Induced pluripotent stem cells (iPSCs) have the capacity to give rise to differentiated progeny representative of all three germ layers of the body: 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 iPSCs supplied by the European Bank of induced pluripotent Stem Cells (EBiSC). Human induced pluripotent stem cell (iPSC) lines are different from 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 before starting, including the sections on required reagents, thawing, passaging, precautions, and troubleshooting tips.
- Make sure all necessary reagents are available prior to thawing the cells.
- Use the correct media and matrix combination. iPSCs require specialized media and culture conditions.
- Make sure your equipment is calibrated regularly and no reagents have expired.
The certificate of analysis (CofA) makes recommendations for thawing cells in media and matrix that are specific to each cell line. Where required, the matrix and media used can be changed to an alternative during passaging only. Cells may require time to adapt following a change of media or matrix. No guarantees can be given regarding cell viability or quality when the recommended tissue culture system is not used.
Extracellular Matrix Preparation
Preparation of Basement Membrane Extract (BME)
Stem Cell Qualified ECM Gel Matrix (CC131) is a soluble basement membrane extract (BME) purified from Engelbreth-Holm-Swarm (EHS) tumors that has been pre-qualified for human ES/iPS cell culture. The matrix polymerizes at 20-40 °C to form a reconstituted basement membrane. The product contains laminin, collagen IV, entactin and heparan sulfate among other ECMs. The matrix eliminates the need for time-consuming prescreening and lot identification, provides an optimized ECM coating necessary for long term culture of pluripotent human ES/iPS cells, and supports the culture of human ES/iPS cells in multiple serum-free/feeder free human ES/iPS cell expansion media (i.e. mTeSR®, PluriSTEM™). Below are general guidelines for the coating of 6-well plates using the Stem Cell Qualified ECM Gel Matrix. All procedures should be performed under aseptic conditions in a biological safety cabinet.
- Thaw the ECM Gel Matrix (CC131) in the 2-8 °C fridge one day before use. Once thawed, maintain ECM Gel on ice at all times and use pre-cooled medium and pipettes to avoid gelling of the product.
- Dilute the ECM Gel Matrix (CC131) 1:80 with cold DMEM/F12 (D6421) medium. Scale up or down according to the volumes required. Below is an example for coating a 6-well plate with 1.5 mL of 1:80 diluted ECM Gel.
To make a 1:20 dilution, add 0.5 mL ECM Gel to 9.5 mL cold DMEM/F12 or DMEM medium into a 15 mL conical tube. Total volume = 10 mL.
A further 4-fold dilution is required to make the final 1:80 dilution. Add 2.5 mL of 1:20 diluted ECM Gel to 7.5 mL cold DMEM/F12 medium. Total volume = 10 mL
NOTE: The recommended dilution is 1:80, however more concentrated Stem Cell Qualified ECM Gel may be used if desired.
- Add 1.5 mL of the 1:80 diluted ECM Gel Matrix (CC131) to each well of a 6-well plate. Swirl the culture plates to spread the ECM Gel Matrix evenly across the surface of the plate. Store in a 2 – 8 °C fridge overnight or at least 2 hours in the fridge before use. If not used immediately, parafilm wrap the ECM coated culture plates and store at 2 - 8 °C until ready to use. Use the ECM coated culture plates within 3-4 days.
- Prior to seeding the cells, bring the plate back to room temperature for 10-15 minutes, remove the coating solution and add 3 mL/well of human ES/iPSC growth media (SCM130). Cells can now be plated onto the newly coated plates.
IMPORTANT: Do not allow the plates to dry out.
Preparation of Vitronectin
- Upon receipt, store Vitronectin (CC130) at -80 °C. Prior to use, thaw the stock vial of Vitronectin at room temperature and prepare 60 μL aliquots in sterile polypropylene tubes. Freeze the aliquots at -80 °C or use immediately. One 60 μL aliquot is sufficient for coating all wells of a 6-well plate.
- To prepare Vitronectin at a working concentration of 0.5μg/cm2, dilute the Vitronectin 1:100 by gently mixing 6 mL of room temperature PBS (D8537) with 60 μL of Vitronectin. Add 1 mL of diluted Vitronectin to each well of a 6-well plate.
- 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.
- 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.
Cell Culture Media Preparation
- When required, remove the mTeSR™1 supplement (5x) from the freezer and thaw overnight at 2-8 °C prior to use. Do not thaw at 37 °C.
- Aseptically add 100 mL of mTeSR™1 supplement (5x) to 400 mL of cold (2-8 °C) basal medium.
- Aliquot medium into volumes required for 1 week of culture work.
- Complete mTeSR™1 may be stored at 2-8 °C for 1 week or at -20 °C for 6 months. Frozen complete mTeSR™1 may be thawed once. Do not repeatedly freeze thaw medium. Prior to use, warm mTeSR™1 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
- 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.
- Aseptically remove 10 mL of E8 basal medium to leave 490 mL.
- Add 10 mL of E8 supplement (50x) to the 490 mL of basal cold (2-8 °C) medium.
- Aliquot medium into volumes required for 1 week of culture work.
- 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® medium (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 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 2-8 °C and used within two weeks.
Thawing of Human iPSCs
- 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% ethanol (793213) or an equivalent disinfectant before opening.
- Using a 5 mL sterile pipette, transfer the cryoprotectant/cells mix from the cryovial into a 15 mL centrifuge tube. Care should be taken not to physically damage cells.
- Slowly, drop by drop, add 10 ml of appropriate medium at room temperature to the cells in the 15 mL centrifuge tube. Gently rock the 15 mL centrifuge tube back and forth while adding drops of medium. This is a crucial step than minimizes osmotic shock to the cells and helps to ensure that cells are treated as gently as possible.
- Check tube to ensure all cell contents are removed. If needed, rinse with 1 mL of appropriate medium.
- A small amount of cells can be used for performing a cell count. A single cell suspension should be created using trypsin or other appropriate cell detachment medium. As a general guideline, the seeding density range for one well of a 6-well plate is between 2x105 - 1x106 viable cells. Refer to CofA for guidelines for any specific EBiSC cell line lot number.
- Centrifuge the cells at 200 x g for 2 minutes. Remove and discard the supernatant.
- Prepare culture vessels by adding an appropriate amount of medium (for example 1.5 – 2 mL per one well of a 6-well plate).
- Gently tap the 15 mL centrifuge tube to dislodge the cell pellet, then gently add 1 mL 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 overaspirate the cells, as this will lead to decreased viability due to generation of a single cell suspension.
- Gently rock plate side to side, and back and forth to spread the cells evenly across the well.
- It is advisable to record images of cells immediately post-thaw, at 48 hours and at approximately 70-80% confluence.
Culturing of Human iPSCs
- It is good practice to observe iPSC lines daily under phase contrast microscopy (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: