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Freezing monolayers of cells without gap junctions.

W J Armitage1, B K Juss

  • 1Division of Ophthalmology, Bristol Eye Hospital, University of Bristol, Lower Maudlin Street, BS1 2LX, Bristol, United Kingdom. w.j.armitage@bristol.ac.uk

Cryobiology
|April 11, 2003
PubMed
Summary

Monolayer cells, unlike dispersed cells, often suffer more freezing damage due to intracellular ice. However, MDCK Type II cells lacking gap junctions showed reduced freezing injury, suggesting gap junctions enhance susceptibility to freezing.

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Area of Science:

  • Cell biology
  • Cryobiology
  • Biophysics

Background:

  • Cells cultured as monolayers are generally more susceptible to freezing injury than dispersed cells.
  • This enhanced susceptibility is often attributed to intracellular ice formation, potentially facilitated by gap junctions allowing ice propagation between cells.

Purpose of the Study:

  • To investigate the role of gap junctions in freezing injury of cells in monolayers.
  • To determine if MDCK Type II cells, which lack gap junctions, exhibit altered freezing responses compared to cells with gap junctions.

Main Methods:

  • MDCK Type II cells were cultured as monolayers and frozen at various rates (0.2–10 °C/min) with cryoprotectants like propane-1,2-diol, dimethyl sulfoxide, and glycerol.
  • Cell survival rates were assessed post-freezing to evaluate the influence of cooling rate and cryoprotectant type.

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Main Results:

  • Monolayers in propane-1,2-diol or dimethyl sulfoxide showed minimal impact of cooling rate on survival, indicating no enhanced intracellular freezing in the absence of gap junctions.
  • Conversely, monolayers frozen in glycerol exhibited increased cell damage at higher cooling rates (>0.5 °C/min).

Conclusions:

  • The absence of gap junctions in MDCK Type II cell monolayers appears to mitigate enhanced susceptibility to intracellular freezing.
  • Glycerol's potential to promote intracellular freezing may explain the observed damage in monolayers, independent of gap junction presence.