Engineering a Microfluidic Platform to Cryopreserve Stem Cells: A DMSO-Free Sustainable Approach

Adv Healthc Mater. 2024 Nov;13(29):e2401264. doi: 10.1002/adhm.202401264. Epub 2024 Aug 17.

Abstract

Human adipose-derived stem cells (hASCs) are cryopreserved traditionally using dimethyl sulfoxide (DMSO) as the cryoprotectant agent. DMSO penetrates cell membranes and prevents cellular damage during cryopreservation. However, DMSO is not inert to cells, inducing cytotoxic effects by causing mitochondrial dysfunction, reduced cell proliferation, and impaired hASCs transplantation. Additionally, large-scale production of DMSO and contamination can adversely impact the environment. A sustainable, green alternative to DMSO is trehalose, a natural disaccharide cryoprotectant agent that does not pose any risk of cytotoxicity. However, the cellular permeability of trehalose is less compared to DMSO. Here, a microfluidic chip is developed for the intracellular delivery of trehalose in hASCs. The chip is designed for mechanoporation, which creates transient pores in cell membranes by mechanical deformation. Mechanoporation allows the sparingly permeable trehalose to be internalized within the cell cytosol. The amount of trehalose delivered intracellularly is quantified and optimized based on cellular compatibility and functionality. Furthermore, whole-transcriptome sequencing confirms that less than 1% of all target genes display at least a twofold change in expression when cells are passed through the chip compared to untreated cells. Overall, the results confirm the feasibility and effectiveness of using this microfluidic chip for DMSO-free cryopreservation of hASCs.

Keywords: DMSO‐free cell banking; cryopreservation; mechanoporation; microfabricated chip; microfluidics; stem cell freezing; trehalose delivery.

MeSH terms

  • Adipose Tissue / cytology
  • Adipose Tissue / metabolism
  • Cryopreservation* / methods
  • Cryoprotective Agents* / pharmacology
  • Dimethyl Sulfoxide* / chemistry
  • Dimethyl Sulfoxide* / pharmacology
  • Humans
  • Lab-On-A-Chip Devices
  • Microfluidics / instrumentation
  • Microfluidics / methods
  • Stem Cells* / cytology
  • Stem Cells* / metabolism
  • Trehalose* / chemistry
  • Trehalose* / pharmacology

Substances

  • Trehalose
  • Dimethyl Sulfoxide
  • Cryoprotective Agents