Culturing Human iPSCs

Reagents

Media

Human iPSCs can be cultured in a range of media; in this lab, we mainly use mTeSR-Plus. The medium has two components: the basal medium and the 5X supplement. The supplement is stored at -20°C; the basal medium is stored at 4°C. To make the complete medium, add an entire bottle of supplement to the bottle of basal medium. The complete medium is stored at 4°C. The exact amount of medium needed should be aliquotted and warmed before use.

Important

Do not warm the entire bottle of mTeSR-Plus!

Dissociation Reagents

There are two main dissociation reagents we use:

  1. ReLeSR: non-enzymatic dissociation of cells in clumps; better for regular passaging so as not to disturb the cells too much

  2. Accutase: enzymatic dissociation to single cells; necessary for counting and seeding

Other Reagents (Use bottles designated “iPSC Only”)

  • Geltrex: basement membrane for coating plates; 50X stored at -20°C in 120 µL aliquots, enough each for one 6-well plate (see GelTrex Aliquoting for more information)

  • ROCK inhibitor: Y-27632, a chemical inhibitor of ROCK activity used to prevent differentiation upon thawing and passaging; 1000X stored at -20°C

  • DMEM/F12 for coating plates and spinning down cells

  • PBS

  • FBS for freezing

Geltrex coating plates

Important

Plates require at least 1 hour to coat, so start early or make extras up to a week ahead of time!

  1. Thaw Geltrex on ice (use the designated TC styrofoam for ice) or at room temperature.

    • Thaw one tube (50X, 120 µL aliquot) per 6-well plate to coat. It is recommended to only do ~2 at a time to avoid unintentional gelling.

    • Flick the tube to mix as it thaws and to speed up the process.

    • While the Geltrex is thawing, prepare other materials in the BSC: 6-well plate(s), 15mL conical, DMEM/F12.

  2. Prepare at 15 mL conical with 6 mL DMEM/F12. Immediately once the Geltrex has thawed, pipet it into the prepared 15 mL conical. Pipet 1 mL of the DMEM/F12 mix back into the Geltrex tube to wash out any residual solution and return the liquid to the conical.

  3. With a serological pipette, pipet up and down once, then dispense 1 mL per well into the 6-well plate. Rock the plate to coat the entire surface.

  4. Let the plate sit to coat:

    • If using same-day: leave the plate at 37°C for at least 1 hour

    • If preparing ahead: parafilm the plate, then leave it at 4°C overnight. Plates can be stored at room temperature for at least a week.

Note

The official recommendation is to store coated plates at 4ºC for up to 2 weeks, and then equilibrate them at room temp before plating.

  1. Immediately before seeding cells, aspirate the liquid from the coated well(s). Leave the liquid on any unused wells; these can be aspirated and used at a later date.

Thawing

Estimated time

~30 min

  1. Remove cryovial(s) of cells from liquid nitrogen and thaw quickly in the 37°C bead bath.

Important

Thaw cells quickly and spin down as soon as all ice is gone to limit the cells’ exposure to DMSO.

  1. Pipet the contents of the cryovial into a 15 mL conical. Then, GENTLY add 10 mL of DMEM/F12. Optionally, pipet 1 mL of liquid from the conical around in the cryovial to remove any residual cells, then return the liquid to the conical.

  2. Spin down cells at 40g for 4 min. If cells were not frozen in big enough clumps, they may not pellet at this low speed. In that case, you can try 400g, but cells frozen in these smaller clumps may not recover as well.

  3. Aspirate the media and gently resuspend the cells in the culture medium (i.e., mTeSR) and plate. Typically, iPSCs are frozen such that 1 vial goes to 1 to 6 wells of a 6-well plate. Decide how many wells to use based on the size of the pellet.

  4. Within 24 hours (e.g., the next day), media change to fresh mTeSR. Then, culture as normal.

General Culturing Tips

  • Always culture human iPSCs on Geltrex-coated plates (or other similar matrix coating).

  • Typically, all culturing is done in 6-well plates. Use at least 2 mL of media per well.

  • Change to fresh media every day to keep the cells healthy. iPSCs are very metabolically active and can easily spontaneously differentiate, so if the media looks very yellow try refreshing with a larger volume (e.g., 3 mL).

  • Some amount of cell death is expected, but media changes every 24 hrs with enough fresh media should mitigate this.

  • To maintain optimal cell health, be sure to passage cells before they are 100% confluent. Typically, healthy cells that are passaged 1:6 will need to be passaged again 2 days later.

  • Splits of 1:6 to 1:12 are recommended. Larger dilutions may impede growth if the cells are too sparse, and lower dilutions will require very frequent passaging.

  • iPSCs grow in colonies with a cobblestone-like cell morphology. The edges of the colonies should be smooth, not spiky.

  • Be extra attentive with sterile technique (i.e., don fresh gloves before beginning, use separate glass pipet aspirators for different cell lines) to avoid contamination.

Passaging with ReLeSR

Estimated time

~15 min — don’t forget to coat plates at least 1 hr ahead of time!

To passage for general cell culture maintenance, use ReLeSR to dissociate cells in clumps. This is less disruptive and may improve the long-term integrity of the iPSCs. For seeding or other applications that require counting, dissociate with Gentle Cell Dissociation Reagent (preferred) or Accutase to achieve a single-cell suspension (see Dissociating with Gentle Cell Dissociation Reagent (GCDR) and Dissociating with Accutase below).

