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Chemically Crosslinked Methylcellulose Substrates for Cell Sheet Engineering.

Lorenzo Bonetti1, Luigi De Nardo1,2, Silvia Farè1,2

  • 1Department of Chemistry, Materials and Chemical Engineering "G. Natta", Politecnico di Milano, Via Luigi Mancinelli 7, 20131 Milan, Italy.

Gels (Basel, Switzerland)
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Summary

This study enhances methylcellulose (MC) hydrogels using citric acid (CA) crosslinking for improved stability in cell sheet engineering (CSE). The modified hydrogels successfully supported cell growth and enabled easy detachment of cell sheets for regenerative medicine applications.

Keywords:
cell sheet engineering (CSE)citric acidcrosslinkingmethylcellulosethermoresponsive hydrogels

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Area of Science:

  • Biomaterials Science
  • Tissue Engineering
  • Regenerative Medicine

Background:

  • Methylcellulose (MC) hydrogels are used in cell sheet engineering (CSE) for temperature-triggered cell detachment.
  • Pristine MC hydrogels suffer from poor physical stability and mechanical properties, limiting their applications.
  • Crosslinking strategies are needed to enhance MC hydrogel performance for CSE.

Purpose of the Study:

  • To develop thermoresponsive MC hydrogels with improved mechanical stability using citric acid (CA) crosslinking.
  • To evaluate the suitability of these enhanced MC hydrogels as substrates for cell sheet engineering.
  • To demonstrate the successful harvesting and subsequent use of cell sheets derived from these hydrogels.

Main Methods:

  • MC hydrogels were crosslinked with varying concentrations of citric acid (CA).
  • Mechanical properties (Young's modulus, E) of the crosslinked hydrogels were measured.
  • Cytotoxicity of CA was assessed using L929 fibroblast cells.
  • Cell adhesion, proliferation, and detachment from hydrogel surfaces were evaluated at different temperatures.
  • Detached cell sheets were transferred to new surfaces to assess their viability and functionality.

Main Results:

  • Citric acid crosslinking significantly improved the mechanical performance of MC hydrogels (11-fold increase in E) without inducing cytotoxicity.
  • Murine fibroblast cells (L929) adhered to and proliferated on the crosslinked MC hydrogels.
  • Cell sheets were successfully harvested by simply lowering the temperature below the transition temperature (Tt) of the MC hydrogels.
  • Detached cell sheets retained their adhesive and proliferative capabilities on new culture surfaces.

Conclusions:

  • Citric acid crosslinking is an effective method to enhance the mechanical stability and applicability of methylcellulose hydrogels for cell sheet engineering.
  • The developed thermoresponsive hydrogels provide a promising platform for generating functional cell sheets for regenerative medicine.
  • This approach offers a simple, temperature-controlled method for cell sheet harvesting and transplantation.