Development of Suspension Adaptation, Scale-up cGMP Banking and Cell Characterization Technologies for hESC Lines

Development of Suspension Adaptation, Scale-up cGMP Banking and Cell Characterization Technologies for hESC Lines

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01057
Approved funds: 
$882,929
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
hESCs represent an important source of cell therapies in regenerative medicine and the study of early human development. A number of hESC-based therapies are nearing clinical trials. To bring these to clinical trials requires the scale-up production, or “banking”, of large numbers of the desired hESC cell. The current lack of large scale hESC culture methods presents a serious challenge to ensuring progression of new therapies into clinical testing. In addition, current characterization methods are inadequate to monitor genetic and epigenetic changes that may occur during the long term culture required for banking. Finally, the lack of well characterized hESC research banks limits the comparability of research between laboratories. We propose to address these issues by adapting three representative cell lines to scalable suspensions culture, develop epigenetic and genetic “fingerprinting” methods and generate well characterized Master Cell Banks of the three hESC lines for use by all CIRM investigators. Procedures typically used in adherent hESC cell banking involve feeder cell layers, undefined media and/or mechanical manipulation. Banking of cell lines for anticipated clinical studies with existing technology is impractical, expensive and time consuming. The development of robust large scale banking technology will accelerate the speed of development of hESC therapeutics. In addition, monitoring pluripotency of hESC cell lines during culture adaptation and banking processes is critical. While the ultimate measure of hESC pluripotency is their ability to form teratomas in animal models, this method is insensitive, time consuming and costly, and is not feasible as an in-process or final product test method. Improved in-process characterization methods with demonstrated correlation to pluripotency are needed. While phenotypic and gene expression parameters have been defined for a number of lines, little is known about the fundamental genetic and epigenetic characteristics of these cells as they are maintained in culture. It is becoming apparent that the self-renewal and differentiation potential of hESCs may be impacted by the genetic and epigenetic status of the cells. Further, it is likely that the genetic and epigenetic status of the cells will be important predictors of safety for clinic studies. Correlating a genetic and epigenetic “fingerprint” of hESC lines during long term cell culture with pluripotency as measured by teratoma formation will provide a novel method of predicative in-process monitoring of cultures during banking and would facilitate comparison of processes between various laboratories. The unprecedented cGMP cell banking facilities along with the collective expertise of the assembled team, offers an opportunity to advance the hESC field by establishing suspension adaptation techniques, epigenetic fingerprinting, cGMP scale-up processes & cGLP banking of hESC lines not fundable under current NIH rules and policies.
Statement of Benefit to California: 
An advantage of human embryonic stem cells (hESCs) in research is their ability to self-renew indefinitely. hESCs can provide an inexhaustible source of well-defined human cells and represent an important source of material for therapies in regenerative medicine, for the study of early human development and for many other areas of biomedical research. For the first time, it will be possible to grow large quantities of hESC that can meet the requirements of the FDA. As a number of hESC-based therapies developed in California near clinical trials, there is a pressing need for a source of high quality, well defined hESCs. However, translating bench science into clinical reality involves large scale cell banking and predictive cell characterization and release testing that correlate well with the pluripotent properties of these cells. Current procedures for the culture of these cells are limited to work from research laboratories where the focus is on research and not on methods for large scale production or the Good Manufacturing Practices (cGMP) required to meet FDA standards. While several groups have produced small scale cell banks using current technologies, the methods are neither practical or cost effective, and are not amendable to large the scale expansion and banking required for clinical development. One of the major limitations to growing large amounts of hESCs is that current procedures require manual isolation of cell colonies –a time consuming process. A second limitation is that hESC grow only when adhered to surfaces with “feeder” cells and/or specialized coatings. As different laboratories employ their own methods, each may bias cells to be slightly different from the parent cell line. Lack of standardization of cell lines used by researchers is a concern in comparing research across the field. We propose to address these issues by 1. developing procedures to adapt three hESC lines to suspension culture, 2. create well characterized cell banks of these lines, using GMP processes suitable for preclinical and clinical use and 3. develop epigenetic and genetic fingerprinting for cell bank testing and monitoring of cell lines. This proposal brings together nationally recognized leaders in cell culture, assay development and cGMP banking to develop hESC culture conditions that can be scaled up, to design and implement novel assays for characterizing cells obtained at each stage of production, and to create Master Cell Banks of important hESC lines. These cell banks and assay methodologies will be available to all CIRM investigators and will be a key resource for all investigators in the state of California. The unprecedented cGMP cell banking facility along with the collective expertise of the assembled team, offers an opportunity to advance the hESC field by establishing suspension adaptation techniques, epigenetic fingerprinting, cGMP processes & cGLP banking of hESC lines not funded nor fundable under current NIH policies.
Progress Report: 

