Yang J et al. (2025), The TissueTractor: A Device for Applying Large ...

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Small Methods 2025 Aug 09;98:e2500136. doi: 10.1002/smtd.202500136.

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The TissueTractor: A Device for Applying Large Strains to Tissues and Cells for Simultaneous High-Resolution Live Cell Microscopy.

Yang J, Hearty E, Wang Y, Vijayraghavan DS, Walter T, Anjum S, Stuckenholz C, Cheng YW, Balasubramanian S, Dong Y, Kwiatkowski AV, Davidson LA.

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Mechanical strain substantially influences tissue shape and function in various contexts from embryonic development to disease progression. Disruptions in these processes can result in congenital abnormalities and short-circuit mechanotransduction pathways. Manipulating strain in live tissues is crucial for understanding its impact on cellular and subcellular activities, unraveling the interplay between mechanics and cells. Existing tools, such as optogenetic modulation of strain, are limited to small strains over limited distances and durations. Here, a high-strain stretcher system, the TissueTractor, is introduced to enable simultaneous high-resolution spatiotemporal imaging of live cells and tissues under strain applications varying from 0% to over 100%. We use the system with organotypic explants from Xenopus laevis embryos, where applied tension reveals cellular strain heterogeneity and remodeling of intracellular keratin filaments. To highlight the device's adaptability, the TissueTractor is also used to study two other mechanically sensitive cell types with distinct physiological roles: human umbilical vein endothelial cells and mouse neonatal cardiomyocytes, revealing cell morphological changes under significant strain. The results underscore the potential of the TissueTractor for investigating mechanical cues that regulate tissue dynamics and morphogenesis.

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Species referenced: Xenopus laevis

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	Figure 1Configuration of the stretcher system. A) A schematic of the stage-top stretcher system on an inverted microscope. B) A disposable cassette. Scale bar = 1 cm. C) Top and side section views of the assembled cassette. D) The cassette has a top PES shim with two square cut-outs for stretcher blocks, a dumbbell-shape PDMS substrate with a 2 mm exposed region after assembly, and a bottom PES shim to provide xyz-stability. E) An exploded view of all components in the stretcher system. From top to bottom: two picomotor piezo linear actuators, an “H-bridge” with extended cantilevers controlled by the movement of actuators, a stage top that has an H-bridge rest to ensure a tight fit, a disposable cassette, and a stage bottom that has two motor mounts to mount the linear actuators, including a stage chamber with a glass coverslip bottom that can be filled with liquid. F) Top view of the assembled stage insert at a relaxed state (top) with H-bridge crossbeams bending inward, and at a stretched state (bottom) with H-bridge crossbeams straightened. G) A schematic of the cassette at a relaxed state with linear actuators pushing in to bend the H-bridge crossbeams (top left); as the arms of the actuators retracted, H-bridge crossbeams become straight and stretch the cassette (top right). A schematic of a tissue sample attached to the PDMS substrate of the cassette at a relaxed state (middle left) and at a maximum stretched state (middle right). A side view of a cassette being stretched and imaged on an inverted scope, with the tissue sample faces toward the objective lens (bottom).
	Figure 2Characterization of the strain rate and strain profiles of the PDMS substrate. A) A schematic of the PDMS substrate coated with fluorescent beads. The Center (the magenta box) is indicated on the schematic. B) Representative fluorescent beads were traced at 0, 60, and 120 min; yellow arrows showing position changes between two beads. Scale bar = 200 µm. C) From left to right: (see article for formula) and (see article for formula) strain rate maps at 60 min, (see article for formula) and (see article for formula) strain rate maps at 120 min. D) From left to right: cumulative εxx and εyy strain maps at 60 min, cumulative εxx and εyy strain maps at 120 min. A phase lookup table is applied, where blue indicates contraction and red indicates elongation of the material. E) Quantifications of strain rates (see article for formula) and (see article for formula) at the center of the PDMS every 5 min during the stretch. F) Quantifications of cumulative strain εxx and εyy at the center of the PDMS every 5 min during the stretch. Error bars, standard deviation. N = 1 cassette. (Replicates, Figure S7, Supporting Information).
