Elsevier

Biomaterials

Volume 33, Issue 32, November 2012, Pages 8040-8046
Biomaterials

Light activated cell migration in synthetic extracellular matrices

https://doi.org/10.1016/j.biomaterials.2012.07.013Get rights and content

Abstract

Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels.

Introduction

Cell migration is fundamental to many biological processes including tissue development, regeneration, and repair [1], [2]. It is a highly integrated multi-step process involving cell polarization, protrusion, adhesion, translocation, and rear retraction [3]. A full understanding of the local driving forces and regulating factors of cell migration remains a challenge today, particularly in three dimensional (3D) environments. The primary, non-contact techniques available for single cell manipulation are field gradient traps including optical tweezers [4], magnetic tweezers [5], and dielectrophoretic traps [6]. Unfortunately, these methods for cell manipulation are limited to a liquid medium in a two dimensional (2D) environment which bears little resemblance to the native cell surrounding of the extracellular matrix. In the case of engineered 3D environments, chemical and mechanical gradients can stimulate migration of cell populations but do not allow manipulation of single cells [7], [8], [9]. Less is known about the effects of cell adhesion, matrix stiffness, or geometry on cell motility in 3D [10]. Here, we present a strategy for manipulating individual cells in a synthetic extracellular matrix through selective optical activation of signaling inside the cell to investigate stem cell motility with single cell resolution.

Biomaterial scaffolds are efficient tools for culturing cells in a 3D environment that mimics, to varying degrees, the native in vivo environment [11]. The physical structure and chemical functionality of a synthetic biomaterial scaffold can be manipulated to create highly defined microenvironments in which to evaluate cell response. While cell migration is observed in and around many biomaterials in vivo, similar in vitro cell migration in these 3D environments has not been achieved. Hence, the use of optical tools to precisely control the migration of single cells [12], [13] holds great promise to probe materials response.

In this work, a visible laser was applied to guide directional migration of mammalian stem cells in 3D hydrogels. Mesenchymal stem cells (MSCs) were transfected with a genetically encoded photoactivatable Rac1 (PA-Rac) [14]. Rac is a subfamily of Rho GTPases that plays a pivotal role in cell migration by stimulating actin polymerization [15]. On exposure to visible light locally in defined regions of the cell, subcellular activation of the PA-Rac occurs. The locally augmented Rac activation then stimulates migration of the cell towards the light. Repeated application of the light promotes continuous cell migration. The unique ability to direct the motility of mammalian stem cells in defined, synthetic environments allows investigation into how the local adhesive context, stiffness, and geometry in a material influences migration with single cell resolution to elucidate both fundamental biological principles and materials design parameters.

Section snippets

Cell culture

Human mesenchymal stem cells (hMSCs) were derived from human embryonic stem cells (hESCs) (Hues9) as reported previously [16]. The hMSCs were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone), 2 mm l-glutamine (GIBCO), 100 units/ml of penicillin, and 100 μg/ml of streptomycin (GIBCO). Cells were incubated at 37 °C and 5% CO2 and passaged every 4–5 days using 0.025% trypsin-n-EDTA (Clonetics Biowhittaker) when the cells

Photoactivated cell migration by periodic subcellular activation

Photoactivated cell migration was accomplished by periodic subcellular activation of PA-Rac within stem cells encapsulated in hydrogels. Cells transfected with PA-Rac responded to light of 458 nm rapidly and reversibly (Fig. 1A). Precise spatiotemporal control of the Rac activity was obtained by modulating the Rac1-LOV interactions using focal illumination (Fig. 1B) [14]. In this way, the overall Rac activity in the cellular region exposed to light was increased due to the local activation of

Conclusion

We have used a combination of new techniques to manipulate single stem cells in synthetic 3D matrices using a photoactivatable Rac. Photoactivated cell migration in biomaterials allows for detailed characterization of fundamental cell responses to local environmental physical and biological cues critical in processes such as development, regeneration, and cancer. These studies demonstrate that cell motility can be tuned and guided by adjusting both internal signaling events and external

Acknowledgments

The authors gratefully acknowledge the generous support from the Arthritis Foundation Postdoctoral Fellowship (QG), the Jules Stein Professorship (JE), and NIH GM46425 (DM).

References (26)

  • R.J. Petrie et al.

    Random versus directionally persistent cell migration

    Nat Rev Mol Cell Biol

    (2009)
  • E. Hadjipanayi et al.

    Guiding cell migration in 3D: a collagen matrix with graded directional stiffness

    Cell Motil Cytoskeleton

    (2009)
  • M.H. Zaman et al.

    Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis

    Proc Natl Acad Sci U S A

    (2006)
  • Cited by (25)

    • Smart Instructive Polymer Substrates for Tissue Engineering

      2019, Smart Polymers and Their Applications
    • Engineered Proteins Program Mammalian Cells to Target Inflammatory Disease Sites

      2017, Cell Chemical Biology
      Citation Excerpt :

      Currently, the lowest number of genes required to program this ability into a cell (such as human embryonic kidney 293 [HEK293]) that has no natural propensity to migrate toward TNFα sources is unclear. In cells with no natural propensity, directed migration has been achieved for specific stimuli such as light (Guo et al., 2012; Kim et al., 2014; Mills et al., 2012; Wu et al., 2009) or biologicals such as vascular endothelial growth factor (VEGF) (Mills et al., 2012; Mills and Truong, 2011; Park et al., 2014; Yoo et al., 2010) by overexpressing engineered Rac1 or RhoA proteins, both members of a family of proteins involved in cytoskeletal changes that lead to cell migration. Previously, we have engineered Ca2+-activated proteins to control the formation of cellular blebs and lamellipodia through RhoA (Mills and Truong, 2011) (named CaRQ) and Rac1 (Lin et al., 2012; Mills et al., 2012) (named Racer), respectively.

    • Synthetic mechanobiology: Engineering cellular force generation and signaling

      2016, Current Opinion in Biotechnology
      Citation Excerpt :

      Optical induction also allows for precise spatial control over protein activation, and can target single cells or a specific region within a cell. Optical signals are minimally invasive and can reach cells embedded in three-dimensional materials and living tissue [41–43]. Light-gated ion channels offer a particularly elegant system for studying calcium-induced mechanobiological phenotypes such as muscle cell contraction.

    • 3D Photo-Fabrication for Tissue Engineering and Drug Delivery

      2015, Engineering
      Citation Excerpt :

      This dynamic system was explored as 2D culture platforms of variable elasticity in order to assess the influence of matrix elasticity (~32 kPa to ~7 kPa upon 5 min of light irradiation) and gradients of elastic modulus on the fibroblast-myofibroblast differentiation process. Another relevant application of photodegradation involves the localized photocleavage of hydrogel crosslinks in order to guide cell functions in either 3D or 2D environments [32, 71, 160, 163]. This approach was elegantly demonstrated by DeForest and Anseth, who used multiple wavelengths of light to independently control the functionality and architecture of four-arm PEG-based hydrogels produced by a copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) reaction [160].

    • 3D in vitro cell culture models of tube formation

      2014, Seminars in Cell and Developmental Biology
    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text