Year 1

A number of promising hESC derived cell products are or will be moving into pre-clinical studies over the next few years. In anticipation of this, we have been focusing on cell culture optimization in order to address the imminent requirement for cGMP-compliant scale-up manufacture of hESC parental cell banks. Current standard laboratory cell culture practice for hESC involves such components such as mouse fibroblast feeder layers, serum and poorly defined media and reagents that are not suitable for manufacture of precuts for human clinical use. In addition most laboratory work with hESC is performed at very small scales, and often processes effective at laboratory scale do not perform well when scaled up to support pre-clinical and clinical studies. Our goal has been to develop cell culture adaptation processes to allow for production of hESC cell banks in support of ongoing and imminent CIRM-funded pre-clinical and clinical projects throughout California. We have specifically focused on establishing adherent culture conditions that eliminate all animal derived factors (mouse feeder layers, serum) and poorly defined reagents (e.g. many of the available hESC growth media). In addition, scale up in adherent cell culture is limited by the ability to manipulate many, or large, cell culture flasks and plates. In order to achieve large scale banking capability, we have focused on the development suspension cell culture. However, hESC may be prone to genetic and epigenetic instability during long term culture or from stress induced by passage in defined animal-free conditions. To address this, we are developing sophisticated epigenetic monitoring technologies that will allow us to detect shifts in expression patterns before these changes are detectable with current technology. These techniques will allow us to monitor the stability of cell cultures during culture adaption and maintenance, but may also allow us to assess the purity, and thereby safety, of differentiated cell products derived from our banks. The specific aims of this project include: 1: Adapt three hESC lines to feeder layer-free, serum-free suspension culture, 2: Establish and optimize propagation protocols for suspension culture adapted hESC lines and generate cGLP banks, and 3: Establish hESC profiling panel including epigenetic and genetic fingerprinting and correlate with pluripotency. We have made significant progress toward achieving each of these objectives. We have adapted multiple lines to feeder-free and xeno-free conditions and have successfully scaled up these cultures to produce development cell banks of up to 500 vials. Viability and growth rate are readily maintained at high levels and pluripotency is confirmed using a panel of QT-PCR and flow cytometric analysis assays developed in our program. In addition, we have demonstrated the feasibility of maintaining and expanding these lines in suspension culture by achieving over one million fold expansion of each line while maintaining adherent culture levels of growth rate, viability and pluripotency. Significant progress has also been made in developing epigenetic analysis of the hES cells maintained under different culture conditions and at various differentiation states. The preliminary result indicates that epigenetic profiles may be helpful in predicting the state of differentiation of the hES cells. Progress made in each area is summarized below under each specific aims. Adaptation to feeder free and xeno-free culture conditions was readily achieved using commercially available, but well defined, reagents and media. While ROCK inhibitors, increasingly reported as beneficial in hESC cell culture, appear to provide increased survival of cells during passage and cryogenics, we have found them unnecessary during routine propagation of our adapted cultures. In contrast, we explored numerous conditions before successfully establishing a suspension culture condition that provided cell growth, viability and maintenance of pluripotency comparable to adherent cultures. Numerous growth factors, protectants, inhibitors and other factors were tested. In addition, we tested, and optimized, a variety of parameters before defining passage frequency, culture media and factors, enzyme choice and other factors that influence aggregate size and culture vitality. To achieve sustained viability and meaningful cell growth rates, and to maintain pluripotency, various additives were tested using a common culture medium as a starting point. Two approaches were tested to adapt hES cells in suspension. One was to grow cultures as single cells, or as small loose clusters, and the other approach was to maintain the cells as aggregates of defined size. Efforts to establish single cell suspension conditions were performed at both COH and in collaboration with our collaborators. Numerous conditions were evaluated but none proved effective. Efforts focused on suspension adaptation o

Year 2

A number of promising hESC derived cell products are or will be moving into pre-clinical studies over the next few years. In anticipation of this, we have been focusing on cell culture optimization in order to address the imminent requirement for cGMP-compliant scale-up manufacture of hESC parental cell banks. Current standard laboratory cell culture practice for hESC involves such components such as mouse fibroblast feeder layers, serum and poorly defined media and reagents that are not suitable for manufacture of precuts for human clinical use. In addition most laboratory work with hESC is performed at very small scales, and often processes effective at laboratory scale do not perform well when scaled up to support pre-clinical and clinical studies. Our goal has been to develop cell culture adaptation processes to allow for production of hESC cell banks in support of ongoing and imminent CIRM-funded pre-clinical and clinical projects throughout California. We have specifically focused on establishing adherent culture conditions that eliminate all animal derived factors (mouse feeder layers, serum) and poorly defined reagents (e.g. many of the available hESC growth media). In addition, scale up in adherent cell culture is limited by the ability to manipulate many, or large, cell culture flasks and plates. In order to achieve large scale banking capability, we have focused on the development suspension cell culture. However, hESC may be prone to genetic and epigenetic instability during long term culture or from stress induced by passage in defined animal-free conditions. To address this, we are developing sophisticated epigenetic monitoring technologies that will allow us to detect shifts in expression patterns before these changes are detectable with current technology. These techniques will allow us to monitor the stability of cell cultures during culture adaption and maintenance, but may also allow us to assess the purity, and thereby safety, of differentiated cell products derived from our banks. The specific aims of this project include: 1: Adapt three hESC lines to feeder layer-free, serum-free suspension culture, 2: Establish and optimize propagation protocols for suspension culture adapted hESC lines and generate cGLP banks, and 3: Establish hESC profiling panel including epigenetic and genetic fingerprinting and correlate with pluripotency. We have made significant progress toward achieving each of these objectives. We have adapted multiple lines to feeder-free and xeno-free conditions and have successfully scaled up these cultures to produce development cell banks of up to 500 vials. Viability and growth rate are readily maintained at high levels and pluripotency is confirmed using a panel of QT-PCR and flow cytometric analysis assays developed in our program. In addition, we have demonstrated the feasibility of maintaining and expanding these lines in suspension culture by achieving over one million fold expansion of each line while maintaining adherent culture levels of growth rate, viability and pluripotency. Significant progress has also been made in developing epigenetic analysis of the hESCs maintained under different culture conditions. Preliminary results indicate that epigenetic profiles may be helpful in predicting the state of differentiation of the hESCs.

© 2013 California Institute for Regenerative Medicine