	Figure 3Tissue and cellular strains in Xenopus laevis organotypic explants. A) A schematic of mounting Xenopus laevis organotypic explant on the fibronectin-coated cassette. An animal cap organotypic explant is microsurgically removed from the animal pole of a gastrula stage embryo attached to the PDMS substrate of the cassette and cultured to the desired stage. The cassette is flipped back and put into the microscope stage insert for stretching and imaging. B) A Stage 13 animal cap organotypic explant labeled with membrane-mNeonGreen at relaxed state (0 min, left) and stretched state (16 min, right). The red dashed line outlines the region-of-interests, where the same region was traced and imaged through 8 stretch steps over 16 min in total. Scale bar = 35 µm. * indicates the same cell before and after stretching. C) εxxTstep step strain map overlayed with cell outlines represents continuous tissue step strain over two consecutive stretch steps. D) εxxCstep step strain mapped using individual cellular strain, which strain was calculated based on individual cell shape, with no variation of strain within the single cell. N = 81 cells. E) A concordance/discordance map is represented by the differences between εxxCstep and εxxTstep. A magenta-cyan phase lookup table is applied, where both magenta and cyan indicate discordance behaviors between tissue and the individual cell, and white represents concordance. * indicates the same cell across the frames. “D” represents cells that divided throughout stretching. F) Quantification of both tissue and cellular step strains εxx, step between each two consecutive stretch steps. Error bars, standard deviation. n.s., not significant, P > 0.05, Multiple t-tests were used as statistical analysis to compare tissue and cell step strains at each time point. G) Quantification of cumulative tissue and cellular strains εxx across the 8 stretch steps over 16 min in total. Error bars, standard deviation. n.s., not significant, P > 0.05, Multiple t-tests were used as statistical analysis to compare tissue and cell cumulative strains at each time point. H) Quantification of both tissue and cellular strain rates between each two consecutive stretch steps. Error bars, standard deviation. n.s. not significant, P > 0.05, Multiple t-tests were used as statistical analysis to compare tissue and cell strain rates at each time point. I) Tissue-cell concordance/discordance by plotting differences between εxxCstep and εxxTstep against absolute values of their sum. The grey region represents insignificant movement of both tissue and the cell. The yellow region represents tissue, and the cell strained concordantly. The schematic in the yellow box shows the concordant scenario when the tissue and the cell strain similarly. The magenta region represents discordance that the cell strained larger than the tissue. The schematic in the magenta box shows the discordant scenarios when the cell experiences a larger strain than the tissue. The cyan region represents discordance in that the tissue is strained larger than the cell. The schematic in the cyan box shows the discordant scenarios when the cell experiences a smaller strain than the tissue or even experiences a negative strain when the tissue experiences a positive strain. Tissue is represented by the tan oval; Cell is represented by the purple hexagon. σC + T = ±0.081, standard deviation of \|εxxCstep\| + \|εxxTstep\|. σ = ±0.057, standard deviation of εxxCstep − εxxTstep. N = 81 cells at 8 stretch steps (Replicates, Figure S9, Supporting Information).
	Figure 4High-resolution live imaging of various model systems on the TissueTractor. A) A Stage 11 animal cap organotypic explant labeled with mem-mNeonGreen and keratin8-mCherry at relaxed state (top) and stretched state (bottom). * indicates the same cell across frames. The last column shows keratin filaments in a single cell. The green dashed line outlines the single cell. The magenta arrow highlights tortuous filaments that straightened at the stretched state. The cyan arrow highlights the separation between the filaments and the cell junction. Scale bar = 10 µm. B) Filament orientation at the relax state (purple) and at the stretch state (pink). Keratin filaments were significantly more aligned with the stretch axis (0 degrees) after the stretch. N = 3 explants. The chi-square test was used to compare distributions of filament orientations before and after stretch. p < 0.0001 **. C) Filament straightness at the relax state (purple) and at the stretch state (pink). Straightness was calculated by dividing absolute distance d (red dashed line) by filament length L (black line). Fractions of filaments with straightness values <0.9 decreased while those with values more than 0.9 increased at the stretched state, indicating filaments straightened at the stretched state. N = 3 explants. A two-tailed t-test was used to compare filament straightness at relaxed and stretched states. P = 0.0141 . D) A schematic of seeding cardiomyocytes or HUVECs on fibronectin- or collagen-coated cassette. A droplet of suspended cardiomyocytes or HUVECs is pipetted onto the PDMS substrate of the cassette with the respective medium at the bottom of the petri dish. The cassette with cells at the desired state is flipped back and put into the microscope stage insert before imaging. E) Cardiomyocytes labeled with membrane and nucleus markers were stretched and imaged for 12 min over 6 stretch steps. White arrows indicate straining of the cell membrane. Scale bar = 10 µm. F) εxxCMstep step strain map of cardiomyocytes during stretching. The contraction (shown in blue) was potentially due to the natural beating of cardiomyocytes. G) HUVECs labeled with membrane and nucleus markers were stretched and imaged for 12 min over 6 stretch steps. Red arrows highlight the tearing of cell–cell contacts. Scale bar = 20 µm. H) A single HUVEC at relaxed (left) and stretched (right) states. Red arrows highlight the fenestrated holes in the cell membrane. White arrows highlight the detachment of the cell membrane to the PDMS substrate. The orange dashed box outlines the zoomed-in region shown in I. I) The zoomed-in view of the cell membrane. Red arrows highlight the fenestrated holes before and after stretching. The magenta arrow and bar indicate the area corresponding to the X–Z view shown in J. The direction of the view is marked from A to B. J) X–Z view of two fenestrated holes before and after stretching. Red arrows indicate the sizes of the holes. Scale bar = 10 µm.
