Apr 30, 2026
Version 1
hiPSC-CMs Freezing, Thawing, Recovery, and Regrowth Protocol V.1
- 1Max Delbrück Center for Molecular Medicine
Protocol Citation: Yuichiro Ueda 2026. hiPSC-CMs Freezing, Thawing, Recovery, and Regrowth Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbjb23vpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 29, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 315957
Keywords: workflow for the cryopreservation, cryopreservation, osmotic buffering during thaw, induced pluripotent stem cell, pluripotent stem cell, cryomedium, rate freezing, cryostor cs10, thawing, cms freezing, derived cardiomyocyte, refined seeding density guidance, rock inhibitor treatment
Abstract
This protocol details an optimized workflow for the cryopreservation, thawing, and subsequent regrowth of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). It builds on the foundational approach using 10% DMSO / 90% KSR cryomedium with VIA Freeze controlled-rate freezing (adapted from Miller et al., 2020) and incorporates comprehensive evidence-based optimizations across all stages: cryomedium benchmarking against CryoStor CS10 and osmolyte-containing formulations (7.5% DMSO + 2.5% trehalose), advanced cooling profiles with controlled nucleation, pre-freeze ROCK inhibitor treatment, osmotic buffering during thaw, blebbistatin-mediated contraction inhibition post-thaw, optimized centrifugation, expanded replating medium options (including defined HSA-based medium), refined seeding density guidance (80,000-120,000 cells/cm²), ECM optimization, and functional recovery metrics (beating percentage, calcium transients, MEA/impedance readouts). The protocol is structured to support systematic Design of Experiments (DoE) for further optimization. All key steps include rationales and practical alternatives for head-to-head comparison.
Guidelines
1. Prepare Cryomedium**: Prepare a mixture of 10% (v/v) DMSO and 90% (v/v) KSR. This can be made fresh or stored for up to two weeks at 4°C.
2. Harvest Cells**: Dissociate hiPSC-CMs from their culture vessels (typically between days 11 and 28 of differentiation).
3. Resuspension**: Resuspend the dissociated hiPSC-CMs in the KSR-DMSO cryomedium to achieve an optimized intermediate density of 4 – 15 × 10^6^ cells/ml.
4. Aliquot**: Dispense 0.25 – 0.5 ml of the cell suspension into each 1.8 – 2 ml cryovial.
**Step 2: Controlled-Rate Freezing**
1. Device Loading**: Transfer the prepared cryovials to the VIA Freeze system.
2. Cooling Cycle**: Program the VIA Freeze to execute a near-linear cooling rate of approximately -1 to -2°C/min down to -100°C.
3. Storage**: Once the temperature reaches -100°C, quickly transfer the cryovials on dry ice to the liquid nitrogen (LN2) vapor phase for long-term storage (ensure a minimum of 24 h storage before thawing).
**Step 3: Thawing and Immediate Recovery**
1. Dry Ice Transfer**: Transfer cryovials from LN2 to dry ice (-79°C) for at least 30 minutes (20 minutes with lids on, 10 minutes with lids off to vent).
2. Water Bath Thaw**: Thaw vials in a 37°C water bath for no longer than 2 minutes, ensuring ice crystals are still floating.
3. Dilution**: Add the cell suspension into at least 10x volume of pre-warmed (37°C, strictly *not* RT) thawing medium (RPMI only, without any additional reagents). For example, add 500 μL of cells into 5 mL of medium, or 1000 μL into 10 mL.
4. First Centrifugation**: Centrifuge very mildly at 100 × g for 3 minutes.
5. Resuspension**: Carefully aspirate the supernatant and add at least 2 mL of pre-warmed (37°C, strictly *not* RT) replate medium (RPMI + B27 + 10% KSR + 10 μM ROCKi).
6. Gentle Mixing**: Pipette gently 5-6 times using a 1000 μL tip.
7. Viability Check Preparation**: Prepare 10 μL Trypan Blue in a conical 96-well plate. Add 10 μL of the well-mixed cell suspension drop-wise, and mix again drop-wise 3 times.
