Fig. 1. Schematic of a DLP 3D printer, highlighting the design and mechanical factors as well photopolymerization parameters which influence print resolution. Digital light processing 3D printers project UV or violet light through optically clear sheets (usually Teflon) into a vat of photopolymerizable resin (pink). In locations where the light is projected, the resin crosslinks to form a solid structure. Exposure and crosslinking are performed layer by layer on the base plate, which lifts up as each concurrent layer is formed. Production of a clean print is dependent on instrumental, environmental, chemical, and design elements that impact either the print surface (base plate), mechanics (print orientation, design specifics), or chemical reaction (resin composition, light source, exposure settings, temperature, or humidity).
Here we present an applied, data-supported guide to the key factors that should be considered for design and fabrication of SL 3D printed biomicrofluidic devices, written especially for groups that are new to this growing field. Types of instrumentation and resin formulations have been reviewed and characterized in depth recently, and will not be presented in detail here.8,16,42–44 This tutorial follows the order of a typical workflow by first considering resin selection and then demonstrating how to increase print resolution by using simple changes in feature design and printer settings. Following printing, cytotoxicity of materials is addressed, particularly the extent and effectiveness of post-treatment strategies for applications involving contact with primary cells. Finally, preparation and considerations for use with fluorescent microscopy is outlined with data displaying autofluorescence and optical clarity of materials. We include the results of systematic, head-to-head comparisons of printing and post-treatment conditions designed to optimize the integrity of a printed piece, the resolution of interior channels, and the biocompatibility of the part. It is our hope that this work will streamline the adoption of 3D printed devices by more specialized biomedical research fields, bioanalytical laboratories, and others new to 3D printing.
2. Materials and Methods
2.1 3D Design and Printing
All printed pieces used for this work were designed either in Autodesk Fusion 360 2020 or Autodesk Inventor 2018 and exported as an .stl file. DWG files of prints shown in figures can be found in the supplementary information. Files were opened in the MiiCraft Utility Version 6.3.0t3 software, where pieces would be converted into sliced files with appropriate layer heights. The files were converted to the correct file format to include settings optimized for either the CADworks3D M50−405 printer (MiiCraft, CADworks3D, Canada), which had a 405 nm light source, or for the CadWorks 3D Printer P110Y, which had a 385 nm source. All prints were printed in one of three resins: FormLabs BioMed Clear V1 (FormLabs, USA), FormLabs Clear V4 resin (FormLabs, USA) or MiiCraft BV007a Clear resin (CADworks3D, Canada). These resins were chosen as representative of those formulated for biocompatibility (FL BioMed), standard clear printing (FL Clear), or high-resolution microfluidics (MC BV007a).
After printing, all materials were rinsed with isopropyl alcohol using the FormWash from FormLabs, following the wash suggestions from the FormWash online guide. 45 Alternatively, materials were placed in a container with IPA and placed on a rocker for longer periods, extending times by 5 minutes for low viscosity materials and by 1 hour for more viscous resins. Once residual resin was rinsed from the prints, the pieces were dried with compressed air and post-cured with additional UV dosages using the FormCure (FormLabs, USA). Specific print and post-print settings for each resin and printer can be found in Table S1.
2.2 Viability testing with primary murine lymphocytes
2.2.1 Primary cell preparation
Animal work was approved by the Institutional Animal Care and Use Committee at the University of Virginia under protocol #4042 and was conducted in compliance with guidelines from the University of Virginia Animal Care and Use Committee and the Office of Laboratory Animal Welfare at the National Institutes of Health (United States). Following isoflurane anesthesia and cervical dislocation, spleens were harvested from female and male C57BL/6 mice between 8-12 weeks old. The spleens were collected into complete media consisting of RPMI (Lonza, Walkersville, MD, USA) supplemented with 10% FBS (VWR, Seradigm USDA approved, Radnor, PA, USA), 1× l-glutamine (Gibco Life Technologies, Gaithersburg, MD, USA), 50 U/mL Pen/Strep (Gibco, MD, USA), 50 μM beta-mercaptoethanol (Gibco, MD, USA), 1 mM sodium pyruvate (Hyclone, Logan, UT, USA), 1× non-essential amino acids (Hyclone, UT, USA), and 20 mM HEPES (VWR, PA, USA).
