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Staining image of ACE2-expressing iPS cells (after cryopreservation with STEMCELLBANKER-EX) infected with SARS-CoV-2.
Courtesy of Kazuo Takayama and AMSBIO

Exciting Advances in Mammalian Cell & Tissue Cryopreservation

Innovating and overcoming critical barriers to the translation of cell therapies from bench to bedside

Phillip Pridham, PhD

Phillip Pridham Field, PhD, is cryopreservation business unit manager at AMSBIO.

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Erik Miljan, PhD

Erik Miljan, PhD, is a pioneer in the development of cellular therapies for a range of degenerative and disease conditions. He holds a PhD in biochemistry from Hong Kong University.

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Published:Nov 19, 2021
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Phillip Pridham Field, PhD, is the cryopreservation business unit manager at AMSBIO. Erik Miljan, PhD, is a pioneer in the development of cellular therapies for a range of degenerative and disease conditions. He holds a PhD in biochemistry from Hong Kong University.


Staining image of ACE2-expressing iPS cells (before cryopreservation with STEMCELLBANKER-EX) infected with SARS-CoV-2. Green: SARS-CoV-2 N protein, Red: OCT3/4, Blue: DAPI. Image courtesy of Kazuo Takayama (CiRA, Kyoto University, Japan).
Courtesy of Kazuo Takayama and AMSBIO

Q: WHY IS CRYOPRESERVATION IMPORTANT IN STEM CELL RESEARCH & DEVELOPMENT?

Cryopreservation is often overlooked in stem cell research simply because scientists take for granted that 50-year-old techniques are well-established. As sophisticated knowledge has developed around stem cell biology and regenerative medicine, scientists often do not fully appreciate the parallel progress in cryopreservation techniques. Cryopreservation is a critical step because without it, every experiment would start and end with fresh cells. Not changing or updating techniques could limit any advances made with the stem cell products themselves.

Q: WHAT IS THE DIFFERENCE BETWEEN COMPTEMPORARY AND STATE-OF-THE-ART CRYOPRESERVATION TECHNOLOGIES?

Typically, researchers add DMSO to the culture medium or serum to cryopreserve cells—incorrectly thinking that this is an optimized approach. With these classic approaches, if you freeze the tissue too quickly, ice crystals can kill cells—but freezing too slowly can dehydrate the cells. In addition, animal components and other research-grade ingredients render the cells unfit for clinical applications.

Advances in commercial cryopreservation media include proprietary formulations that remove the need for cell-specific freezing conditions. More importantly they are available as certified xeno-free, chemically defined products manufactured to GMP guidelines, streamlining the inclusion of the cryopreservation media into clinical trial applications.

Q: WHY IS CRYOPRESERVATION CRITICAL IN THE DEVELOPMENT & MANUFACTURE OF CELLULAR THERAPIES?

Cryopreservation efficacy is balanced with cytotoxicity of the cryopreservation agent. Damage to cells during the cryopreservation phase cannot be undone after the fact. Impacts on cell viability, morphology, and adhesion may be immediately obvious upon revival; however, less obvious are documented changes in gene and cytokine expression and differentiation of stem cells because of cytotoxicity from the cryopreservation agent.

Q: WHAT ARE THE QUALITY AND REGULATORY IMPLICATIONS OF THE CRYOPRESERVATION AGENT WHEN DEVELOPING CELLULAR THERAPIES?

Drug regulations for advanced therapies are constantly evolving. It is prudent to use innovative techniques because older approaches are often superseded with new and often more restrictive regulations. A challenge in developing cell therapies is that most of the reagents used are not approved for clinical use. The only way to minimize the risk of regulatory changes that impact translation to the clinic is to use cryopreservation agents that are approved for injection in patients. Moreover, using a cryopreservation agent that is approved for injection would eliminate the need to wash the cells before clinical administration.

Staining image of ACE2-expressing iPS cells (after cryopreservation with STEMCELLBANKER-EX) infected with SARS-CoV-2. Green: SARS-CoV-2 N protein, Red: OCT3/4, Blue: DAPI. Image courtesy of Kazuo Takayama (CiRA, Kyoto University, Japan).
courtesy of Kazuo Takayama and AMSBIO   

This is a critical point because it is not feasible to wash away the cryopreservation agent at the point-of-care because of release testing requirements. A GMP manufactured cryopreservation media that has a master file registered with the FDA and formulated with clinically approved excipients for injection will eliminate these risks.

Q: CAN YOU AFFORD TO OVERLOOK RECENT ADVANCES IN CRYOPRESERVATION?

The significance of the cryopreservation media in developing cell & tissue engineered products cannot be underestimated. Where cryopreservation is concerned, ultimately you finish with what you start with. Efforts to optimize manufacturing processes can be negated by using outdated cryopreservation techniques. Therefore, it is critical to choose a state-of-the-art cryopreservation agent to avoid both regulatory safety issues and the risks of deleterious effects on your cells, organoids, and tissues.