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Rapid spheroid clearing on a microfluidic chip.

Tomas Silva Santisteban1, Omid Rabajania, Iana Kalinina

  • 1Microfluidic and Biological Engineering, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. matthias.meier@imtek.de.

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Summary
This summary is machine-generated.

This study introduces a microfluidic chip for rapid tissue clearing of 3D spheroids. The engineered system significantly reduces clearing time from 14 days to 5 hours using pH-controlled osmotic flow.

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Area of Science:

  • Biotechnology and Bioengineering
  • Microfluidics and Lab-on-a-Chip Systems
  • 3D Cell Culture and Tissue Engineering

Background:

  • Three-dimensional (3D) spheroids bridge the gap between animal models and 2D cell cultures.
  • Microfluidics offers precise control over the chemical environment for 3D cultures.
  • Developing analytical systems for spheroid analysis, particularly tissue clearing, remains a significant challenge.

Purpose of the Study:

  • To engineer a microfluidic large-scale integration (mLSI) chip platform for efficient tissue clearing and imaging of spheroids.
  • To develop a rapid prototyping procedure for pneumatic membrane valves (PMVs) enabling larger microchannels for spheroid handling.
  • To integrate and automate the CLARITY tissue clearing method on-chip for 3D spheroid analysis.

Main Methods:

  • Developed a rapid prototyping method using milled poly(methylmethacrylate) (PMMA) molds for scalable PMV fabrication with 750 μm microchannel heights.
  • Engineered an mLSI chip to miniaturize, automate, and integrate CLARITY steps: fixation, hydrogel infiltration, polymerization, lipid extraction, and immunofluorescence staining.
  • Utilized pH-jump-induced swelling and shrinkage of hydrogel-embedded spheroids to create an osmotic pump for accelerated clearing.

Main Results:

  • Achieved precise fluidic control for localized hydrogel polymerization within spheroids.
  • Demonstrated reversible spheroid volume changes (108% shrinkage at pH 5.5) with high rate constants.
  • Reduced spheroid clearing time from 14 days to 5 hours (for 200 μm diameter spheroids) through 30 pH cycles in 10 minutes.

Conclusions:

  • Developed a novel physicochemical method using an osmotic pump mechanism to drastically decrease hydrogel-embedded tissue clearing times.
  • Successfully integrated the CLARITY method onto an mLSI chip, enabling miniaturization and automation.
  • The on-chip CLARITY platform holds potential for high-throughput imaging of 3D tissue cultures.