Evaluation and Optimization of PolyJet 3D-Printed Materials for Cell Culture Studies

Abstract

Use of 3D printing for microfluidics is a rapidly growing area, with applications involving cell culture in these devices also becoming of interest. 3D printing can be used to create custom designed devices that have complex features and integrate different material types in one device; however, there are fewer studies studying the ability to culture cells on the various substrates that are available. This work describes the effect of PolyJet 3D printing technology on cell culture of two cell lines, bovine pulmonary artery endothelial cells (BPAECs) and Madin-Darby Canine Kidney (MDCK) cells, on two different types of printed materials (VeroClear or MED610). It was found that untreated devices, when used for studies of 1 day or more, led to unsuccessful culture. A variety of device treatment methodologies were investigated, with the most success coming from the use of sodium hydroxide/sodium metasilicate solution. Devices treated with this cleaning step resulted in culture of BPAECs and MDCK cells that were more similar to what is obtained in traditional culture flasks (in terms of cell morphology, viability, and cell density). LC-MS/MS analysis (via Orbitrap MS) was used to determine potential leachates from untreated devices. Finally, the use of a fiber scaffold in the devices was utilized to further evaluate the treatment methodology and to also demonstrate the ability to perform 3D culture in such devices. This study will be of use for researchers wanting to utilize these or other cell types in PolyJet-based 3D printed devices.

Introduction

The use of 3D printing for manufacturing micro- and milli-fluidic devices is a rapidly evolving field that has the potential to supplant traditional fabrication techniques (i.e. soft lithography with PDMS). This is due to the ability to rapidly print many devices at once, to print materials of different composition in one device, to easily share CAD designs with other researchers, and to integrate real-world interconnects (fittings, tubing, etc) [1–6]. While there are a variety of different techniques that can be used to perform 3D printing, the majority of fluidic devices have been manufactured by fused deposition modeling (FDM, with materials such as Acrylonitrile Butadiene Styrene or ABS), some form of stereolithography (SLA, with acrylate or epoxide-based materials), or MultiJet/PolyJet printing (acrylate-based materials with photoinitiators) [4, 6–8]. PolyJet printing involves jetting photopolymer droplets onto a tray, with each layer being cross-linked with UV light. A key advantage of PolyJet printing over other methods is the wide variety of materials that are available to be printed in one device [3, 9]. While there are trade-offs in terms of printer cost, resolution, material properties, build size, and ability to incorporate multiple materials, each technique has its unique advantages and thus one has not yet emerged to be the leading method to 3D print fluidic devices.

The use of microfluidic devices for cell culture studies is well-established, as this approach can be used to replicate in vivo conditions (i.e. shear stress), utilize multiple-cell types (for cell-to-cell communication studies), and integrate 3D culture as well as analytical detection schemes [10–12]. Not surprisingly, recent research has investigated the use of 3D printed devices for cell culture studies [10, 13–15]. This topic has been reviewed [15], with some examples including the use of a SLA-based perfusion device for multicellular spheroids [16], PolyJet printed devices that accept transwell inserts for drug transport studies [1], a microfluidic blood brain barrier (BBB) model using Veroclear material (PolyJet printing) [17], and a perfusion device with integrated fiber scaffolds in a ABS device [18].

One concern has been the biocompatibility of 3D printed materials for cell culture studies, with several studies investigating this possible issue. These have mainly focused on SLA and PolyJet printed devices and possible leaching of materials over time (due to incomplete polymerization of photo-reactive resins) [14]. One approach for devices printed with MED610 (PolyJet printing), which is advertised as a biocompatible material, found that a detailed cleaning procedure which includes sonication with isopropanol led to successful culture of primary mouse myoblasts [19]. Another study investigated the culture of 3 different cell types on devices created by 3 different printing methods (SLA, PolyJet and laser sintering) [20]. Results showed a mixture of compatibility for each cell type when cultured on the different printed parts. For example, VeroClear (PolyJet printing) devices cleaned with isopropanol and methylated spirits (mixture of ethanol and methanol) were found to support proliferation for skeletal muscle cells (C₂C₁₂) and differentiation for neuronal cells (SH-SY5Y) but such devices had no compatibility with hepatic cells (HepG2). The authors used FT-IR and NMR spectroscopy to observe bands that correlate with uncured acrylate materials in the leachate (but no specific identification) [20]. Other research has investigated the use of coatings to improve biocompatability [13, 21, 22]. These studies show that the specific printing method as well as the specific cell type have to be considered when determining the ability to perform successful culture on 3D printed devices.