	Supplemental Video 1
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	Figure 1. Configuration of the stretcher system. A) A schematic of the stage‐top stretcher system on an inverted microscope. B) A disposable cassette. Scale bar = 1 cm. C) Top and side section views of the assembled cassette. D) The cassette has a top PES shim with two square cut‐outs for stretcher blocks, a dumbbell‐shape PDMS substrate with a 2 mm exposed region after assembly, and a bottom PES shim to provide xyz‐stability. E) An exploded view of all components in the stretcher system. From top to bottom: two picomotor piezo linear actuators, an “H‐bridge” with extended cantilevers controlled by the movement of actuators, a stage top that has an H‐bridge rest to ensure a tight fit, a disposable cassette, and a stage bottom that has two motor mounts to mount the linear actuators, including a stage chamber with a glass coverslip bottom that can be filled with liquid. F) Top view of the assembled stage insert at a relaxed state (top) with H‐bridge crossbeams bending inward, and at a stretched state (bottom) with H‐bridge crossbeams straightened. G) A schematic of the cassette at a relaxed state with linear actuators pushing in to bend the H‐bridge crossbeams (top left); as the arms of the actuators retracted, H‐bridge crossbeams become straight and stretch the cassette (top right). A schematic of a tissue sample attached to the PDMS substrate of the cassette at a relaxed state (middle left) and at a maximum stretched state (middle right). A side view of a cassette being stretched and imaged on an inverted scope, with the tissue sample faces toward the objective lens (bottom).
	Figure 2. Characterization of the strain rate and strain profiles of the PDMS substrate. A) A schematic of the PDMS substrate coated with fluorescent beads. The Center (the magenta box) is indicated on the schematic. B) Representative fluorescent beads were traced at 0, 60, and 120 min; yellow arrows showing position changes between two beads. Scale bar = 200 µm. C) From left to right: ε˙xx and ε˙yy strain rate maps at 60 min, ε˙xx and ε˙yy strain rate maps at 120 min. D) From left to right: cumulative ε xx and ε yy strain maps at 60 min, cumulative ε xx and ε yy strain maps at 120 min. A phase lookup table is applied, where blue indicates contraction and red indicates elongation of the material. E) Quantifications of strain rates ε˙xx and ε˙yy at the center of the PDMS every 5 min during the stretch. F) Quantifications of cumulative strain ε xx and ε yy at the center of the PDMS every 5 min during the stretch. Error bars, standard deviation. N = 1 cassette. (Replicates, Figure S7, Supporting Information).
