Tape-assisted fabrication method for constructing PDMS membrane-containing culture devices with cyclic radial stretching stimulation

Abstract

Advanced in vitro culture systems have emerged as alternatives to animal testing and traditional cell culture methods in biomedical research. Polydimethylsiloxane (PDMS) is frequently used in creating sophisticated culture devices owing to its elastomeric properties, which allow mechanical stretching to simulate physiological movements in cell experiments. We introduce a straightforward method that uses three types of commercial tape-generic, magic and masking-to fabricate PDMS membranes with microscale thicknesses (47.2 µm for generic, 58.1 µm for magic and 89.37 µm for masking) in these devices. These membranes are shaped as the bases of culture wells and can perform cyclic radial movements controlled via a vacuum system. In experiments with A549 cells under three mechanical stimulation conditions, we analysed transcriptional regulators responsive to external mechanical stimuli. Results indicated increased nuclear yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) activity in both confluent and densely packed cells under cyclically mechanical strains (Pearson's coefficient (PC) of 0.59 in confluent and 0.24 in dense cells) compared with static (PC = 0.47 in confluent and 0.13 in dense) and stretched conditions (PC = 0.55 in confluent and 0.20 in dense). This technique offers laboratories without microfabrication capabilities a viable option for exploring cellular behaviour under dynamic mechanical stimulation using PDMS membrane-equipped devices.

Keywords: PDMS membrane; YAP/TAZ; microfabrication; microphysiological systems.

Figure 1.

Fabrication of the one-piece PDMS construct. (a–d) Schematic illustration of the fabrication process. (a) Plastic board with a smooth finish is used as the raw material for the CNC machining process. (b) Plastic mould is created with a smooth finish on the surface for the central post, which is level with the surrounding walls, to mould the bottom of cell culture wells. (c) Fabrication of the one-piece moulded PDMS construct involves placing adhesive tapes around the mould, pouring PDMS into the mould and covering it with a plastic sheet for clamp moulding. (d) The cross-sectional view shows the thickness of the PDMS membrane (not to scale), with a glass board placed on top of the plastic sheet to squeeze out excess PDMS along the flat surface under clamping pressure. (e) The mould designed for producing 4-well PDMS constructs. (f) The final product is a one-piece PDMS construct featuring four culture wells with bottoms made of elastic membranes.

Figure 2.

Characterization of the tape-thick PDMS membrane. (a) The sectional view of a PDMS construct well, with membrane thickness measured at three positions (indicated by red circles) and averaged (scale bar = 1 mm). A schematic view of the cut slice for measurement is shown above. (b) Moulded PDMS replicas arranged from left to right, created with hole diameters of 250, 500, 1000 and 2000 µm in the frames (scale bar = 10 mm). (c) Strain measurements of replicas: the white dotted line represents the length of the strained membrane, and the red bottom line indicates the original length of the flat membrane without straining (scale bar = 200 µm). A schematic view of the cut slice for measurement is shown above. (d) The schematic of alveolar-like stretching formation: (d(i)) shows a sectional view of the membrane in a static state (no vacuum applied); (d(ii)) depicts the membrane in a stretched state (with vacuum applied).

Figure 3.

The assembled setting for cell experiments.

Figure 4.

Comparison of thicknesses for commercial tapes and PDMS membranes produced using a tape-assisted method. (a) Images of commercial tapes arranged from left to right: a generic brand of transparent tape (generic), 3M Scotch Magic 810 (magic) and 3M Scotch Masking 244 (masking). (b) Thickness comparison of commercial tapes according to specifications (in black columns), measurements obtained using microscopic analysis software (in dark grey columns) and PDMS membranes fabricated with different tapes (n = 5; mean with standard deviation displayed).

Figure 5.

PDMS membrane stains and curvature. (a) Plot of the linear regression trendline on the hole diameter of the frames versus strain (n = 5). (b) Plot of the linear regression trendline on the vacuum strength versus the strain (n = 5). (c) Plot of the linear regression trendline on the hole diameter of the frames versus curvature (n = 5). (d) Plot of the linear regression trendline on the vacuum strength versus curvature (n = 5).

Figure 6.

Influence of seeding density on YAP/TAZ translocation. (a) Various A549 seeding densities displayed from left to right: 2.5 × 10⁴, 5 ×10⁴, 7.5 ×10⁴, 10 ×10⁴, 12.5 ×10⁴ and 15 × 10⁴ cells per well in a PDMS construct. After 2 days of culture, cells were fixed and immunostained with anti-ZO1 (red), anti-YAP/TAZ (green) and Hoechst stain. Representative images from each density are shown with a white scale bar of 20 µm at the bottom right of each image. Scatter plots for each density were analysed using ImageJ, with Pearson’s coefficients displayed at the top right of the plots. (b) Comparison of nuclear to cytoplasmic fluorescence intensity across different seeding densities. (c) Comparison of Pearson’s coefficients across seeding densities, with three sets of fluorescent images analysed for each group (mean with s.d.).

Figure 7.

Impact of mechanical states on confluent cells (2.5 × 10⁴ cell seeding density for 2-day culture per well). (a) Representative immunofluorescent images and scatter plots of confluent cells subjected to static, stretched and dynamic mechanical conditions. (b) Comparison of nuclear to cytoplasmic fluorescence intensity ratios across different mechanical states. (c) Comparison of PCs for the three mechanical states, with analyses performed on seven sets of imaging data for each condition (mean with s.d.; *p < 0.05, **p < 0.005, ***p < 0.001).

Figure 8.

Influence of mechanical states on dense cells (10 × 10⁴ cells seeding density for 2-day culture per well). (a) Representative immunofluorescent images and scatter plots of dense cells in the static, stretched and dynamic mechanical states. (b) Comparison of the nuclear/cytoplasmic fluorescence intensity of different seeding densities. (c) Comparison of PCs for the three mechanical states, with analyses performed on seven sets of imaging data for each condition (mean with s.d.; *p < 0.05, ***p < 0.001, ****p < 0.0001).