Induced Pluripotent Stem Cell Culture Protocols

Introduction

Induced pluripotent stem cells (iPSCs), have the capacity to give rise to differentiated progeny representative of all three germ layers of the body including: ectoderm, endoderm, and mesoderm. The ability to expand human iPSCs in vitro and subject them to cell-type specific differentiation protocols is critical for generating patient derived “disease-in-a-dish” cellular models for basic stem cell research and drug-discovery applications

This protocol guide details steps on how to thaw, culture and cryopreserve human induced pluripotent stem cells (iPSCs) supplied by the European Bank of induced pluripotent Stem Cells (EBiSC). Human induced pluripotent stem cell (iPSC) lines are different to any other established cell line. If you are not familiar with culturing iPSCs make sure you read the following instructions carefully.

Key Points for Success

  • Read these instructions carefully, including the sections on required reagents, thawing, passaging, precautions and troubleshooting tips before starting.
  • Make sure all necessary reagents are available prior to thawing the cells.
  • Use the correct media and matrix combination. iPSCs requires specialized media and culture conditions.
  • Make sure your equipment is calibrated regularly and no reagents have expired.

Methods

It is recommended to thaw cells in media and matrix outlined in the cofa of each cell line. Where required, the matrix and media used can be changed to an alternative during passaging only. There might be a time of adaptation when switching media or matrix. No guarantees can be given regarding cell viability or quality where the advised tissue culture system is not used.

Extracellular Matrix Preparation

Stock vials of Matrigel® and Cultrex® should be thawed overnight on ice or within a refrigerator (2-8°C) prior to use. Culture vessels, tubes and pipette tips should be pre-chilled prior to making aliquots or coating. All manipulations of Matrigel and Cultrex must be carried out on ice-packs to avoid premature gelling. Do not repeatedly freeze thaw stock or working vials of Matrigel or Cultrex.

Preparation of Matrigel

  1. Upon receipt, store Matrigel at -20°C.
  2. Protein concentration within the Matrigel stock vial is batch dependent and must be obtained from its accompanying certificate of analysis. Use this concentration to calculate the volume of Matrigel required for 2mg protein/working vial.
  3. Transfer 2mg of protein into pre-chilled 15ml tubes. These tubes are working aliquots and should be stored at -20°C until required.
  4. When required, thaw the working Matrigel aliquot in the fridge (2-8°C) overnight. Add 6ml of cold (2-8°C) DMEM (D5796) to the vial and mix by pipetting up and down thoroughly. This is enough for a whole 6-well plate as 1 ml of the Matrigel/DMEM mix is required for one well of a 6-well plate. Chilled, diluted Matrigel must be used immediately to coat ice-cold tissue culture vessels. Then the vessel should be incubated at 37°C/5% CO2 for 1 hour and equilibrated to room temperature 30 minutes prior to use. Alternatively, vessels can be sealed with Parafilm (P7793) after the incubation at 37°C/5% CO2 and stored at 2-8°C for a maximum of one week. Ensure stored vessels are equilibrated to room temperature 30 minutes prior to use.
  5. Prior to use, aspirate Matrigel from the vessel and wash vessel with 1ml of DMEM (D5796)  and replace with an appropriate volume of culture medium (2ml of medium per well of a 6-well plate). Vessels are now ready for cell culture use.

Preparation of Cultrex

  1. Upon receipt Cultrex (3532-005-02, 3533-005-02) should be stored at -80°C. After thawing, mix 5ml of Cultrex stock solution with 5ml of ice-cold DMEM-F12 (D6421). Make 180μl aliquots of diluted Cultrex in pre-chilled 15ml tubes. These tubes are working stocks and should be stored at -20°C.
  2. When required, thaw the working Cultrex aliquot at 2-8°C overnight. To dilute the working stock for use, add 8.82ml of ice-cold DMEM-F12 (D6421) to the vial using a chilled pipette and mix by pipetting up and down thoroughly. This is enough for a whole 6-well plate as 1.5 ml of chilled, diluted Cultrex is required for one well of a 6-well plate.
  3. After adding the diluted Cultrex to the required wells the vessel should be incubated at 37°C/5% CO2 for 1 hour and equilibrated to room temperature 30 minutes prior to use. Alternatively, vessels can be sealed with Parafilm (P7793) after the incubation at 37°C/5% CO2 and stored at 2-8°C for a maximum of two weeks. Ensure stored vessels are equilibrated to room temperature 30 minutes prior to use.
  4. Prior to use aspirate the solidified Cultrex from the vessel, wash the vessel with 1ml of DMEMF12 (D6421) and replace with an appropriate volume of culture medium (for example 2ml of medium per well). Vessels are now ready for tissue culture use.

