Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

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.

Cell sheet engineering for regenerative medicine: Current challenges and strategies

“…None MSC [25], ADSC [38], Cardiac Stem Cell [39], ESC-derived cardiomyocyte [40], iPSC-derived cardiomyocyte [41] Magnetic harvesting of the cell assembly by dispase, which leads to cell sheet detachment via digestion of several ECM proteins but not cell-junction proteins [6]. The cell sheet graft bioprocessed by this method has been clinically proven as a treatment for deep dermal or full thickness burns [2].…”

“…This noninvasive cell sheet can be engrafted at the desired transplantation site without suturing the cell sheet because the ECM deposited underneath the cell sheet works as an adhesive agent [12]. Pre-clinical studies demonstrate that cell sheet engineering has been successful with various types of cells: epithelial cells [13][14][15][16][17][18][19][20], non-epithelial cells [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37], stem cells [25,38,39], and pluripotent stemderived cells [40,41]. These results suggest there is a high potential for cell sheet technologies to contribute to the creation of tissues or organs when combined with other innovative technologies.…”

“…Cost performance is one of the most essential elements for industrialization and widespread acceptance of cell sheet products. We have designed a 3D cell suspension culture system meeting these requirements [40,41]. A few improvements to the system were obtained using two types of bioreactors (1 L and 100 mL scales) allowing us to obtain 8 × 10 7 cells/100 mL vessel constituting around 80% of cardiomyocytes differentiated by human iPSCs, leading to great savings in both effort and consumable supplies: approximately 80 and 90% reductions of time spent at work and total culture medium consumed, respectively.…”

A multistep procedure to prepare pre-vascularized cardiac tissue constructs using adult stem sells, dynamic cell cultures, and porous scaffolds

“…Exploiting cell sheet technology, our group and others successfully achieved the preparation of 3D cardiac tissues in the absence of scaffolds by different cell types having a cardiac significance (Haraguchi et al, 2006; Forte et al, 2011; Matsuura et al, 2012; Sekine et al, 2013). Nonetheless, the scaling up of solid engineered tissues to obtain a critical size substitute with therapeutical relevance is limited by the diffusion of oxygen, nutrients and waste products to and from the inner portion of the construct, impairing the survival of the newly formed tissue.…”

Elimination of Remaining Undifferentiated Induced Pluripotent Stem Cells in the Process of Human Cardiac Cell Sheet Fabrication Using a Methionine-Free Culture Condition

“…23 Primer pairs and TaqMan MGB probes were designed for human OCT3/4, NANOG, MYL2, MYL7, TNNT2, COL1A1, COL3A1, NPPA, NPPB, ACTB, and GAPDH with the TaqMan gene expression assay (Table 1) (Applied Biosystems [ABI], Foster City, CA). Quantitative PCR was performed with a 7300 Real-Time PCR System (Applied Biosystems).…”