Wax patterned microwells for stem cell fate study

Abstract

The fabrication of cost effective paper-based analytical devices by wax printing has recently become popular, by and large, using cellulose filter papers. Paper-based devices need higher temperature to form hydrophobic barrier across paper substrate, rely on large working channels (≥500 μm) for liquid handling, and exhibit lower efficiency (∼50%) of sample mobility. Such limitations confine applications of wax based fabrication. In this work, we report printability, fidelity, and application of wax micropatterns on a non-cellulosic, non-fibrous, and non-porous polyethylene terephthalate based substrate (mPET). Resolution of wax printing on mPET was found to be 120 μm for line and 60 μm for channel micropatterns. The wax micropatterns can sustain heat and retain their structural integrity at melting temperature of wax and above (≥120 °C). In application, wax microwells were patterned on the new substrate in a high throughput fashion, which formed a suitable niche for mouse embryonic stem cell (mESC) culture either to maintain self-renewal or direct differentiation. This study will open a new direction in wax printing applications not only as a low-cost but a multipurpose fabrication tool.

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Introduction

Wax printing has recently been widely adopted to fabricate low cost paper-based analytical devices for biological applications, primarily paper-based diagnostics,^1–5 since its first emergence in 2009.^6,7 A variety of simple and low-cost procedures, such as solid wax printing,⁶ liquid wax inkjet printing,^8–10 wax screen-printing,¹¹ wax lithography,⁷ and wax dipping,¹² were employed to fabricate 2D^6–9,11,12 and 3D^13–15 format devices. The popular substrate to fabricate wax patterned devices has been cellulose/nitrocellulose based filter paper because of its well-known properties. It is interesting to note that other commercially available papers (for e.g. laser printing papers) cannot compete due to presence of undisclosed additives added during manufacturing. Nevertheless, paper-based wax patterned devices are exposed to melting temperature (120 °C) to create hydrophobic barrier across thickness of paper substrate, while the lateral flow of wax causes damage to desired features and fine boundaries.^16,17 Paper substrates have limitations, as discussed elsewhere,^9,18 which has limited the applications of wax printing to prototyping of point-of-care testing.

In this communication, we report the printability, fidelity, and application of wax micropatterns on a non-cellulosic, non-fibrous, and non-porous polyethylene terephthalate (PET) based substrate. Wax micropatterns, lines and channels, were printed using solid wax printer followed by characterization under optical microscope. Fidelity of wax micropatterns at higher temperature was studied by measuring dimensions of micro-features in x–y plane using optical microscope and in z-axis by scanning electron microscopy. Finally, high density wax microwells were patterned to study mouse embryonic stem cell (mESC) showing either self-renewal or differentiation starting from single mESC.

Experimental

Materials

Xerox ColorQube 8580/DN color printer and solid wax ink was purchased from an online vendor. Surface modified polyethylene terephthalate (mPET) 8.5′′ × 11′′ substrate, also known as Novele™, was obtained from Novacentrix (Austin, Texas). Surface modification of the Novele™ substrate is propriety of the manufacturer, Mitsubishi Imaging (MBM) Inc. High glucose-Dulbecco's modified Eagles medium, 15% ES-qualified fetal bovine serum, L-glutamine, MEM nonessential amino acid solution, sodium pyruvate, and TrypLE™ were obtained from Invitrogen. While, β-mercaptoethanol and penicillin–streptomycin were purchased from Sigma, and 1000 U ml⁻¹ ESGRO® LIF from EMD Millipore.

Wax microprinting and characterization

Inkscape 0.91 vector based drawing software (available free online) was used to draw micropatterns, channels and lines, having widths between 10 μm and 300 μm. The wax micropatterns were printed in horizontal, vertical, and diagonal orientation using Xerox ColorQube 8580/DN color printer on mPET without any prior pretreatment of the substrate. The wax patterned substrates were then heated on a hot plate between two glass slides at 80 °C, 120 °C, and 160 °C for 10 minutes followed by microscopic characterization. Glass slides were used to prevent curling of the substrate during heating, which may cause non-uniform heating of the pattern.

