James Kusena PhD

Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates… Show more

Standardization is crucial when culturing cells including human embryonic stem cells (hESCs) which are valuable for therapy development and disease modeling. Inherent issues regarding reproducibility of protocols are problematic as they hinder translation to good manufacturing practice (GMP), thus reducing clinical efficacy and uptake. Pluripotent cultures require standardization to ensure that input material is consistent prior to differentiation, as inconsistency of input cells creates end-product variation. To improve protocols, developers first must understand the cells they are working with and their related culture dynamics. This innovative work highlights key conditions required for optimized and cost-effective bioprocesses compared to generic protocols typically implemented. This entailed investigating conditions affecting growth, metabolism, and phenotype dynamics to ensure cell quality is appropriate for use. Results revealed critical process parameters (CPPs) including feeding regime and seeding density impact critical quality attributes (CQAs) including specific metabolic rate (SMR) and specific growth rate (SGR). This implied that process understanding, and control is essential to maintain key cell characteristics, reduce process variation and retain CQAs. Examination of cell dynamics and CPPs permitted the formation of a defined protocol for culturing H9 hESCs. The authors recommend that H9 seeding densities of 20,000 cells/cm2, four-day cultures or three-day cultures following a recovery passage from cryopreservation and 100% medium exchange after 48 hours are optimal. These parameters gave ~SGR of 0.018 hour−1 ± 1.5x10−3 over three days and cell viabilities ≥95%±0.4, while producing cells which highly expressed pluripotent and proliferation markers, Oct3/4 (>99% positive) and Ki-67 (>99% positive). Show less

Work undertaken using the embryonic carcinoma 2102Ep line, highlighted the requirement for robust, well-characterized and standardized protocols. A systematic approach utilizing 'quick hit' experiments demonstrated variability introduced into culture systems resulting from slight changes to culture conditions (route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (route B).Results… Show more

Work undertaken using the embryonic carcinoma 2102Ep line, highlighted the requirement for robust, well-characterized and standardized protocols. A systematic approach utilizing 'quick hit' experiments demonstrated variability introduced into culture systems resulting from slight changes to culture conditions (route A). This formed the basis for longitudinal experiments investigating long-term effects of culture parameters including seeding density and feeding regime (route B).Results demonstrated that specific growth rates (SGR) of passage 59 (P59) cells seeded at 20,000 cells/cm2 and subjected to medium exchange after 48h prior to reseeding at 72h (route B2) on average was marginally higher than, P55 cells cultured under equivalent conditions (route A1); whereby SGR values were (0.021±0.004) and (0.019±0.004). Viability was higher in route B2 over 10 passages with average viability reported as (86.3%±8.1) compared to route A1 (83.3±8.8). The metabolite data demonstrated both culture route B1 (P57 cells seeded at 66,667 cells/cm2) and B2 had consistent-specific metabolite rates (SMR) for glucose, but SMR values of route B1 was consistently lower than route B2 (0.00001 mmol, cell-1.d-1 and 0.000025).Results revealed interactions between phenotype, SMR and feeding regime that may not be accurately reflected by growth rate or observed morphology. This implies that current schemes of protocol control do not adequately account for variability, since key cell characteristics, including phenotype and SMR, change regardless of standardized seeding densities. This highlights the need to control culture parameters through defined protocols, for processes that involve culture for therapeutic use, biologics production, and reference lines. Show less

Currently, there is scant evidence of policies that adequately outline the process for therapies and technologies in the regenerative medicine (RM) field, known in the EU as Advanced Therapy Medicinal Products (ATMPs). Areas such as payment mechanisms, pricing and reimbursement schemes still remain largely elusive. Clear policies are pertinent due to ATMPs such as KYMRIAH™ and YESCARTA® that have been approved, but how they and future ATMPs will be sustainably paid for remains unclear for many… Show more

Currently, there is scant evidence of policies that adequately outline the process for therapies and technologies in the regenerative medicine (RM) field, known in the EU as Advanced Therapy Medicinal Products (ATMPs). Areas such as payment mechanisms, pricing and reimbursement schemes still remain largely elusive. Clear policies are pertinent due to ATMPs such as KYMRIAH™ and YESCARTA® that have been approved, but how they and future ATMPs will be sustainably paid for remains unclear for many healthcare systems globally. Also, their long-term effects are not yet known, and social, ethical and legal issues have not always been adequately considered.

The scarcity in defined reimbursement and adoption policies for ATMPs prompted the development of an international workshop on the ‘Challenges in the Adoption of Regenerative Medicine Therapies (CHART)’. Co-hosted by Medicine by Design (a regenerative medicine initiative at the University of Toronto and funded by the Canada First Research Excellence Fund), CCRM, Toronto Health Economics and Technology Assessment (THETA) Collaborative, and the Centre for Biological Engineering at Loughborough University UK, CHART focused on the post-market approval challenges associated with economic evaluation, reimbursement and adoption of regenerative medicine products. The challenges were explored in the context of the Canadian and UK healthcare systems as provision of health in both countries is largely via publicly funded systems. The workshop was attended by 37 experts from institutions and companies from Canada, the UK, and the USA that included representatives of the Ontario government, National Institute for Health and Care Excellence (NICE), Canadian Agency for Drugs and Technologies in Health (CADTH), Takeda Canada, Gilead Sciences Canada, Inc., Office of Health Economics (OHE), Centre for Health Economics (CHE), University of York, and Oxford Academic Health Science Network (OAHSN) amongst others. Show less

Current cell therapy product limitations include the need for in-depth product understanding to ensure product potency, safety and purity. New technologies require development and validation to address issues of production scale-up to meet clinical need; assays are required for process control, validation and release. Prior to clinical realization, an understanding of production processes is required to implement process changes that are essential for process control. Identification of key… Show more

Current cell therapy product limitations include the need for in-depth product understanding to ensure product potency, safety and purity. New technologies require development and validation to address issues of production scale-up to meet clinical need; assays are required for process control, validation and release. Prior to clinical realization, an understanding of production processes is required to implement process changes that are essential for process control. Identification of key parameters forms the basis of process tolerances, allowing for validated, adaptive manufacturing processes. This enables greater process control and yield while withstanding regulatory scrutiny. This report summaries key milestones in specifically for ventral midbrain dopaminergic neuroprogenitor differentiation and key translational considerations and recommendations to enable successful, robust and reproducible current cell therapy product-manufacturing. Show less