PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models
<p>Schematic overviews of: (<b>A</b>) the BBB-on-chip; and (<b>B</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 <a href="https://biorender.com" target="_blank">https://biorender.com</a> (accessed on 20 August 2022).</p> "> Figure 2
<p>Slowly and carefully placing the transparency film over the uncured, PDMS-filled mold minimizes formation of bubbles: (<b>A</b>–<b>D</b>) 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.</p> "> Figure 3
<p>PDMS Moat Fabrication Steps: (<b>A</b>) Assembled chips are placed into a flat, smooth bottom dish. (<b>B</b>) PDMS is poured into the dish ensuring that the chips are completely surrounded. (<b>C</b>) 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. (<b>D</b>) A weight is used to weigh the chips down within the liquid PDMS. (<b>E</b>) 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.</p> "> Figure 4
<p>Illustration of chip design and highlighting of critical features. (<b>A</b>) 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>B</b>) illustration of corner alignment features to assist manual mating of chip halves during bonding. (<b>C</b>) Illustration of liquid-PDMS reinforcement of fluidic port seal via pipetting of uncured PDMS into top-clamp cutouts. (<b>D</b>) Highlighting of punch guides to assist in the accurate positioning and clear formation of manually punched through-holes for fluidic connection.</p> "> Figure 5
<p>Comparing the performance of rapidly prototyped master molds. (<b>A</b>,<b>B</b>) comparison images captured of PDMS devices superimposed upon fine-point text to highlight optical clarity: (<b>A</b>) device cast from Form2-SLA-printed mold; (<b>B</b>) device cast from MiiCraft-DLP-printed mold. The device cast from the DLP-printed mold shows improved optical clarity. (<b>C</b>,<b>D</b>) 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 (<b>A</b>,<b>B</b>). (<b>E</b>) representative topographic images obtained through AFM characterization of DLP and SLA 3D printed master molds (top) and cast PDMS (bottom); color scale corresponds <span class="html-italic">z</span>-axis height (surface profile), illustrating a more uniform surface obtained on the DLP-printed mold and cast PDMS. Scale bar represents 2.5 µm.</p> "> Figure 6
<p>PDMS device fabrication with the transparency film method improves the flatness and uniformity of the top PDMS surface. Comparison of uniformity in PDMS thickness: (<b>A</b>) in absence of transparency film and; (<b>B</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. (<b>C</b>) Comparing the impact of the transparency film on the measured thickness at several points on replicate PDMS devices and; (<b>D</b>) 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 (<b>C</b>) and the standard deviation of the five measurements corresponding to each device is plotted in (<b>D</b>) (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. (<b>E</b>) 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.</p> "> Figure 7
<p>(<b>A</b>) When employing the transparency film during casting, the improved PDMS thickness uniformity facilitates straightforward reversible PDMS bonding using a compression setup; (<b>B</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.</p> "> Figure 8
<p>Investigating PDMS device sealing method. Sealing with PDMS moat prevents delamination during cell culture period. Comparison of degradation of sealing methods over 24 h: (<b>A</b>) 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>B</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 (<b>C</b>) epoxy and (<b>D</b>) PDMS moat (<b>D</b>) 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.</p> "> Figure 9
<p>Illustration of blood–brain barrier chip cell culturing protocol and representative results: (<b>A</b>) Configuration with five chips in a cell culture incubator with medium reservoirs and pumping set-up; (<b>B</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; (<b>C</b>) 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.</p> "> Figure 10