8. Counting**: Cast the mixture into a hemocytometer for the Countess 3 (verify the Trypan Blue color is evenly distributed). Record the Counts [Mil/mL], Viability [%], Live average diameter [μm], Dead average diameter [μm], and calculate the Recovery rate [%, Live/Frozen cell numbers].
9. Seeding**: Seed cells at 50,000 cells/cm² (e.g., 480k per well in a 6-well plate, *optimized from 21,000 - 764,000 cells/cm²). Wait at least 20 minutes at room temperature to allow CMs to settle evenly to the bottom of the wells before transferring to the incubator (to prevent cells from gathering strictly in the center of the wells).
**Step 4: Medium Maintenance and Day 7 Dissociation**
1. Day 1 Medium Change**: Perform a full medium change after 24 hours to completely remove any remaining DMSO.
2. Day 3 and 6 Maintenance**: Perform a 50% volume medium change on days 3 and 6.
3. Day 7 Washing and Dissociation**: On day 7 after seeding, perform 2x washes with PBS(-). Add 10x TrypLE and incubate with shaking (65 rpm) for 5-7 minutes at 37°C.
4. Harvesting**: Use a 1000 μL tip to pipette 3-4 times per well to dislodge cells. Collect the suspension into a 15 mL or 50 mL falcon tube.
5. Collection Wash**: Wash the wells with 2 mL of pre-warmed (RPMI only) medium and pool the collection into the falcon tube.
6. Second Centrifugation**: Centrifuge very mildly at 100 × g for 3 minutes.
7. Regrowth Resuspension**: Carefully aspirate the supernatant and add at least 2 mL of pre-warmed (37°C, strictly *not* RT) replate medium (RPMI + B27 + 10% KSR + 10 μM ROCKi).
8. Gentle Mixing**: Pipette gently 5-6 times using a 1000 μL tip.
9. Regrowth Viability Preparation**: Prepare 10 μL Trypan Blue in a conical 96-well plate. Add 10 μL of the well-mixed cell suspension drop-wise, and mix again drop-wise 3 times.
10. Regrowth Counting**: Cast the mixture into a hemocytometer for the Countess 3. Record the Counts [Mil/mL], Viability [%], Live average diameter [μm], Dead average diameter [μm], and calculate the final Regrowth rate [%, Live/Seeded cell numbers].
Materials
- hiPSC-CMs (Day 11 to 28 of differentiation)
- KnockOut Serum Replacement (KSR)
- Dimethyl sulfoxide (DMSO)
- CM Suspension medium (containing KSR)
- RevitaCell supplement or 10 μM ROCK inhibitor (ROCKi)
- RPMI/B27 maintenance medium
- RPMI (basal medium without additional reagents)
- 10x TrypLE
- PBS(-)
- Trypan Blue
- Geltrex or Matrigel-coated culture plates (e.g., 6-well plates)
- VIA Freeze controlled-rate freezing system
- 1.8–2 ml cryovials
- 15 ml and 50 ml sterile centrifuge tubes
- Hemocytometer (Countess 3 compatible)- CryoStor CS10 (BioLife Solutions) [cGMP-grade cryomedium, optional benchmark]
- Trehalose (e.g., D-(+)-Trehalose dihydrate, Sigma-Aldrich) [osmolyte additive for cryomedium and/or thaw medium]
- Sucrose [alternative osmolyte for thaw medium, 25-50 mM]
- Y-27632 (ROCK inhibitor, 10 mM stock) [for pre-freeze recovery and replating]
- Blebbistatin or para-nitroblebbistatin (photostable analog) [optional, 2.5-5 µM for first 24 h post-thaw]
- Human Serum Albumin (HSA, pharmaceutical grade) [optional, for defined replating medium]
- Dead Cell Removal Kit (density gradient or magnetic bead-based) [optional, for pre-plating dead cell clearance]
Freezing Protocol
Prepare Cryomedium: Prepare a mixture of 10% (v/v) DMSO and 90% (v/v) KSR as the standard cryomedium. This can be made fresh or stored for up to two weeks at 4°C. Alternative options to benchmark in parallel: (A) CryoStor CS10 (BioLife Solutions): a cGMP-grade, protein-free, defined 10% DMSO cryomedium; use side-by-side against KSR/DMSO to assess post-thaw viability and regulatory suitability. (B) 7.5% DMSO + 2.5% trehalose (w/v) + 90% KSR: a reduced-DMSO variant with osmolyte support that may improve post-thaw functional recovery while lowering cytotoxic DMSO exposure. Keep all other variables identical when comparing cryomedia.
