PVA-Coated 3D-Printed Molds for Rapid Prototyping of PDMS Microdevices for Stem Cell Culture

doi:10.21203/rs.3.rs-8258207/v1

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PVA-Coated 3D-Printed Molds for Rapid Prototyping of PDMS Microdevices for Stem Cell Culture

https://doi.org/10.21203/rs.3.rs-8258207/v1

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3D printing has emerged as a versatile approach for fabricating molds used in PDMS-based microdevices. However, residual photoinitiators from photocurable resins often inhibit PDMS curing, while layer-by-layer printing induces surface roughness that hampers device performance in cell culture applications. Here, we present a facile surface modification strategy using PVA dip coating to enhance the compatibility of 3D-printed molds with PDMS replication. Systematic characterization revealed that PVA concentration governs film viscosity and thickness, with the 3–18% (w/w) range yielding uniform and reproducible coatings. PVA-coated molds effectively suppressed PDMS curing inhibition and reduced surface roughness by up to 80%, enabling high-fidelity replication of microstructures. Furthermore, PDMS microwell arrays fabricated from PVA-coated molds supported efficient and uniform embryoid body (EB) formation from human iPS cells, with a significantly increased frequency of single EB per well compared to uncoated molds. These findings demonstrate that PVA coating provides a facile, biocompatible, and geometry-preserving post-treatment to overcome key limitations of 3D-printed molds. The proposed method offers a robust and accessible pathway for the rapid prototyping of PDMS-based microdevices for stem cell culture and broader biomedical applications.

3D-Printing

PDMS

Polyvinyl alcohol

3D culture

iPS cell

Embryoid Body

Bio-microfluidic devices incorporating microscale features ranging from tens to hundreds of micrometers offer precise control over fluid dynamics and cell culture conditions within confined environments. These capabilities facilitate the recreation of physiologically relevant microenvironments and enable high-sensitivity biological analyses, finding broad utility in applications such as cell culture, tissue modeling, and diagnostics [1–6]. In recent years, the growing accessibility and resolution of 3D printing technologies have driven increased interest in their use for microfluidic device fabrication. Two predominant approaches have emerged: (i) direct fabrication of microfluidic structures via 3D printing, and (ii) indirect fabrication, wherein a 3D-printed mold is used to replicate structures in polydimethylsiloxane (PDMS).

While direct printing offers design flexibility and rapid prototyping, it is often hindered by challenges such as cytotoxicity of photopolymer resins and autofluorescence that interferes with optical imaging [7–9]. In contrast, PDMS, commonly used in indirect molding, is highly suitable for biological applications due to its biocompatibility, optical transparency, gas permeability, and low autofluorescence [10, 11]. Accordingly, indirect molding using 3D-printed molds has gained prominence for cell culture applications. However, challenges remain in optimizing the mold surface to ensure compatibility with PDMS curing and cell experiments.

Photopolymerization-based 3D printing, including stereolithography (SLA), constructs complex three-dimensional structures layer-by-layer from 3D-CAD data. Although conventional photolithography remains the gold standard for microfabrication, it requires cleanroom facilities, expensive equipment, and trained personnel, and is limited in geometrical complexity. In contrast, SLA-based desktop 3D printers offer low-cost, rapid fabrication of intricate designs, democratizing access to microfabrication by enabling reproducibility through shareable digital files. While high-resolution systems such as two-photon polymerization can achieve submicron precision [12], their high cost and low throughput limit their utility for routine biological experimentation. SLA printers thus strike a practical balance between resolution, speed, and cost, making them attractive for fabricating cell culture devices [13].

Nevertheless, 3D-printed molds present two key issues that limit their integration with PDMS: (i) inhibition of PDMS curing due to residual photoinitiators and additives leaching from the resin, and (ii) anisotropic surface roughness, particularly vertical stepping artifacts from the layer-by-layer process, which can influence cellular behavior [14]. To address curing inhibition, various resin-specific post-treatments and surface coatings such as parylene deposition have been explored [15, 16]. Meanwhile, the mitigation of surface roughness—especially in the Z-direction—has garnered attention for its importance in cell-microenvironment interactions [17].