To produce a splenocyte suspension, harvested spleens were crushed through a 70-µm Nylon mesh filter (Thermo Fisher, Pittsburgh, PA, USA) into 10 mL of complete media. The cells were then centrifuged for 5 minutes at 400 xg. The pellet was resuspended into 2 mL of ACK lysis buffer, which consisted of 4.15 g NH4CL (Sigma-Aldrich, St. Louis, MO, USA), 0.5 g KHCO4 (Sigma, MO, USA), 18.7 g Na2EDTA (Sigma, MO, USA) into 0.5 L MiliQ H2O (Millipore Sigma, Burlington, MA, USA). The cells were lysed for 1 minute before being quenched by bringing up the solution to 10 mL with complete media and immediately centrifuging again. The pellet was resuspended into 10 mL of complete media, and density determined by trypan blue exclusion. To prepare for cell culture, the suspensions were diluted with complete media to a concentration of 1x106 cells/mL of media.
2.2.2 Print preparation for biocompatibility studies
Disks with a diameter of 15 mm and a height of 1 mm, designed to fit snugly against the base a 24-well plate, were printed in all three representative resins (BioMed, Clear, and BV007a) following the print settings outlined in Table S1. These pieces were divided into “non-treated,” with no further post-treatment, or “treated”. The latter prints were post-treated by soaking in sterile 1x phosphate-buffered saline without calcium and magnesium (PBS, Prod. No. 17-516F, Lonza, USA) for 24 hours at 37°C for BV007a or at 50°C (BioMed and Clear resins) to mitigate cytotoxicity, with similar treatments shown to be effective in previous works.46
To compare post-treatment strategies for the BioMed resin, disks were printed as above. The posttreatments included a 24-hr PBS soak at room temperature within a biosafety cabinet; 24-hr incubation in a 37°C cell culture incubator while dry or soaked in PBS; or autoclaving for 30 minutes at 120°C gravity cycle. In all cases, both treated and untreated pieces were rinsed again with IPA, dried, and UV sanitized for an additional 10 minutes before use.
2.2.3 Analysis of cell viability
Aliquots of suspended splenocytes (1 mL, 106 cells/mL) were added to two 24-well plates containing samples of either treated resins or non-treated resins as previously outlined in section 2.2.2. Wells that did not contain any resins were reserved for plate controls. The cell cultures were incubated for 4 hours at 37°C with 5% CO2. Following the culture period, the viability of the splenocytes was assessed by flow cytometry using a previously established protocol.47 Concisely, 500 µL of the cultured samples were stained using Calcein AM (eBioscience, San Diego, CA, USA) at 67 nM in 1x PBS for 20 min at 37°C. The stained samples were centrifuged at 400 xg for 5 min and resuspended in flow buffer (1 x PBS with 2% FBS), after which 4 µL of 1 mg/mL 7-AAD (AAT Bioquest, Sunnyvale, CA, USA) was added. Calcein-AM single stains were prepared using live cells, and 7-AAD single stains were prepared using cells pre-treated for 20 min with 70% ethanol added in a 1:1 v/v ratio to the culture. Additional controls included unstained cells and an ethanol-treated double-stained control. All samples and controls were run on a Guava 4-color cytometer (6-2L) and analyzed with Guava® InCyte™ Software. Live cells were defined as being high in Calcein-AM and low in 7-AAD signal, while dead cells were defined as the inverse.
2.2.4 Analysis of the viability after direct and indirect contact with treated resin Indirect contact was defined as cell culture in media that had been conditioned by incubation with printed resin, whereas direct contact was defined as cell incubation in physical contact with the printed resins. To test indirect viability, treated BioMed disks were prepared for cell culture as noted in section 2.2.2. Following treatment, the disks were added to a 24-well plate and incubated in complete media for 24 hours at 37C. After incubation, 1 mL of suspended splenocytes at 106 cells per mL were spun down and brought back up in 500 µL of resin-conditioned media. All samples were then cultured for 45 minutes, 4 hours, and 24 hours. Viability was analyzed as in section 2.2.3 to determine the percent of live cells present for each sample.
Direct viability was tested in a similar manner. Treated BioMed disks were added to a 24-well plate, and 1 mL aliquots of suspended splenocytes at 106 cells per mL in fresh complete media were added to sample and control wells. Viability was analyzed after 45 minutes, 4 hours, and 24 hours.
2.3 Characterization of material properties of printed pieces
2.3.1 Autoclave compatibility and heat tolerance
To test heat stability of printed pieces, small pieces with square channels (as used for print resolution tests, .DWG files are included in the supplement) were 3D printed in each of the three resin types using setting from Table S1 and autoclaved at 120°C for a 30-minute gravity cycle. Following autoclaving, the pieces were visually evaluated for cracks, delamination, or other alterations to the original design. Similar tests were conducted by leaving the prints overnight in ovens at 37°C, 70°C, and 120°C and then visually assessing the prints for discrepancies after 1, 3, and 7 days.