Harvest Cells: Aspirate old culture medium completely and perform 2x PBS(-) washes (at least RT or 37°C pre-warmed, at least 200 µl/cm²). Dissociate hiPSC-CMs with 10x TrypLE (37°C pre-warmed, 100 µl/cm²) from their culture vessels on the shaker (65 rpm, 25 mm Throw) for 4-7 min. Harvest cells typically between days 11-28 of differentiation; standardize to the window where CMs show stable spontaneous beating, consistent cell size, and minimal proliferative contaminants — lock this window in as your standard once identified. Note: cell growth phase and confluence at freezing significantly affect post-thaw recovery.
Pre-freeze Recovery (Optional but Recommended): After dissociation and initial centrifugation (Step 4), resuspend cells in warm RPMI+B27 (37°C) containing 10 µM Y-27632 (ROCK inhibitor) and incubate at 37°C for 1-2 hours before adding cryomedium. This brief recovery period: (1) reduces dissociation-induced apoptosis via ROCK inhibition, (2) allows cells to re-equilibrate after enzymatic stress, and (3) improves post-thaw viability. Keep dissociation-to-freezing time and temperature tightly controlled when standardizing this step. If time is critical, at minimum add 10 µM ROCKi to the resuspension medium and proceed directly.
Dissociation: Gentle Mixing: Pipette gently 5-6 times using a 1000 μL tip to dissociate the hiPSC-CMs from the bottom and transfer to 15 or 50 ml falcon and add fresh culture medium (4°C, at least 200 µl/cm2) to rinse the well and transfer to 15 or 50 mL falcon.
Centrifugation: 100 xg for 3 min (at RT or 4°C) and aspirate the supernatant carefully and resuspended with fresh culture medium adjust the range of 1-3 x 106/ml for precise counting.
Viability Check Preparation: Prepare 10 μL Trypan Blue in a conical 96-well plate. Add 10 μL of the well-mixed cell suspension drop-wise, and mix again drop-wise 3 times.
Resuspension: Resuspend the dissociated hiPSC-CMs in the chosen cryomedium to achieve an optimized density of 5 – 8 × 10⁶ cells/ml (narrowed from the 4–15 × 10⁶ range to avoid high-end density effects: excessive local DMSO and metabolic waste accumulation during cooling). Avoid densities above 10 × 10⁶ cells/ml unless experimentally validated for your line and cryomedium combination.
Aliquot: Dispense 0.5 ml (preferred standard) of the cell suspension into each 1.8 – 2 ml cryovial. Standardizing to 0.5 ml fill (vs. 0.25 ml) reduces headspace, leading to more reproducible cooling profiles within the VIA Freeze system. Avoid overfilling (>0.5 ml) as this can slow heat transfer and create ice formation heterogeneity.
Device Loading: Transfer the prepared cryovials to the VIA Freeze system.
Cooling Cycle: Standard profile: Program the VIA Freeze to execute a near-linear cooling rate of approximately -1 to -2°C/min down to -100°C. Advanced profile (to test for improved recovery): (1) Cool at -1°C/min to -6 to -8°C, (2) apply a controlled nucleation/ice seeding step, (3) then cool at 2-5°C/min to -40°C, (4) followed by 5-10°C/min to -100°C. Rationale: too-slow cooling throughout can exacerbate cellular dehydration; controlled nucleation followed by moderate-fast cooling can reduce this. Compare profiles with all other variables identical and assess live/dead counts and Day 7 regrowth.
Storage: Once the temperature reaches -100°C, quickly transfer the cryovials on dry ice to the liquid nitrogen (LN2) vapor phase for long-term storage (ensure a minimum of 24 h storage before thawing). Note: Where logistics allow, consider transferring directly from the VIA Freeze to LN2 vapor phase without the dry ice intermediary step, as each temperature transition (LN2 → dry ice → water bath) adds risk of partial warming and recrystallization. If dry ice transport is used, enforce consistent timing and ensure vials do not warm above -50°C during transfer.