In this study, we investigated the ability to culture 2 cell lines directly on PolyJet printed devices (printed with VeroClear or MED610) without the use of adhesion factors. Two different cell types were investigated, bovine pulmonary artery endothelial cells (BPAECs), which are commonly used to study vasodilation and red blood cell/endothelial cell interactions [23, 24], and Madin-Darby Canine Kidney cells (MDCK cells), which are commonly used in permeability screening methods [25, 26]. To improve the culture of cells vs. an untreated device, a variety of treatment/cleaning methodologies were investigated, with the most success coming from the use of sodium hydroxide/sodium metasilicate solution. Devices treated with this cleaning step resulted in culture of BPAECs and MDCK cells that were more similar to what is obtained in traditional culture flasks. LC-MS/MS analysis (via Orbitrap MS) was used to determine potential leachates from untreated devices. Finally, the use of a fiber scaffold in the devices was utilized to further evaluate the treatment methodology and to also demonstrate the ability to perform 3D culture in such devices.

Experimental

3D Printing

Stratasys J735 and Eden 260V PolyJet printers were used to print the devices used in this study. VeroClear resin was printed with the J735 printer while the MED610 resin was printed with the Eden 260V printer. MED610 is approved for permanent skin contact and limited mucosal membrane contact for up to 24 hours [27]. Design for the cell culture devices in this paper were completed in Autodesk Inventor Professional 2020. For the majority of studies, the dimensions of the cell culture devices were based on a 24 well plate (Figure 1A). Devices were either printed to have three wells (depth: 6 mm, diameter: 17 mm) or were printed as individual wells (depth: 2.0 mm, diameter: 18 mm) for confocal imaging. For studies involving determining cell density, devices were the same as a 35 mm culture dish (Eppendorf, Hamburg, Germany) so that a direct comparison could be made (depth: 2 mm, diameter: 35 mm). Standard high quality print settings were used with the printer being operated in support mode. The devices themselves were designed and processed not to print support material in the voids. Only the carpet layer included support material, which was removed manually before processing or use of devices. Several cleaning/treatment methods were explored. Devices that were treated in water were completely covered by DI water and were allowed to soak overnight. Devices that were treated in the Super DT3 CleanStation (PM Technologies, Inc., Osseo, MN) were placed in the sodium hydroxide/sodium metasilicate bath (according to manufacturer [28], the concentrations are 10–30% NaOH, 1–3% Na₂SiO₃, 60–80% Na₂CO₃) overnight, followed by thorough rinsing with water before use. Another cleaning method that was explored involved placing the devices in a bath containing 2% NaOH overnight, followed by thorough rinsing with water before use.

Figure 1.

Initial cell culture results of BPAECs cultured on PolyJet 3D printed devices using VeroClear and MED610 resins. (A) CAD rendering of PolyJet 3D printed well plate, based on dimensions for a 24 well plate. (B) Printed devices in VeroClear (left) and MED610 (right). Brightfield images of BPAECs cultured directly in a 35 mm polystyrene culture dish (C), untreated VeroClear (D), and untreated MED610 (E). Confocal images of BPAECs stained with acridine orange in a petri dish (F), untreated VeroClear (G), and untreated MED610 (H). Scale bar: 100 μm. Images were collected after incubating for 24 hours.

