Enabling Cell Therapies with improved Cryopreservation

Sperm frozen storage in liquid nitrogen tank

What is cell banking?
Cell banking is the process of preserving functional and structural biological systems, by using lower temperature storage techniques. The advantages of cryopreservation have been demonstrated extensively across many cell-based processes, with cell banks now playing a central role in biomanufacturing.

The practice of cell cryopreservation dates back to the 1940s, with the discovery of glycerol as a Cryo-Protective Agent (CPA) that maintained the survival of spermatozoa from different species after freezing at temperatures of -79°C 1. Today, cell banks are used routinely in bioproduction to support cell line development, upstream production processes for biologics, and as a key enabler in the development and delivery of cell-based therapies (e.g., Master Cell Banks (MCB), Working Cell Banks (WCB) or End-of-Production Cell Banks (EoPC)).

What are the benefits of cryopreservation?
The Cell Therapy (CT) sector has experienced unprecedented growth in the last 5 years, but the reality is that significant barriers to adoption still remain. Delivery of these medicines from bench to beside in a timely and commercially viable way, without compromising product efficacy, is key to success.

Cell cryopreservation via the slow cooling method, has played an essential role in the development of the CT and regenerative medicine fields, enabling the storage and use of cells for clinical purposes in early trials. In 2014, 80% of Mesenchymal Stromal Cell (MSC) clinical trial product proposals submitted to the FDA reported the use of cryopreservation as an integral part of the cell manufacturing process, specifically to store and transport final products 2.

Some of the benefits of cryopreservation and cell banking are:
Long-term storage: allowing CT developers to avoid maintaining cells in long-term culture with increased risk of genetic mutation, preserving cells from aging and loss of therapeutic function. Long-term storage allows for development of cell banks to provide preparations of consistent quality as well as retention of testing and patient samples.
Extended product shelf-life and consistency: allowing banking of large quantities of allogeneic cells for distribution, or storage of single production runs to improve consistency in repeat treatments.
Improved CT logistics: development of storage and transport procedures (cryochain) that allow for flexibility, supply, availability and ultimately reduced cost due to less product loss.

Is your cell bank stable and well characterised?
Since discovery in the 1940s, the field of cryobiology has evolved significantly, refining the process of cell cryopreservation and banking. Even so, high process variability persists, with poor understanding of the effect of cryopreservation on CTs. These shortcomings become especially clear as CT trials progress towards approval.

It is now understood that the success of cryopreservation is dependent on both intrinsic and extrinsic factors, like the quality and specificity of the biological starting material, the composition of the CPA, the adopted protocol of cooling and thawing and the container type. A comprehensive and standardised testing programme is needed to fully characterise CT products and minimise variability.

Typical testing systems used to ensure cell bank quality evaluate sterility of the sample (e.g. mycoplasma and endotoxins) and cell recovery. Targeted analyses of cell viability, growth, genomic stability, and functional/potency assays serve to inform on cell quality and direct decisions on that cell population. Ideally, the characterisation panel is repeated at every stage of cell banking, from MCB to WCT and EoPC, to ensure cells remain sterile and unchanged from their original sources. These methods are laborious, and costly, in terms of time and money.

What does the data tell us?
Evaluating the effects of cryopreservation on a CT calls for therapy specific potency assays that relate to the product mechanism of action (MOA) or cell functionality. This is a limitation in itself, as the industry has struggled to develop robust and MOA specific potency assays for many years. While MSCs remain one of the most studied and clinically trialled cell types (with over 1000 trials to date 3), there is still a lack of functionally relevant cell characterisation tools for the field.

A recent review from Bahsoun et al., (2019) 4 on cryopreservation of Bone-Marrow derived MSCs (BM-MSCs) highlighted the areas of deviation and agreement on cell properties and function following cryopreservation. Within the 41 studies assessed, cells were harvested from different species, and cryopreserved using different freezing reagents (CPAs and cell culture supplements), and protocols (freezing rates and associated equipment). Interestingly, many of the International Society for Cellular Therapy (ISCT) criteria used to define MSCs where unchanged. Cryopreservation did not appear to impact proliferation, immunophenotype, differentiation or morphology, suggesting that these cell attributes might not be the best indicators of cell health or function.