In this study, we introduce a surface modification strategy for 3D-printed molds using polyvinyl alcohol (PVA) dip coating. PVA, a water-soluble, biodegradable, and non-toxic polymer derived from the hydrolysis of polyvinyl acetate, is widely employed in biomedical, packaging, and electronic industries due to its excellent film-forming properties, strong adhesion, and compatibility with biological systems [18–24]. By dip-coating 3D-printed molds with PVA, we aim to simultaneously suppress PDMS curing inhibition and reduce surface roughness (Fig. 1). The PVA film serves as a physical barrier, preventing direct contact between PDMS and the inhibitory resin surface, while also filling micro-grooves generated during printing to enhance smoothness.

Dip coating is a widely utilized thin-film deposition technique, wherein a substrate is immersed in a coating solution and withdrawn vertically at a controlled rate, followed by solvent evaporation or film curing. The film thickness obtained can be predicted by classical models such as the Landau–Levich–Derjaguin equation, which relates film thickness to surface tension, viscosity, density, gravitational acceleration, and withdrawal speed [25–29]. Importantly, the water solubility of PVA allows for facile removal after PDMS molding, ensuring the native surface properties of PDMS are retained, which is useful for biological applications.

We first characterize the relationship between PVA concentration, viscosity, and resulting film thickness, then assess the effectiveness of the coating in mitigating PDMS curing inhibition and improving mold surface smoothness. To validate the utility of this method for biological applications, we fabricate PDMS-based microwell arrays using PVA-coated 3D-printed molds and evaluate their performance in the formation of embryoid bodies (EBs) from induced pluripotent stem (iPS) cells.

iPS cells are stem cells established by reprogramming somatic cells and characterized by their capacity for self-renewal and pluripotency to differentiate into the three primary germ layers—ectoderm, mesoderm, and endoderm. iPS cell technology offers significant advantages over ES cells, such as overcoming ethical concerns and avoiding immune rejection through autologous transplantation. Consequently, iPS cells are highly anticipated for applications in regenerative medicine, drug screening, and disease modeling [30–33]. The formation of EBs is a widely employed intermediate step for directing the differentiation of iPS cells into specific cell types. EBs are 3D aggregates formed by self-organization of iPS cells, providing an in vitro environment that recapitulates early embryogenesis. Therefore, this process is a critical stage in efficient differentiation protocols for obtaining target cells and organoids [34–36]. Uniform EB formation is critical for reproducibility, and microwell-based techniques—wherein cells sediment and aggregate in confined wells—have proven effective for generating homogeneous EBs [37–39].

In this study, we demonstrate the fabrication of such microwell structures using our PVA-coated mold strategy and evaluate their performance in EB formation, thereby underscoring the potential of this approach to enhance the utility of 3D-printed molds in microfluidic and cell culture applications.

2.1 Characterization of PVA coating

Polyvinyl alcohol (PVA; molecular weight: 31,000–50,000, Sigma-Aldrich, 363138) was dissolved in deionized water at elevated temperatures to prepare solutions at concentrations ranging from 3% to 30% (w/w). The viscosity of each PVA solution was measured at 60–65°C using a vibration viscometer (VM-10A-L, Toki Sangyo), maintained in a temperature-controlled water bath.

For dip-coating, glass substrates (76 × 26 mm, thickness: 0.8–1.0 mm, Matsunami Glass) were immersed in PVA solutions at room temperature for 10 minutes and then vertically withdrawn at a constant speed of ~ 15 mm/s. After withdrawal, substrates were left to stand for 10 minutes to allow drainage of excess solution, followed by drying in a convection oven (WFO-420, EYELA) at 80°C for 24 hours. The thickness of the dried PVA films was measured using a contact-type stylus profiler (DektakXT-S, Bruker AXS). For each condition, three glass substrates were coated, and the film thickness was measured at consistent locations to determine the mean and standard deviation, detailed in Supplementary information (Fig. S1 and S2).