Disks with a diameter of 15 mm and a height of 1 mm were printed in each representative resin. A square piece of PDMS was used as a control. All images were collected on a Zeiss AxioZoom macroscope (Carl Zeiss Microscopy, Germany) with Zeiss filter cube channels including Cy5 (Ex 640/30, Em 690/50, Zeiss filter set #64), Rhodamine (Ex 550/25, Em 605/70, #43), EGFP (Ex 470/40, Em 525/50, #38), and DAPI (Ex ~320-385 nm, Em 445/50, #49). A 500 ms exposure time was used for all images. Following imaging, analysis was performed using Image J v1.530 (imagej.nih.gov). On each image, three 1 x 1 in2 regions were analyzed for mean gray value in each channel. Background regions were also measured from the borders in each image (outside of the printed parts) and subtracted from each sample measurement individually. The mean gray intensity was calculated for each resin piece and the PDMS control; higher mean gray intensities represented higher autofluorescence of the pieces.
2.3.3 Optical clarity
Disks with a diameter of 15 mm and a height of 5 mm were printed in the FormLabs Clear resin following the print settings listed in Table S1. Several post-processing methods were compared to determine which had the greatest improvement on optical clarity of printed devices. These included printing on glass, a nail polish coating, a resin coating, sanding, and buffing the pieces. A non-processed piece was used as a control, and a glass slide (0.17 mm thick) was used as a benchmark for optimal material clarity.
To prepare the printed-on-glass piece, a print was set up as previously described by Folch, et al.38 First, small drops of resin were applied to the baseplate using a transfer pipette. A large cover glass with dimensions of 1.42” x 2.36” and a thickness of 0.13-0.17 mm (Ted Pella, Inc. USA) was attached to the baseplate by lightly pressing the slide over the resin then using a UV flashlight to quickly cure the resin between the slide and the baseplate. In the printer software, the initial layer height (“gap height”) was increased by the thickness of the slide (1.7 mm) prior to printing to account for the change in the z-position of the first layer. After printing, the piece was removed from the glass slide with a razor blade and postcured typically. The glass slide and adherent resin drops were also easily removed with a razor blade.
For acrylate coating, a baseplate-printed piece was coated with generic clear nail polish from a convenience store. The top was coated using the polish applicator, allowed to dry for ~15 minutes, and the process was repeated on the bottom of the piece. Similarly, a pipette tip was used to apply a thin layer of FormLabs Clear resin to a separate piece on both the top and bottom, with both sides being UV cured for 10 minutes.
For the sanding method, 3M WetorDry Micron Graded Polishing Paper (ZONA, USA) was used. The piece was sanded on both sides starting at a 30 µm grit paper and followed by 15 µm, 9 µm, 3µm, and finally 1 µm grit. Moderate pressure was used to press the piece into the polishing paper in a circular motion to smooth the surface of the piece. Similarly, a generic 4-sided nail buffer (similar products, Walmart, USA) was used to evaluate the impact buffing could have on the printed piece.
Following post-processing, all pieces were imaged to determine optical clarity. All images were collected in the brightfield under transmitted light on a Zeiss AxioZoom macroscope (Carl Zeiss Microscopy). The intensity of light that passed through each piece was measured using Image J for three 1 x 1 inch2 sections on each image. The average intensity and standard deviation were recorded for n = 3 regions per sample, with the background subtracted from each measurement individually. The relative transmittance, T, of each sample was calculated according to Equation 1,
Resins for SL printing are comprised of a photocrosslinkable polymer base, photoinitiators to initiate crosslinking, and (optionally) additives such as photoabsorbers, dyes, and plasticizers.54–56 Common photoinitiators such as Irgacure compounds or lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) are activated by UV (365 or 385 nm) or violet (405 nm) light.55 While commercial photocrosslinkable resins for SL printing usually keep their exact compositions as a trade secret, the fundamental chemistry can be found in MSDS documentation. All three test resins in this work contained monomers and oligomers of an acrylate or methacrylate polymer base.50,51,53 Many SL resins for microfluidics are fairly similar in their basic composition and can be used across different printers, especially with printers that allow for exposure setting adjustments
Resins may be formulated with additives to achieve a large increase in print resolution or other desired properties. For example, addition of photoabsorbers reduces the effect of scattered light between layers and dramatically improves z-resolution.32 Addition of plasticizers lowers the viscosity of the pre-cured resin, which improves print resolution of hollow internal features by facilitating drainage of uncured resin from the feature during printing and cleaning steps. MiiCraft’s microfluidic BV007a resin, with the highest percent of additives, has a manufacturer-reported viscosity about 10-fold lower than that of the other listed resins (Table 1), and superior resolution for microscale channels. In highly viscous materials like the BioMed resin, undrained resin is easily retained inside the internal features, where it may be crosslinked by light that has been transmitted or scattered in excess, especially as subsequent layers of resin are cured above the hollow features.
