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Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments
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Transfection in the third dimension.

Anandika Dhaliwal1, Victor Oshita, Tatiana Segura

  • 1Biomedical Engineering Interdepartmental Program, University of California at Los Angeles, Los Angeles, USA.

Integrative Biology : Quantitative Biosciences From Nano to Macro
|August 10, 2013
PubMed
Summary

Gene transfer mechanisms differ between 2-D and 3-D cell cultures. This study reveals distinct endocytosis pathways, cytoskeletal dynamics, and RhoGTPase signaling roles in non-viral gene delivery for tissue regeneration applications.

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Area of Science:

  • Biomaterials Science
  • Cell Biology
  • Gene Therapy

Background:

  • Gene transfer in 2-D cultures is influenced by cell area, length, integrin expression, and RhoGTPase signaling.
  • Understanding 3-D gene transfer is crucial for developing effective gene delivery systems for tissue regeneration and therapy.

Purpose of the Study:

  • To investigate if cationic polymer-mediated gene transfer mechanisms differ between 2-D and 3-D cell cultures.
  • To examine the endocytosis pathways, cytoskeletal dynamics, and RhoGTPase involvement in non-viral gene transfer in both 2-D and 3-D environments.

Main Methods:

  • Utilized mouse mesenchymal stem cells (mMSCs) in both 2-D and 3-D culture models.
  • Investigated the impact of inhibiting clathrin- and caveolae-mediated endocytosis on transgene expression.

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  • Assessed the effects of actin and microtubule polymerization inhibitors, as well as RhoGTPase inhibitors (ROCK, PAK1), on gene transfer efficiency.

    • Main Results:

    • Inhibition of clathrin- and caveolae-mediated endocytosis more severely reduced transgene expression in 3-D than in 2-D cultures.
    • Microtubule depolymerization enhanced 2-D transfection but inhibited 3-D transfection.
    • ROCK effector inhibition decreased transgene expression and polyplex internalization in 3-D but not 2-D, while PAK1 inhibition increased expression in both dimensions.

    Conclusions:

    • Gene transfer mechanisms mediated by cationic polymers differ significantly between 2-D and 3-D cell cultures.
    • Endocytosis pathways, cytoskeletal dynamics, and RhoGTPase signaling play distinct roles in non-viral gene delivery depending on the cellular microenvironment (2-D vs. 3-D).
    • These findings provide critical insights for designing advanced gene delivery scaffolds for in vivo tissue engineering.