Preparation of Vitronectin

  1. Upon receipt, store Vitronectin (CC130) at -80°C. Prior to use, thaw the stock vial of Vitronectin at room temperature and prepare 60μl aliquots in sterile polypropylene tubes and freeze the aliquots at -80°C or use immediately. One 60μl aliquot is sufficient for coating all wells of a 6-well plate.
  2. To prepare Vitronectin at a working concentration of 0.5μg/cm2, dilute the Vitronectin 1:100 by gently mixing 6ml of room temperature PBS (D8537) with 60μl of Vitronectin. Add 1ml of diluted Vitronectin to each well of a 6-well plate.
  3. Incubate the coated culture vessels at room temperature for 1 hour. If storage is required, vessels can be sealed with Parafilm (P7793) and stored at 2-8°C for up to 3 days. Allow the vessel to equilibrate to room temperature for 1 hour prior to use.
  4. To prepare the vessel for culture, remove the excess Vitronectin from the culture vessel and discard. It is not necessary to wash the culture vessel after the removal of Vitronectin.

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Cell Culture Media Preparation

mTeSR™-1 Media

  1. When required, remove the mTeSR1 supplement (5x) from the freezer and thaw overnight at 2-8°C prior to use. Do not thaw at 37°C.
  2. Aseptically add 100ml of mTeSR1 supplement (5x) to 400ml of cold (2-8°C) basal medium.
  3. Aliquot medium into volumes required for 1 week of culture work.
  4. Complete mTeSR1 may be stored at 2-8°C for 1 week or at -20°C for 6 months. Frozen complete mTeSR1 may be thawed once. Do not repeatedly freeze thaw medium. Prior to use, warm mTeSR1 to room temperature, do not leave medium at room temperature for longer than 2 hours per day and avoid exposure to light to avoid degradation of medium components.

Essential 8™ (TESR-E8™) Media

  1. When required, remove the E8 supplement (50x) from the freezer and thaw overnight at 2-8°C prior to use. Do not thaw at 37°C.
  2. Aseptically remove 10ml of E8 basal medium to leave 490ml.
  3. Add 10ml of E8 supplement (50x) to the 490ml of basal cold (2-8°C) medium.
  4. Aliquot medium into volumes required for 1 week of culture work.
  5. Complete E8 may be stored at 2-8°C for 1 week or at -20°C for 6 months. Frozen complete E8 may be thawed once. Do not repeatedly freeze thaw medium. Prior to use, warm E8 to room temperature, do not leave medium at room temperature for longer than 2 hours per day and avoid exposure to light to avoid degradation of medium components.

PluriSTEM™ Human ES/iPSC Media

PluriSTEM (SCM130, SCM132) is a complete small molecule based serum-free medium that enables feeder-free culture of human ES/iPS cells and allows for media exchanges every other day without compromising the morphology or long term functionality. The media is complete and does not require further supplementation. When required, remove the media from the freezer and thaw overnight at 2-8°C prior to use. Do not thaw at 37°C. Once thawed, PluriSTEM media should be stored at 2-8C and used within two weeks.