Cell Culture

Bovine pulmonary artery endothelial cells (BPAEC, Cell Applications, San Diego, CA), between the passages of 5–25, were cultured in Corning T-75 flasks (Corning, NY) using high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC, Manassas, VA) containing 10% FBS (Millipore Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Lonza, Walkersville, MD). Media was changed every 72 hr. Cells were cultured at 37 °C and 5% CO₂ until 80% confluence was achieved. Cells were passaged with the aid of a trypsin-EDTA solution (Millipore Sigma, St. Louis, MO). Seeding density was determined after counting cells using a cell hemocytometer. Cells were seeded in 3D printed devices at ~4,500 cells per mm². Cells were placed in an incubator and monitored over 24 hr. No adhesion factor was used in these studies.

Madin-Darby canine kidney cells (MDCK NBL-2, ATCC, Manassas, VA) between the passages 20–35, were cultured in T-75 flasks (Corning, NY) using Eagle’s Minimum Essential Medium (EMEM, Millipore Sigma, St. Louis, MO) containing 10% FBS and 1% penicillin-streptomycin. Media was changed every 72 hr. Cells were cultured and passaged in similar conditions as sub-culturing for BPAECs. Cells were seeded in 3D printed devices at ~3,500 cells per mm². Cells were placed in incubator and monitored over 24 hr. No adhesion factor was used in these studies in order to focus on direct surface effects and to eliminate as many variables as possible with this study.

Effects of embedded fibrous scaffolds in the cell culture devices for 3D culture was also investigated. Polystyrene nanofibers ranging between 200–400 nm in size were prepared by electrospinning [29]. A 12% (w/v) polystyrene solution was prepared by dissolving polystyrene pellets (280 kDa M.W., Millipore Sigma, St. Louis, MO) and 1% Tetrabutylammonium bromide (Millipore Sigma, St. Louis, MO) in dimethylformamide (DMF). The solution was allowed to completely dissolve by being placed on a shaker overnight. A pressure of 4 psi was applied (using helium gas) to a glass vial with a septum containing about 2 mL of the dissolved polystyrene solution to induce flow through a 15 cm, 150 μm I.D. fused silica capillary. High voltage between 15–17 kV was applied to the solution via a platinum wire (0.5 mm diameter; Alfa Aesar, Ward Hill, MA) connection through the septum. The electrospinning setup was located in a customized plexiglass chamber. Optimal spinning conditions were feasible with the temperature and humidity inside the temperature kept at 22 °C and less than 40%, respectively. This solution was electrospun on a 10 cm × 18.5 cm sheet of Whatman lens cleaning tissue (Grade 105; GE Healthcare Life Sciences, Marlborough, MA). This sheet was wrapped around a grounded mandrel (14 cm long by 6 cm in diameter) that rotated at 300 rpm and would slide horizontally over 12 cm span at 6 cm/s. Fibers were collected after 90 minutes and were characterized using Scanning Electron Microscopy (SEM). Fibers were laser cut to create 19 mm² diameter circular scaffolds and were peeled from the lens paper to be placed in the 3D printed wells. Fibers were plasma treated for 30 seconds at medium RF before being used for cell culture [29]. BPAECs were seeded at the same seeding density as devices without fibers. A second seeding of BPAECs was performed 24 hours after the initial seeding. Cells were then imaged after an additional 24 hours of incubation with this second seeding using acridine orange as an intracellular dye.

Mass Spectrometry Analysis of Leachate

Devices were printed in Veroclear and MED610 using the J735 and Eden 260V PolyJet printers. Devices were either left untreated or treated/cleaned with NaOH and sodium metasilicate. A 10 mM ammonium formate buffer was prepared as the leaching buffer. A volume of 750 μL was added to each well for VeroClear and MED610 devices. Devices were placed in a cell incubator for a 24 hr period. An aliquot volume of 150 μL was taken after 2 and 24 hr from each well and was reserved for analysis by LC-MS/MS.

