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Disaccharide-assisted inkjet freezing for improved cell viability.

Tomona Takigawa1, Hiroki Watanabe2, Yoshitake Akiyama3

  • 1Department of Biomedical Engineering, Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano, 386-8567, Japan.

Cryobiology
|June 26, 2024
PubMed
Summary

Adding disaccharides like trehalose and sucrose to inkjet-frozen cells improves cryopreservation. Optimal concentrations prevent ice crystal formation, enhancing cell viability in tiny droplets.

Keywords:
Cell cryopreservationCritical cooling rateInkjet printingSucroseSuperflash freezingTrehalose

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Area of Science:

  • Cryobiology
  • Cellular cryopreservation
  • Biotechnology

Background:

  • Non-permeable disaccharides are used as cryoprotectant agents (CPAs) but are insufficient extracellularly.
  • Cryoprotectant agent-free cryopreservation via inkjet-freezing is challenging due to vitreous water instability.

Purpose of the Study:

  • To evaluate the effectiveness of trehalose and sucrose in inkjet freezing of 3T3 cells.
  • To determine optimal disaccharide concentrations for cell viability.
  • To analyze the relationship between disaccharide concentration, droplet size, and ice crystal formation.

Main Methods:

  • Inkjet-freezing of 3T3 cells in droplets ranging from 70 pL to 450 pL.
  • Evaluating cell viability at various trehalose and sucrose concentrations (up to 0.57 M).
  • Analyzing intracellular and extracellular ice crystal formation in relation to droplet size and CPA concentration.

Main Results:

  • Trehalose and sucrose at 0.57 M yielded over 90% cell viability in 70 pL droplets.
  • Higher disaccharide concentrations led to decreased viability due to dehydration.
  • Trehalose inhibited intracellular ice in 450 pL droplets, but 0.57 M was needed extracellularly for 70 pL droplets.

Conclusions:

  • Extracellular ice crystal suppression is critical for improving cell viability in inkjet superflash freezing.
  • Optimized disaccharide concentrations enhance cryoprotection in inkjet freezing.
  • Inkjet freezing with disaccharides offers a promising method for high-viability cell preservation.