Isolation of bovine cardiomyocytes for reprogramming studies based on nuclear transfer

Area of Science:

• Reproductive Biology
• Cell Biology
• Biotechnology

Background:

• Somatic cell nuclear transfer (SCNT) requires efficient donor cell preparation.
• Bovine cardiomyocytes offer unique advantages for studying gene reprogramming due to their differentiated state and gene expression patterns.

Purpose of the Study:

• To establish and validate a protocol for preparing bovine cardiomyocytes from slaughterhouse material for nuclear transfer.
• To optimize cell isolation and handling for subsequent nuclear transfer experiments.

Main Methods:

• Bovine fetal heart tissue was stabilized using Custodiol perfusion.
• Enzymatic dissociation with collagenase in calcium-free medium yielded individual cardiomyocytes.
• Cardiomyocytes were subsequently treated with Ca2+ to induce rounding for nuclear transfer.

Main Results:

• An optimized protocol successfully yielded individual contractile cardiomyocytes from bovine fetuses.
• Nuclear transfer using cardiomyocytes maintained at 37°C resulted in a lower blastocyst rate (14.0%) compared to adult fibroblasts (32.7%).
• Storage of cardiomyocytes at 4°C prior to nuclear transfer inhibited blastocyst development.

Conclusions:

• A validated protocol for preparing bovine cardiomyocytes from slaughterhouse material is presented.
• Maintaining cardiomyocytes at 37°C is critical for successful blastocyst development post-nuclear transfer.
• This system provides a valuable tool for investigating gene reprogramming in SCNT.