Frozen Does Not Mean Stable: Rethinking Cryopreservation in Cell and Gene Therapy Manufacturing

Cell and gene therapies (CGTs) are built on living systems. From early research through commercial distribution, the integrity of those systems depends on the ability to preserve cells without compromising viability, potency, or safety. Cryopreservation underpins this process. 

However, frozen does not automatically equal stable.

Even at ultra-low temperatures, cryopreserved cells are not in suspended animation. Ice crystals can shift, solutes can concentrate in damaging ways, and small temperature changes can alter the environment surrounding each cell.

What ultimately determines cell performance is how these factors play out over storage time and during shipment of cryopreserved products. This distinction becomes especially important as CGT’s are developed at commercial scale.

Cryopreservation under real-world stress

In controlled laboratory settings, established freezing protocols deliver relatively predictable performance. However, commercial manufacturing and handling introduces variables that are difficult to eliminate, like extended storage durations, freezer cycling, shipping transfers, inventory checks, and handling events across multiple facilities.

These routine activities can create what are known as transient warming events (TWEs), which are brief, often small increases in temperature that occur when frozen material is moved, accessed, or transported. TWEs are inherent to real-world workflows and represent a persistent operational challenge.

Importantly, this damage accumulates silently through routine operations. It is not solely a function of staff speed or adherence to standard operating procedures. Well-trained teams following validated protocols cannot fully eliminate the thermodynamic realities of frozen systems responding to temperature fluctuation.

Protecting against transient warming

Modern CGT manufacturing demands strategies that account for transient warming, rather than assuming it can be eliminated. As supply chains scale and products move across facilities, brief temperature shifts become part of the operating environment. The question then becomes how do you design the product to withstand them?

Until now, the primary response has been tighter control on temperature in the form of more standardized procedures, more monitoring, and clearer handling guidance. These steps are critical, but they wrongfully assume that variability can be fully managed through process discipline.

That assumption starts to fall apart at commercial scale. Cells move between facilities, storage periods stretch from weeks to months, freezers are frequently opened, shipments occur over long distances, and much more. Not every real-world condition can be anticipated, and not every temperature fluctuation can be prevented. If some degree of warming is inevitable, then the product itself needs a higher level of variability tolerance. 

Emerging approaches, including use of ice recrystallization inhibitors (IRIs), reflect a broader shift in thinking. Instead of relying solely on procedural controls, they aim to build resilience directly into the product itself. By addressing the root cause of instability – ice recrystallization – use of IRIs signal a move away from relying solely on process discipline toward designing durability into the therapy itself.

Ice recrystallization is one of the key drivers of functional loss during transient warming. Even brief temperature shifts can allow ice structures within the frozen product to reorganize in ways that compromise cell integrity. IRIs are designed to slow or limit that restructuring, helping preserve cellular performance across handling, storage, and transport.

What makes this meaningful at a commercial scale is that IRIs do not depend on perfect conditions. They provide protection at the very moments that are hardest to control. In experimental models, supplementing standard cryoprotectants with an IRI preserved post-thaw recovery and potency under warming stress.

In other words, IRIs represent an acknowledgment that variability is part of real-world manufacturing instead of assuming warming can be eliminated.

Importantly, this does not replace good manufacturing discipline. Controlled-rate freezing, validated storage conditions, and rigorous SOPs remain foundational. But IRIs add a complementary layer of protection. Their use acknowledges that variability cannot be eliminated entirely and instead reduces the biological consequences when it occurs.

This layered approach of process control combined with formulation-level resilience offers a more realistic path to maintaining consistency as CGT supply chains grow more complex.

Designing for variability

As cell-based therapies grow more complex and manufacturing networks expand, resilience can’t be an afterthought, and cryopreservation can no longer just be a step that gets validated during development and left alone. It needs to play an ongoing role in product consistency, potency, and reliability.

Variability is part of real-world manufacturing. The goal should not solely be focused on efforts to eliminate it entirely, but to design products that can tolerate it. That shift in mindset may ultimately determine how reliably therapies perform outside the laboratory.

Photo: ipopba, Getty Images

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