All wax micropatterned samples were characterized by optical and scanning electron microscope (SEM). The dimensions of the printed micropatterns were observed and measured on x–y plane of the substrate using Leica Microsystems DM IRB inverted microscope equipped with CCD camera and 10× objective lens. The electron microscopic images of the wax modified substrates in z-axis were obtained by a scanning electron microscope (SEM), Quanta 450 FEG (FEI), located in the Image Center facility at SIUC. Elemental composition of the surfaces were obtained using energy-dispersive X-ray spectroscopy (EDS) mode in SEM.

Water contact angle measurements on mPET and wax printed microwells were performed using Goniometer (Model CAM100, KSV Instruments). Wax printed microwells were also incubated with 500 μl water droplet for 12 hours at 4 °C in a sealed chamber followed by contact angle measurement.

ESC culture for self-renewal or differentiation

For cell culture study, square shaped array of 250 μm × 250 μm microwells were patterned on 1 cm × 1 cm area of the mPET substrate. The micropatterns were sterilized by UV light before cell plating. For experiments, cells were plated on microwells coated both with type I collagen (40 μg ml⁻¹) or fibronectin (5 μg ml⁻¹) and monitored up to 5 days in the presence of Leukaemia Inhibitory Factor (LIF). A transgenic cell line, called OGR1, which expresses EGFP under the Oct3/4 promoter was used in this study.^19,20 Complete ES medium as described previously^19,20 was used in the presence of LIF. In short, ES cell culture medium consisted of high glucose-Dulbecco's modified Eagles medium along with 15% ES-qualified fetal bovine serum, 2 mM L-glutamine, 0.1 mM MEM nonessential amino acid solution, 0.1 mM β-mercaptoethanol, 50 mg ml⁻¹ penicillin–streptomycin, 1 mM sodium pyruvate, and 1000 U ml⁻¹ ESGRO® LIF. Cells were routinely cultured on plates coated with 0.1% gelatin at 37 °C with 5% CO₂. Cell passaging was done every 3 days at a ratio of 1 : 10 using TrypLE™.

Fluorescence microscopy

For quantification of self-renewal or differentiation of mouse ES cells on either Col-I or FN coated microwell surfaces, a transgenic mouse ES cell line, named OGR1, expressing enhanced GFP (EGFP) under the promoter of Oct3/4 (Oct3/4::EGFP) was utilized in this study.²⁰ Differential Interference Contrast (DIC) and corresponding fluorescence images were captured by Leica DMi8 widefield epifluorescence microscope with ORCA-FLASH 4.0 V2+ camera (Hamamatsu Photonics, Japan) and 20× objective at different time points. Statistical analysis of total fluorescence quantification was carried out by Student's t-test.

Results and discussion

Scheme 1 shows the channel and line micropatterns of various widths printed on polyethylene terephthalate-based substrate (mPET) using solid wax printer. Resulting widths were measured under optical microscope. mPET substrate—originally designed for printing electronic inks—is a flexible, translucent, and heat resistant substrate with good adhesion for inks. SEM mapping was performed to estimate the elemental composition of mPET substrate. Since mPET is a multilayer substrate, substantial difference in composition was observed between the surface and the base layers. Fig. S1† shows the abundance of Si (35%) in the surface layer, C (62%) in the base layer, and O in both layers comprising 52% and 27% respectively (Table S1†). This clearly proves that the surface of mPET is hydrophilic due to the presence of substantial amount of polar groups. Moreover, water contact angle measurement verifies the hydrophilicity of the mPET surface with 52° ± 0.7, whereas wax printed on mPET is significantly hydrophobic (114° ± 2.8), as expected.

Scheme 1 Wax printing of channel and line micropatterns of various widths on mPET substrate using solid wax printer. Resulting widths were measured under optical microscope before and after heating the wax patterned mPET on a hot plate.