LC-MS analysis: A 17.5 cm × 50 μm i.d. capillary column was fabricated as previously described [30]. Briefly, a photopolymerized frit and emitter tip were created in a 50 μm inner diameter fused silica capillary (Polymicro Technologies, Phoenix, AZ), then packed with 3 μm Atlantis T3 C₁₈ particles (Milford, MA, USA). Samples were analyzed on a Thermo Q-Exactive mass spectrometer coupled to a Vanquish LC system and a flow splitter. Flow rate from the LC was 175 μL/min with an injection volume of 3 μL. This flow was split to direct 120 nL/min towards the capillary column, resulting in an injection volume of 2 nL. Mobile phases A and B were 0.1% formic acid in water and acetonitrile, respectively. The gradient was as follows: 0 – 2 min, 0% B; 10 min, 98% B; 12 min, 98% B; 12.1 min, 0% B; 23 min, 0% B.

The MS system was operated in polarity switching mode, acquiring spectra at a resolution of 70K in each polarity. Spectra were acquired from m/z 90 – 1350 with a maximum injection time of 200 ms, AGC target of 1e⁶, a capillary temperature of 200 °C, and a spray voltage of 1.75 kV in each polarity. Quantitation of n-butylbenzenesulfonamide (NBBS) was performed using a selected ion monitoring method and a calibration curve from 0.1 to 5.0 μM using the commercially available standard (Millipore Sigma). A top 5 data dependent fragmentation was performed with an inclusion list of all significant features with MS¹ resolution of 35K, MS² resolution of 17.5K, and a maximum injection time of 50ms.

MS-DIAL version 4.24 was used for feature detection, alignment, and MS² analysis [31]. MS-DIAL settings for peak detection and MS² identification are provided in Table S1. Features with a mean intensity < 1e⁴ or less than 5-fold higher than the blank were removed in addition to naturally occurring isotopes. Further data analysis was performed in excel and R version 4.0.5. Statistical comparison of treatments for each material and timepoint was performed using an unpaired t-test with p < 0.05 indicating significance.

Confocal Imaging

Cells were washed with 1 mL of 1X Phosphate buffered saline (pH = 7.0, Cytiva, Marlborough, MA) after 24 hr of incubation on 3D printed devices. Both BPAECs and MDCK cells were stained with 100 μM acridine orange (Millipore Sigma, St. Louis, MO) for fluorescent imaging (5 minute incubation), followed by another rinse with 1 mL of 1X PBS. Fluorescent images were obtained of cells directly on the devices using a Leica SP8 confocal microscope (10X). Images were processed using the manufacturer’s software (Leica Application Suite X) and further analyzed using FIJI/ImageJ.

Cell Assays

Quantification of cell viability was determined by performing a viability assay using 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT; Millipore Sigma, St. Louis, MO) and cell density was determined through cell counting using confocal microscopy. Cells were cultured on untreated and treated 3D printed devices, printed with VeroClear and MED610 resins, for 24 hours before adding the proper reagents and lysing. The MTT assay was performed following the manufacturer’s protocol using BPAECs and MDCKs [32]. Samples were transferred to a 96 well plate to measure absorption at 550 nm using a plate reader (SpectraMax iD3, Molecular Devices). Cell viability using the MTT kit was done by normalizing the resulting absorption values to cells cultured in a traditional Falcon 24 well plate (Corning, Corning, NY) that is the same surface area as the 3D printed devices. Cell density was determined for BPAECs and MDCKs seeded in the treated VeroClear and MED610 devices. Cells were cultured for 24 hours on these devices before being rinsed with 1X PBS and were stained with acridine orange (see procedure described earlier). Confocal imaging was performed to collect images of stained cells from different regions of the device. FIJI/ImageJ and the “Analyze Particles” function was used to count the number of cells (with each analysis being manually verified). Cell density was calculated as the number of cells/cm².

