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Spatial considerations during cryopreservation of a large volume sample.

Peter Kilbride1, Stephen Lamb2, Stuart Milne2

  • 1Institute for Liver and Digestive Health, Royal Free Hospital Campus, UCL, London, NW3 2PF, UK; Asymptote Ltd. St. John's Innovation Centre, Cowley Road, Cambridge, CB4 0WS, UK.

Cryobiology
|June 4, 2016
PubMed
Summary

Large volume cryopreservation impacts cell recovery, with cells in early and late freezing zones showing impaired function. Faster thawing improved post-thaw viability in encapsulated liver spheroids.

Keywords:
Bioartificial liverHepG2Large volume cryopreservationProgressive solidification

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Area of Science:

  • Cryobiology
  • Biopreservation
  • Tissue Engineering

Background:

  • Limited research exists on large-volume (liters) cryopreservation compared to smaller volumes (up to 2 ml).
  • Large-scale cryopreservation often involves progressive solidification, posing unique challenges for cell viability.
  • The Bioartificial Liver Device necessitates cryopreserving 2 L in a cylindrical geometry, where progressive solidification is unavoidable.

Purpose of the Study:

  • To investigate the effects of progressive solidification on encapsulated liver hepatocyte spheroids during large volume cryopreservation.
  • To develop a reliable method for sampling different regions within cryopreserved samples.
  • To understand the impact of spatial location and thawing rates on cell recovery in large-volume cryopreservation for Bioartificial Liver Devices.

Main Methods:

  • Encapsulated liver hepatocyte spheroids were subjected to large volume cryopreservation with progressive solidification.
  • A novel sampling method was developed to assess cells from various regions of the frozen core.
  • Post-thaw viability and function were evaluated 24 hours after thawing.

Main Results:

  • Spatial location within the cryopreserved sample significantly affected post-thaw recovery.
  • Cells in the initial and final solidification zones showed impaired function.
  • Regions solidifying during the main phase of the process exhibited better post-thaw outcomes.
  • Faster thawing rates correlated with higher post-thaw viability (75.7 ± 3.9% vs. 62.0 ± 7.2%).

Conclusions:

  • Progressive solidification in large-volume, geometrically constrained cryopreservation negatively impacts cell viability.
  • Cellular location and thawing speed are critical factors for successful cryopreservation of Bioartificial Liver Devices.
  • Findings provide crucial insights for optimizing large-volume cryopreservation protocols for clinical applications.