Periodic Reporting for period 2 - HoliFAB (Holistic digital-to-physical prototyping and production pilot for microfluidic MEMS) Reporting period: 2019-05-01 to 2021-04-30 Summary of the context and overall objectives of the project Numerous key study systems have to deal with very small sizes of dynamic elements, particularly to mimic living systems, with actors on a scale of micro- to pico-meters: this is the field of microfluidic. The HoliFAB project now applies to this one a holistic approach combining manufacturing technologies for prototyping and large series fabrication of the chips and design of instruments for commercialization. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far HoliFAB consortium releases to market 3 complementary industrial lines based on (1) 3D printing and bioprinting for small to medium scale production of devices and microphysiological systems, (2) injection moulding for large series chip production, and (3) instrument engineering for system design.3D printing pilot line (PL1) CNRS-LAAS hosts in its FabLab (“MultiFab”) platform the PL1 that is devoted to the rapid 3D printing and bioprinting. Several equipment have been developed and optimised based on commercial printers from the Kloé Dilase family for high resolution (up to 3 µm), multimaterial and bioprinting or DWS Systems for low resolution (typically 30 µm) and high speed printing. A custom CNRS-LAAS prototype called LAMP (Light Assisted Microfluidic Printing) Technology has also been implemented for bioprinting (with resolution down to 20µm). These machines use polymer and composite materials which have been optimised based on the experience of MicroResist . The partners established a material library that includes inorganic-organic hybrid polymers and regular organic photopolymers, or are newly developed materials like hydrogel materials for cell culture. In addition, with DCU, we now have responsive hydrogels, undergoing photoactuation, i.e. irradiation induced swelling and contraction for active element integration inside microfluidic chips. Finally a software suite developed by Sculpteo was developed to control and manage all the steps required for device fabrication in PL1. The software suite contains features ranging from material selection, 3D models handling, hosting, optimization, preparation for printing, to production monitoring and management. Injection moulding pilot line (PL2)HoliFAB enabled the development of standalone technologies for microfluidic device production by injection moulding. MYPA worked on the design and fabrication of steel injection moulds for the replication of hard plastic parts that contain diverse microfluidic figures. The feature sizes range is wide, from millimetres to micrometres, and for those devices with high aspect ratio, that are based on nickel shims manufactured by microLIQUID. To precisely replicate these moulds by injection molding, dynamic temperature control is implemented to help in meeting this micrometric challenge. The integration of reagents in microfluidic devices is also a key point for chip functionalization An equipment assists in the dispensing of reagents into these injection moulded cartridges, and led to a new OEM instrument dispensing up to 8 different reagents. EVG has developed a fully automated production bonding equipment for injection-moulded microfluidic chips. Addressing the need for room-temperature bonding due to reagent deposition prior to bonding, they have focused on adhesive bonding techniques. They manufactured bonding equipment capable of utilizing the adhesive layer transfer technique. In addition, the equipment can process adhesive tape bonding and is prepared for a special solvent assisted thermal bonding technique. Finally, a custom-made quality control equipment was built by microLIQUID to optically inspect key parts of microfluidic devices at different processing stages of their manufacturing. Its precision stage along with the swift interface and processing, make this equipment a suitable instrument for the detailed measurement of microfluidic devices at the different stages of processing.Instrument integration pilot line (PL3)The PL3 is an adaptation of an existing pilot line proposed by Fluigent that aims to convert microfluidic systems into complete instruments. Microfluidics enables an enhancement of biochemical applications with miniaturization, reagent and sample volume/cost reduction, automation, application portability/accessibility, etc. However, the microfluidic chips rely on external instrumentation and when evolving towards an integrated instrument, those elements are often assembled together rather than designed together. Reducing the time-to-market, by reducing R&D design efforts and production time are the main goals: In that context a new software based on CAD solutions has been developed in order to first design and optimize the system before focusing on the product design. The PL3 is evaluated with indicators of improvement which compare the performances to the development process and final product levels of the pilot line between the end and the beginning of the project. For example, following Fluigent achievements, the R&D engineer time (the highest cost of an instrument development) has been drastically reduced by a factor 2 thanks to the support of the microfluidic system dedicated software and the Solidworks™ plugin that Fluigent developed that allows respectively to design and optimize a microfluidic system in a few clicks and hours, and to transfer it automatically to the mechanical design process. Application demonstrators In HoliFAB, thanks to the work realised on the three PLs, two devices for cancer diagnosis and cancer cells characterization were developed. The first one, dedicated to circulating tumour DNA, used chips developed within PL2. It also involved an instrument developed in PL3, showing a reduction of the total volume footprint of the experiment by a factor 20. The second instrument faced difficulties regarding the availability of injection moulded chips, but this was mitigated by the development of an alternate hot embossing protocol. Both systems were validated with model and patient samples. In addition, 3 “organ on chip” applications were developed successfully with PL1 and PL2, and reached the specifications regarding resolution and duration of viable cell culture. An instrument for environment control was also developed within PL3, and is now the basis of a commercialized product. Finally, 2 microfluidic chips/cartridges for environment control of phosphates, nitrates and nitrites were developed with PL1: they reached validation by deployment in the field, and showed performances in terms of sampling frequency and stability significantly superior to state of -the- art sampling methods. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In short:The PL1 offers a resolution of 3D printing that is increased by a factor of at least 10, down to 3μm, with a throughput of 10 to 100x higher than the current high-resolution 3D printing machines. This one uses new soft, bio-, environment-friendly and/ or active materials developed in HoliFAB..The PL2 addresses large-scale markets requiring mass production at the lowest cost with a fully integrated pilot line, streamlining injection moulding of raw chips, reagents and components integration, sealing and quality control..The PL3 enables the use of chips produced in PL1 and PL2 in an operational environment by end-users with lab-on-chip applications in health (cancer diagnosis, organ-on-chip) and environment (water control).