In contrast, significant changes where observed post thaw in cell viability, attachment, metabolism, apoptosis and immunomodulatory capacity. Here, consistency of results is highlighted as an issue; for example, of the 8 manuscripts reviewed where BM-MSC immunomodulatory capacity was assessed, exactly half reported impaired cell function following cryopreservation while the rest described no effect 4. Possible reasons for these inconsistencies include variation in cell handling and cryopreservation methods, as well as variation in the characterisation assays themselves. Consider that assessing the impact of MSCs on T-cell proliferation also requires consistency in T-cell quality, introducing another layer of complexity and potential source of variation to the system.

How do we improved cryopreservation for Cell Therapies?
To establish and maintain a trustworthy cell bank, or release a therapeutically active and consistent cryopreserved product, the CT industry needs to align on when and how to assess cell quality post thaw.
WHEN is relevant as immediate cell viability assessment post thaw (most common approach) is not always indicative of cell quality hours later when the cell has been infused into the patient, and is required to be therapeutically active.
HOW is even more challenging, as methods not only need to measure cell function as it relates to the therapies MOA, but also need to be quick, easy and suitable for clinicians to perform at the point of thaw and infusion.

At ValitaCell we are working to better understand the analytical requirements for assessing, developing and optimising cryopreservation strategies, with the overall goal of providing faster and more efficient testing paradigms. We are investigating the use of our ChemStress® and CellAi® Technology platforms for better insight into the effects of cryopreservation on cell quality.

ChemStress® Fingerprinting is an information-rich, analytical assay that provides data on the functional quality of cells. The technology draws on the unique biological signature that a given cell population generates in response to small molecule stressors that simulate specific stress response pathways relevant to large-scale expansion. ChemStress® can be deployed post thaw to ‘stress test’ cell preparations and compare back to pre-preserved fingerprints.

CellAi® is an Artificial Intelligence (AI) powered image-analysis software application designed to process cell image data and extract insights into cell morphology, function, identify and quality. This technology replaces slow, complex, and destructive staining workflows with Deep Learning (DL) analysis of fast and non-destructive label-free (unstained) brightfield images of cells, allowing users to extract greater insights per image. CellAi® could be used to monitor cell quality post-thaw in a quick and simple format.

Connect with us to learn more about this work.

References

1. Coriell, L. L., Greene, A. E. & Silver, R. K. Historical development of cell and tissue culture freezing. Cryobiology 1, 72–79 (1964).
2. MSC-Based Product Characterization for Clinical Trials: An FDA Perspective | Elsevier Enhanced Reader. https://bit.ly/30v5CeW
3. García-Bernal, D. et al. The Current Status of Mesenchymal Stromal Cells: Controversies, Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy. Frontiers in Cell and Developmental Biology 9, 609 (2021).
4. Bahsoun, S., Coopman, K. & Akam, E. C. The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: A systematic review. Journal of Translational Medicine 17, 1–29 (2019).

Dr. Stephanie Davies
Stephanie Davies, PhD
Head of Cell Therapy

Stephanie Davies leads the application of all Valitacell technology into The Cell Therapy Space, with particular focus on their integration into advanced MSC, & Exosome manufacturing platforms. Stephanie has a primary degree in Genetics and PhD in Molecular Cell Biology from University College Cork with advanced training in cell biology, biochemistry and genetics with extensive experience in the design and optimisation of cell-based systems.

Dr. Alessandra Prinelli
Alessandra Prinelli, PhD
Product Development Scientist Cell Therapy

Alessandra Prinelli joined ValitaCell after completing an industrial PhD in primary human immune cell biology and cell-based assays development. With her passion for applied sciences, in her current role as Product Development Scientist, Alessandra is leading the development of the Chemstress® platform for stem cells characterization in the cell therapy manufacturing space, aiming to provide analytical tools to assess the complexity of function of cellular therapy products.

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