PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models

Abstract

The PDMS-based microfluidic organ-on-chip platform represents an exciting paradigm that has enjoyed a rapid rise in popularity and adoption. A particularly promising element of this platform is its amenability to rapid manufacturing strategies, which can enable quick adaptations through iterative prototyping. These strategies, however, come with challenges; fluid flow, for example, a core principle of organs-on-chip and the physiology they aim to model, necessitates robust, leak-free channels for potentially long (multi-week) culture durations. In this report, we describe microfluidic chip fabrication methods and strategies that are aimed at overcoming these difficulties; we employ a subset of these strategies to a blood-brain-barrier-on-chip, with others applied to a small-airway-on-chip. Design approaches are detailed with considerations presented for readers. Results pertaining to fabrication parameters we aimed to improve (e.g., the thickness uniformity of molded PDMS), as well as illustrative results pertaining to the establishment of cell cultures using these methods will also be presented.

Keywords: cell culture; microfluidic; organ-on-chip; rapid prototyping.

Figure 1

Schematic overviews of: (A) the BBB-on-chip; and (B) the airway-on-chip. Key features of the BBB-on-chip include making molds of higher gauge (smaller outer diameter) needles before adding the I/O needles and sealing the chip using a PDMS moat. Both chips incorporate a porous membrane to separate the channels, with the BBB-on-chip seeding endothelial cells basally and the airway-on-chip seeding epithelial cells apically. Portions of this figure were created using https://biorender.com (accessed on 20 August 2022).

Figure 2

Slowly and carefully placing the transparency film over the uncured, PDMS-filled mold minimizes formation of bubbles: (A–D) depict the different stages of slowly lowering the transparency onto the convex PDMS surface, starting from the top left corner. A red dotted outline is used to depict the area of the transparency film that is in contact with the PDMS.

Figure 3

PDMS Moat Fabrication Steps: (A) Assembled chips are placed into a flat, smooth bottom dish. (B) PDMS is poured into the dish ensuring that the chips are completely surrounded. (C) An acrylic sheet is placed on top of the chips to ensure the area of interest of the chip (central channel) remains clear of PDMS. (D) A weight is used to weigh the chips down within the liquid PDMS. (E) Once cured, PDMS block is removed from dish. The chips are now embedded in PDMS block and able to be immediately connected to fluidics system.

Figure 4

Illustration of chip design and highlighting of critical features. (A) exploded view of chip components (created using SolidWorks): i: 1/4-inch-thick acrylic top clamp, ii: 8–32 brass flanged thumb nut, iii: 8–32 oversized washer, iv: PDMS chip (top half), v: 8–32 threaded rod (1.5” length), vi: PDMS chip (bottom half), vii: 1/4-inch thick acrylic bottom clamp, viii: PET membrane. (B) illustration of corner alignment features to assist manual mating of chip halves during bonding. (C) Illustration of liquid-PDMS reinforcement of fluidic port seal via pipetting of uncured PDMS into top-clamp cutouts. (D) Highlighting of punch guides to assist in the accurate positioning and clear formation of manually punched through-holes for fluidic connection.

Figure 5

Comparing the performance of rapidly prototyped master molds. (A,B) comparison images captured of PDMS devices superimposed upon fine-point text to highlight optical clarity: (A) device cast from Form2-SLA-printed mold; (B) device cast from MiiCraft-DLP-printed mold. The device cast from the DLP-printed mold shows improved optical clarity. (C,D) boxplot summarizing RMS surface roughness of SLA and DLP molds alongside corresponding cast PDMS. Individual data points are represented as black dots superimposed on a boxplot, where the box height represents the interquartile range, the whiskers the total range, and the central line the median value. While statistical significance was not achieved, a trend towards lower roughness associated with DLP-printed molds and corresponding cast PDMS was exhibited—manifesting through the improved optical clarity illustrated over Subfigures (A,B). (E) representative topographic images obtained through AFM characterization of DLP and SLA 3D printed master molds (top) and cast PDMS (bottom); color scale corresponds z-axis height (surface profile), illustrating a more uniform surface obtained on the DLP-printed mold and cast PDMS. Scale bar represents 2.5 µm.