On the other hand, photoinitiators and plasticizers, as well as acrylate monomers, can increase cytotoxicity with various cell types due to factors including oxidative stress, enzymatic inhibition, and lipophilic reactions with cell membranes.56–59 Deliberately leaching these toxic components from the printed materials after post-curing can increase cytocompatibility. 29,30 However, we have found that in some cases it also decreases material stability, causing cracking or a decrease in material strength and flexibility as the plasticizers are removed. This problem was particularly prevalent with BV007a prints, which peeled apart when leached in PBS for longer than 48 hours, presumably due removal of plasticizers that were essential to the structural stability of the print. For this reason, selecting a more biocompatible material (e.g. with fewer leachable toxic additives) to begin with may better address this problem when working with sensitive cells. 48
As heat stability is an important factor for chips that will be subject to autoclave sanitation or extended cell culture, we tested the heat stability of each resin (Table 1). The FL BioMed material was autoclavable and also stable overnight at 120°C, allowing for thorough sterilization should any prints need to be reused or prepped for use with live biomaterials. In contrast, MC BV007a withstood mild sanitation procedures, e.g. alcohol rinses or UV sanitation, but high heat (>50°C) delaminated the material over time, although it was stable for 7-day incubation at 37°C when dry, or for 48-hrs in PBS. The FL Clear resin was found to withstand conditions heating conditions up to 70°C for 7 days dry or in solution and also mild sanitation procedures (i.e. UV curing, solvent rinses).
3.2 Physiochemical and environmental factors that influence print resolution
In addition to resin composition, the functionality and printability of a piece are also influenced by other factors that affect photopolymerization and thus the resolution and the mechanical integrity of the part (Fig.1).
3.2.1 Choosing wavelength and exposure settings to mitigate light scattering and bleed-through
Internal features such as enclosed microchannels are imperative to many 3D printed microfluidic devices. Most commercial resin materials yield features at the scale of millimeters or hundreds of micrometers on standard printers with 30-40 µm pixel size, and it is possible to improve the print resolution of internal features through strategic selection of resins, light sources and exposure settings, and number of repeat exposures. 32 The central requirement is to avoid unintentional crosslinking of uncured resin inside the feature, which would lead to blocked features.
Light source wavelength, intensity, and exposure time have a large impact on resolution by modulating on the rate and extent of crosslinking.31,32,60 Common light sources include a laser, LED, or UV lamp, and typically emit at 385 nm or 405 nm in commercial printers.61 Though most commercial resins can be printed at either wavelength, more efficient reactions are achieved by matching the wavelength of the light source with the excitation and absorbance peaks of the photoinitiators and photoabsorbers, respectively. Doing so decreases the exposure time and intensity required to achieve crosslinking and reduces unwanted scattered and transmitted light through print layers, as documented by Nordin and Wooley32 as well as Pontoni.61 To briefly demonstrate the impact of wavelength on resolution of internal features, test pieces containing six internal channels of decreasing square cross-section (0.2 - 1.2 mm side-length; Fig. 2A) were printed in FL Clear resin, using either a 405-nm or 385-nm printer (Fig. 2B vs 2C). The X,Y-resolution (effective pixel resolution) was similar between the two printers, at 30 and 40 µm for the 405- and 385-nm printers, respectively. In this resin, the crosslinking reaction was more efficient with the 385 nm light source, enabling reduced light dosage (shorter times and lower intensities; Table S1), which assisted in diminishing bleed-through light allowing uncured resin to drain more easily from channels. Consistent with this, channels were printable at sizes ~0.2 mm smaller with the 385 nm printer (Fig. 2C) versus the 405 nm source (Fig. 2B).
