Ex vivo simulated ischemia reperfusion

Ischemic injury is a pervasive, pathological mechanism that underlies many debilitating conditions, such as cardiac arrest, myocardial infarction, or stroke. Currently, no therapies exist to reduce or reverse ischemic damage. Thus, identifying strategies that may protect tissues from ischemic damage is critical. Here, we provide a step-by-step protocol to measure cell survival following ex vivo ischemic reperfusion injury in human induced pluripotent stem cell-dervied cardiomyocytes (hIPSC-CMs), including how to acquire hIPSC-CMs, hIPSC-CM cell culture, hIPSC-CM differentiation, sample and reagent preparation, and cell survival analysis. The protocol described here may be applicable to other cell types that are sensitive to ischemic injury.

Keywords: Cardiomyocytes, Ischemia, Ischemia-repurfusion, Ischemic Protection, Oxygen

Ischemia reperfusion (I/R) injury is a pathological mechanism that occurs when the blood supply to an organ is disrupted, and then restored, and underlies many common disorders, such as heart attack or stroke (Yellon DM, 2007). Intracellular mechanisms underlying I/R injury include oxidative stress, calcium overload, metabolic dysfunction, and inflammation (Eltzschig HK, 2011, Timmers L, 2012). Effective therapies to reduce or reverse tissue injury following ischemia have not been identified. Therefore, idenitfying novel endogenous ischemic protectant molecules and their specific mechanisms that preserve tissue function is of significant interest.

Any improvement in the identification of putative therapeutic targets and drug development in the setting is I/R injury can only be based on detailed mechanistic understanding of the processes that drive I/R injury. While many compounds have shown promise in reducing I/R injury in preclinical studies, there have been none identified that have demonstrated benefit in large clinical trials. This failure is likely multifactual, in part likely due to heavy reliance on small animal models that do not fully recapitulate human physiology (Hamlin RL, 2011, Gibbs CL, 2003). However, isolated cell studies, such as with the use of human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) offer the ability to recapitulate human cellular physiology in vitro (Sharma A, 2018). Here, we describe an ex vivo ischemia reperfusion cell survival assay in hIPSC-CMs with detailed information regarding reagents, procedure, and analysis. We have successfully used this strategy to understand a model of remote ischemic preconditioning (RIPC), a phenonmena whereby a transient ischemic insult can temporarily protect a tissue from a future, much larger ischemic injury. Specficially, RIPC promotes the production of kynurenic acid that activates an orphan receptor, G-Protein Coupled Receptor 35 (GPR35) expressed on the cardiomyocyte (Wyant GA, 2022).

Materials and Reagents

hiPSCs

Matrigel (hESC-qualified; BD Sciences, cat. no. 354277)

DMEM/F12 medium (Thermo Fisher Scientific, cat. no. 11320033)

RPMI 1640 medium (Thermo Fisher Scientific, cat. no. 11835055)

Glucose-free RPMI 1640 medium (Thermo Fisher Scientific, cat. no. 11879-020)

B27 Supplement Minus Insulin (Thermo Fisher Scientific, cat. no. A1895601)

B27 Supplement with Insulin (Thermo Fisher Scientific, cat. no. 17504-044)

CHIR99021 (Thermo Fisher Scientific, cat. no. 508306)

Wnt-C59 (Biorbyt, cat. no. orb181132)

10-cm tissue culture-treated plates

Standard cell culture laminar flow hood

mTeSR1 medium (StemCell Technologies, cat. no. 05850)

Rho kinase inhibitor (ROCKi; Tocris cat. no. 1254)

0.5M EDTA (Thermo Fisher Scientific, cat. no. 15575020)

Deoxyglucose (Sigma, D8375)

NaCl (Sigma, S3014)

KCl (Sigma, P9541)

KH₂PO₄ (Sigma, P5655)

MgSO₄ (Sigma, M2643)

CaCl₂ (Sigma, C5670)

NaHCO₃  (Sigma, S5761)

Sodium lactate (Sigma, 71716)

Hepes (Sigma, H4034)

0.4um Filter (Fisher Scientific, 09-761-118)

PBS (Sigma, 806544)

TryLE (Fisher Scientifiic, 12-605-036)

Equipment

Milli-Q IQ Water Purification System (Sigma, ZIX7003T0C)

