Abstract
Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.
Highlights
Two-dimensional (2D) tissue culture has developed into an integral toolset for studying fundamental cell biology in vitro
Hydrogels are ideal materials to recapitulate the endogenous microenvironment of the extracellular matrix (ECM) in vivo due to their tissue-like mechanical properties and water-swollen structure that enables rapid transport of nutrients and signaling factors[7,8]
The elastin-like protein (ELP) used in this protocol are comprised of five regions: a T7 tag, His[6] tag, enterokinase (EK) cleavage site, a bio-active region, and an elastin-like region (Figure 1)
Summary
Two-dimensional (2D) tissue culture has developed into an integral toolset for studying fundamental cell biology in vitro. Criteria for an ideal 3D hydrogel matrix include simple, non-cytotoxic cell-encapsulation as well as independent tunability of physiologically relevant mechanical properties and mimics of native cell-adhesive motifs Both synthetic (e.g., polyethylene glycol, polylactic acid, poly(glycolic acid)) and naturally-derived (e.g., alginate, collagen, Matrigel) hydrogels have advantages over 2D in vitro culture platforms; they have significant shortcomings which limit their applicability. In addition to matrix stiffness and adhesive ligand tunability, recombinant hydrogels offer the capability to design specific material degradation profiles, which is necessary for cell spreading, proliferation, and migration within a 3D context[4,9] This degradation is afforded by cell secretion of proteases that target either the extended 'RGDS'9 or elastin-like sequence[25]. We further present the methodology for down-stream fluorescent labeling and confocal microscopy of encapsulated cells
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