Fig. 1 shows resulting widths of vertically printed line and channel micropatterns versus printing widths and effect of printing orientation. Previous studies involving solid wax printing on cellulose/nitrocellulose based paper substrates reported resolution of 100 μm for line and 50 μm for channel wax patterns.²¹ However, we found the resulting resolution slightly higher than previous reports, i.e. 120 μm for line (Fig. 1a) and 60 μm for channel patterns (Fig. 1b). This may be attributed to the physical nature of the substrate used. Wax printing involves molten wax ink (maintained at 135 °C) sprayed onto a drum by an inkjet printhead, from where it is transferred (off-set) onto paper substrate. When paper substrate is pressed against the rotating drum, the molten wax ink fuses and solidifies quickly on the cool printing surface.²² Since paper substrates are porous therefore molten wax slightly penetrates into pores and causes less lateral spreading, whereas mPET is a non-porous therefore there is slightly higher spreading of molten wax before solidification. Moreover, we are unable to see perfectly linear relationship between resulting and printing width in contrary to other report²¹ showing perfect linearity after 100 μm width. The imperfect linearity found in this study is rational because these printers are not designed to handle differences at <100 μm level. Fig. 2c and d compare the effect of printing orientation. It is evident that printing in horizontal and diagonal orientation result in irregular boundaries of micropatterns and affecting the resulting widths with a trend of vertical < horizontal < diagonal for ‘line’ while vertical > horizontal > diagonal for ‘channel’ micropattern. This can be rationalized with respect to fixed alignment of nozzles on printhead, i.e. patterns not aligned with nozzles orientation can be expected to have irregular boundaries. Surface of the wax pattern is also affected due to printing orientation as one can easily see highly smooth surface in Fig. 2d for vertically printed patterns while the diagonal printing seems result of irregular fusion of wax droplets.

Fig. 1 Resolution of wax micropatterns and effect of printing orientation. Resulting width versus printing width of vertically printed line (a) and channel (b) micropattern. Effect of printing orientation (vertical, horizontal, and diagonal) on 100 μm wide line and channel micropattern by bar graph (c) and optical microscope (d). ‘L’ and ‘C’ symbolize line and channel.

Fig. 2 Effect of temperature on wax pattern in x–y plane and z-axis. (a) Optical image of line and channel micropattern on mPET at various temperatures. (b) Resulting width of vertically printed 100 μm wide line and channel pattern on mPET at various temperatures. (c) SEM images of the wax layer and (d) wax thicknesses on mPET at various temperatures.

Fig. 2 shows the effect of temperature on x–y plane and z-axis (layer thickness) of wax pattern on mPET substrate. Temperature study on the wax micropattern is essential because potential applications and multilayer fabrication procedures may involve exposure to temperatures higher than melting temperature of wax. All the wax patterned mPET samples were heated at particular temperature sandwiched between two glass slides and cooled down at room temperature before optical measurements. It is evident from the optical images (Fig. 2a) that there is a slight change in substrate color (slightly darker) and micropatterns appear to be slightly wider at ≥120 °C. Nevertheless, integrity of micropatterns was found to be conserved on x–y plane in contrast to paper-based substrates where pattern spreads several folds greater than its actual dimensions and narrow patterns become completely fused. Fig. 2b shows the resulting width of vertically printed 100 μm line and channel pattern at various temperatures. The resulting widths before heating were 225 μm for line and 125 μm for channel, which slightly decreased when temperature was raised to 80 °C. This behavior may be attributed to shrinking allowance property of wax.²³ However, pattern width increased slightly when heated up to 120 °C (wax melting temperature). Moreover, change above the melting temperature was found to be insignificant, i.e. pattern remained in their original shape and dimensions within limits even temperature upto 160 °C, perhaps due to non-porous and non-fibrous nature of the substrate. The impact of temperature on the thicknesses of wax layer (z-axis) was studied using scanning electron microscopy (SEM) as shown in Fig. 2c. SEM images reveals that mPET is a multilayer substrate and confirms its non-porous and non-fibrous structure. Wax layer on top of the substrate at 25 °C and 80 °C is distinguishable due to relatively rough texture as shown by arrow, however, at 120 °C and 160 °C layer becomes significantly smoother and perfectly aligned with the top layer of the substrate. This also seems to fill any cavity within the wax layer that is analogous to thermosetting of viscoelastic materials. Fig. 2d summarizes the results of change in wax thicknesses versus temperature. The wax layer at room temperature was found to be 28.42 ± 0.96 μm. When comparing with Fig. 2b, wax pattern contracted on x–y plane at 80 °C that can be presumed to increase in wax thickness along the z-axis. The assumption was confirmed, as shown in Fig. 2d, by SEM characterization where wax thicknesses increased to 33.05 ± 3.65 μm while there was ∼10 μm decrease in wax thickness at 120 °C, which is in line with the result in Fig. 2b. Further rise in temperature could not cause significant difference from the effect of 120 °C. Thus, temperature study concludes that wax micropatterns on mPET substrates can survive at higher temperatures without significant loss in integrity compared to cellulose based paper substrates.

