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. 2015 Aug:59:39-52.
doi: 10.1016/j.biomaterials.2015.04.036. Epub 2015 May 15.

Three-dimensional, soft neotissue arrays as high throughput platforms for the interrogation of engineered tissue environments

Michael Floren  1 , Wei Tan  2
Affiliations

Three-dimensional, soft neotissue arrays as high throughput platforms for the interrogation of engineered tissue environments

Michael Floren et al. Biomaterials. 2015 Aug.

Erratum in

  • Biomaterials. 2015 Oct;67:204

Abstract

Local signals from tissue-specific extracellular matrix (ECM) microenvironments, including matrix adhesive ligand, mechanical elasticity and micro-scale geometry, are known to instruct a variety of stem cell differentiation processes. Likewise, these signals converge to provide multifaceted, mechanochemical cues for highly-specific tissue morphogenesis or regeneration. Despite accumulated knowledge about the individual and combined roles of various mechanochemical ECM signals in stem cell activities on 2-dimensional matrices, the understandings of morphogenetic or regenerative 3-dimenstional tissue microenvironments remain very limited. To that end, we established high-throughput platforms based on soft, fibrous matrices with various combinatorial ECM proteins meanwhile highly-tunable in elasticity and 3-dimensional geometry. To demonstrate the utility of our platform, we evaluated 64 unique combinations of 6 ECM proteins (collagen I, collagen III, collagen IV, laminin, fibronectin, and elastin) on the adhesion, spreading and fate commitment of mesenchymal stem cell (MSCs) under two substrate stiffness (4.6 kPa, 20 kPa). Using this technique, we identified several neotissue microenvironments supporting MSC adhesion, spreading and differentiation toward early vascular lineages. Manipulation of the matrix properties, such as elasticity and geometry, in concert with ECM proteins will permit the investigation of multiple and distinct MSC environments. This paper demonstrates the practical application of high through-put technology to facilitate the screening of a variety of engineered microenvironments with the aim to instruct stem cell differentiation.

Keywords: 3-Dimensional cell culture; Extracellular matrix; High-throughput screening; Stem cell differentiation.

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Figures

Figure 1
Figure 1
Illustration of ECM Neotissue Fabrication and Utility for Multivariate Cell Culture Platforms.
Figure 2
Figure 2
Electrospun PEGDM fibrous hydrogel characterization. (A) Mid-range IR identifies methacrylate conversion with UV exposure. (B) Fibrous architecture was investigated using confocal laser microscopy and scanning electron microscopy in both wet and dry states. (C) Shear stress vs. shear strain relationships for several PEGDM substrates prepared under different UV exposures. (D) Translation of shear-strain relationships into elastic modulus using a Poisson ratio ν ~ 0 [16].
Figure 3
Figure 3
Protein Microdot Optimization. (A) Array layout depicted through color dye control. (B) Optimization of buffer glycerol content achieves ideal spotting. Inset images scale bar 50 μm. (C) Serial dilution of two model proteins (Albumin-Cy3, Streptavidin-Cy5) demonstrating distinct dot deposition and periodicity. Scale bar 500 μm. (D) Confocal microscopy rendering of albumin-Cy3 deposition illustrating 3-dimensional dot presentation. Scale bar 50 μm. (E) Printing optimization techniques allow for global array deposition onto PEGDM substrates. Scale bar 1 mm. Inset image scale bar 500 μm.
Figure 4
Figure 4
Design and Characterization of Combinatorial Protein Neotissue Array. (A) Design of a combinatorial ECM matrix with 6 proteins yielding 64 unique spotting combinations: C1 (collagen I), C3 (collagen III), C4 (collagen IV), L (Laminin), Fn (Fibronectin), E (Elastin). (B) Serial dilutions of Collagen I are retained after several rinsing stages and detectable at concentrations as low as 15 μg/ml (n=8). Immunostaining of combinatorial ECM matrix for collagen I (C) and collagen IV (D) after deposition and rinsing. Scale bar 1 mm.
Figure 5
Figure 5
Rat mesenchymal stem cells (MSCs) adhered on neotissue slides (E = 4.6 kPa substrate). (A) Confocal montage image of neotissue array after 24 h cell culture with distinct cellular islands visible for all protein spotting conditions (scale bar 1 mm)(green, f-actin; blue, DAPI). (B) Magnification of a4 subarray depicting cellular dot circular geometry and periodicity (scale bar 500 μm). (C, D) Confocal 3-dimensional rendering of cell loaded subarray (C)(scale bar 250 μm) and of a single cellular dot (D). (E) Nuclear staining of MSCs seeded on the neotissue arrays for image analysis and quantification. (F) 3-dimensional bar graph representing the average nuclear pixel intensities for all protein combinations for image (A). (G, H) Sorted average pixel intensities for adhesion (G) and spreading (H) for all protein combinations after 24 h cell culture (n = 3 neotissue arrays); Insets depict cellular dot images for adhesion and spreading of representative protein conditions (scale bars 100 μm).
Figure 6
Figure 6
Cultured MSCs display differential adhesion and spreading characteristics in response to protein combination and matrix elasticity. (A, B) Cellular structures, nuclei and f-actin, are stained, imaged (A) and quantified using software to produce an average intensity map (B) for distinct elastic environments (4.6 kPa; 20 kPa). (C, D) Comparison of average adhesion (C) and spreading (D) of MSCs on each protein condition for soft (4.6 kPa) and stiff (20 kPa) neotissue substrates. Conditions denoted by blue or red significantly favor adhesion or spreading for soft (4.6 kPa) or stiff (20 kPa) matrix elasticity respectively, purple denotes both. Insets depict adhesion and spreading of representative protein conditions screened from the arrays in (A) (scale bars 100 μm). (E, F) Results of 27 full factorial ANOVA of significant (p < 0.01) main and interaction effects for DAPI (E) and f-actin (F) intensities supporting either soft (4.6 kPa) or stiff (20 kPa) matrix conditions (n = 3 neotissue arrays). Blue and red dots represent prominent protein conditions supporting significant effects only on soft (4.6 kPa) or stiff (20 kPa) or both (purple dot) substrates respectively.
Figure 7
Figure 7
Cultured MSCs differentiate in response to protein combination and matrix elasticity on neotissue arrays. (A) Confocal montage images of neotissue arrays stained for nuclei, f-actin and PECAM for both elasticities investigated. Scale bar 1mm. (B) Differentiation marker PECAM average intensities obtained from neotissue arrays (A) and rendered into intensity maps for distinct elastic environments (4.6 kPa; 20 kPa). (C) Confocal images of cell nuclei (DAPI), f-actin (green) and PECAM (red) of MSCs cultured on specific protein conditions and matrix elasticity. Scale bar 50 μm. (D) Average PECAM intensity of MSCs after 24 h for each protein condition compared against soft (4.6 kPa) and stiff (20 kPa) neotissue substrates. Conditions denoted by blue or red significantly favor PECAM expression for soft (4.6 kPa) or stiff (20 kPa) or both (purple dot) matrix elasticity respectively. (E–G) Results of 27 full factorial ANOVA of significant (p < 0.01) main and interaction effects for PECAM intensities (G) and compared against either soft (4.6 kPa) or stiff (20 kPa) matrix conditions (E)(n = 3 neotissue arrays). Blue and red dots represent prominent protein conditions supporting significant effects only on soft (4.6 kPa) or stiff (20 kPa) or both (purple dot) substrates respectively. (F) Significant effects of matrix elasticity on all cellular phenomenon investigated (adhesion, spreading, PECAM) as reported from 27 full factorial ANOVA.
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