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Thawing of Human iPSCs

  1. Cells should be thawed rapidly by placing the cryovial in a water bath set to maintain 37°C. Swirl the cryovial gently in the water bath to ensure rapid thaw but do not submerge the cap of the cryovial. Disinfect the cryovial with 70% alcohol (793213) or an equivalent disinfectant before opening.
  2. Using a 5ml sterile pipette, transfer the cryoprotectant/cells mix from the cryovial into a 15ml centrifuge tube. Care should be taken not to physically damage cells.
  3. Slowly, drop by drop, add 10ml of appropriate medium at room temperature to the cells in the 15ml centrifuge tube. Gently rock the 15ml centrifuge tube back and forth while adding drops to minimize osmotic shock to the cells. This is a crucial step and cells should be treated as gently as possible.
  4. Check tube to ensure all cell contents are removed and if not, rinse with 1ml of appropriate medium.
  5. A small amount of cells can be used for performing a cell count. A single cell suspension should be created using trypsin or similar. As a general guideline the seeding density range for one well of a 6-well plate is between 2x105 - 1x106 viable cells. Refer to CoA for guidelines for a specific EBiSC cell line lot number.
  6. Centrifuge the cells at 200xg for 2 minutes. Remove and discard the supernatant.
  7. Prepare culture vessels by adding an appropriate amount of medium (for example 1.5 - 2ml per one well of a 6-well plate)
  8. Gently tap the 15ml centrifuge tube to dislodge the cell pellet then gently add 1ml of appropriate medium and seed into 2 wells of a coated 6-well plate (adjust if using other culture formats or if advised differently in the Certificate of Analysis). Do not over aspirate the cells as this will lead to decreased viability due to generation of a single cell suspension.
  9. Gently rock plate side to side, back and forth to spread the cells evenly across the well.
  10. It is advisable to record images of cells immediately post-thaw, at 48 hours and at approximately 70-80% of confluence.

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Culturing of Human iPSCs

  1. It is good practice to observe iPSC lines daily under phase contrast microscope (4x, 10x, 20x and 40x magnification) to check for iPSC-like morphology, the presence of differentiated cells and confluence. A typical scoring is outlined below:
Grade Description
A Optimal, compacted iPSC colonies with defined edges; morphology uniform across colonies
B Acceptable iPSC colonies with some differentiation around the edges, cells more loosely packed within colonies
C Good adherence with small iPSCs colonies emerging
D Poor adherence and no obvious iPSCs


Scoring of iPSC Colonies

Scoring of iPSC Colonies



Differentiation Levels within iPSC Cultures

Differentiation Levels within iPSC Cultures

  1. Cells are fed by removing 95% of the medium from the wells using an aspirator pipette. Do not completely remove the medium; a thin film of medium should cover the cell layer to avoid drying out the cells.
  2. Aseptically add 2ml of fresh medium per 1 well of a 6-well plate by gently adding to the side of the well. Incubate cells at 37°C/ 5% CO2.
  3. Typically, medium exchanges occur daily on six of seven days with increased volume of media (1.5x-2x the normal amount; cell density dependent) if cells need to be left for longer periods between media changing. Do not leave more than two days between medium exchanges.

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Passaging of Human iPSCs

EZ-LiFT Reagent Passaging Protocol

  1. Warm EZ-LiFT Reagent (SCM139) to 37°C before starting.
  2. Aspirate the culture medium and wash wells twice with 1.5 mL EZ-LiFT Reagent (SCM139).
  3. Add 1 mL of EZ-LiFT reagent (SCM139) to each well.  Incubate the plate at 37°C for 4 minutes. 
  4. After 4 minutes, tap rapidly on the bottom of the plate (i.e. 20-25 taps in 5 secs). 
  5. Place the plate back in the 37°C incubator for an additional 4 minutes.
  6. After 4 minutes, tap rapidly on the bottom of the plate (i.e. 20-25 taps in 5 secs).
  7. Perform a quick microscopic inspection of the well(s). 
    1. If a significant number of detached clumps are visible proceed to step 8.
    2. If no obvious detachment is observed, repeat steps 4-7 except that in step 5, the 37°C incubation should be for 2 instead of 4 minutes.  Proceed to step 8.
  8. Gently collect the cell suspension (~1 mL) and transfer to a 15 mL conical tube.  Neutralize with 5 mL of culture medium by gently adding the medium to the cell suspension.  Do not pipette up and down as this may break cell clumps into single cells suspension. 
  9. Centrifuge at 800 rpm for 3 minutes.  Aspirate the supernatant.
  10. Gently resuspend the cell pellet in 1 mL pluripotent medium such as PluriSTEM (SCM130).  Do not pipette up and down more than two times.  Over-pipetting may result in single cell dissociation. 
  11. Passage dissociated cell clumps to newly coated 6 well plates. Split ratio should be 1:6 up to 1:30. Monitor cells daily. Cells will typically reach 60-80% confluence in 6-8 days depending upon the split ratio.

