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Related Experiment Video

Updated: Mar 7, 2026

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation
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Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent

Kathryn Pollock1, Rebekah M Samsonraj2, Amel Dudakovic2

  • 11 Department of Biomedical Engineering, University of Minnesota , Minneapolis, Minnesota.

Stem Cells and Development
|February 10, 2017
PubMed
Summary

Novel freezing solutions using sugars and sugar alcohols improve mesenchymal stromal cell (MSC) function after thawing. These DMSO-free methods preserve cell viability, differentiation potential, and key gene expression for therapeutic applications.

Keywords:
amino acidscryopreservationepigeneticmesenchymal stromal cellssaccharides

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Area of Science:

  • Cell Biology
  • Biotechnology
  • Regenerative Medicine

Background:

  • Current cryopreservation methods for mesenchymal stromal cells (MSCs) lead to reduced post-thaw functionality, limiting their clinical use.
  • Dimethylsulfoxide (DMSO) is a common cryoprotectant but can cause cellular toxicity and impair cell function.
  • There is a need for improved cryopreservation techniques to maintain MSC viability and therapeutic potential.

Purpose of the Study:

  • To develop and evaluate novel, DMSO-free freezing solutions for MSCs.
  • To assess the impact of these solutions on post-thaw cell function, including attachment, cytoskeleton integrity, and differentiation capacity.
  • To investigate the molecular mechanisms underlying improved cell preservation, including epigenetic modifications and gene expression profiles.

Main Methods:

  • MSCs were frozen using novel solutions containing sugars, sugar alcohols, and small-molecule additives, compared to traditional DMSO-based solutions.
  • Post-thaw cell attachment, actin cytoskeleton alignment, and osteogenic/chondrogenic differentiation potential were evaluated.
  • Genomic analysis of DNA hydroxymethylation and RNA sequencing were performed to assess epigenetic and gene expression changes.
  • Quantitative real-time PCR validated the expression of key cytoprotective and adhesion genes.

Main Results:

  • MSCs frozen in novel solutions showed improved post-thaw attachment and more normal actin cytoskeleton alignment compared to DMSO-treated cells.
  • Cell differentiation potential for osteogenesis and chondrogenesis was retained in MSCs preserved with the new solutions.
  • Genomic analysis revealed differential effects of freezing media on DNA hydroxymethylation levels.
  • RNA sequencing indicated upregulation of cytoprotective genes (e.g., galanin, BCL2) and adhesion molecules (e.g., CD106, CD54) in DMSO-free solutions.

Conclusions:

  • DMSO-free freezing solutions containing sugars, sugar alcohols, and additives are effective alternatives for MSC cryopreservation.
  • These novel solutions enhance post-thaw cell function, preserve differentiation capacity, and modulate gene expression and epigenetic profiles.
  • Osmolyte-based freezing solutions represent a promising new paradigm for the preservation of therapeutic cells.