Application of Three-Dimensional Culture Method in the Cardiac Conduction System Research

Bhishek Mishra, Kishore B.S. Pasumarthi

Research output: Contribution to journalArticlepeer-review

Abstract

Congenital heart defects (CHD) are the most common type of birth defects. Several human case studies and genetically altered animal models have identified abnormalities in the development of ventricular conduction system (VCS) in the heart. While cell-based therapies hold promise for treating CHDs, translational efforts are limited by the lack of suitable in vitro models for feasibility and safety studies. A better understanding of cell differentiation pathways can lead to development of cell-based therapies for individuals living with CHD/VCS disorders. Here, we describe a new and reproducible 3-D cell culture method for studying cardiac cell lineage differentiation in vitro. We used primary ventricular cells isolated from embryonic day 11.5 (E11.5) mouse embryos, which can differentiate into multiple cardiac cell types including VCS cells. We compared 3-D cultures with three types of basement membrane extracts (BME) for their abilities to support E11.5 ventricular cell differentiation. In addition, the effects of atrial natriuretic peptide (ANP) and an inhibitor for its high affinity receptor were tested on cell differentiation in 3-D cultures. Following the cell culture, protocols for immunofluorescence imaging, cell extraction and protein isolation from the 3-D culture matrix and in-cell western methods are described. Further, these approaches can be used to study the effects of various ligands and genetic interventions on VCS cell development. We propose that these methodologies may also be extended for differentiation studies using other sources of stem cells such as induced pluripotent stem cells.

Original languageEnglish
Article number50
JournalMethods and Protocols
Volume5
Issue number3
DOIs
Publication statusPublished - Jun 2022

Bibliographical note

Funding Information:
Funding: This research was funded by a Grant-in-Aid from the Heart and Stroke Foundation of Canada, grant number G-18-0022140.

Funding Information:
Acknowledgments: We thank Sarita Chinni for excellent administrative and technical assistance. Abhishek Mishra is the recipient of a Nova Scotia Graduate Scholarship and the Dalhousie Medical Research Foundation MacDonald Graduate Studentship.

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

ASJC Scopus Subject Areas

  • Biotechnology
  • Structural Biology
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

PubMed: MeSH publication types

  • Journal Article

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