<p>Illustration of airway-on-chip cell culturing protocol and representative results: (<b>A</b>) configuration of six chips in a cell-culture incubator with medium reservoirs and pumping set-up; (<b>B</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; (<b>C</b>) 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. (<b>D</b>) three representative ROIs captured at 60× magnification along the length of the membrane (scale bar: 50 µm).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fabrication and Design of the Master Molds
2.1.1. Fabrication of the Master Molds
Design of the BBB Chip
Design of the Airway-On-Chip
2.2. Fabrication and Assembly of the Microfluidic Chips
2.2.1. PDMS Preparation and Bonding
Oxygen Plasma Bonding—BBB Chip
Partial Curing—Airway-On-Chip
2.2.2. Fluidic Integration and Reinforcement
BBB Chip
Airway-On-Chip
2.3. Microfluidic Cell Culture
2.3.1. BBB Chip
2.3.2. Airway-On-Chip
2.4. Cell Culture Analysis
2.4.1. BBB Chip
2.4.2. Airway-On-Chip
2.5. Imaging
2.6. PDMS Thickness Uniformity Characterisation
2.7. PDMS and Mold Surface Roughness Characterisation
3. Results
3.1. Surface Roughness of 3D Printed Molds
3.2. PDMS Thickness
3.3. PDMS Encasement for Leakage Prevention: Comparison with Traditional Epoxy
3.4. Success Rate of BBB Chips
3.5. Cell Proliferation and Morphology
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Design Aspect | Airway-On-Chip | BBB Chip | Comments |
---|---|---|---|
PDMS Bonding | Partial cure | Oxygen plasma | Oxygen plasma treatment is the most commonly used option and is well characterized but requires an oxygen plasma cleaner [35]. Partial curing does not require additional hardware and has been shown in some reports to provide higher bond strength [22]. It, however, requires tight control over temperature for bond-strength reproducibility and may pose difficulties when manipulating more flexible and tacky partially cured PDMS layers. |
I/O location | Top-loaded | Side-loaded | Side loaded fluidic ports are more amenable to dual-sided imaging |
I/O port formation | Biopsy punch (0.5 mm OD) | Needle mold (0.5 mm OD) | Side loaded fluidics require sacrificial molding of ports, while top loading enables punching of I/O ports. |
Fluidic seal reinforcement | PDMS seal + compressive clamp | PDMS encasement (“moat”) | See Section 2.2.2 |
Description | Common and Suspected Failure Modes | ||
---|---|---|---|
Sample size of chips between checkpoints 1–2 | 17 | ||
Checkpoint 1 | Successful PDMS-PDMS bonding based on complete transparency of PDMS pieces and manual leak test | 100% | N/A |
Checkpoint 2 | Viable chips at the end of the experiment | 88% | Tubing coming undone from needle on syringe that is dispensing media. |
Sample size of chips between checkpoints 3–4 | 8 | ||
Checkpoint 3 | Chips with cell attachment | 88% | Cell detachment due to uneven ECM coating, air bubbles in the channel, or insufficient media supply. |
Checkpoint 4 | Chips with a confluent monolayer | 57% | Washing and fixing steps and disassembly of chips caused cells to lift before mounting membranes onto glass slides. Cell detachment due to uneven ECM coating. |
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Cameron, T.C.; Randhawa, A.; Grist, S.M.; Bennet, T.; Hua, J.; Alde, L.G.; Caffrey, T.M.; Wellington, C.L.; Cheung, K.C. PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models. Micromachines 2022, 13, 1573. https://doi.org/10.3390/mi13101573
Cameron TC, Randhawa A, Grist SM, Bennet T, Hua J, Alde LG, Caffrey TM, Wellington CL, Cheung KC. PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models. Micromachines. 2022; 13(10):1573. https://doi.org/10.3390/mi13101573
Chicago/Turabian StyleCameron, Tiffany C., Avineet Randhawa, Samantha M. Grist, Tanya Bennet, Jessica Hua, Luis G. Alde, Tara M. Caffrey, Cheryl L. Wellington, and Karen C. Cheung. 2022. "PDMS Organ-On-Chip Design and Fabrication: Strategies for Improving Fluidic Integration and Chip Robustness of Rapidly Prototyped Microfluidic In Vitro Models" Micromachines 13, no. 10: 1573. https://doi.org/10.3390/mi13101573