Accutase Passaging Protocol

  1. Aliquot sufficient PluriSTEM (SCM130), Accutase (A6964) and DMEM/F12 (D6421) to passage the cells. Warm reagents at room temperature.
  2. One hour before the cells are to be passaged, add ROCK Inhibitor, Y-27632 (SCM075) to each well of the 6-well plate at a final concentration of 10 μM.
  3. After 1 hour, use a dissection microscope to visually inspect the plate containing human pluripotent cells to be passaged. Remove areas of spontaneous differentiation.
  4. Aspirate the medium containing the scrapped areas from the well. Rinse with 2mL per well with DMEM/F-12 (D6421) medium or 1X PBS (D8537).
  5. Aspirate and replace with 1 mL of Accutase (A6964) per well of a 6-well-plate. Incubate at 37°C for 8-10 minutes.
  6. Quench the Accutase reaction by adding 1 mL PluriSTEM (SCM130) for each mL of Accutase used. Gently detach cells using a sterile 1000-μL pipette tip.
  7. Collect the dissociated cells to a 15 mL conical tube. Rinse the wells with an additional 2 mL of PluriSTEM medium (SCM130) to collect any remaining cells. Add the rinse to the 15 mL conical tube.
  8. Centrifuge the 15 mL conical tube containing the cell suspension at 300 x g for 5 minutes at room temperature.
  9. Aspirate the supernatant. Resuspend the cells in fresh PluriSTEM (SCM130)  containing 10 μM ROCK Inhibitor, Y-27632 (SCM075).
  10. Count the number of cells using a Scepter or hemocytometer. Ensure that the cells are in a single cell suspension. Determine the cell viability using Trypan Blue (T8154) exclusion.
  11. Set up a titration of different cell densities ranging from 0.5 –1x104 cells/cm2. This corresponds to 50,000 – 100,000 cells per well of a Matrigel-coated 6-well plate in PluriSTEM medium containing 10 μM ROCK Inhibitor, Y-27632.
  12. The next day, replace with fresh PluriSTEM media (SCM130). Replace with fresh PluriSTEM medium (SCM130) every 2 days (3 mL volume per well). Cells can be passaged every 5-7 days.

Dispase Passaging Protocol

  1. Aliquot sufficient Dispase II, 1 mg/ml (CC130) and DMEM/F12 (D6421) to passage the cells. Warm reagents at room temperature.
  2. Use a dissection microscope to visually inspect the plate containing human pluripotent cells to be passaged. Inspect the colonies for areas of spontaneous differentiation.
  3. Use a sterile p200 pipette tip attached to a p200 pipetman to scrape away areas of spontaneous differentiation.
  4. Aspirate the medium containing the scrapped areas from the well. Rinse with 2mL per well of DMEM/F-12 (D6421) medium or 1X PBS (D8537).
  5. Add 1 mL Dispase II, 1 mg/ml (CC130) per well of the 6-well plate containing pluripotent human ES or iPS cells to be passaged.
  6. Incubate at 37°C for 6-7 minutes. After incubation, visually inspect the colonies under a microscope. The edges of the colonies may appear slightly rounded up and folded back but the overall colony should still be attached to the plate.
  7. Aspirate the Dispase II (CC130) and gently rinse each well two times with 2 mL 1X PBS (D8537) or DMEM/F12 (D6421) medium.
  8. Add 1.5–2 mL PluriSTEM medium (SCM130) to each well. Gently detach the colonies using a cell scraper.
  9. Use a 5 mL serological pipette to collect the cell aggregates to a 15 mL conical tube. Minimize pipetting up and down as this may break up the colonies to suboptimal small pieces.
  10. Rinse the wells with an additional 2 mL of PluriSTEM medium (SCM130) per well to collect any remaining cell aggregates. Add the rinse to the 15 mL conical tube.
  11. Centrifuge the 15 mL conical tube containing the cell aggregates at 300 x g for 5 minutes at room temperature.
  12. Aspirate the supernatant. Resuspend the cell aggregates in an appropriate volume of PluriSTEM (SCM130) for passaging. Split the cells 1:3 to 1:6 depending on number of cell clumps.
  13. Place the plate in a 37°C incubator. Agitate the plate gently from side to side and forward and backwards to ensure that the cell aggregates are evenly distributed across the surface of the well.
  14. The next day, replace with 3 mL per well of fresh PluriSTEM medium (SCM130). Cells can be passaged every 5-7 days.