Hypoxia Chamber (Coy Lab)-Oxygen control system (including sensor), Purge Airlock, Circulation Fan, Gas Regulator, Gloveless Sleeves, Humidified Incubation Box, Cell Culture System including Temperature Control, CO2 Control System, High Accuracy Calibration System, Polymer Glove Box

Laminar flow hood class II, a CO₂ incubator for cell cultivation (with settings at 37°C and 5% CO₂), vacuum pump for media removal using sterile glass pasteur pipettes

Vi-CELL XR Cell Analyzer (Beckman Coulter, 175478)

Procedure

Differentiate hIPSCs to hIPSC-Cardiomyocytes

hIPSCs can be made using established reprogramming protocols. Expressing the hiPSC reprogramming factors OCT4, SOX2, KLF4, and MYC to reprogram peripheral blood mononuclear cells (PBMCs) to pluripotent stem cells is suitable. Alternatively, low-passage hiPSCs can be obtained from commercial vendors or academic institutions, such as the Stanford University Cardiovascular Institute Biobank (http://med.stanford.edu/scvibiobank.html). Expanding acquired hIPSCs for freezing newly obtained hIPS lines is critical to reduce the possible impact of common cell culture issues, such as cell line contamination, overgrowth, or frequent, unnecessary passaging. To ensure successful hiPSC differentiation into cardiomyocytes, hIPSCs must be growing optimally at time of use. hIPSCs will exhibit tighly packed colony structure and protein expression of characteristic pluripotent stem cells, such as NANOG and TRA-1-81. Once hIPSCs are obtained, they may be passaged almost indefinitely.   

1. Prepare 10cm plate coated with Matrigel extracellular matrix, on which hiPSCs will grow in feeder cell-free format prior to differentiation. Thaw Matrigel on ice and add 125 μL Matrigel per 50 mL of cold DMEM/F12 (1:400 dilution). Add 5 mL of Matrigel DMEM/F12 mixture to 10cm plate to coat for at least 1 hour before use and place in 37°C cell culture incubator. Matrigel-coated plates can be prepared in advance and are stable at 37°C for 1 month. 

2. Following Matrigel coating, remove Matrigel mixture from 10cm plate, and wash 1X with PBS and add hiPSC. Resuspend hiPSCs in mTeSR1 pluripotent stem cell growth medium containing 10 uM rho kinase inhibitor and plate on Matrigel-coated 10cm plate. 

3. After 24 hours of plating, replace media with fresh mTeSR1 without rho kinase inhibitor. Allow cells to reach ~80-100% confluency and replace mTeSR1 daily. It is imperative the media is changed daily. 

4. Begin CHIR99021 GSK3B inhibitor (12 uM) treatment. This is day 0 of differentiation. Remove mTeSR1 medium, add RPMI 1640 medium, supplemented with B27 supplement (without insulin), and 12 uM CHIR99021 and place in 37°C incubator for 24 hours. 

5. Following 24 hours CHIR99021 treatment, aspirate RPMI 1640 medium (with B27 supplement without insulin) containing CHIR99021 and replace with fresh RPMI 1640 medium (with B27 supplement without insulin). Place cells back at 37°C for 48 hours. 

6. After 48 hours, aspirate RPMI 1640 medium and replace with fresh RPMI 1640 (with B27 supplement without insulin) containing 2 uM Wnt-C59. Place back in 37°C for 48 hours. 

7. Following 48 hours, aspirate RPMI1640 medium containing Wnt-C59 and replace with fresh RPMI 1640 medium (with B27 supplement without insulin) and place back in 37°C for 48 hours. 

8. Following 48 hours, aspirate RPMI1640 medium and replace with fresh RPMI 1640 medium (with B27 supplement without insulin) and place back in 37°C for 72 hours. Continue growing cells on RPMI 1640 medium (with B27 supplement without insulin) and replace media every 48 hours. Cardiomyocyte differentiation will be become visible at this stage. 

9. Purify hIPSC-cardiomyocytes from non-cardiomyocytes via metabolic selection. Aspirate media and add fresh RPMI 1640 medium without glucose (with B27 supplement with insulin). Place back in 37°C incubator for 48 hours, replenishing media every 48 hours. Non-cardiomyocytes will die following 24 hours and leave in media for 48 hours total. Following 48 hour metabolic purification, replace media with RPMI 1640 medium containing glucose (with B27 supplement with insulin). These purified hiPSC-cardiomyocytes are now ready for downstream experimental analysis.  

