Julio Aleman, a Bioengineering PhD student at the US-based Swanson School of Engineering, University of Pittsburgh, is conducting research into microphysiological systems. Like many researchers in his field, Aleman required microwell arrays that had individual wells of a specific size, shape and density. The microwell arrays that he needed would serve as master moulds for replicate moulds used in the micro-injection moulding of hydrogels.
Traditionally, photolithography is most often used to produce microwell master moulds. The material of choice is usually polydimethylsiloxane (PDMS), a biocompatible silicone. First, spin coating is used to deposit a negative photoresist on a wafer to establish mould thickness and microwell depth. Then, photopatterning is achieved via light exposure through specialised, high-resolution masks and finalised by chemical development. Lastly, replicas of the master mould are produced by casting pre-polymers over the negative template and polymerising them by thermal curing.
Injection moulding is also used to produce microwell master moulds, but the tooling is expensive and time-consuming to produce.
Typically, the photolithography and injection moulding processes require specialised cleanroom facilities and equipment for the production of master moulds and masks.
An alternative to photolithography and injection mouldingBoston Micro Fabrication (BMF) is a US-based provider of projection microstereolithography (PμSL) 3D printers and printing services. PμSL is a 3D printing process that uses a single flash of ultraviolet (UV) light to rapidly photopolymerise layer upon layer of resin at microscale resolution. It is an alternative to the traditional mould and pattern creation methods of photolithography, injection moulding, etching and deposition. Most of these methods limit a researcher’s ability to create complex 3D channels and, as highlighted above, some are expensive and time-consuming.
PμSL can be used to rapidly produce small prototypes and end-use parts that have fine features and tight tolerances. Furthermore, it is able to achieve ultra-high resolutions of ~2 to 50 µm and tolerances of +/-5 to ~25 µm mould-free and mask-free. Aleman said that he considered other 3D printing processes, but found them to be comparatively slow and expensive, and unable to compete in terms of the critically important levels of accuracy, precision and resolution required.
Project requirements
Aleman’s project required master moulds for replicate PDMS moulds that, in turn, could be used to produce biomaterial-based microwells. Normally, such moulds must be produced in a cleanroom and can be challenging to characterise. Furthermore, researchers cannot perform profilometer measurements until the moulds are baked, and a mould that has incorrect dimensions inevitably means lost time and money. The project also demanded high-volume production and fast turnaround times.
Multiple PDMS replicate moulds cured at 80°C were needed. The master mould would be securely fixed in a substrate and repeated processes on this mould could have little to no impact on its dimensions. The replica PDMS moulds needed to allow the bottoms of the hydrogel microwells to have constant and defined amounts of biomaterial once injected and polymerised onto a glass slide.
Alleman asked BMF to produce master moulds that were square with a defined hole pattern. There were two different master moulds. The first mould was for a 64-microwell array. It had an overall area of 20 x 20 mm and a thickness of 1.65 mm. The diameter for each hole and the distance between holes was 600 μm, or 0.6 mm, and the base of each of the microwells had a height of 50 μm from the surface. The second mould was for a 484-microwell array. The overall area was the same as for the first mould, but the thickness was significantly less at 1 mm. There were also more holes, which were smaller, measuring just 150 μm, or 0.15 mm, in diameter, with a similar base height of 50 μm.
Expert knowledge
BMF reviewed Aleman’s master mould designs. The company suggested increasing the thickness of the second mould to greater than 1 mm, since a mould that is too thin may experience part distortion or internal stresses during the printing process. So, Aleman revised the thickness of the second mould design and resent it. BMF then created sample moulds using GR, a high-performance, engineering resin that is black, has good impact strength and heat-resistance (being able to withstand temperatures of up to 102°C), and supports autoclave sterilisation.
The COVID-19 pandemic limited Aleman’s access to university facilities, forcing him to evaluate the moulds using only microscopy techniques. Any part produced in a dark resin is likely to be challenging to inspect using microscopy techniques, so Aleman asked BMF to produce the moulds again in a resin that allowed their features to be more clearly seen. BMF decided to use HLT, a resin that is yellow and therefore offers greater transparency, but still affords good impact strength and heat-resistance, and supports autoclave sterilisation.
Speed, accuracy and other benefits of PμSL
PμSL does not involve multiple fabrication steps like photolithography or require tooling like injection moulding, therefore the master moulds could be produced much more quickly. Aleman was impressed by how BMF only took a fortnight to deliver a master mould for each microwell array. This was a significant improvement on the months previously spent awaiting the arrival of a single injection-moulded mould. Moreover, he was able give a mould to every member of the research team.
The master moulds exhibited impressive dimensional stability. Aleman hard-baked some of the moulds overnight in an oven, then attached them to a surface using double-sided electrical tape. He found that they had reduced in size by only 10 to 20 μm, and thought that this may have been caused by pressing them against the surface or the tape while they were still malleable from baking. Aleman also reported that the moulds had not bent or deformed after approximately 10 cycles of PDMS moulding.
The replicate PDMS moulds were salinated using vapour phase deposition (VPD) to prevent sticking. This treatment, which is also used with the moulds in photolithography, permitted the easy detachment of the PDMS moulds from the master moulds. Aleman stated that the master mould with larger features required over one hour of exposure, while the master mould with smaller features required five hours or overnight exposure.
PμSL for proof-of-concept projects
Aleman’s project clearly demonstrated that PμSL can be used to speed microwell fabrication. BMF offers a comprehensive PμSL 3D printing service for master moulds and various other parts, possessing the necessary expertise to advise on key factors such as design and material selection.
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A hydrogel mould of a 64-microwell array.
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A hydrogel mould of a 484-microwell array.
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A 3D printed mould for a 64-microwell array.
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A 3D printed mould for a 484-microwell array.
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A polydimethylsiloxane (PDMS) replica of a 64-microwell array.
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A PDMS replica of a 484-microwell array.
Swanson School of Engineering, University of Pittsburgh
BMF