In application, array of square shaped wax printed microwells (250 μm × 250 μm) were patterned on mPET substrate and used to design a suitable niche for mES cell culture either to maintain self-renewal or direct differentiation. There were 110 patterns printed on single sheet of mPET where each pattern occupies 1 cm² area and carries 441 microwells (21 × 21 columns vs. rows). First, hydrophobicity of the wax patterned microwells was tested at room temperature without pretreatment and also mimicking collagen (Col-I) or fibronectin (FN) coating condition, i.e. incubating wax microwells with 500 μl droplet of water for 12 h at 4 °C in a humid chamber. Prior to treatment, water contact angle was found to be 118° ± 4.0 while 105° ± 2.0 after incubation. Decrease in contact angle confirms that the mPET base of the microwells are functionalizable with FN and Col-I despite of the hydrophobic walls. Then, following the UV sterilization, the substrates were coated with either type I collagen (Col-I) or fibronectin (FN) coating and then transferred to standard 6-well cell culture plates. A total of 6000 cells were plated per 6-well cell culture plates. The cell seeding density was adjusted such that single ES cells were placed on each grid (Fig. S2†). Under the aforementioned cell seeding condition, around 75% of the microwells housed single cells, which were monitored for several days. After 3 days or 5 days (Fig. 3c) of cell culture, ES cells exhibited round multicellular morphology (Col-I) or spread colonies (FN). The multicellular round morphology and progressive cell growth, as observed on type I collagen (Fig. 3c), is a classic indicator of self-renewing mES population which started from single cells. In contrast, cells plated on fibronectin coated microwells began to spread out and exhibited slower proliferation rate indicating cell differentiation (Fig. 3c, Fig. S3†). Moreover, no cytotoxic effect was observed from the wax patterns. Initial results showed that cells were also growing in cavities within wax layer, which is solved by smoothening the wax layer at 120 °C before cell seeding.

Fig. 3 (a) Optical image of wax patterned array of square shaped microwells on mPET substrate. After 3 days (b) or 5 days (c) of single ES cell plating either on type I collagen or fibronectin, ES cells exhibit round morphology (Col-I) or spread colony (FN) indicating self-renewal or differentiation respectively. The insets show zoom-in view of the colonies. Enlarged images show colonies present in various microwells. Scale bar, 250 μm.

Type I collagen and fibronectin target different integrin receptors. As such these natural extracellular matrix ligands have been known to be potent regulators of ES cell behavior. The role of such physical cues, mechanical environment, and mechanical forces determining cell fate decisions is very evident.^19,24–27 At present, there is no mechanical niche that can be easily customized and utilized to track single ES cells over time in a high-throughput manner. This study demonstrated the feasibility of utilizing our wax patterned microwells to track ES cell self-renewal or differentiation in a high-throughput fashion. Moreover, printing method used for the study is simpler, inexpensive, and has less footprint than other study used for similar application.²⁸

Conclusion

Wax printing has a real potential for fast, customized, and low-cost fabrication. In order to harness its potential, we presented successful printability, fidelity, and application of wax micropatterns on a non-cellulosic, non-fibrous, and non-porous polyethylene terephthalate (mPET) based substrate. Resolution of wax micropattern on mPET is comparable to paper-based substrate, however, patterns are highly stable at temperatures above the wax melting temperature. As a proof of principle, high density wax patterned platform was built to design a suitable niche for mouse embryonic stem cell (mESC) renewal or differentiation. Stem cells were grown for several days under chemical microenvironment of fibronectin and collagen-I with no sign of toxicity. Our wax patterned high-throughput platform are promising to investigate screening strategies to identify necessary conditions for preclinical applications of pluripotent cells. We anticipate that wax patterning on unique substrates will increase the applications of wax patterning and eventually will reduce the cost and time significantly involved in making such platforms.

Acknowledgements

Authors acknowledge the Elevating Research 2.0 Seed grant from Southern Illinois University Carbondale to make this research possible.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra22422a

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