Generation of strip-format fibrin-based engineered heart tissue (EHT)

Methods Mol Biol. 2014:1181:121-9. doi: 10.1007/978-1-4939-1047-2_11.

Abstract

This protocol describes a method for casting fibrin-based engineered heart tissue (EHT) in standard 24-well culture dishes. In principle, a hydrogel tissue engineering method requires cardiomyocytes, a liquid matrix that forms a gel, a casting mold, and a device that keeps the developing tissue in place. This protocol refers to neonatal rat heart cells as the cell source; the matrix of choice is fibrin, and the tissues are generated in rectangular agarose-casting molds (12 × 3 × 3 mm) prepared in standard 24-well cell culture dishes, in which a pair of flexible silicone posts is suspended from above. A master mix of freshly isolated cells, medium, fibrinogen, and thrombin is pipetted into the casting mold and, over a period of 2 h, polymerizes and forms a fibrin cell block around two silicone posts. Silicone racks holding four pairs of silicone posts each are used to transfer the fresh fibrin cell blocks into new 24-well dishes with culture medium. Without further handling, the cells start to remodel the fibrin gel, form contacts with each other, elongate, and condense the gel to approximately ¼ of the initial volume. Spontaneous and rhythmic contractions start after 1 week. EHTs are viable and relatively stable for several weeks in this format and can be subjected to repeated measurements of contractile function and final morphological and molecular analyses.

MeSH terms

  • Animals
  • Fibrin / chemistry
  • Fibrin / pharmacology*
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / drug effects*
  • Rats
  • Sepharose / chemistry
  • Tissue Engineering / methods*
  • Tissue Scaffolds / chemistry

Substances

  • Fibrin
  • Sepharose