Defining optimal enzyme and matrix combination for replating of human induced pluripotent stem cell-derived cardiomyocytes at different levels of maturity

Exp Cell Res. 2021 Jun 15;403(2):112599. doi: 10.1016/j.yexcr.2021.112599. Epub 2021 Apr 10.


Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications.

Keywords: Cardiac differentiation; Cardiomyocytes; Cell dissociation; Dissociation enzymes; Extracellular matrix; Human induced pluripotent stem cells; Replating.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Biomarkers / metabolism
  • Cell Culture Techniques*
  • Cell Differentiation / drug effects
  • Cellular Reprogramming / drug effects
  • Cellular Reprogramming / genetics
  • Collagen / pharmacology
  • Collagenases / pharmacology
  • Culture Media / chemistry
  • Culture Media / pharmacology*
  • Drug Combinations
  • Female
  • Fibronectins / pharmacology
  • Gene Expression
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / drug effects*
  • Induced Pluripotent Stem Cells / metabolism
  • Insulin / analogs & derivatives
  • Insulin / pharmacology
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / metabolism
  • Laminin / pharmacology
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / metabolism
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects*
  • Myocytes, Cardiac / metabolism
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / metabolism
  • Primary Cell Culture
  • Proteoglycans / pharmacology
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • Pyridines / pharmacology
  • Pyrimidines / pharmacology
  • SOXB1 Transcription Factors / genetics
  • SOXB1 Transcription Factors / metabolism


  • B27 Lys destripeptide insulin
  • Biomarkers
  • Chir 99021
  • Culture Media
  • Drug Combinations
  • Fibronectins
  • Insulin
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Laminin
  • MYC protein, human
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Proteoglycans
  • Proto-Oncogene Proteins c-myc
  • Pyridines
  • Pyrimidines
  • SOX2 protein, human
  • SOXB1 Transcription Factors
  • matrigel
  • Collagen
  • Collagenases