The Journey to a Manufactured Flow Insert + Protocol for Use

Introduction

The modular μSiM is based on a 96-well plate format with a 100 μl open well. While these devices have proven extremely useful for various barrier models including the blood-brain barrier¹ and the tendon-vascular interface², there is a need to transform the open well into a fluidic channel. A closed channel device would enable us to introduce perfusion and fluidic shear stress, and presents additional advantages for static culture. For leukocyte trafficking studies, adding cells to an open well results in a period of settling that manifests as de-focused rings. The flow channel would eliminate this challenge by enabling the introduction of a rolling cell suspension or even circulating immune cells under flow with a pumping system. In addition, media in the open well often forms a meniscus that creates phase imaging shadows unless a flat media interface is maintained.

Now, with the innovation of Mansouri et al.³ in developing a flow channel geometry for the modular μSiM, we sought to develop a manufactured flow insert component. The ultimate goal was to create a component equally as accessible and modular as Component 1 and 2 of the modular μSiM. After about a year of prototyping and validation testing in collaboration with ALine, we have arrived at a mass-produced, peel-and-stick flow insert. For more detailed background information on the manufactured flow insert and the initial design process please see this post.

Initial Design Considerations

I. Design Iterations

As detailed in Figure 1 below, our initial designs for a manufactured flow insert included complex geometries and a larger overall device thickness, which we deemed too complex for high-throughput manufacturing methods.

ALine follows specified design constraints to reduce risk throughout the manufacturing process. They prefer 1 mm of edge-to-edge spacing between features. Given that we were limited by the membrane dimensions, we established the channel length at 4.4 mm, thus pushing the edge-to-edge spacing recommendation by 0.5 mm. As shown in Figure 2, there is 0.5 mm of bonding space between the membrane and the right/left edges of the flow insert.

II. Material Considerations

Similar to the design of the original flow module, we wanted to produce the manufactured flow insert with PDMS due to its gas permeability, biocompatibility, and ease of fluidic integration. ALine traditionally works with thin layers of materials since they are easier to source and incorporate in a laminated device format. The maximum layer thickness for ALine’s silicone material is 1.5 mm. Therefore, we would need to stack multiple silicone layers to achieve the 3 mm height of the flow insert. This may present leakage risks in addition to the difficulties related to laser cutting thick layers of silicone. 

Due to these initial concerns related to the scalability PDMS parts, I tested an acrylic insert fabricated in the lab (Figure 3). As expected, the acrylic did not form a tight seal between the flow needles. Moreover, the small footprint poses a barrier to including barb microfluidic connectors. Thus the most viable route was to create a mold for the 3 mm thick PDMS portion and laminate the PSA on the bottom of the PDMS component.

Final Design for Mass Production

The final flow insert design for manufacturing includes a 3 mm thick PDMS part with flow ports connected to a 200 μm tall flow channel. This flow channel has a pressure-sensitive adhesive (PSA) on the bottom to enable peel-and-stick capabilities.

To improve the efficiency of the manufacturing process, the components are molded in a 5×5 array and laminated with the PSA/PET/PSA as an array. The schematic of the 5×5 array and key dimensions are indicated in Figure 5 below.

V1-V4 Validation Tests

V1

A previous post detailed the validation tests for the V1 manufactured flow inserts. In summary, this first round of prototypes looked promising in regards to a leak-proof seal and alignment with component 1. However, the major concerns included cell death after 24 hours of initial static culture, cloudy PDMS, as well as surface roughness that affected image quality.

In addition, we observed poor cell growth after 24 hours of static culture. I investigated the potential source of toxicity by placing the inserts in a 24-well plate and culturing HUVECs for 24 hours. As shown in Figure 7, the cells appeared viable in both the ALine manufactured inserts as well as the in-house fabricated inserts. I reasoned that this may be due to the sterilization protocol that I used with the initial devices that resulted in cell death. I had rinsed the inserts with ethanol and then put them under UV for 15 minutes before proceeding with seeding. For this well plate experiment, I rinsed with ethanol but then rinsed 3 times with PBS before seeding. In the previous experiment, there may have been trace amounts of ethanol present in the inserts leading to a loss of viability. Thus the PBS washes are necessary to avoid these problems in the future.