Further Optimized Thawing and Regrowth Protocol
Dry Ice Transfer (Optional): Transfer cryovials from LN2 to dry ice (-79°C) for at least 30 minutes (20 minutes with lids on, 10 minutes with lids off to vent). Note: This step is useful for safety and logistics but introduces an additional temperature transition that risks partial warming and recrystallization. Where possible, go directly from LN2 to the 37°C water bath. If dry ice transfer is used, enforce strict timing and transport to prevent vials from warming above -50°C.
Water Bath Thaw: Thaw vials in a 37°C water bath for no longer than 2 minutes. Standardize the endpoint: remove vials when exactly one small sliver of ice (1-2 mm) remains floating in the vial — this minimizes DMSO exposure time while preventing over-thawing and recrystallization at room temperature. Avoid fully melting before transfer. Use gentle swirling motion (do not vortex) during thaw.
Dilution: Add the cell suspension into at least 10x volume of pre-warmed (37°C, strictly *not* RT) thawing medium. Standard thawing medium: RPMI only (without additional reagents) + 10 µM Y-27632 (ROCKi). For example, add 500 µL of cells into 5 mL of medium, or 1000 µL into 10 mL. Pour the suspension slowly down the tube wall or use wide-bore tips to minimize shear stress. Osmotic buffering option (recommended): add trehalose or sucrose to the thawing medium at 25-50 mM for the first 5-10 min post-thaw before proceeding with centrifugation; this reduces osmotic shock due to the unusual volume drop hiPSC-CMs show upon resuspension in standard medium. If an osmolyte-containing cryomedium (e.g., 7.5% DMSO + trehalose) is used, match some osmolyte content in the thaw medium to reduce abrupt osmotic shifts.
Blebbistatin Treatment (Optional, Recommended for stressed cells): Add 2.5-5 µM blebbistatin (or a photostable analog, e.g., para-nitroblebbistatin) to the replating medium for the first 24 hours post-thaw. Rationale: spontaneous contraction immediately post-thaw exacerbates cell death in stressed hiPSC-CMs; transient myosin ATPase inhibition reduces contraction-induced mechanical stress and improves early attachment and spreading. Remove blebbistatin completely at the Day 1 medium change (24 h) to restore normal electromechanical function. Note: keep blebbistatin-treated plates away from direct light exposure to prevent phototoxicity.
First Centrifugation: Centrifuge very mildly at 100 × g for 3 minutes (standard). If cell loss in the supernatant is observed, test 150-200 × g for 2.5-3 minutes; slightly higher g forces can improve cell pellet recovery without increasing cell death provided handling remains gentle. Assess viability and regrowth when adjusting centrifugation conditions.
Resuspension: Carefully aspirate the supernatant and add at least 2 mL of pre-warmed (37°C, strictly *not* RT) replate medium. Standard replate medium: RPMI + B27 + 10% KSR + 10 µM ROCKi + (if used) blebbistatin. ROCKi duration: include for 24-48 h post-thaw; some lines benefit from 48 h ROCKi exposure post-thaw; taper by switching to standard RPMI+B27 without ROCKi after this period. Defined/reduced-serum alternative (to compare): RPMI + B27 + 5% KSR + 2-5% human serum albumin (HSA, pharmaceutical grade) + 10 µM ROCKi; reduces batch variability vs. 10% KSR. Short-term commercial CM medium (optional): use a commercial cardiomyocyte maintenance medium for the first 24-48 h after thaw, then switch back to RPMI+B27; this may provide additional survival signals. ECM: Ensure Geltrex or Matrigel coating is fresh, from a consistent batch, at standardized dilution, incubation time, and temperature. Consider using a higher ECM density (1.5-2× standard concentration) for post-thaw replating to improve attachment of stressed cells. For MEA cultures, pre-patterned ECM stripes or micro-contact printing can improve uniform spreading.
Gentle Mixing: Pipette gently 5-6 times using a 1000 μL tip.