Cryopreservation of Human iPSCs

  1. Keep reagents and freezing container (e.g. Mr. Frosty) chilled during the cryopreservation procedure.
  2. Cells must be cryopreserved when in their log phase of growth to enhance survival upon thaw. The optimal time for harvest is normally when cells are approximately 70-80% confluent
  3. The type of cryoprotectant medium used depends on culture conditions and laboratory preferences. Use either commercially available Cryostor CS10 (C2874) or DMSO (D2650) based freeze mix (10% DMSO in FBS and culture medium). Cryostor is supplied ready to use and is stored at 2-8°C. To prepare DMSO based cryoprotectant, mix 40% FBS with 10% DMSO, then mix with 50% appropriate medium.
  4. Remove spent medium from the tissue culture vessel and wash the vessel twice with the recommended volume of wash buffer depending on culture conditions (Wash buffer for Cultrex/Matrigel is 0.5mM EDTA, wash buffer for Vitronectin is PBS).
  5. To lift the cells from the tissue culture plastic, add 1ml of 0.5mM EDTA (03690) to the tissue culture vessel. Incubate the cells for the recommended time and temperature, depending on matrix used. Aspirate the EDTA from the well. Care must be taken as the colonies are very loosely attached to the plastic.
  6. Afterwards add 1 ml of cryoprotectant per 1 well. Gently wash the cryoprotectant over the vessel with a 1ml sterile pipette to dislodge the cells from the plastic. Do not aspirate more than 3 times to avoid breaking the cell clumps into single cells. Place the cryoprotectant and cell mix into an appropriately labelled cryovial.
  7. If cryopreservation of more wells is desired, cells from the same passage number and culture condition should be pooled together. An aliquot of pooled cells can be used for a cell count. Centrifuge harvested cells at 200xg for 2 minutes, aspirate spent medium and gently resuspend the cell pellet in an appropriate volume of cryoprotectant. One well of a 6-well plate gives rise to approximately 1-2x106 cells. It is recommended to freeze around 1-2x106 cells per cryovial. Use 1ml of cryoprotectant-cell mix per cryovial.
  8. Immediately place the cryovials into a pre-chilled Mr Frosty tub (2-8°C) then immediately transfer the Mr Frosty tub to a -80°C freezer. Allow the cells to remain at -80°C overnight (16-36 hours). Once frozen transfer the cells, on dry ice, to an ultra-low temperature storage vessel (LN2 or -150°C freezer).

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Characterization of Human iPSCs

The undifferentiated state of the iPSCs is characterized by high level of expression of alkaline phosphatase (SCR004) and the stem cell transcription factors Nanog, Oct-4 and Sox-2. These cells also exhibit marked differences from their murine counterparts in regards to their expression of stage-specific embryonic antigen (SSEA1, 3, 4) and Podocalyxin (TRA-1-60, TRA-1-81) markers. Cells can be analyzed by antibody ICC staining using stem cell characterization kits (SCR001, SCR002, SCR078) or by PCR analysis.