To address the imaging quality issues, ALine explored different mold materials and performed finer passes while machining the mold. This decreased the appearance of machining marks on the PDMS to achieve a smooth top channel surface. As shown in Figure 8 below, these changes resulted in a material clarity similar to the in-house parts.

V2

While the imaging clarity was improved and machining marks reduced, the V2 flow inserts ports appeared larger than the specified 0.8 mm diameter. With the new molding procedure, the ALine engineers decided to re-punch the ports after removing the PDMS from the mold to ensure that a complete via was formed. However, this caused the port diameters to be larger than 0.8 mm. 

Despite this, I performed validation testing with 18 gauge flow needles and confirmed endothelial cell shear conditioning.

Scale Up – 10×10 Attempt

Since the port issue was a straightforward fix, we proceeded with efforts to scale up the production process. Up until this point, ALine was producing the inserts in 3×3 arrays. To drive costs down to a reasonable unit price, we needed to fabricate a larger array. ALine created a similar mold as before but as a 10×10. The resulting components are shown in Figure 11. While the flow ports were the correct diameter and peel-and-stick functionality worked well, the overall scrap rate was too high. Many of the parts that passed QC still had excess material around the edges of the part and ports, making it difficult to reliably pipette through the channel. It was clear that this method would not yield reproducible parts on a large scale.

V3

Fortunately, ALine identified a company that specializes in PDMS production to outsource the molding step. ALine then proceeded with the PSA lamination process after receiving the PDMS components. This partnership proved successful, as we received a clean 10×10 array with a 95% yield.

I confirmed a leak-proof seal after 24 hours of flow (500 μl/min) on the benchtop, biocompatibility, phase imaging clarity, and endothelial junctional protein expression after 72 hours of either static or fluidic culture at 1.5 dynes/cm². These results are summarized in Figure 13.

While these early results looked very promising, we soon uncovered leakage issues with many of the parts during flow in the incubator. In an effort the maintain an alignment tolerance between the edge of the ports and flow channel, ALine had decided to lengthen the channel. This decreased the minimum bonding distance between the membrane and the insert from 0.5 mm to 0.15 mm. Due to thermal expansion of PDMS at 37 ºC, this created a gap for media to leak out of the flow channel. It was difficult to catch this change before ALine had proceeded with production as they had not specified the channel length in the spec sheet.

V4

After discussing this issue with ALine, we determined that maintaining our original channel length (4.4 mm) without leaving tolerance between the channel and ports was worth the risk of potential channel/port overlap. We reasoned that a small area of exposed PSA would not harm the cells because there are currently areas of exposed PSA in the open well configuration. After performing COMSOL simulations to evaluate the flow profile in 200 μm offset channels, we decided to proceed with production.

ALine altered the QC process by including a misalignment measurement step. Any part exceeding 200 μm misalignment would fail QC. Despite this, the success yield was maintained at 95% with the first shipment of 100 parts.

With this adjustment back to our original dimensions, the leakage issue was completely eliminated. ALine is now producing the parts in a 5×5 array while maintaining a cost per part similar to that of the 10×10 array.

Side Note

Since the flow channel volume is so small (~2 ul), it was standard practice to add a droplet of media on top of the insert during static culture to prevent the channel from drying out. I recently tested devices without including a droplet of media (but still including a moistened kim wipe in the petri dish) and found no significant effect on cell moropholgy after 24 hours of static culture.