Replate hIPSC-CMs for ex vivo simulated ischemia-reperfusion 

1. Begin with a purified population of hiPSC-cardiomyocytes no older than 20 days following differentiation as this improves cell dissociation.

2. Prepare either 6-well or 10-cm plates for hiPSC-cardiomyocyte plating with 1:400 dilution Matrigel/RPMI 1640 medium. Allow plates to coat for at least 1 hour at 37°C prior to use. 

3. While plates are coating with Matrigel, prepare hiPSC-cardiomyocyte passaging medium and TrypLE dissocation enzyme. Warm TrypLE at 37°C for 1 hour. 

4. Prepare RPMI 1640 Medium (with B27 supplement with insulin) with 20% fetal bovine serum. Add 10 uM rho kinase inhibitor to RPMI solution. This is hiPSC-Cardiomyocyte passaging medium. 

5. Wash metabolically selected hiPS-cardiomyocytes with 1xPBS three times. Add pre-warmed TrypLE to cell-culture plate of metabolically selected hiPSC-cardiomyocytes and place cells in 37°C incubator for 5 minutes. 

6. Add pre-warmed cardiomyocyte passaging medium to deactivate TrypLE. Manually dissociate hiPSC-cardiomyocytes from cell culture plate with pipette. Pipette the hiPSC-cardiomyocytes for ~50 repetitions to ensure hiPSC-cardiomyocytes are in a single-cell suspension. Count hiPSC-cardiomyocytes using ViCELL-XR cell counter. Use 1 mL volume in ViCELL-XR cell counting tube.

7. Aspirate Matrigel from 6-well or 10-cm plates that were prepared prior. Add cardiomyocyte passaging medium to either plate format. These plates will receive the newly passaged hiPSC-cardiomyocytes. 

8. Plate hiPSC-cardiomyocytes in the cardiomyocyte passaging medium at a density of 2.4x10^4 cells/cm to the number of plates needed for simulated Ischemia-reperfusion experiment as needed and return the hiPSC-cardiomyocytes to 37°C incubator for 48 hours. 

9. After 48 hours, hiPSC-cardiomyocytes are ready for Ischemia-reperfusion.

Simulated Ex vivo Ischemia Reperfusion 

For ex vivo simulated ischemia reperfusion injury of hiPS-derived cardiomyocytes, ischemia was modeled by growth in ischemia-mimetic media (20 mM deoxyglucose, 125 mM NaCl, 8 mM KCl, 1.2 mM KH₂PO₄, 1.25 mM MgSO₄, 1.2 mM CaCl_2, 6.25 mM NaHCO₃, 5 mM Sodium Lactate, and 20 mM Hepes pH 6.6) in 1% O₂ for 6 hrs followed by growth in hIPS-cardiomyocyte media and 21% O₂ overnight. Cell survival was determined by manual cell counting using a Vi-Cell XR Cell Counter (Beckman Coulter).  

1. Prepare Ischemia-mimetic media at least 24 hours in advance to planned experiment. Ischemia-mimetic media is comprised of (20 mM deoxyglucose, 125 mM NaCl, 8 mM KCl, 1.2 mM KH₂PO₄, 1.25 mM MgSO₄, 1.2 mM CaCl_2, 6.25 mM NaHCO₃, 5 mM Sodium Lactate, and 20 mM Hepes pH 6.6 in Milli-Q H2O)

2. Sterile filter ischemia-mimetic media in 0.4um Filter in sterile laminar hood and pre-warm media in warm bath.

3. Set Coy Labs Hypoxia Chamber to 1%O₂ at least 24 hours prior to experiment to let chamber to equilibrate to needed oxygen concentration. Oxygen levels are altered within chamber by replacing oxygen content with 95% N₂and 5% CO₂ gas mixture.

4. In laminar hood, aspirate hiPSC-cardiomyocyte passaging media and wash cells three times with 1xPBS.

5. Following PBS washing, replace media with either hiPSC-cardiomyocyte passaging media or ischemia-mimetic media.

6. Cells of which received ischemia-mimetic media are then placed in Coy Lab Hypoxia Chamber pre-equilibrated to 37°C and 1% O₂ for 6 hours.