2.2 Evaluation of PDMS curing inhibition

Molds were fabricated using a digital light processing (DLP) 3D printer (Saturn 3 Ultra, Elegoo), equipped with a 12K monochrome LCD (11,520 × 5120 pixels), providing an XY resolution of 19 × 24 µm and a minimum Z-axis resolution of 10 µm. Layer thickness was set between 10 and 200 µm. Slicing was performed using Formware 3D (FormWare), and printing was conducted using 8K Standard Resin (Space Grey, Elegoo). During the layer-by-layer printing process, photopolymerization of the resin induced by UV exposure served as the first UV-curing step. The mold design consisted of linear arrays of square cross-sections (250–1500 µm width) to evaluate the replication fidelity of PDMS structures (Fig. S3 in Supplementary information).

Post-printing, molds were detached from the build platform, and support structures were removed. Molds were washed in isopropyl alcohol (IPA) using a Form Wash unit (Formlabs) for 10 minutes, followed by air drying at room temperature. UV post-curing was performed using Form Cure (Formlabs) at 60°C for 15 minutes. In select conditions, a second UV-curing step was omitted to intentionally retain uncured resin components as a negative control for curing inhibition assessment.

For PVA treatment, 15% (w/w) aqueous PVA solution was poured over the mold surface, incubated for 10 minutes at room temperature, and the excess was drained. Molds were subsequently dried at 80°C for 24 hours. PDMS (base:curing agent = 10:1, w/w) was mixed using a planetary centrifugal mixer (AR-100, THINKY), with 1-minute mixing and 1-minute degassing. The PDMS mixture was poured into the coated molds, degassed again, and cured at 80°C for 24 hours.

After curing, PDMS replicas were demolded and immersed in a 50°C water bath for 10 minutes, followed by ultrasonic cleaning (CPX3800H-J, Branson) for 30 minutes to remove residual PVA. Surface morphology was evaluated using scanning electron microscopy (SEM; JSM-IT800, JEOL), and dimensional analysis was performed using ImageJ. Feature depth and width of the PDMS replicas were measured according to the method described in [15]. To obtain cross-sectional specimens, the mold structures were cut using a power scroll saw, while the PDMS samples were cut with a utility knife. Cross-sectional areas of mold features and their PDMS counterparts were calculated to determine replication accuracy (Figs. S4 and S5 in Supplementary information).

2.3 Measurement of surface roughness

Mold fabrication followed the same protocol as Section 2.2. To characterize vertical surface roughness, molds were printed at a 45° tilt relative to the build platform (Fig. S6). We also tested the molds printed with 30° tilt angle, which are detailed in Supplementary information (Figs. S6 and S7). Following post-curing, dip-coating was conducted using PVA solutions at concentrations of 6%, 15%, and 30% (w/w). As a negative control, molds were dipped in deionized water without PVA.

PDMS was cast and cured as described previously. After demolding, residual PVA was removed via ultrasonic cleaning. Surface profile of the PDMS replicas was measured using a stylus profilometer (DektakXT-S, Bruker AXS). Measurements were performed over 10 mm scan lengths perpendicular to the layer direction, with 15 random locations per sample. The arithmetic average surface roughness (R_a) was calculated according to the standard method [40].

2.4 Culturing iPS cell-derived embryoid bodies

Microwell structures were fabricated based on our previously developed EB culture platform (EBCP, Fig. S9 in Supplementary information) [41], consisting of 5 × 5 well arrays. Molds were printed using a Form 3 SLA printer (Formlabs) with Clear Photopolymer Resin. Post-printing steps included IPA washing for 20 minutes, support removal, UV post-curing at 60°C for 20 minutes, and thermal drying at 60°C for 12 hours. Surface hydrophilization was performed using a UV–ozone cleaner (UV253V8, Filgen) with 10 minutes of oxygen flow and 30 minutes of UV exposure.

