonies periodically under microscope during incubation.
5. Stop the incubation when the edges of the colonies are starting to pull away from the plate.
6. Remove the culture dish from the incubator, aspirate the Collagenase IV solution, and gently rinse the dish with DPBS without Ca2+ and Mg2+.
7. Add 2 mL of iPSC culture medium or DMEM/F-12 and gently dislodge the cells off the surface of the
   culture dish using a sterile pipette or a cell scraper. Transfer the cells to a sterile 15-mL centrifuge tube. Rinse the dish with additional iPSC medium or DMEM/F-12 to collect any leftover colonies.
8. Centrifuge the cells at 200 × g for 2–4 minutes at room temperature.
9. Discard the supernatant, gently tap the tube to dislodge the cell pellet from the tube bottom, and resuspend the cells in freezing medium A. After the cell clumps have been uniformly suspended, add an equal volume of freezing medium B to the cell suspension in a drop-wise manner while gently swirling the cell suspension to mix.
   Note: At this point, the cells are in contact with DMSO, and work must be performed efficiently with no or
   minimum delays. After the cells come into contact with DMSO, they should be aliquoted and frozen within 2–3 minutes.
10. Aliquot 1 mL of the cell suspension into each cryovial.
11. Quickly place the cryovials containing the cells in a cryo freezing container (e.g., Mr. Frosty) to freeze the
    cells at 1°C per minute and transfer them to –80°C overnight.
12. After overnight storage at –80°C, transfer the cells to a liquid nitrogen tank vapor phase for long term
    storage.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

MAN0006712           22-Mar-2012