Results and Discussion

Untreated devices with cell culture

The material jetting, or PolyJet, printers utilized in this research are manufactured by Stratasys and utilize proprietary materials. Structures are formed through acrylate cross-linking chemistry using a mixture of acrylate oligomers and monomers along with a photoinitiator [7]. The material is jetted onto a surface, followed by UV-initiated polymerization. Due to their rigidity and transparent nature, this study utilized VeroClear and MED610; the latter is marketed as a biocompatible material. VeroClear resin has been reported to contain isobornyl acrylate, acrylic monomer, acrylate oligomer, acrylic acid ester, and photoinitiator [20, 33]. MED610 has less published information but is classified as an acrylic formulation that contains acrylic acid and a similar number of proprietary materials [34]. In this study, cells were cultured directly on these materials using a device with dimensions similar to a 24 well plate (Figure 1A–B). When using the aforementioned BPAECs and MDCK cell lines on devices printed with this material for longer term studies (~24 hr), it was found that both types of cells started to lose their native morphology and detach from the surface. As seen in Figures 1C and 1F, healthy cell culture for BPAECs is characterized by proliferation and branching out of cells. At least 80% confluency is reached 24 hours after subculturing the cells. After seeding BPAECs on devices that were used directly from the printer, cell culture was not sustainable after 24 hours. Figures 1D and 1G illustrate this effect for devices printed using VeroClear. Similar results were seen using the claimed biocompatible material MED610 (Figures 1E and 1H). Due to these findings and previous studies using Veroclear with other cell lines [20], we sought to investigate potential treatment methods to enable culture studies in PolyJet-based devices. It should be noted that this involved direct culture on the devices without any adhesion factor.

Comparison of treatment methods

Treatment methods that were explored were based on approaches that would reduce effects of potential chemicals that may leach from the material during incubation. Use of different solvents and additional exposure of these devices to a UV flood source were investigated as methods to improve the cell compatibility and possibly decrease the amount of leachate. A simple water treatment was first performed which had involved soaking VeroClear and MED610 devices in a DI water bath for overnight. As seen in Figure 2A, there was no improvement seen in cell culture after 24 hours. Cells were not adherent to the surface of the devices or showed poor confluency for both VeroClear and MED610 materials. Additional UV exposure was also considered due to potentially cure any UV-dependent components in the material that had not been fully cured during the print. Devices were placed in a UV flood chamber for 8 hours at 3.4 mW/cm² (~98 J/cm²) before use with cells. Figure 2B shows the cell culture results using these devices. Similar results were seen with no adherent cells and poor confluency. Soaking newly printed devices in a 2% NaOH solution overnight was also explored. Cell culture was also not improved under these conditions with either material, as seen in Figure 2C where the cell characterization was similar to untreated devices shown in Figure 1 (D,G and E,H).

Figure 2.

Brightfield images of BPAECs cultured on both VeroClear and MED610 devices using different post-printing treatment methods. Devices were either soaked in water overnight (A), exposed to UV light for 8 hr. (B), or soaked in a 2% NaOH solution overnight (C). UV exposure was 97.1 J/cm². Cells were incubated for 24 hours before imaging. Scale bar = 100 μm.

These results indicate that there is some source of cytotoxicity still present in the material after applying the treatments described above. A different treatment approach had to be investigated to reduce or eliminate these leachates and improve the success of cell culture. An available resource to the lab was access to a Super DT3 CleanStation support cleaning system that contains a solution with varying concentrations of NaOH and sodium metasilicate. This solution is agitated for a pre-determined amount of time. The purpose of this system is to remove support material that can be deposited during the print. Even though these devices used here only have support material deposited as the carpet layer and not within voids of the device that contacts cells, they were placed in this bath for an overnight time period to see if the cleaned devices had less leachate and more successful cell culture. Results for cell culture on these devices are shown in Figure 3. Both materials demonstrated healthy and successful cell culture shortly after cells were seeded on the devices and showed similar characteristics as seen in the control (Figure 1C and F). Cells continued to show compatibility with the devices for over 24 hours.

Figure 3.

(A) Side-by-side views of untreated devices (left) and devices treated in the NaOH/sodium metasilicate bath (right). Top row is devices printed with VeroClear and the bottom row is devices printed with MED610. (B) Brightfield and confocal micrographs of BPAECs cultured directly on VeroClear and MED610 devices that were treated in the NaOH/sodium metasilicate bath overnight. Characteristics of these cells were highly similar to the control and cell culture appeared healthy for over 24 hours. Scale bar = 100 μm (C) Viability results of MTT assays comparisons for BPAEC (blue) and MDCK (yellow) after 24 hours of culture. Error bars displayed as standard error of the mean. Cells cultured in a Falcon 24 well plate served as the control (red bar). (D) Cell density comparisons for treated VeroClear and MED610 devices using BPAEC (blue) and MDCK (yellow) after 24 hours of culture. Error displayed as standard error of the mean. Cells cultured in a 35 mm polystyrene culture dish served as the control (red bar).