To improve single-EB formation efficiency, molds were dip-coated with aqueous PVA solutions at concentrations of 3–18% (w/w). Molds were immersed for 15 minutes, then inverted and allowed to drain naturally for 30 minutes at room temperature. They were subsequently dried at 60°C for 12 hours. As controls, molds were soaked in pure water instead of PVA solution.

PDMS was poured into the molds and cured as described above. After demolding, devices were sterilized by autoclaving and dried at 80°C for 3 hours. Surface hydrophilization was repeated using UV–ozone treatment. To render the PDMS surfaces non-cell-adhesive, devices were coated with Pluronic F-127 (P2443-250G, Sigma-Aldrich), a block copolymer of hydrophilic polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO). The PEO segments inhibit protein adsorption and integrin-mediated cell adhesion [42, 43]. This surface modification has been widely employed in stem cell research [44–49].

Devices were immersed in 2% (w/v) Pluronic F-127 in PBS overnight, a concentration known to modulate adsorption without altering coating thickness [50]. Prior to cell seeding, devices were rinsed thoroughly with PBS and filled with mTeSR Plus Basal Medium (STEMCELL Technologies).

Human iPS cells were dissociated into single cells using 0.5× TrypLE Select (12563011, Gibco™, Thermo Fisher), resuspended in medium, and seeded into the microwells at a density of 2.0 × 10³ cells/well. Y-27632 (ROCK inhibitor, APExBIO) was added at a final concentration of 10 µM to enhance cell viability. EB formation was monitored at 36 and 84 hours post-seeding using an inverted microscope (CKX53, OLYMPUS) equipped with a digital camera (ILCE-7SM2, SONY). EB formation efficiency and diameters were quantified using ImageJ.

3.1 PVA thin film formation

To evaluate the characteristics of PVA films prepared via dip coating, the relationships among PVA concentration, solution viscosity, and resulting film thickness were analyzed (Fig. 2). As expected, solution viscosity increased exponentially with increasing PVA concentration (Fig. 2a), consistent with polymer chain entanglement at higher concentrations. Correspondingly, film thickness also increased with PVA concentration (Fig. 2b). Within the concentration range of 3–18% (w/w), film formation was stable and reproducible. However, concentrations exceeding 21% resulted in markedly increased variability, with a maximum thickness of 61.0 µm observed at 30% PVA.

This sharp increase in both thickness and variability at high concentrations can be attributed to enhanced polymer chain entanglement and the corresponding increase in solution retention during substrate withdrawal. Additionally, elevated viscosity hinders uniform drainage of excess solution, leading to localized accumulation and reduced film uniformity—an effect widely reported in dip-coating studies [51]. In contrast, the 3–18% range offers a favorable balance of viscosity and surface wetting, yielding uniform films with good reproducibility [52]. Thus, this range is considered optimal for forming controllable and homogeneous PVA coatings via dip coating.

To further analyze the relationship between viscosity and dried film thickness, the data were fitted to the empirical model Eq. (1):

$$\:\begin{array}{c}h=a\cdot\:{\eta\:}^{\frac{1}{2}}\#\left(1\right)\end{array}$$

where h, η, and a are the film thickness, solution viscosity, and a fitting constant, respectively (Fig. 2c). This square-root dependence deviates from the classical Landau–Levich–Derjaguin (LLD) theory, which predicts a viscosity exponent of 2/3 for liquid film thickness immediately after withdrawal [28, 29]. The discrepancy likely arises because the LLD theory applies to wet film thickness, whereas our measurements reflect the final dried solid film. Factors such as solvent evaporation, polymer shrinkage during drying, and the non-Newtonian behavior of concentrated polymer solutions can contribute to this deviation. Indeed, similar square-root dependencies have been experimentally reported by Cisneros-Zevallos et al. [51] and Snoeijer et al. [53]. Zhang et al. have also pointed out significant differences between theoretical and actual dried film thicknesses [54]. These findings support the validity of our empirical model and underscore the importance of accounting for post-deposition dynamics in polymer film formation.