Summary of Human iPSC Markers

Cell Type Alkaline Phosphatase Oct-4 Sox-2 Nanog SSEA-1 SSEA-4 TRA-1-60 TRA-1-81
Human iPSC + + + + - + + +
Mouse iPSC + + + + + - - -

 

Pluripotent iPS cells express pluripotent markers

Figure 1. Pluripotent iPS cells express pluripotent markers, alkaline phosphatase (40x) (A), Oct-4 Alexa 488 (MAB4401A4, 400x) (B), Sox-2 Cy3 (MAB4423C3, 100x) (C), Nanog Alexa 488 (MABD24A4, 400x) (D), TRA-1-60 Cy3 (MAB4360C3, 100x) (E), and TRA-1-81-Cy3 (MAB4381C3, 100x) (F). Nuclei were counterstained with DAPI: blue (D9542).

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Troubleshooting Tips

Problem Observation Solution
Low viability of iPSCs
after thaw
• Little to no colonies visible
within 4 days after recovery
• Ensure that cryovials are thawed quickly and that medium is added to the cells very slowly (dropwise while gently swirling the tube)
• Add 10μm ROCK inhibitor at thaw but do not use routinely
• Ensure that cells were banked at log phase of growth with low levels of differentiation
• Let small colonies grow until robust and passage with low split ratio (1:1 or 1:2)
Low viability after
passage
• Cells do not attach properly
• Non-typical morphology
• High levels of cell death
• Cells do not proliferate
• Use lower split ratio and maintain a more confluent culture
• Ensure cells are in log phase of growth at passaging
• Work quickly or reduce incubation time of EDTA as clump size could be influenced by too long an exposure to EDTA
• Increase incubation time of EDTA if cells do not come off easily. This is to avoid having to harshly rinse cells off thereby creating too small aggregates/ single cell suspension
• Check if plates were coated correctly, matrix is within expiry date and check batch with manufacturer if this issue occurs on a regular basis and other reasons have been excluded
Spontaneous differentiation • Colonies do not have defined edges
• Cells within the colonies are
less compact
• Cells appear flattened and
bigger or fibroblastic
• Ensure cells are being cultured using recommendations (i.e. daily feeding of cells)
• Ensure that reagents are freshly prepared (i.e. used within two weeks)
• Avoid leaving plates outside the incubator to decrease temperature fluctuation and exposure to light.
• Decrease colony density by plating fewer cell aggregates per cm2 during passaging
• If good iPSC colonies persist between differentiated areas manual picking of colonies with good iPSC morphology using a pipette tip can be considered. It is recommended to select several colonies and cut them in pieces with a
pipette tip, lift them, aspirate them and then pass them to a fresh 1:6 well.
• Removal of differentiated cells by scraping the differentiation away with a pipette tip leaving iPSC colonies intact can be considered. Care must
be taken not to disturb the iPSC colonies and not to scrape away too much of the matrix layer in this process.
Non-uniform
distribution of colonies
within plate
• Areas with too high a density of iPS cells and where cells start to differentiate from the middle. Additionally to areas with hardly any colonies • Make sure that the whole surface area of the tissue culture vessel is coated with the
appropriate matrix
• Ensure that the cell aggregates are evenly distributed by gently rocking the plate back and
forth and side to side
• Take care when placing plate into the incubator and leave undisturbed for 24 hours
Significant scraping is
required to dislodge
cells
• Colonies do not come off the plate with 2- 3 rinses with a 1ml pipette • Ensure that incubation time and temperature of EDTA are in accordance with matrix
• Increase incubation time of EDTA
• Do not let cells become more than 70% confluent
• Do not let colonies become overgrown in the centers, sometimes it is necessary to passage a less confluent plate with fewer but robust colonies, using lower split ratio
Poor attachment and
significant increase in
cell death post passage
• Cells start to lift off even
though they seemed to attach
after passage
• Rather than exchanging medium top up wells with fresh medium to ensure sufficient amount of nutrients and leave cells undisturbed for an additional 24 hours to allow aggregates to fully attach
• Exchange medium very gently, do not subject colonies to excessive shear forces by rapid addition of medium

Trademarks

Matrigel® is a registered trademark of Corning, Inc.
Cultrex® is a registered trademark of Trevigen, Inc.
MTESR™ is a registered trademark WARF
Essential 8™ is a registered trademark of Thermo Fisher Scientific
TESR-E8™ is a registered trademark of Stem Cell Technologies
PluriSTEM™ is a registered trademark of Merck KGaA, Darmstadt, Germany