Mastering
Biopreservation
Best practices and key insights
Biopreservation methods, including cryogenic and hypothermic preservation, play a crucial role in cell therapy manufacturing, impacting both the starting material and the final product. Proper biopreservation ensures that cells maintain their viability and recover their function with minimal impact due to processing, storage, and transport. Below, we explore Biopreservation Best Practices, key concepts, and expert recommendations for effective cell and tissue cryopreservation.
Cryopreserved vs. fresh cells
A common misconception in cell therapy is that “fresh” cells always outperform frozen ones. In reality, studies show that properly cryopreserved cells perform on comparably with freshly prepared cells — and not all “fresh” products are truly equal.
Cells that are stored or shipped only at chilled temperatures (not cryopreserved) can rapidly lose viability and potency over time. While certain handling methods can help slow that decline, the loss may be inevitable. In contrast, cryopreserved cells remain stable during storage and shipment until they’re thawed.
If your current process isn’t delivering consistent post-thaw performance, our experts can help you refine and optimize your approach.
Biopreservation modes
Each mode of cell preservation serves a different purpose depending on the application.
- Hypothermic (+2°C to +8°C Storage): Short-term preservation of cells, tissues and organs.
- Lyophilization (Room Temperature Storage): Freeze-drying process where samples are first frozen (below -40°C), then dried by vacuum sublimation. Primarily used for proteins, vaccines, enzymes and small molecules for extended shelf life.
- Subzero Hypothermic (-20°C to 0°C Storage): Preserves large tissues and whole organs for several days.
- Ultra-low temperature (-80°C Storage): Primarily used to preserve biological materials for medium to long-term storage in research and clinical settings.
- Vitrification (-196°C to -80°C Storage): Generally achieved via ultra-fast cooling to achieve a glass-like state without ice crystal formation. Used mainly for oocytes, embryos, and small tissue samples with indefinite storage potential.
- Cryopreservation (-196°C to -130°C Storage): Ideal for cell suspensions, ensuring long-term viability.
Not sure where to start? Speak to an expert about your biopreservation needs.
The science behind biopreservation
As cells cool, changes in cell membrane permeability combined with the disruption of ion pumps, slower energy metabolism, increased reactive oxygen species (ROS) generation and pH shift, consequently impairs the cell function. This may lead to stress-induced cell death via apoptosis (programmed cell death).
HypoThermosol® FRS (HTS-FRS) was developed to mitigate these adverse effects of hypothermia by maintaining:
- Ionic balance
- Osmotic support
- pH stability
- Free radical scavenging
Cryopreservation: avoiding cell damage
Cryopreservation requires balancing the freezing rate and cryoprotectant agents (CPA) use to prevent cell lysis due to intracellular ice formation or excessive dehydration.
Key considerations:
- Cooling too fast: Internal ice formation leads to cell rupture (lysis).
- Cooling too slow: Ice formation in extracellular milieu results in significant elevation of salt ions, leading to a flooding of intracellular milieu through the cold-induced permeable cell membrane resulting in denaturation of protein structure and disruption of intracellular signaling. Adding an intracellular-like balance of salts and ions will significantly reduce these stresses during cooling.
- Under hypothermia, ion pumps have lower capacity to regulate the intracellular balance resulting in interruptions of intracellular signaling, salinity, and osmolality.
- Contrary to common belief, the main role of DMSO or other CPAs is to reduce salt toxicity and prevent excessive osmotic shrinkage.
- A freezing rate of -1°C per minute is generally considered optimal for most mammalian cells. However, depending on cell type and DMSO concentration, the optimal freezing rate may vary.
Processing and storage considerations
For cell therapy manufacturing workflows, every step may have a significant impact on final product quality:
- Processing of starting materials
- Cell isolation/selection/activation/transduction
- Wash/Concentration
- Formulation/Fill/Finish: Manual vs Automation
- Controlled Freezing: Optimize cooling rates
- Storage & Transport: Minimize transient warming effects
- Thaw & Delivery: Standardize thawing rate and post-thaw assessment timing
BioLife Solutions has assisted numerous academic and commercial groups in optimizing the critical process parameters (CPPs) of various steps.
Biopreservation
Solutions
BioLife Solution’s biopreservation media has been optimized to support various CGT manufacturing process steps, like:
DMSO-free cryopreservation: challenges and alternatives
While some processes seek dimethyl sulfoxide (DMSO)-free formulations, alternative cryoprotectants (e.g., ethylene glycol, glycerol) may introduce new challenges:
- Increased viscosity may affect flow rates through tubing.
- Balancing dehydration and salt toxicity is complex.
- New reagents lack the clinical and regulatory support that DMSO has amassed in the past 60 years of usage.
- Mole per mole, DMSO may be the most effective cryoprotective agent (CPA). Alternative CPAs often require high concentrations to deliver the same effectiveness.
Tell us about your process so we can best help improve your outcomes.
Thawing Solutions: what to expect at thaw
Cells experience significant stress during cryopreservation and thawing processes, akin to a “punch to the face.” Key post-thaw considerations are:
- Viability Testing: Best assessed 24 hours post-thaw rather than immediately.
- Delayed Onset Cell Death: Some damage is not visible immediately after thaw.
- Markers of cell stress and damage should be assessed beyond membrane integrity alone.
Partner with us to optimize your bioprocesses
Optimizing biopreservation requires careful control of numerous process parameters including cryogenic media formulation and cell thawing protocols, as well as the choice of container closure and freezing rate. If your process isn’t achieving the desired results, let’s collaborate to refine your workflow.
Tell us about
your process
Do you have
other questions?