Controlled ATMP freezing: the role of cooling rate and phase transition control in cell viability

Cryopreservation of ATMPs requires precise control of the freezing process to maintain cell viability. This article presents a study conducted by Single Use Support GmbH in collaboration with Management Center Innsbruck examining the effect of different controlled cooling rates (−0.68, −1.15, and −4.28 °C/min) on Chinese Hamster Ovary (CHO)-K1 cell viability, alongside an uncontrolled freezing comparator. Results demonstrated that controlled freezing at approximately −1 °C/min achieved the highest viability (~95% by live/dead assay), and that phase transition control is a critical determinant of cell survival, independent of cooling rate. The study also highlights the role of user‑defined, recipe‑driven freezing systems in implementing controlled cryopreservation workflows within GMP manufacturing environments.

Controlled-rate freezing at ~−1 °C/min achieves ~95% post-thaw viability in CHO-K1 cells, yet phase transition control – not cooling rate alone – is the critical survival determinant: uncontrolled static freezing yields below 50% viability at a comparable rate, highlighting the need for recipe-driven systems in GMP ATMP manufacturing.

What you will learn

01

Why the −1 °C/min benchmark only holds within controlled-rate systems with active phase transition management – not in static freezers that happen to reach the same rate

02

How phase transition control reduces variability ~14-fold vs static freezing (CV 2.3% vs 32.6%), and why this interval is the period of greatest osmotic and mechanical stress on cells

03

How plate-based and cryogenic LN2 freezing systems with user-defined recipes deliver reproducible, high-viability cryopreservation and reduce cryoconcentration in GMP workflows

Controlled freezing workflow

1

Cell preparation & cryoprotectant addition (5% DMSO)

2

Recipe-defined cooling with controlled phase transition (~40 min)

3

Post-transition cooling to target temperature (−150 °C or −80 °C)

4

Thawing & multi-assay viability assessment (trypan blue, live/dead, XTT)

Key findings

Freezing at −1.15 °C/min achieved ~95% post-thaw viability; the slowest rate (−0.68 °C/min) yielded only ~67%, even under controlled conditions

Uncontrolled static freezing produced below 50% viability despite a rate comparable to controlled Run 1 (~88%), confirming phase transition – not cooling rate – as the decisive variable

Plate-based freezing reached −70 °C ~3× faster than a static freezer (1:08 h vs 3:13 h) with substantially reduced cryoconcentration

Key interests

Cryopreservation

ATMP Manufacturing

Controlled-Rate Freezing

Phase Transition Control

Cell Viability

CHO-K1 Cells

GMP Compliance

Cold-Chain Management