Fig. 7. Viability of primary murine splenocytes in contact with 3D printed materials. Primary splenocytes from male and female mice (Nmice = 2 per experiment) were evaluated by flow cytometry after live/dead staining with CalceinAM/7AAD. (A) Cell viability after 4 hr of direct physical contact with PBS+incubation treated (T) or non-treated (N) resins compared with well plate control (C). Treated BioMed and Clear prints maintained relatively high viability compared to the well plate, while BV007 did not. Bars show mean ± SD. One-way ANOVA per data set, ns > 0.06, *p > 0.01, **p = 0.01, ****p < 0.0001. (B) Multiple post-treatment methods (PBS, incubation at 37 °C, PBS+incubation, and autoclavation) were evaluated using BioMed resin and culturing for 4 hours with direct contact. Bars show mean ± SD. One-way ANOVA, ns > 0.2, *p > 0.02. ****p < 0.0001. (C) Viability of direct contact (i.e. culture with resin) and indirect contact (i.e. culture with resin leachate) of cells with treated BioMed prints at 45 min, 4 hours, and 24 hours showed a decrease in viability over time. Values show mean ± SD. One-way ANOVA at final time point, ****p < 0.0001.
We found that for experiments under 4 hours, overnight heat and saline leaching treatment was sufficient to increase viability of primary murine splenocytes in contact with 3D printed materials (Fig. 7A). Splenocytes cultured in direct physical contact with either of the treated FL resins retained viability high enough (>60%) after 4 hr to enable short on-chip experiments, whereas untreated materials resulted in lower viability on average. For the MC BV007a resin, the treatment did improve viability over the nontreated resin, but the resin was still largely cytotoxic for these cells at 4 hours. The higher cytotoxicity is consistent with the greater quantity of potentially toxic additives in BV007a compared to other resins (10- 15%, Table 1). Furthermore, BV007a had lower heat stability and could not be leached at the same temperature as the FL resins without mechanical damage (Section 3.1). Therefore, for short experiments with primary cells in suspension, we concluded that the Biomed or Clear resins are more suitable than BV007a and should be pre-treated to minimize release of toxic components into the culture.
Next, we compared a number of common leaching treatments head-to-head: heat (37°C incubation, 24 hr), saline (sterile PBS soak at room temperature, 24 hr), a combination of the two (sterile PBS soak at 37°C, 24 hr), or autoclaving (gravity, 120°C, 30 min) to leach unbound toxins out of solid prints (Fig. 7B). Using the BioMed resin, we found all overnight saline (PBS) and incubation (37°C heat) treatments increased splenocyte viability at 4 hours, with non-significant differences between the live plate control and treated materials. Autoclaving was slightly less effective, and though this treatment takes less time (~45 minutes), it is not recommended, at least for this resin.
Finally, as many experiments require longer term culture than just 4 hr, we tested splenocyte viability over time when cultured in direct physical contact with treated FL BioMed resin or in “indirect contact,” i.e. cultured in contact with media that was pre-conditioned by incubation with the resin. Direct contact with resin is likely to occur in a fully integrated bioanalytical microchip with on-chip cell culture, while indirect contact may occur when microdevices are used to prepare media or drug solutions (e.g. mixers or droplet microfluidics) or to deliver media components to cultures (e.g. flow channels or bubble traps). We again found that splenocyte viability was high for both contact conditions within 0-4 hr, but decreased overnight (24 hrs) for both contact conditions compared to plate controls (Fig. 7B). Others have reported longer culture times for hardier cell lines. 18,25,27–29,33,38,80,81 This result indicates that at least for primary murine splenocytes and perhaps for other fragile cells, more work is needed to identify best practices for post-print treatments and/or more biocompatible resin formulations.
3.5 Optical components of resins
Microfluidics is frequently integrated with on-chip imaging. Autofluorescence is a potential limitation of polymeric chips, whereas PDMS is often praised for its optical compatibility. Therefore, we quantified the autofluorescence of each of the representative resins in four standard fluorescence channels. In the Cy5 and Rhodamine channels, all three resins showed relatively low autofluorescence and were comparable to PDMS (Fig. 8). However, UV excitation (DAPI channel) elicited high levels of autofluorescence from the two FormLabs resins. The Clear resin also showed moderate autofluorescence in the GFP channel. Autofluorescence at short wavelengths is consistent with the use of photoabsorbers intended to absorb at short wavelengths. 76 On the other hand, MC BV007a had negligible autofluorescence in all channels, similar to PDMS. Thus, optical compatibility in the intended fluorescence channels should inform material choice for microfluidics devices.