Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes
Abstract
:1. Introduction
2. Large Scale Expansion Culture of Human Pluripotent Stem Cells
2.1. Adherent Culture of Human Pluripotent Stem Cells
2.2. Suspension Culture of Human Pluripotent Stem Cells
2.2.1. Application of Microcarriers
2.2.2. Formation of Carrier-Free 3D Aggregates
2.2.3. Hydrogels
2.2.4. Functional Polymers for Suspension Culture of hiPSCs without Agitation
3. Application of Human Pluripotent Stem Cells for Cardiomyocyte Differentiation
3.1. Cardiomyocyte Differentiation as Monolayer Culture
3.2. Cardiomyocyte Differentiation by Suspension Culture Using Microcarriers
3.3. Cardiomyocyte Differentiation by Applying Carrier-Free Cell Aggregates
3.4. Cardiomyocyte Differentiation by Applying Hydrogels
4. Strategies for Cardiomyocyte Maturation
5. Conclusions and Future Perspective
Author Contributions
Funding
Conflicts of Interest
References
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hPSCs Culture Format | Characteristics | Method | Advantage | Disadvantage | Efficiency | References |
---|---|---|---|---|---|---|
Microcarrier | Matrigel-coated cellulose microcarriers for human embryonic stem cells (hESCs) culture |
|
|
| Achieved 3.5 × 106 cells/mL in 50-mL spinner flask compared to 1.5 × 106 cells/mL in static microcarrier culture and 0.8 × 106 cells/mL in 2D colony cultures | Oh et al., 2009 [42] |
Microcarrier | A xeno-free subtract (human vitronectin) and chemically defined medium (Essential 8) culture system for human induced pluripotent stem cells (hiPSCs) |
|
|
| Achieved 1.4 × 106 cells/mL after 10 days | Badenes et al., 2016 [43] |
Microcarrier | Dissolvable microcarriers for scalable expansion of hiPSCs under xeno-free conditions |
|
|
| Achieved maximum 8.81 × 105 cells/mL using dissolvable Synthemax coated microcarriers at high recovery rate of 92% | Rodrigues et al., 2019 [49] |
Carrier-free cell aggregates (3D sphere) | Suspension culture of hPSCs in static and dynamic culture, using interleukin and bFGF with serum-free media |
|
|
| 25-fold in 10 days | Amit et al., 2010 and 2011 [50,56] |
Carrier-free cell aggregates (3D sphere) | Sphere culture of hPSCs with single cell dissociation |
|
|
| Cell numbers increase by six-fold within 4 days | Olmer et al., 2010 [51] |
Carrier-free cell aggregates (3D sphere) | A scalable GMP compliant suspension culture system for hESCs |
|
|
| An average expansion rate of 4-fold can be obtained per passage of 3–4 days | Chen et al., 2012 [53] |
Hydrogel | Use a thermo-responsive hydrogel to culture hPSCs |
|
|
| In single cell passage, cell fold increase is 10 after 4 days | Lei et al., 2013 [70] |
Hydrogel | Apply plant-derived nanofibrillar cellulose (NFC) hydrogel to culture hPSCs |
|
|
| N/A | Lou et al., 2014 [69] |
Functional polymers | Apply two functional polymers to change the viscosity of medium in the culture hPSCs |
|
| Seeding at 13.2 × 106 cells/bag, yielded 1.4 × 108 cells/bag which corresponds to a 12.5-fold | Otsuji et al., 2014 [71] |
hPSCs Culture Format | Characteristics | Culture Vessel | Medium for hPSCs Expansion | Yield | Culture Medium and Format for CMs Differentiation | Efficiency of CMs Differentiation | Advantage | Disadvantage | References |
---|---|---|---|---|---|---|---|---|---|
Monolayer culture |
| 4-layer or 10-layer of 632 cm2 culture plates |
| Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer and 1.7 × 109 hiPSCs in 10-layer culture plates |
|
| High efficiency in generation of pure hiPSCs-CMs since all cells are evenly exposed to purification medium. |
| Tohyama et al., 2017 [78] |
Monolayer culture |
| N/A |
| N/A |
|
| Approach by co-differentiation to get cardiac identity-endothelial cells prior to microtissue formation helps promote CMs maturation |
| Giacomelli et al., 2017 [84] |
Microcarriers | Expansion of hESC followed by CMs differentiation in a homogenous process | Ultra low attachment T-25 flask with rocker culture |
| Seeding of 2 × 105 cells/mL yielded 3.74 × 106 cells/mL after 7 days |
| Yield 2.45 × 106 CM/mL with 65.73% expression of cTnT after 12 days differentiation | Integrate hPSCs expansion and CMs differentiation in a continuous process | CMs were separated from microcarrier by enzymatic dissociation and filter through 40 µm cell strainer | Ting et al., 2014 [103] |
Microcarriers | hPSC expansion, differentiation, and purification using microcarriers | 500 mL controlled bioreactor |
| 3.66 × 106 cells/mL after 7 days culture (18-fold increase) |
| 1.33 × 106 CMs/mL with 83.1% expression of cTnT after 23 days culture and purification | A high yield of CMs can be obtained using a large volume bioreactor | Removal of microcarriers before further applying CMs for transplantation, drug screening and disease modeling | Steve Oh et al., 2017 [91] |
Carrier-free cell aggregates (3D sphere) | Two methods to form aggregates
|
|
| 60 × 106 cells in 100 mL bioreactor |
| In bioreactor, efficiencies are 85%, 54%, 68% (n = 3) after 10 days of differentiation Erlenmeyer ~ 60.4% | Applying carrier-free cell aggregates facilitates cell harvesting compared to microcarriers | Maintenance of cell aggregates required ROCK inhibitor during culture process may change hPSCs metabolome profile. | Kempf et al., 2014 [94] |
Carrier-free cell aggregates (3D sphere) | The culture process is defined and standardized in compliance with GMP regulations. | 6-well plate; 125, 500, and 1000 mL spinner flasks |
| Seeding at 2.5 × 105 cells/mL yielded 1 × 106 cells/mL after 3 days |
| >90% CM purity after 25 days of differentiation Yield 1.5 to 2 × 109 CM/L |
| Maintenance of cell aggregates required ROCK inhibitor during culture process may change hPSCs metabolome profile. | Chen et al., 2012;Chen et al., 2015 [95] |
Hydrogel | hiPSCs were encapsulated in PEG-fibrinogen hydrogels and differentiated into CMs continuously | Prepare PDMS mold on acrylated glass, put in 6-well plate | mTeSR1 with ROCK inhibitor supplemented for the first 24 h | Cells were seeded at 5.5 × 105 hiPSCs per tissue |
|
|
| Kerscher et al., 2016 [102] |
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Le, M.N.T.; Hasegawa, K. Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes. Bioengineering 2019, 6, 48. https://doi.org/10.3390/bioengineering6020048
Le MNT, Hasegawa K. Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes. Bioengineering. 2019; 6(2):48. https://doi.org/10.3390/bioengineering6020048
Chicago/Turabian StyleLe, Minh Nguyen Tuyet, and Kouichi Hasegawa. 2019. "Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes" Bioengineering 6, no. 2: 48. https://doi.org/10.3390/bioengineering6020048
APA StyleLe, M. N. T., & Hasegawa, K. (2019). Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes. Bioengineering, 6(2), 48. https://doi.org/10.3390/bioengineering6020048