- Poor trypsinization leads to incomplete cell dissociation and clump formation, creating a heterogeneous cell suspension. This initial problem cascades into multiple issues during the cryopreservation process, significantly affecting cell viability upon revival.
- The primary challenge with cell clumps is their reduced surface area-to-volume ratio, which creates uneven distribution of cryoprotective agents (CPAs). While cells on the outer edges of clumps receive adequate protection, those in the center remain poorly protected. This uneven exposure to CPAs makes inner cells particularly vulnerable to freezing damage and ice crystal formation.
- During the freezing process, cell clumps create areas of high cell density with uneven freezing rates. Ice crystals preferentially form in the center of clumps, causing physical damage through mechanical stress. The cells in these central regions often rupture or die due to ice crystal formation and inadequate cryoprotection.
- The thawing process presents additional challenges. Cell clumps experience uneven warming rates and osmotic stress, with outer cells shrinking or swelling differently than inner cells. This leads to membrane damage and reduced viability. The limited surface area of clumps also hinders the exchange of nutrients and gases during thawing.
- Post-thaw recovery is significantly compromised in clumped cells. Large aggregates restrict oxygen and nutrient diffusion, leading to increased cell death through apoptosis or necrosis. Surviving cells often struggle to attach and spread properly, resulting in poor cell growth and functionality.
- These issues collectively reduce the reliability and reproducibility of cryopreserved samples, particularly affecting their use in cell culture, regenerative medicine, and biobanking. To optimize cryopreservation outcomes, proper enzymatic dissociation to achieve a single-cell suspension is essential before freezing.
Was this post helpful?
Let us know if you liked the post. That’s the only way we can improve.