Conclusions + Lessons Learned

We initiated the first conversation with ALine about the manufactured flow insert in January 2023. Since then we have met with ALine engineers to work through design changes and troubleshoot issues that arose during validation testing. The engineers provided useful suggestions for adapting our design for mass production. Since prototyping costs add up quickly, we often tested these initial ideas in the lab to determine feasibility before proceeding with testing at ALine. By doing so we decreased risk and discovered solutions more efficiently.  

The primary ALine engineer working on the project changed between the V2 and V3 iterations. Due to this, there was a lack in communication leading to the lengthened channel and ultimate leakage issues. It is imperative to request and review spec sheets before each build and check that all dimensions match the agreed upon design.

The peel-and-stick capability has proven extremely useful in the quick adoption of the fluidic configuration by others within and outside the lab. Collaborators have begun to use the manufactured inserts in applications such as T-cell migration and iPSC-EC culture. Looking forward to future developments, it will now be possible to connect multiple fluidic μSiM devices to increase throughput and study crosstalk between different organ chip systems, such as between the brain and lung.

Table Summary of Manufacturing Process

	V1	V2	10×10 Attempt	V3	V4
Notes			ALine made 10×10 PDMS mold	Collab with PDMS manufacturer ALine lengthened channel for manufacturing tolerances	Collab with PDMS manufacturer Revert to original channel length
Array Layout	3×3	3×3	10×10	10×10	5×5
Successes	1.       Leak proof	1. Transparent PDMS 2. No machining marks 3. Biocompatible		1. Transparent PDMS 2. Great optical clarity 3. Biocompatible 4. Correct port diameter	1. Transparent PDMS 2. Great optical clarity 3. Biocompatible 4. Correct port and channel dimensions 5. Fully leak proof
Problems	1. Cloudy PDMS 2. Surface roughness/machining marks due to mold material 3. Biocompatibility concerns in devices	1. Some surface roughness but cells are visible 2. Ports are blown out	1. ALine PDMS method not transferable to 10×10 – most parts unusable due to excess material	1. Leakage with flow in incubator (seals well in static conditions)

 

Protocol for Use

1. Avoid handling inserts outside of a sterile cell culture hood.
2. Spray down flow inserts with ethanol and place in hood.
3. Using a sharp razor blade or scissors, cut out the flow inserts from the array by removing the support areas.
4. Place inserts with channel side facing up in a sterile petri dish and UV in hood for 15 minutes.
5. After UV sterilization, carefully remove protective liner to expose the adhesive.
6. Insert tweezers in the two ports and align the insert in the open well of the device. Alternatively, hold the insert with tweezers on opposing sides as indicated in Figure 18 step 5 and align in the open well.
7. Apply pressure with the flat side of the tweezers around the perimeter of the flow insert, making sure to avoid the membrane window area.
8. Pipette sterile PBS (~50 ul) through the channel to rinse.
9. Pipette off any excess PBS.
10. The device is now fully-sealed and ready for cell seeding and flow experiments.

References

1. McCloskey MC, Kasap P, Ahmad SD, Su S-H, Chen K, Mansouri M, Ramesh N, Nishihara H, Belyaev Y, Abhyankar VV, Begolo S, Singer BH, Webb KF, Kurabayashi K, Flax J, Waugh RE, Engelhardt B, McGrath JL. The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro. Advanced Healthcare Materials. 2022;11(18):2200804. doi: https://doi.org/10.1002/adhm.202200804.
2. Awad H, Ajalik RE, Alenchery RG, Linares I, Wright T, Miller B, McGrath J. Human tendon-on-a-chip for modeling vascular inflammatory fibrosis. Research Square. 2023;Version 1. doi: 10.21203/rs.3.rs-3722255/v1.
3. Mansouri M, Ahmed A, Ahmad SD, McCloskey MC, Joshi IM, Gaborski TR, Waugh RE, McGrath JL, Day SW, Abhyankar VV. The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow. Advanced Healthcare Materials. 2022;11(21):2200802. doi: 10.1002/adhm.202200802.