7. Following 6 hours of ischemia conditions, ischemic-mimetic media is aspirated in Coy Lab Hypoxia Chamber and cells are washed once with PBS and followed by addition of hiPSC-cardiomyocyte passaging media. Cells are then removed from Coy Lab Hypoxia Chamber and placed back in cell culture incubator (21% O₂, 37°C, and 5% CO₂) for the next 18 hours. 

8. Next morning, hiPSC-cardiomyocytes are analyzed for survival following simulated ischemia-reperfusion. hiPS-cardiomyocytes are washed with 1xPBS three times. Add pre-warmed TrypLE to cell-culture plate of hiPSC-cardiomyocytes and place cells in 37°C incubator for 5 minutes.

9. Following TrypLE treatement, manually dissociate hiPSC-cardiomyocytes from cell culture plate with pipette. Pipette the hiPSC-cardiomyocytes for ~50 repetitions to ensure hiPSC-cardiomyocytes are in a single-cell suspension. Count hiPSC-cardiomyocytes using ViCELL-XR cell counter.

Data analysis

1. Simulated Ischemia-Reperfusion must be done in triplicate and cells are counted using ViCELL-XR Cell counter.

2. Each experiment must contain a triplicate of full-media controls (hIPSC-cardiomyoctes grown in cardiomyocyte passaging media at 21% O₂, 37°C and 5% CO₂) grown in cell culture incubator to compare against ischemia-reperfusion conditions.

Notes (optional)

If hIPS-Cardiomyocytes are not manually dissociated to a single cell suspension, cell counting variability will be much larger. ViCELL-XR allows the user to review each image taken to allow the user to identify whether cells are uniformly single-cell or still within clumps. If cells are still clumped, manually dissociate further and recount with ViCELL-XR.

ViCELL-XR provides a significant advantage over the classic trypan blue exclusion assay typically done using a haemocytometer and manual cell counting. First, accurate pipetting is crucial to manual cell counting as a haemocytometer slide volume is ~1nL. To report viable cell concentration per mL, the haemocytometer cell count must be multiplied by a factor of 10⁶, therefore any small deviation in pipetting will lead to large changes in cells calculated. Similarly, manual trypan blue addition contains the same issues as manual cell counting with a haemocytometer. Small variations in volume ultimately affect effective penetration of the dye and dye dilution. 

While hIPSC-CMs offer a unique tool to study human cardiac cell biology in vitro, it is understood hIPSC-CMs are more similar to fetal cardiomyocytes rather the adult stage cardiomyocytes.

Recipes

Ischemia-mimetic media (20 mM deoxyglucose, 125 mM NaCl, 8 mM KCl, 1.2 mM KH₂PO₄, 1.25 mM MgSO₄, 1.2 mM CaCl_2, 6.25 mM NaHCO₃, 5 mM Sodium Lactate, and 20 mM Hepes pH 6.6)

Matrigel-based Media (125 μL Matrigel per 50 mL of cold DMEM/F12, 1:400 dilution)

References

Eltzschig, H. K. & Eckle, T. Ischemia and reperfusion–from mechanism to translation. Nature Med. 17, 1391–1401 (2011)

Gibbs CL. Cardiac energetics: sense and nonsense. Clin Exp Pharmacol Physiol. 2003 Aug;30(8):598-603. 

Hamlin RL, Altschuld RA. Extrapolation from mouse to man. Circ Cardiovasc Imaging. 2011 Jan;4(1):2-4. 

Sharma A, Toepfer CN, Schmid M, Garfinkel AC, Seidman CE. Differentiation and Contractile Analysis of GFP-Sarcomere Reporter hiPSC-Cardiomyocytes. Curr Protoc Hum Genet. 2018 Jan 24;96:21.12.1-21.12.12.

Timmers, L. et al. The innate immune response in reperfused myocardium. Cardiovasc. Res. 94, 276–283 (2012)

Wyant GA, Yu W, Doulamis IP, Nomoto RS, Saeed MY, Duignan T, McCully JD, Kaelin WG Jr. Mitochondrial remodeling and ischemic protection by G protein-coupled receptor 35 agonists. Science. 2022 Aug 5;377(6606):621-629.

Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury. N. Engl. J. Med. 357, 1121–1135 (2007)