Figure 6

PDMS device fabrication with the transparency film method improves the flatness and uniformity of the top PDMS surface. Comparison of uniformity in PDMS thickness: (A) in absence of transparency film and; (B) when employing transparency film method, scale bar (black) represents 2 mm. Insets depict warping of the surface of the PDMS device fabricated without the transparency method, in regions surrounding the through-hole structures in the mold (visible as reflections and optical aberrations). In contrast, the surface of the device fabricated using transparencies is flat and does not impart these optical aberrations. (C) Comparing the impact of the transparency film on the measured thickness at several points on replicate PDMS devices and; (D) on the intra-device variability in PDMS thickness measurements taken at different regions on PDMS devices. The 15 raw thickness measurements for each set of devices is plotted in (C) and the standard deviation of the five measurements corresponding to each device is plotted in (D) (the asterisk denotes statistical significance at the 5% level). Individual data points are represented as black dots superimposed on a boxplot, where the box height represents the interquartile range, the whiskers the total range, and the central line the median value. (E) Illustration describing application of PDMS: microchannels molded into PDMS are sealed against a silicon wafer device to be subjected to flow via compression of a rigid, laser-cut, acrylic piece where non-uniformities in PDMS thickness may undermine the PDMS seal and flow path integrity.

Figure 7

(A) When employing the transparency film during casting, the improved PDMS thickness uniformity facilitates straightforward reversible PDMS bonding using a compression setup; (B) Upon compression of the device against a flat surface to enclose the channels, increased channel deformation is evident under comparable force when employing a device fabricated without a transparency film.

Figure 8

Investigating PDMS device sealing method. Sealing with PDMS moat prevents delamination during cell culture period. Comparison of degradation of sealing methods over 24 h: (A) Samples are submerged in 70% ethanol to accelerate the degradation seen in other aqueous liquids such as cell culture media. When initially placed in ethanol there are no visual signs of degradation in chips sealed with epoxy and chips sealed with a PDMS moat. (B) After 24 h, the ethanol in which the chip seal with epoxy is submerged exhibits a discoloration indicating a degradation of the epoxy. The chip sealed with PDMS moat shows no signs of discoloration or breakdown. Visual inspection of chips sealed with (C) epoxy and (D) PDMS moat (D) after 24hrs of submersion in ethanol. Over 24 h of submersion, the Epoxy-PDMS interface begins to delaminate and separation between the two layers can be seen macroscopically. This separation results in limited support for preventing leakage in chips when higher pressures are experienced within the device.

Figure 9

Illustration of blood–brain barrier chip cell culturing protocol and representative results: (A) Configuration with five chips in a cell culture incubator with medium reservoirs and pumping set-up; (B) Schematic illustrating culture environment of single chip, with apical channel static and syringe pump flow through basal channel of the BBB chip, created with BioRender.com; (C) Stitched microscopy image comprised of images capturing three regions of interest (ROIs) of the membrane post extraction and immunofluorescence staining (scale bar: 200 um), the green stain is phalloidin, and the blue stain is Hoechst 33342.

Figure 10

Illustration of airway-on-chip cell culturing protocol and representative results: (A) configuration of six chips in a cell-culture incubator with medium reservoirs and pumping set-up; (B) Schematic illustrating culture environment of single chip, with apical channel static and peristaltic-driven flow through basal channel (absent: luer-luer connections between microfluidic tubing and Ismatec 1.02 mm ID 2-stop peristaltic tubing), created with BioRender.com; (C) stitched microscopy image comprised of 10× magnification images capturing the entirety of a representative membrane post extraction and immunofluorescence staining (scale bar: 1.25 mm); green represents Alexa-fluor-488 Phalloidin, staining F-actin, while blue represents DAPI, present in the mounting medium. (D) three representative ROIs captured at 60× magnification along the length of the membrane (scale bar: 50 µm).