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Abstract
Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×108 cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×108. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.
Highlights
Within the rapidly advancing field of regenerative medicine, cell-based therapies have emerged as a promising alternative to cardiac transplantation for the management of damaged heart tissue
A single cell suspension of mouse embryonic stem (mES) cells (EMG7) expressing GFP under the control of the amyosin heavy chain promoter were seeded into the culture vessel of the bioreactor system, and cells were cultured without leukemia inhibitory factor and stirred
We previously reported creating the cardiac tissue by layering cardiac cell sheets derived from neonatal rat cardiomyocytes
Summary
Within the rapidly advancing field of regenerative medicine, cell-based therapies have emerged as a promising alternative to cardiac transplantation for the management of damaged heart tissue. Recent evidence suggests that the paracrine effects, angiogenesis and cardioprotection mediated by secreted factors derived from the transplanted cells might be the major mechanisms underlying the cell transplantation-mediated improvement in cardiac function [6,7]. These effects might be a prerequisite to creating transplantable bioengineered thickened cardiac tissue that directly contributes to contraction
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