Another cell line of interest was MDCK cells and similar studies were performed to determine if they were compatible with these materials. As seen in Figure S1A, culturing MDCK cells directly on the devices without any treatments did not indicate proliferation and healthy cell culture. The same treatments described previously were applied and similar results were seen with little indication of healthy cell culture. However, successful cell culture was attained with devices that were soaked in the NaOH/sodium metasilicate support removal bath illustrated in Figure S1B. Confocal images were obtained for MDCK culture on the cleaned devices as shown in Figure S1C.

Futher characterization of treated devices

Quantification of the success of cell culture between the untreated and treated devices was analyzed by MTT assays, to determine percent cell viability, and counting stained cells, to determine cell density. Results were compared to use of a traditional culture dish or well plate of the same surface area. Ideally, the treated devices would provide successful cell culture, as seen in Figure 3B, that would mimic cell culture on polystyrene dishes. Adhesion factors can be used to improve the success of cell culture [35–37] but they were not employed here, as we wanted to eliminate as many variables as possible with this study.

A method for determining cell viability includes the MTT colorimetric assay, which can be used to measure the metabolic activity of living cells by monitoring the reduction of MTT to form formazan (purple in color) [32]. The reaction can only be completed by living cells, so differences in solution color (absorbance) can be measured to determine cell viability relative to a control. For this study, the MTT assay is of interest due to the significant differences in cell culture results observed between uncleaned and cleaned 3D printed devices. For the MTT assays, the results are normalized to culture in a traditional Corning 24 well plate. Since this normalization is done for each trial, the control does not have any error bars. The MTT assay was used for BPAECs and MDCKs cultured on untreated and treated devices. In both cases, cells were allowed to incubate for 24 hours before adding assay reagents. As is shown in Figure 3C, devices that were treated had increases in percent viability for devices made in both VeroClear and MED610. This supports the images obtained for untreated and treated devices printed with both resins (Figure 3B). Cells cultured on untreated VeroClear and MED610 devices displayed poor results for the MTT assay but the viability was improved after treatment.

Determining cell density via microscopy provides information on cell confluence, with this comparison being done for treated VeroClear and MED610 devices (vs. polystyrene culture dish) using BPAECs and MDCKs. Since the confocal microscope used in these studies could not image a 24 well plate, the dimensions of the 3D printed devices were increased to have the same surface area as a 35 mm polystyrene culture dish. Three different areas of the device were analyzed using confocal microscopy and a cell counting. Results are displayed in Figure 3D with comparison to a polystyrene culture dish. For BPAEC and MDCK, the cell density of the treated devices were not statistically different (at the 95% confidence level) than the culture dish control. These results indicate that the NaOH/sodium metasilicate treatment is an effective method that enables much improved cell culture on PolyJet materials. Results with this treatment are comparable to cell culture results with a polystyrene dish.

Clearly, the NaOH/sodium metasilicate cleaning procedure has shown to be an effective method for improving the biocompatibility of PolyJet materials for culturing these two different cell lines. Results showed that cell culture of BPAECs and MDCKs on the printed devices are similar to cultured in a Corning polystyrene 24-well plate. The overall cleaning method is less rigorous than previously described cleaning methods and is readily available to labs that have PolyJet printers. Indication of successful cell culture can be seen by observing the healthy morphology of these cells within two hours after passaging as well as the other assays shown in in Figure 3. Cell culture on VeroClear and MED610 devices that have been cleaned with this solution was shown to be stable for over 72 hours (Figure S2) which may be of interest for long-term analysis. Cells were also able to be successfully cultured directly on the device indicating that scaffolds or coatings/adhesion factors were not required to promote cell culture. This may also simplify the setup for cell culture experiments as little processing has to be completed for these devices to be functional, expanding the potential for culture in microfluidic devices.

