Supplementary guide.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.</div

PDMS soft lithography and biofunctionalisation.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.</div

Comparison of costs for 3D vat polymerisation printing and photolithography with all the required equipment and materials but excluding personnel training costs.

Comparison of costs for 3D vat polymerisation printing and photolithography with all the required equipment and materials but excluding personnel training costs.</p

Microstimulation hardware.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.</div

PDMS substrates cast from 3D-printed devices permit regular-sized embryoid body.

(A) Schematic of design, manufacturing and seeding of IPSCs on microwells. (B) Representative optical profile of 3D-printed microwell device with well sizes of 390 μm length × 350 μm width × 150 μm height. (C) Representative SiR-tubulin live cell dye images of IPSCs before seeding in microwell mould cast. (D) Representative SiR-tubulin live cell dye images of IPSCs seeded on PDMS cast from 3D-printed microwell device compared to flat PDMS substrate after seeding (left) and 2 days culture (right). (E) Representative SiR-tubulin image of fused embryoid bodies on microwell PDMS mould prior to detachment after 4 days in culture (top) and embryoid bodies following washing off the PDMS microwell substrate (bottom left). Quantification of embryoid body diameter detached from microwell PDMS mould demonstrates homogenous size of embryoid bodies (bottom right). Data points can be found in the file 6E-Data in S1 Data.</p

The surface of 3D prints is rough.

(A) Representative optical profiles from PDMS casts demoulded from a single airbrushed 3D-printed mould and a single microfabricated mould. (B) Representation of 5 ROI selection for quantification of surface roughness on PDMS casts. (C) Quantitation of surface roughness of PDMS casts from the same device over time (25 simultaneous casts) between an airbrushed 3D-printed mould and a microfabricated mould. (DOCX)</p

Entirely SOL3D-manufactured hydrogel moulding and culturing chamber system with customised imaging chamber.

(A) Schematic overview of PDMS mould generation for hydrogel moulding and MN seeding. Cells are manually pipetted into a preformed hydrogel moulded in the SOL3D-fabricated PDMS mould. (B) Schematic overview of the PDMS chamber manufacturing process using SOL3D. The chamber has a complex design with a funnel shape within the structure and multiple openings. We used a two-part design for ease of demoulding. (C) Schematic overview of the combined hydrogel with MNs placed in the mould (A) and a diffusion chamber around the construct (B), arrows indicate the flow of medium. (D) Optional media compartments highlighted using food colouring (blue, red) and PBS (top). (E) Representative images of the SOL3D chamber system (Brightfield) with MNs (SiR-Tubulin) in culture in a 6-well plate. (F) Schematic overview of the mounting process of the complex hydrogel samples for fluorescent imaging. A SOL3D manufactured holder allows the transfer of whole constructs without disturbance and mounting close to the coverslip for imaging. (G) Schematic overview of the hydrogel within the holder. The PDMS mould sits in the bottom of the well with the hydrogel and cells on top. The sample is surrounded by mounting medium and covered with a thin glass coverslip for imaging. Due to the custom holder system, the distance between sample and glass is minimised allowing for fluorescent imaging. (H) Representative fluorescent images of MNs cultured for 7 days in the SOL3D hydrogel chamber system and mounted in the SOL3D chamber. MNs (β-III-Tubulin) seeded in the hydrogel extend many axonal processes.</p

Plating devices enable spatiotemporal control of cell plating with different geometries for construction of complex neural circuits.

(A) Schematic overview of alternate seeding of RFP and non-RFP+ motor neurons in the same device and the following live cell staining. (B) Representative line profile of stained across the well showing segregation of individual populations to their designated wells—RFP+ only in wells 1 and 3, but SiR-tubulin+ (here in green) in all wells (top). Representative fluorescence images of stained RFP+/− motor neurons (bottom). (C) Schematic overview of the multi-device protocol for constructing a neural circuit using 2 stencil devices and 3 different cell types (MNs, cortical, and astrocytes) with seeding performed at different time points. (D) Composite of the complete circuit after 19 days of culture. GFP transfected motor neurons = green, Glial Fibrillary Acidic Protein (GFAP) identifies astrocytes, β-III–Tubulin identifies cortical neurons and the tubulin in GFP+ motor neurons. Blue device well shapes overlaid for illustrative purposes. (E) Schematic overview of protocol for manipulating aggregate geometry in combination with existing microgroove. (F) Representative images of SiR-tubulin live-cell stained motor neuron aggregoids at day 2. (G) Boxplot of aggregoid aspect ratio fold change by shape between CAD (blue line) and day 2 of culture from β-III-Tubulin channel (top). (H) Boxplot of aggregoid area fold change by shape between CAD (blue line) and day 2 of culture from β-III Tubulin channel. Data points for G and H can be found in the files 3G-Data and 3H-Data in S1 Data.</p

Fluidic seal for cell plating devices.

(A) Schematic (top) and representative image (bottom) of clamping approach to ensure fluid seal when devices are placed on PDMS microgroove substrates. (B) Comparison of liquid seal integrity of clamping strategy (top) compared to open curing (bottom) on PDMS microgroove substrate with different well sizes and shapes using dyed liquid. Successful sealing of devices cast with a glass cover (Green zoom). Dye spreads throughout device and grooves using open cured (Red zoom). Arrows highlight liquid spreading. (DOCX)</p

Enamel paint coating facilitates rapid PDMS curing on 3D-printed moulds.

(A) Schematic overview of the investigation strategy to establish a protocol for PDMS curing on 3D-printed moulds. (B) Representative images of PDMS casts removed from printed devices classified as cured or uncured. Arrows highlight liquid PDMS. (C) Representative images of a CAD of 3D-printed moulds, the completed print, and surface optical profiles of layer thickness and feature dimensions. (D) Heatmap of PDMS curing time by resin type for different washing conditions. (E) Heatmap of PDMS curing time by resin type for different PDMS curing temperatures (right y-axis) and different SLA print coatings. (F) Representative SEM images of uncoated (right) and enamel paint coated prints (left), arrows highlight the paint layer. (G) Schematic overview of optimised fabrication, post processing, and PDMS casting protocols with (yellow) and without (purple) enamel coating.</p