Viability Check Preparation: Prepare 10 μL Trypan Blue in a conical 96-well plate. Add 10 μL of the well-mixed cell suspension drop-wise, and mix again drop-wise 3 times.
Counting: Cast the mixture into a hemocytometer for the Countess 3 (verify the Trypan Blue color is evenly distributed). Record the Counts [Mil/mL], Viability [%], Live average diameter [μm], Dead average diameter [μm], and calculate the Recovery rate [%, Live/Frozen cell numbers].
Seeding: Standard: Seed cells at 50,000 cells/cm² (e.g., 480k per well in a 6-well plate). Optimized range for better syncytia formation: 80,000–120,000 cells/cm² (tested from 21,000–764,000 cells/cm²); higher densities within this range often improve functional coupling and survival, though optimal density depends on cell line size and purity — validate for your line. Suggested DoE to optimize: test 40k, 80k, 120k, 160k cells/cm² × ROCKi duration (24 h vs. 48 h) × ECM concentration (standard vs. 2×); read out beating area, live cell counts, and Day 7 regrowth. Wait at least 20 minutes at room temperature to allow CMs to settle evenly to the bottom of the wells before transferring to the incubator (to prevent cells from gathering strictly in the center of the wells). If large numbers of dead cells are observed at seeding, consider dead-cell removal (density gradient or magnetic bead-based) before replating to improve effective live-cell seeding density.
Medium Maintenance and Day 7 Dissociation
Day 1 Medium Change: Perform a full medium change after 24 hours to completely remove any remaining DMSO.
Day 3 and 6 Maintenance: Perform a 50% volume medium change on days 3 and 6 (standard). At higher seeding densities (>80,000 cells/cm²) or if significant metabolic stress/lactate accumulation is observed (e.g., rapid medium color change to yellow), switch to daily 50% medium changes from day 2-5 to maintain pH and nutrient availability. Resume the 50% every-other-day schedule from day 6 onwards if cells appear stable.
Day 7 Washing and Dissociation: On day 7 after seeding, perform 2x washes with PBS(-). Add 10x TrypLE and incubate with shaking (65 rpm) for 5-7 minutes at 37°C.
Harvesting: Use a 1000 μL tip to pipette 3-4 times per well to dislodge cells. Collect the suspension into a 15 mL or 50 mL falcon tube.
Collection Wash: Wash the wells with 2 mL of pre-warmed (RPMI only) medium and pool the collection into the falcon tube.
Second Centrifugation: Centrifuge very mildly at 100 × g for 3 minutes.
Regrowth Resuspension: Carefully aspirate the supernatant and add at least 2 mL of pre-warmed (37°C, strictly *not* RT) replate medium (RPMI + B27 + 10% KSR + 10 μM ROCKi).
Gentle Mixing: Pipette gently 5-6 times using a 1000 μL tip.
Regrowth Viability Preparation: Prepare 10 μL Trypan Blue in a conical 96-well plate. Add 10 μL of the well-mixed cell suspension drop-wise, and mix again drop-wise 3 times.
Regrowth Counting: Cast the mixture into a hemocytometer for the Countess 3. Record the Counts [Mil/mL], Viability [%], Live average diameter [μm], Dead average diameter [μm], and calculate the final Regrowth rate [%, Live/Seeded cell numbers].
Functional Viability Assessment: In addition to Trypan Blue viability and cell diameter metrics (already recorded), assess functional recovery with these additional metrics at Day 7 regrowth: (1) Spontaneous beating rate and percentage of beating area (visual assessment or automated beating analysis software); (2) Calcium transient amplitude and kinetics (if calcium imaging is available; use Ca2+ indicator dye or genetically encoded calcium indicator); (3) Contractility/impedance readout (if impedance-based platform such as xCELLigence RTCA or CardioExcyte is available); (4) MEA field potential parameters: beating frequency, FPD, conduction velocity (for MEA-plated cultures). Note: live/dead counts and regrowth % can sometimes diverge from functional outcome; functional metrics provide a more complete picture of post-thaw recovery quality.
Protocol references
https://doi.org/10.1186/s13287-025-04384-5
https://doi.org/10.3892/etm.2020.8436
https://doi.org/10.1101/2025.09.04.674322