Mass Spectrometry Analysis

LC-MS/MS was employed to investigate if the improved cell culture in treated devices was from some type of surface effect or from decreasing the amount of chemical leachate into the media over time. Devices were printed in Veroclear (VC) and MED610 and either left untreated or treated/cleaned with the NaOH and sodium metasilicate solution. A 10 mM ammonium formate buffer (750 μL) was added to each well, placed in an incubator for a 24 hr period, and aliquots for subsequent analysis taken at 2 and 24 hrs. The results of the LC-MS analysis are summarized in Figure 4. Analysis across both polarities show a majority decrease in detected features when comparing treated to untreated devices across both materials (Figure 4A). For the MED610 material, 1448 features were found to significantly decrease in the treated devices, as compared to the untreated devices. For the VeroClear material, a similar trend was seen (1301 downregulated features in the treated devices). The 24 hr time point (Figure S3A) gave similar results. A feature is a coordinate (m/z, retention time) where a peak is detected and these are derived from the peak picking software used for analysis (MS-DIAL in this study). While one species often creates multiple features due to adducts, in source fragments, and dimerization [38], this analysis clearly shows the treated devices lead to significantly fewer species leaching into the media.

Figure 4.

Mass spectrometry analysis of Veroclear and Med610 devices (either treated or cleaned with NaOH/sodium silicate), with each device being exposed to a 10 mM ammonium formate buffer for various time points (2 or 24 hrs). (A) Volcano plot showing significant features upon treatment across both positive and negative mode which significantly decrease (blue) and increase (red) for each material (2 hr exposure to buffer). Cutoff points shown by dashed lines are p < 0.05 with a fold change greater than 1.5. (B) High resolution MS² spectra of n-butylbenzenesulfonamide (NBBS) in the VeroClear 24-hour extract sample (top) compared to the NBBS standard (bottom); (C) Extracted ion chromatograms of m/z 214.0896 for VeroClear 24-hour extract (top), NBBS standard (middle), and VeroClear extract spiked with NBBS standard (bottom); (D) Micrograph of BPAECs grown in a treated Med610 device for 24 hrs, with 50 μM of NBBS being added to the cell media (Brightfield-top, confocal-bottom; scale bar = 100 μm).

Fragmentation analysis was performed on all significant features (both up and down-regulated) using a data dependent method with an inclusion list. Since the materials employed are proprietary, exact determination is difficult. A comparison across all public MS/MS databases in MS-DIAL revealed n-butylbenzenesulfonamide (NBBS) as a potential match. This feature was explored further to confirm its identity and determine if it was one of the factors effecting cell morphology and viability. A commercially available standard was used for comparing the high resolution MS² spectra to the Veroclear extract, with both spectra being virtually identical (Figure 4B). Figure 4C shows spiking experiments to confirm the identification of NBBS chromatographically. A further comparison of NBBS concentration in extracts from MED610 and VeroClear at 2 and 24 hr time points is given in Figure S3B. BPAECs grown in media that contained 50 mM NBBS were shown to be unhealthy and similar to untreated devices using both VeroClear and MED610 (Figure 4D and Figure S4).

It should be noted that these data show NBBS being only one of many potential toxins that are reduced upon treatment. Since high resolution MS was possible with the Orbitrap system, additional chemical formula predictions were made using exact mass. Features were sorted by p-value, and hits that greater than a 1.5 fold change, with an average intensity greater than 1e5 were selected for manual inspection to create a top 10 list for each polarity, timepoint, and material. The isotope ratio was used to determine carbon number and if any Cl, Br, or S isotopes were present to aid in formula prediction based on exact mass. These results are presented in Tables S2 through S5. Clearly, cleaning the devices with NaOH and sodium metasilicate decreases the amount of leachate, as compared to untreated devices.