3.2 Suppression of PDMS curing inhibition

PDMS replicas demolded from 3D-printed molds under four different surface treatment conditions were evaluated for curing quality and structural fidelity (Fig. 3). The conditions were as follows:

Condition 1: UV post-curing + PVA coating
Condition 2: PVA coating only
Condition 3: UV post-curing only
Condition 4: Untreated control

In Condition 1, no tackiness was observed, and the replication exhibited minimal geometric distortion, indicating complete PDMS curing. Condition 2 also yielded acceptable curing quality, though slight tackiness was sometimes noted. In contrast, Condition 3 (UV only) resulted in significant tackiness and evident structural deformation, particularly in features larger than 750 µm. Condition 4, which involved neither UV treatment nor PVA coating, showed severe inhibition with pronounced collapse of microstructures below 750 µm.

Notably, surface tackiness and feature distortion—such as asymmetrical sidewalls and rounded corners—were prominent in the untreated and UV-only conditions. These defects are consistent with curing inhibition due to the diffusion of uncured acrylate monomers and photoinitiators into PDMS, which are known to deactivate the platinum catalyst required for crosslinking [14–16, 55]. PDMS’s affinity for hydrophobic compounds, including UV resin components, exacerbates this effect [55].

In contrast, PVA-coated molds (Conditions 1 and 2) significantly reduced curing inhibition. PVA is a hydrophilic, semi-crystalline polymer that forms dense films capable of acting as effective diffusion barriers [56]. Moreover, the combination of UV post-curing (which reduces residual unreacted species) and PVA coating (which physically isolates PDMS from resin surfaces) in Condition 1 eliminated inhibition artifacts. These findings demonstrate the synergistic effect of UV curing and PVA coating treatments in suppressing PDMS curing inhibition.

3.3 Surface smoothing of 3D printed molds by PVA coating

To assess the surface smoothing effect of PVA coatings, SEM imaging and surface roughness measurements were performed on PDMS replicas cast from coated molds (Fig. 4). Due to the ~ 45° layer-by-layer printing orientation, uncoated molds exhibited pronounced stair-step artifacts. As PVA concentration increased, these features progressively diminished. At 30% (w/w) PVA, the mold surface became nearly planar, and layer lines were barely discernible (Fig. 4b).

Quantitatively, surface roughness (R_a) decreased significantly with increasing PVA concentration, from 4.14 µm (uncoated) to 0.84 µm at 30% PVA (Fig. 4c), which approaches standards for optical-grade surfaces (R_a < 1 µm) [57]. Even at 15% PVA, roughness was reduced by ~ 57% to 1.76 µm, highlighting the effectiveness of PVA dip coating in mitigating layer-induced artifacts.

This surface smoothing effect is attributed to the self-leveling behavior of viscous PVA solutions during the drying process. Surface tension facilitates the flow of liquid PVA into microgrooves, thereby minimizing topographic variation. However, at very high concentrations (e.g., 30%), excessive viscosity hinders uniform drainage, leading to PVA accumulation at edges and corners. Indeed, 3D scanning revealed rounding and overcoating of sharp features under these conditions (Fig. 4d), resulting in a trade-off between surface smoothness and dimensional fidelity.

Thus, while 30% PVA provides optimal smoothness, 15% PVA offers a more suitable compromise for microfabrication applications requiring both surface quality and geometric accuracy. Future efforts may incorporate localized coating control, masking techniques, or precision withdrawal systems to further refine feature fidelity.

3.4 Application to iPS cell culture for embryoid body formation

To validate the biological compatibility and utility of PVA-coated molds, we fabricated PDMS microwell arrays for EB formation from iPS cells (Fig. 5a). At 36 hours post-seeding, all devices—regardless of PVA concentration—supported EB formation, and no cytotoxic effects were observed (Fig. 5b). The diameter distribution of EBs at 36 hours (Fig. 5c) indicated more uniform and centrally located EBs in the PVA-coated groups (3–18% PVA), typically within 200–280 µm. In contrast, the uncoated group (0% PVA) showed frequent formation of multiple small EBs (50–150 µm) within a single well; upper photograph in Fig. 5(b) shows a typical image.