	Figure 3. Tissue and cellular strains in Xenopus laevis organotypic explants. A) A schematic of mounting Xenopus laevis organotypic explant on the fibronectin‐coated cassette. An animal cap organotypic explant is microsurgically removed from the animal pole of a gastrula stage embryo attached to the PDMS substrate of the cassette and cultured to the desired stage. The cassette is flipped back and put into the microscope stage insert for stretching and imaging. B) A Stage 13 animal cap organotypic explant labeled with membrane‐mNeonGreen at relaxed state (0 min, left) and stretched state (16 min, right). The red dashed line outlines the region‐of‐interests, where the same region was traced and imaged through 8 stretch steps over 16 min in total. Scale bar = 35 µm. * indicates the same cell before and after stretching. C) ε xx Tstep step strain map overlayed with cell outlines represents continuous tissue step strain over two consecutive stretch steps. D) ε xx Cstep step strain mapped using individual cellular strain, which strain was calculated based on individual cell shape, with no variation of strain within the single cell. N = 81 cells. E) A concordance/discordance map is represented by the differences between ε xx Cstep and ε xx Tstep . A magenta‐cyan phase lookup table is applied, where both magenta and cyan indicate discordance behaviors between tissue and the individual cell, and white represents concordance. * indicates the same cell across the frames. “D” represents cells that divided throughout stretching. F) Quantification of both tissue and cellular step strains ε xx, step between each two consecutive stretch steps. Error bars, standard deviation. n.s., not significant, P > 0.05, Multiple t‐tests were used as statistical analysis to compare tissue and cell step strains at each time point. G) Quantification of cumulative tissue and cellular strains ε xx across the 8 stretch steps over 16 min in total. Error bars, standard deviation. n.s., not significant, P > 0.05, Multiple t‐tests were used as statistical analysis to compare tissue and cell cumulative strains at each time point. H) Quantification of both tissue and cellular strain rates ε˙xx between each two consecutive stretch steps. Error bars, standard deviation. n.s. not significant, P > 0.05, Multiple t‐tests were used as statistical analysis to compare tissue and cell strain rates at each time point. I) Tissue‐cell concordance/discordance by plotting differences between ε xx Cstep and ε xx Tstep against absolute values of their sum. The grey region represents insignificant movement of both tissue and the cell. The yellow region represents tissue, and the cell strained concordantly. The schematic in the yellow box shows the concordant scenario when the tissue and the cell strain similarly. The magenta region represents discordance that the cell strained larger than the tissue. The schematic in the magenta box shows the discordant scenarios when the cell experiences a larger strain than the tissue. The cyan region represents discordance in that the tissue is strained larger than the cell. The schematic in the cyan box shows the discordant scenarios when the cell experiences a smaller strain than the tissue or even experiences a negative strain when the tissue experiences a positive strain. Tissue is represented by the tan oval; Cell is represented by the purple hexagon. σC + T = ±0.081, standard deviation of \|ε xx Cstep \| + \|ε xx Tstep \|. σ = ±0.057, standard deviation of ε xx Cstep − ε xx Tstep . N = 81 cells at 8 stretch steps (Replicates, Figure S9, Supporting Information).
	Figure 4. High‐resolution live imaging of various model systems on the TissueTractor. A) A Stage 11 animal cap organotypic explant labeled with mem‐mNeonGreen and keratin8‐mCherry at relaxed state (top) and stretched state (bottom). * indicates the same cell across frames. The last column shows keratin filaments in a single cell. The green dashed line outlines the single cell. The magenta arrow highlights tortuous filaments that straightened at the stretched state. The cyan arrow highlights the separation between the filaments and the cell junction. Scale bar = 10 µm. B) Filament orientation at the relax state (purple) and at the stretch state (pink). Keratin filaments were significantly more aligned with the stretch axis (0 degrees) after the stretch. N = 3 explants. The chi‐square test was used to compare distributions of filament orientations before and after stretch. p < 0.0001 **. C) Filament straightness at the relax state (purple) and at the stretch state (pink). Straightness was calculated by dividing absolute distance d (red dashed line) by filament length L (black line). Fractions of filaments with straightness values <0.9 decreased while those with values more than 0.9 increased at the stretched state, indicating filaments straightened at the stretched state. N = 3 explants. A two‐tailed t‐test was used to compare filament straightness at relaxed and stretched states. P = 0.0141 . D) A schematic of seeding cardiomyocytes or HUVECs on fibronectin‐ or collagen‐coated cassette. A droplet of suspended cardiomyocytes or HUVECs is pipetted onto the PDMS substrate of the cassette with the respective medium at the bottom of the petri dish. The cassette with cells at the desired state is flipped back and put into the microscope stage insert before imaging. E) Cardiomyocytes labeled with membrane and nucleus markers were stretched and imaged for 12 min over 6 stretch steps. White arrows indicate straining of the cell membrane. Scale bar = 10 µm. F) ε xx CMstep step strain map of cardiomyocytes during stretching. The contraction (shown in blue) was potentially due to the natural beating of cardiomyocytes. G) HUVECs labeled with membrane and nucleus markers were stretched and imaged for 12 min over 6 stretch steps. Red arrows highlight the tearing of cell–cell contacts. Scale bar = 20 µm. H) A single HUVEC at relaxed (left) and stretched (right) states. Red arrows highlight the fenestrated holes in the cell membrane. White arrows highlight the detachment of the cell membrane to the PDMS substrate. The orange dashed box outlines the zoomed‐in region shown in I. I) The zoomed‐in view of the cell membrane. Red arrows highlight the fenestrated holes before and after stretching. The magenta arrow and bar indicate the area corresponding to the X–Z view shown in J. The direction of the view is marked from A to B. J) X–Z view of two fenestrated holes before and after stretching. Red arrows indicate the sizes of the holes. Scale bar = 10 µm.