Culture on Fibers in Devices

In addition to chemicals leaching from the printed devices, topography of the printed wells may also affect cell proliferation. Previous methods have described coatings that have been applied to 3D printed materials to limit surface roughness from the resin used and improve cell culture [21]. The success of cell culture could determine if the incompatibility seen previously was purely a chemical leaching issue or also related to topography of the printed material. To investigate this, polystyrene nanofibrous 3D scaffolds were electrospun as described previously [29]. Integrating fibers in these devices could improve cell culture and would also lead to a better representation of the 3D extracellular matrix (ECM) [39, 40]. In this study, polystyrene scaffolds were directly embedded in the cell culture devices that were either directly from the printer or after cleaning the devices with NaOH/sodium metasilicate. As seen in Figure 5A and 5C, untreated VeroClear and MED610 devices showed similar results as to untreated devices without fibrous scaffolds. The treated devices had successful cell culture with fibers after 24 hours (Figure 5B and D) for both materials. This indicated that including another material as a barrier between the device surface and the cells was not sufficient for successful cell culture on PolyJet materials. It is evident from Figure 5A and 5C that some leaching of uncured species from the material was still occurring and affecting cell culture. Cell count and proliferation were improved when the embedded fibers were used with cleaned devices for both VeroClear and MED610. From these observations, it was determined that the NaOH/sodium metasilicate bath treatment is effective in reducing chemicals in the printed resin leachate. Even with a scaffold in place, there were still issues with cell culture in untreated devices. This also demonstrates the ability to integrate 3D scaffolds into these devices.

Figure 5.

Use of electrospun polystyrene fibers embedded in 3D printed well plates for BPAEC culture. Top row represents brightfield micrographs and the bottom row represents confocal micrographs. Fibrous scaffolds were embedded in untreated VeroClear (A), treated VeroClear (B), untreated MED610 (C), and treated MED610 (D). Cells were stained with acridine orange after 24 hours of incubation in the devices. Scale bar =100 μm

Conclusions

Recent work involving microfluidics has been focused on utilizing 3D printing for device fabrication. PolyJet 3D printing technology is of interest because of its high resolution, high throughput, and use of multiple materials. Improving the biocompatibility of PolyJet materials was investigated in order to perform successful cell culture of endothelial and MDCK cells. An effective and efficient treatment to perform successful cell culture was found to be cleaning the PolyJet printed devices with a support removal solution containing NaOH and sodium metasilicate. Cell culture results with cleaned devices were comparable to cell culture in a Corning flask. Possible causes for issues with direct cell culture could be related to surface morphology or leaching of uncured chemicals from the devices. This was investigated through LC-MS/MS analysis which was able to show significant decreases in intensity for numerous hits when the treatment was applied. The findings of these studies should be useful for future work using microfluidic devices for culture of multiple cell types in PolyJet devices.

Biographies

Emily Currens is a M.S. student, working under the direction of Dr. Scott Martin in the Department of Chemistry at Saint Louis University. Her research focuses on utilizing PolyJet 3D-printing technology to fabricate microfluidic devices with embedded features for integrating cell culture with analysis.

Michael Armbruster is a doctoral student, working in the Department of Chemistry at Saint Louis University under the direction of Dr. Jim Edwards. His research focuses on isotope derivatization of metabolites to improve mass spectrometry throughput by multiplexing.

Andre Castiaux is currently an analytical chemist working for BASF in their agriculture products division. Andre previously spent three years as a post-doctoral researcher at Saint Louis University working with Dr. Scott Martin. While there he developed new techniques to fabricate complex microfluidic chips utilizing PolyJet 3D printers and was a founding member of the Saint Louis University Center for Additive Manufacturing (SLU-CAM).

James Edwards is a Professor of Chemistry at Saint Louis University. His research focuses on the development of novel separation and mass spectrometry methods to investigate diabetic complications.

R. Scott Martin is a Professor of Chemistry at Saint Louis University, where he has been on the faculty since 2003. His research is focused on the development of microchip-based systems that integrate cell culture with analysis to study cell to cell communication and disease onset. He is a founding member of the Saint Louis University Center for Additive Manufacturing (SLU-CAM).