  1. Aspirate the old media. Gently wash with 1 mL PBS and aspirate.

  2. Add 1 mL ReLeSR and incubate at room temperature for 1 min.

  3. Immediately after the 1 min is up, aspirate to remove the ReLeSR. At this point, no cells should be lifting off from the plate.

  4. Incubate the empty plate (it essentially has a thin film of liquid) at 37°C for 5-7 min.

  5. When the plate is done incubating, add 1-3 mL media to each well and tap the plate to dislodge the cells.

    • It is convenient to add 0.5 mL of media for each new well you’re passaging into, e.g., 3 mL media for passaging one well 1:6.

  6. Gently pipet up the liquid with a serological and dispense into the prepared wells (with Geltrex aspirated).

    • Use a serological pipette rather than a P1000 to maintain cell clumps.

    • Pipet up and down 1-2 times in the well to resuspend as many cells as possible, since the cells tend to stick to the well. However, don’t pipet too much—this will break up the clumps!

  7. Add additional media to each well to bring the total volume to the desired amount (e.g., 2 mL). Pipetting up and down here is not necessary; rocking back and forth achieves sufficient mixing.

    • Alternatively, dissociated cells from the previous step can be pipetted into a conical to be mixed with fresh media, then transferred from the conical to the new wells.

    • After dissociation, avoid excessive pipetting to maintain cell clumps.

  8. Within 24 hours (e.g., the next day), media change to fresh mTeSR. Then, culture as normal.

Dissociating with Gentle Cell Dissociation Reagent (GCDR)

Estimated time

~15 min — don’t forget to coat plates at least 1 hr ahead of time!

To seed cells for an experiment, use Gentle Cell Dissociation Reagent (GCDR) to dissociate single cells. This allows for more accurate cell counting and seeding. However, ROCK inhibitor (Ri) must be added during seeding to promote survival of the single cells. GCDR is preferred over Accutase because it is non-enzymatic and gentler on the cells.

Note

Addition of Ri leads to cytoskeletal remodeling, so cells will have a spiky morphology the day after passaging. The morphology should return to normal a day or so after the ROCK inhibitor is removed.

  1. Aspirate old media. Gently wash with 1 mL PBS and aspirate.

  2. Add 1 mL GCDR (100 uL for a 96-well plate) and incubate at 37°C for 8-10 min until cells ball up on the plate. You will likely NOT observe cells lifting off.

  3. Gently pipet up and down to dislodge cells. If preparing for flow cytometry, spin down the plate and prep as normal. If preparing to seed cells, add the dissociated cells to a 15 mL conical with DMEM/F12 and spin at 400g.

    • While cells are spinning, prepare 1000x dilution of Ri in mTeSR.

  4. Aspirate the media and gently resuspend the cells in a small amount of mTeSR (e.g., 1 mL).

  5. Count cells as normal and dilute to desired concentration.

  6. Aspirate Geltrex from wells to seed and add cell suspension.

  7. Within 24 hours (e.g., the next day), media change to fresh mTeSR without Ri. Then, proceed with the experiment as normal.

Dissociating with Accutase

Estimated time

~15 min — don’t forget to coat plates at least 1 hr ahead of time!

Accutase can be used to dissociate iPSCs to single cells for seeding. However, Gentle Cell Dissociation Reagent (GCDR) is preferred, since Accutase (enzymatic) is harsher on the cells than GCDR (non-enzymatic).

Note

Addition of Ri leads to cytoskeletal remodeling, so cells will have a spiky morphology the day after passaging. The morphology should return to normal a day or so after the ROCK inhibitor is removed.

  1. Aspirate old media. Gently wash with 1 mL PBS and aspirate.

  2. Add 1 mL Accutase (100 uL for a 96-well plate) and incubate at 37°C for 5-7 min until cells begin to lift off.

  3. Gently pipet up and down to dislodge cells. If preparing for flow cytometry, spin down the plate and prep as normal. If preparing to seed cells, add the dissociated cells to a 15 mL conical with DMEM/F12 and spin at 400g.

    • While cells are spinning, prepare 1000x dilution of Ri in mTeSR.

  4. Aspirate the media and gently resuspend the cells in a small amount of mTeSR.

  5. Count cells as normal and dilute to desired concentration.

  6. Aspirate Geltrex from wells to seed and add cell suspension.

  7. Within 24 hours (e.g., the next day), media change to fresh mTeSR without Ri. Then, proceed with the experiment as normal.

Freezing

  1. Passage cells with ReLeSR through step 4 - incubate empty plate at 37°C for 5-7 min.

  2. Add 0.9 mL of FBS to each well, tap to dislodge cells.

  3. Gently pipet up the liquid with a serological and dispense into a labeled cryovial. 1 well of a 6-well plate per cryovial.

    • Alternatively, dissociated cells from multiple wells can be pooled into a conical tube before aliquoting for freezing.

    • After dissociation, avoid excessive pipetting to maintain cell clumps.

  4. Add 100 uL of DMSO to cryovial to achieve a final concentration of 10% DMSO.

    • If multiple wells were pooled in the previous step, add 100 uL of DMSO per well pooled, and then aliquot 1 mL of final mixture into labeled cryovials.

  5. Transfer tubes to styrofoam boxes in -80 °C freezer for overnight freezing.

  6. The following day, transfer frozen tubes to liquid nitrogen for longe term storage.