A low-cost uniaxial cell stretcher for six parallel wells

Abstract

Cells in the lungs, the heart, and numerous other organs, are constantly exposed to dynamic forces and deformations. To mimic these dynamic mechanical loading conditions and to study the resulting cellular responses such as morphological changes or the activation of biochemical signaling pathways, cells are typically seeded on flexible 2D substrates that are uniaxially or biaxially stretched. Here, we present an open-source cell stretcher built from parts of an Anet A8 3D printer. The cell stretcher is controlled by a fully programmable open-source software using GCode and Python. Up to six flexible optically clear substrates can be stretched simultaneously, allowing for comparative multi-batch biological studies including microscopic image analysis. The cell yield from the cell culture area of 4 cm² per substrate is sufficient for Western-blot protein analysis. As a proof-of-concept, we study the activation of the Yes-associated protein (YAP) mechanotransduction pathway in response to increased cytoskeletal tension induced by uniaxial stretching of epithelial cells. Our data support the previously observed activation of the YAP transcription pathway by stretch-induced increase in cytoskeletal tension and demonstrate the suitability of the cell stretcher to study complex mechano-biological processes.

Keywords: 3D printer; Cell Mechanics; Cell stretcher; Mechanosensing; Uniaxial stretch; YAP.

Graphical abstract

Fig. 1

Hardware. A: Stretcher unit. B: Electronics and controller unit. C: Technical drawing of the casting mold for PDMS substrates.

Fig. 2

Assembly of the stretcher unit. A: Acrylic glass parts (of 6 mm thickness) needed for the stretcher unit. B: Completely assembled carriage with nuts and bearings from the Anet A8 kit. C: Stepper motors in acrylic glass mounting frames. D: Mounting of a lead screw and motor. E: Assembly of the aluminum frame (bottom view). F: Top view of the final stretcher unit.

Fig. 3

Assembly of the electronics and controller unit. A: Acrylic housing without the electronics installed. B: Top view of all parts installed, but unwired. C: Completely assembled controller unit.

Fig. 4

Fabricating PDMS cell substrates. A: Mold parts. B: PDMS mixed from base and crosslinker. C: Cell substrate after curing and removal from the mold.

Fig. 5

Software for operation of the cell stretcher. A: Cycle Stretch window for setting up and starting a cyclic stretch protocol. B: Calibration window for moving the stretcher to absolute or relative positions and performing a zero-point calibration.

Fig. 6

Stretching of PDMS cell substrates. A: Cell substrate inserted into the acrylic glass mountings at 0% stretch and fastened with spacer bolts. B: The same sample stretched to 5 mm. C: Acrylic glass frame mounted to the sample with four spacer bolts. D: After unmounting the sample from the stretcher, the acrylic glass frame maintains the 5 mm stretch amplitude.

Fig. 7

Verification of cyclic stretch. A: Setup for imaging the stretcher from top view. B: Still frame of tracked marker points in the video. C: Comparison of evaluated positional data (orange points) with an ideal triangular cyclic stretch (black line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Uniaxial stretching of PDMS substrates without and with clamps attached. A: Traces of marker points on a PDMS substrate, which was stretched to a total amplitude of 5 mm in steps of 250 µm without clamps attached. The image shown was recorded at 5 mm stretch. B: Measured stretch of marker points for target stretches between 0 and 5 mm (median (orange line), 25% / 75% percentiles (boxes), 1.5 inter-quantile range (error bars)). Dashed line represents the target stretch assuming a baseline length of 25 mm. C: Spatial distribution of the effective stretch as determined by linear regression from the slope of the local stretch in percent versus mm stretcher motion. (D,E,F): Same as (A,B,C), but with clamps attached to the PDMS substrate for a more even load distribution. The dashed line in (E) is the target stretch assuming a baseline length of 21.5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

YAP activation through cyclic uniaxial stretching. A: Immunofluorescence image of MDCK cells. Cells were either not stretched or cyclically stretched with an effective stretch amplitude of 24.8 ± 2.3% at 0.5 Hz for 6 h, with or without blebbistatin treatment. Letters indicate predominant YAP localization in each cell (C: cytoplasmic, P: pancellular, N: nuclear). Scale bars: 10 μm. B: YAP localization (mean ± SEM from 7 to 10 fields of view per condition) in 451 (control), 506 (stretched, no blebbistatin), 327 (stretched, 5 μM blebbistatin), and 350 (stretched, 20 μM blebbistatin) cells. C: Immunoblot analysis of YAP expression levels in MDCK cells treated as in A and B. D: YAP expression levels normalized to GAPDH expression.