Light-driven biological actuators to probe the rheology of 3D microtissues

Abstract

The mechanical properties of biological tissues are key to their physical integrity and function. Although external loading or biochemical treatments allow the estimation of these properties globally, it remains difficult to assess how such external stimuli compare with cell-generated contractions. Here we engineer microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induce local contractions within microtissues, while monitoring microtissue stress and strain. We investigate the regulation of these local contractions and their spatio-temporal distribution. We demonstrate the potential of our technique for quantifying tissue elasticity and strain propagation, before examining the possibility of using light to create and map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to guide the formation of tissues while non-destructively charting their rheology in real time, using their own constituting cells as internal actuators.

Fig. 1. Light-controlled contraction of microtissues.

a Representative top view images, among 21 microtissues over 3 independent experiments, and side view schemes depicting the formation of a microtissue. Over the first 24 h of cultivation, the fibroblasts spread and spontaneously compact the collagen matrix. The two T-shaped cantilevers anchor and constrain the contraction of the collagen matrix to form a microtissue that spans across the top of the pair of cantilevers. b Schematic illustrating the light-stimulation of a microtissue composed of Opto-RhoA fibroblasts. c Schematic of the ARHGEF11(DHPH)-CRY2/CIBN-CAAX optogenetic system to control cellular contractility. Upon blue light stimulation, CRY2 changes conformation and binds to CIBN, bringing the RhoA-activator ARHGEF11 in close proximity of RhoA, which in turn induces a contractility increase. d Upon blue light illumination of its left- or right-half, an opto-RhoA microtissue contracts locally, as shown by the PIV-tracking of the displacements, whereas a WT microtissue is unaffected (e). For readability reasons, only half of the vectors are represented. Scale bar is 100 µm. Temporal evolution of the tension (f) and stress σ_xx (g) generated by Opto-RhoA (in red) or WT (in blue) microtissues upon the simulation of their left- and right-half. Data are the average of n = 20 microtissues over 3 independent experiments ±SD. Source data are provided as a Source Data file.

Fig. 2. The anisotropy of the optogenetically-induced contraction correlates with the microtissue architecture.

a Representative microtissue where the blue disc represents the 50 µm diameter isotropic stimulation area. b Resulting average displacement field (for readability reasons, only half of the vectors are represented) and c polar plot of its orientation. Data are presented as mean ± SD with n = 25 microtissues over 2 independent experiments superimposed with a dot plot of the data distribution. Resulting average strain fields (ε_xx in d, ε_yy in e) and comparison of the average strain amplitudes in the area of stimulation (f). Data are presented as Tukey box plots (i.e. the box extends from the 25th to 75th percentiles, the median is plotted as a line inside the box and the whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the edge of the box) with n = 25 microtissues over 2 independent experiments. ^****P < 0.0001 determined by a two-tailed t-test between ε_xx and ε_yy. g Confocal slice of the fluorescent staining of actin in a representative microtissue and h corresponding color-coded map of the actin orientation. i Polar plot showing the anisotropic orientation of actin fibers along the x-axis. j Confocal slice of the fluorescent staining of collagen in a representative microtissue and k corresponding color-coded map of the collagen orientation. l Polar plot showing the anisotropic orientation of collagen fibers along the x-axis. Data in (i) and (l) are presented as mean ± SD with n = 27 microtissues over 4 independent experiments superimposed with a dot plot of the data distribution. Scale bars are 100 µm. Source data are provided as a Source Data file.

Fig. 3. Light-induced local contractions evidence the viscoelastic properties of microtissues.

Temporal evolution of the average displacement (a) and ε_xx strain field (b) after stimulation of the left half of the tissue by blue light (symbolized by a blue rectangle in a). c Evolution of the stress σ_xx over time and d resulting strain ε_xx decomposed in its positive (stretch, in red) and negative (compression, in blue) components. e Corresponding stress-stretch (in red) and stress-compression (in blue) curves. The dotted lines indicate the maximum stress σ^max (in black), the maximum stretch ${\epsilon }_S^{\max }$ (in red) and the maximum compression ${\epsilon }_C^{\max }$ (in blue). Data are the average of 22 microtissues over 2 independent experiments ±SD. Scale is 100 µm. Source data are provided as a Source Data file.

Fig. 4. Light-driven probing of the apparent elastic modulus of microtissues.

Average displacement (a) and ε_xx strain (b) fields resulting from 20 µm, 50 µm and 100 µm wide light stimulations (symbolized by blue brackets). For readability reasons, only half of the displacement vectors are represented. Scale bar is 100 µm. c Corresponding maximum stress σ^max and stretch ${\epsilon }_S^{\max }$. d Resulting elastic modulus E, reported as σ ^max / ${\epsilon }_S^{\max }$, and time delay τ_S between σ ^max and ${\epsilon }_S^{\max }$ in function of the pulse width. Data are presented as Tukey box plots (i.e. the box extends from the 25th to 75th percentiles, the median is plotted as a line inside the box and the whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the edge of the box) with n = 20 microtissues over 2 independent experiments. ^***P < 0.001 between conditions. n.s. stands for non-significant (i.e. P > 0.05). Statistical significances were determined by one-way analysis of variance (ANOVA) corrected for multiple comparisons using Tukey test. Source data are provided as a Source Data file.

Fig. 5. Optogenetically-mapped mechanical heterogeneities correlate with actin and collagen architecture.

a Representative square microtissue where the red (a) and blue (b) discs represent the centered and eccentric 200 µm diameter stimulation areas, respectively. Corresponding average displacement fields for the centered (c) and eccentric (d) stimulations. For readability reasons, only half of the vectors are represented. e Polar plot of the orientations of the displacement vectors. Data are presented as mean ± SD with n = 15 microtissues over 2 independent experiments, superimposed with a dot plot of the data distribution. Resulting average strain fields for centered (ε_xx in f, ε_yy in g) and eccentric (ε_xx in h, ε_yy in i) stimulations. Comparison of the average strain amplitudes (j) and anisotropy coefficient A.C. k in the areas of stimulation. Data are presented as Tukey box plots (i.e. the box extends from the 25th to 75th percentiles, the median is plotted as a line inside the box and the whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range from the edge of the box) with n = 15 microtissues over 2 independent experiments. ^***P < 0.001 and n.s. stands for non-significant (i.e. P > 0.05), determined by two-tailed t-test. Confocal projection of the fluorescent staining of actin in a representative microtissue (l), magnifications and color-coded map of the actin orientation (m) and polar plot (n) of the orientations of actin fibers in the center (red) or the left side (blue). Confocal projection of the fluorescent staining of collagen in a representative microtissue (o), magnifications and color-coded map of the collagen orientation (p) and polar plot (q) of the orientations of collagen fibers in the center (red) or the left side (blue). Data in (n) and (q) are presented as mean ± SD with n = 16 microtissues over 2 independent experiments superimposed with a dot plot of the data distribution, and the probability of alignment 75°−90° between the center and left conditions are significantly different: ^****P < 0.0001 determined by two-way ANOVA corrected for multiple comparisons using Sidak test. Scale bars are 100 µm. Source data are provided as a Source Data file.