Core Challenges in Graded Cryopreservation of Stem Cells

Stem cells (especially pluripotent stem cells) are extremely fragile and highly sensitive to temperature fluctuations during cryopreservation. Improper cryopreservation can lead to:

Intracellular ice crystal formation: Rapid cooling causes intracellular water to freeze before it can escape,

rupturing cell membranes and resulting in cell death.

Solution effect damage: Slow cooling causes extracellular solution to freeze first, leading to increased extracellular solute concentration and causing osmotic shock and chemical damage to cells.Damage during thawing: The thawing process is equally critical. Excessively slow thawing allows small ice crystals to recrystallize and grow, inflicting secondary damage on cells.

Staged cryopreservation (or programmed cooling) was designed to address these issues. It employs a preset, gradual cooling protocol (e.g., -1°C/minute) to allow intracellular water sufficient time to diffuse out of cells, thereby minimizing intracellular ice crystal formation.

The dual-probe refrigerator plays a crucial role in this process.

Dual-probe refrigerators feature two independent temperature sensors (probes), typically one monitoring ambient air temperature and the other monitoring sample core temperature (via a simulated test tube or a probe placed within the sample). This design offers unparalleled advantages in staged cryopreservation:

1. Probe 1: Monitors ambient air temperature

Function: This probe connects directly to the refrigerator's cooling system to control and maintain the overall temperature within the refrigerator compartment. It ensures the entire cooling program operates stably according to the preset curve (e.g., starting at 4°C and decreasing at -1°C/minute to reach -80°C).

Importance: It serves as the "executor" of the programmed cooling process, guaranteeing the stability of the overall environment.

Probe 2: Monitors the core temperature of the sample

Function: This is the essence of the dual-probe system. The probe is placed in a test tube containing cryopreservation solution, which is maintained in an identical environment to the stem cell sample. Therefore, it measures not the air temperature, but the actual temperature experienced by the sample.

Importance:

Reflects Reality: Due to latent heat released during water crystallization, the sample experiences a brief plateau or even slight temperature rise at the phase transition point (approximately -5°C to -15°C). While an air probe cannot detect this critical plateau, the sample probe can.

Ensures Protocol Validity: The programmable cryostat's algorithm adjusts cooling power based on the sample's actual temperature (not air temperature). Upon detecting the sample entering the phase transition plateau, the system maintains or fine-tunes the cooling rate to ensure complete cellular water efflux.

Provides Process Evidence: The dual-probe system simultaneously records both ambient temperature and sample temperature curves. This documentation serves as critical quality control evidence demonstrating compliance with Standard Operating Procedures (SOPs) for cell cryopreservation, essential for GLP/GMP laboratories and clinical applications.

In the graded cryopreservation of stem cell samples, dual-probe refrigerators are not an "optional accessory" but a "core piece of equipment." By directly monitoring the core temperature of samples, they transform the cryopreservation process from a "black box operation" into a precise scientific procedure that is controllable, monitorable, and traceable. This offers irreplaceable value in ensuring the survival rate and functionality of precious, non-renewable stem cell samples, as well as in meeting stringent regulatory requirements.

In addition to refrigerators, INNOVA also offers vapor-phase liquid nitrogen tanks and programmable cooling controllers for stem cell storage.