Development of a scalable, practical, and transferable GMP-compliant suspension culture-based differentiation process for cardiomyocyte production from human embryonic stem cells.
Human embryonic stem cells (hESC) have the unique capabilities of unlimited replication and differentiation into multiple cell types. We can now generate induced pluripotent stem cells (iPSC) from adult tissues that possess hESC-like properties. Because of their unique features, both hESCs and iPSCs hold great potential for medical applications. Many different cell types have been produced from both hESCs and iPSCs to repair and/or replace diseased/ damaged tissues in treating conditions such as macular degeneration, heart disease, Parkinson’s disease, spinal cord injury and diabetes. This new type of therapeutic approach is called regenerative medicine (RM); we have already produced clinical grade retinal pigmented epithelial cells (RPEs) from hESC to support clinical trials for the treatment of macular degeneration.
A crucial challenge for RM is the ability to generate large quantities of stem cell in excess of those produced by traditional 2 dimensional culture systems. We have established a 3-dimensional (3-D) suspension culture system using spinner flasks for large scale production of hESC and iPSC. In this system, hESC/iPSC form cell aggregates which increase in size during the cultivation period, thereby overcoming the bottleneck of 2 dimensional adherent culture. At CBG, hESCs/iPSCs have been produced in our suspension system and used to produce a number of various cell types for RM applications [RPEs, neural stem cells, dopaminergic neurons and cardiomyocytes (CMs)]. Importantly, we have successfully established a process to produce clinical grade CM in suspension. The same approach may also be applied to the production of other cell types.
While the current suspension system has provided unprecedented production capability for a number of cell products in pre-clinical and clinical studies, it may not be practical to scale up to the level to fulfill the requirement for large clinical trials. To address this hurdle, we have proposed to develop a suspension culture process in the bag-based system to replace spinner flasks for large scale production of hESC/ iPSC and their differentiation. Based on our previous studies using the spinner flask system, we have demonstrated that the cell aggregates are very sensitive to shear force and the culture conditions need to be changed based on the size of each flask due to the differences in shear force caused by the geometry of the vessels. Shear force in the spinner flask system is generated by the rotating propeller inside the flask, while shear force generated by the bag-based system is created by a rocking platform. Based on other studies indicating that the wave bag produces more homogenous gentle shear force, we therefore hypothesize that the bag-based system would be superior to the spinner flask and other propeller-based systems. Specifically, the aim for this project is to resolve the scale-up challenges by adapting our current suspension cell culture system into the bag-based system (which can be more easily scaled up for large scale productions). By achieving this objective, the key barrier will be resolved for cardiac applications in RM and we will have opened the door for large clinical trials and commercialization of other cellular products in RM.
The first year milestones for this project are to complete the establishment of the procedures for large scale production of hESC/iPSC in the bag-based culture system; the second year milestones include the establishment of the CM differentiation in this system. Here we report the completion of all first year milestones; the specific achievements are summarized below.
1. We have successfully adapted hESC to the bag-based system using gas permeable bags and determined agitation conditions (rocking speed and angles), cell density and passage interval, and the concentration of bFGF to be used for optimal results.
2. All process parameters were optimized and established for the production of hESC/iPSC in the bag-based system at both the 500 mL and l L scales.
3. The reliability of the bag-based system has been tested by passaging the cell culture repeatedly for 3-6 passages. The results have shown the reproducibility of the productions from the serial passages of hESC/iPSC using established parameters in the bag-based system and results met all the successful criteria.
4. We successfully performed a 3 L production in the bag-based system that proves the scale up potential of this system for hESC/iPSC. The results have shown 5-fold increases from two continuous passages of hESC with normal karyotype.
5. Cell dissociation procedure is a critical aspect of passaging the cells. We compared the use of GentleMACs (MACS, Mitenyi Biotec inc.) with the traditional manual dissociation process and the resultant viability and recovery rates are comparable between the two methods.
Reporting Period:
Year 2
We have developed GMP-compliant suspension cell culture processes for scalable production of human pluripotent stem cells (hPSCs) and derivatives. These processes have been invaluable in our support of CIRM- and NIH-funded regenerative medicine projects, including those with RPE, NSC, DA neurons and cardiomyocytes (CM), as well as for production of GMP banks of hPSC for various projects. Our GMP-compliant suspension culture CM production process has made pre-clinical animal studies and small early clinical trials practical. However, while the current CM system has been readily transferred to other groups and is meeting current production requirements, the scale requirements for anticipated high dose clinical trials is beyond the practical limitation of our spinner flask-based system. Our experience using bag-based bioreactors for non-hESC products suggested that scale-up in bags will be more controllable and predictable than spinners or stir-tanks reactors. It is also a readily transferred technology. We successfully adapted our suspension hPSC and CM processes to a bag system, and demonstrated scalability at a 3L scale generating highly pure batches of hPSC-derived CMs at the multibillion cell scale. Success in this project removes a key barrier to developing many regenerative medicine products, and in particular those where high human doses are anticipated, such as CM.
Grant Application Details
Application Title:
Development of a scalable, practical, and transferable GMP-compliant suspension culture-based differentiation process for cardiomyocyte production from human embryonic stem cells.
Public Abstract:
As ongoing CIRM-funded development of regenerative medicine (RM) progresses, the demand for increasing numbers of pluripotent stem cells and their differentiated derivatives has also increased. We have established a scalable suspension culture system for the production of large quantities of hESC for banking and to seed production of a number of regenerative medicine cell types, notably retinal pigmented epithelia, neural stem cells, dopaminergic neurons and cardiomyocytes, that support a number of CIRM and NIH-funded groups. In addition, we have adapted this system for the suspension production of several hESC derivative cell types, notably cardiomyocytes. While our system has provided unprecedented production capability for a number of cell products in pre-clinical and imminent clinical studies, it has proven impractical to scale up to the level that will be required for clinical trials for some hESC cell products, notably cardiomyocytes, due to high expected human doses. This project will resolve this scale-up challenge by adapting our suspension cell culture system, that is limited to 1-3L spinner culture flasks, to a more readily scalable and controllable suspension bioreactor system that utilizes “bags” capable of volumes up to 500L. Achieving this objective will remove a key barrier to progressing RM for cardiac applications as well as open the door to large clinical trials and commercialization of other regenerative medicine cell products in the years to come.
Statement of Benefit to California:
We have developed GMP-compliant suspension cell culture processes for scalable production of hPSC and derivatives. These processes have been invaluable in our support of CIRM- and NIH-funded regenerative medicine projects, including those with RPE, NSC, DA neurons and cardiomyocytes (CM), as well as for production of GMP banks of hPSC for various projects. Our GMP-compliant suspension culture CM production process has made pre-clinical animal studies and small early clinical trials practical. However, while our current CM system is readily transferred to other groups and is meeting current production requirements, the scale requirements for anticipated high dose clinical trials is beyond the practical limitation of our spinner flask-based system. hPSC and CM are sensitive to changes in shear encountered at every scale-up step and re-optimizing conditions at each step is prohibitively expensive. Our experience using bag-based bioreactors for non-hESC products suggests that scale-up in bags will be more controllable and predictable than spinners or stir-tanks reactors. It is also a readily transferred technology. We propose to adapt our suspension hPSC and CM processes to a bag system, optimize conditions at a small scale, then demonstrate scalability at a moderate scale. Success in this project will remove a key barrier to developing many regenerative medicine products, and in particular those where high human doses are anticipated, such as CM.