The number of wells containing single EBs was significantly higher in PVA-coated devices at both 36 and 84 hours of culture, suggesting that smoother well surfaces facilitated cell sedimentation and aggregation at the well center (Fig. 5d). In contrast, surface irregularities from 3D-printed layer lines in uncoated molds may have acted as microscale traps, impeding the formation of unified aggregates and leading to multiple smaller EBs per well. The increase of the number of single EB well from 36 to 84 hours indicates the fusion of these aggregates, resulting in non-spherical morphologies that can compromise reproducibility in differentiation assays.

Compared with commercial 96U-well plates, which yield EBs of ~ 300 µm under the same seeding density (Fig. S10 in Supplementary information), our 3D-printed microwells produced slightly smaller EBs (~ 200–280 µm). This size discrepancy likely reflects incomplete cell sedimentation due to residual roughness or microdefects on the PDMS surface. Nonetheless, the reduced variability and enhanced frequency of single-EB formation in the PVA-coated devices underscore the importance of mold surface smoothness in EB culture reproducibility.

Collectively, these findings confirm that PVA coating provides a non-cytotoxic, geometry-preserving, and performance-enhancing strategy for 3D-printed microdevices used in stem cell culture. By improving surface quality and suppressing curing inhibition, this method represents a robust post-processing approach to bridge the gap between low-cost additive manufacturing and the requirements of bio-microdevice fabrication.

In this study, we established PVA dip coating as an effective strategy to address the two challenges associated with 3D-printed molds for PDMS microdevice fabrication: curing inhibition and surface roughness. By characterizing PVA concentration, we achieved coatings that functioned as a diffusion barrier against resin-derived inhibitors while simultaneously smoothing surface artifacts. This dual function enabled the production of PDMS devices with improved structural fidelity and surface quality, suitable for sensitive biological applications. Importantly, when applied to the fabrication of microwell arrays, PVA-coated molds enhanced the reproducibility of iPS cell-derived EB formation, demonstrating both biological compatibility and functional benefits. Taken together, our results highlight PVA coating as a facile post-processing technique that bridges the gap between low-cost additive manufacturing and the demands of cell culture microdevices. This approach paves the way for broader adoption of low-cost desktop 3D printing in biomedical device prototyping, offering an accessible tool for stem cell research, tissue engineering, and lab-on-a-chip technologies.

Author Contribution

K.T. conceived the study. Y.A. and D.F. designed the devices, performed the experiments, and analyzed the data. T.T. and N.F. conducted the iPS cell–derived embryoid body formation experiments and analyzed the results. Y.A. wrote the manuscript. All authors read and approved the submitted manuscript.

Data Availability

All data supporting the findings of this study are included in the main text and the Supplementary Information. The Supplementary Information provides detailed descriptions of PVA coating characterization methods (Figs. S1–S2), mold designs and evaluation procedures for PDMS curing inhibition and surface roughness (Figs. S3–S8), as well as the design of the 5×5 embryoid body culture plate (Fig. S9). Additionally, results of embryoid body formation using a 96-well round-bottom plate are provided (Fig. S10).

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No competing interests reported.

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PVA-Coated 3D-Printed Molds for Rapid Prototyping of PDMS Microdevices for Stem Cell Culture

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Characterization of PVA coating

2.2 Evaluation of PDMS curing inhibition

2.3 Measurement of surface roughness

2.4 Culturing iPS cell-derived embryoid bodies

3 Results and Discussion

3.1 PVA thin film formation

3.2 Suppression of PDMS curing inhibition

3.3 Surface smoothing of 3D printed molds by PVA coating

3.4 Application to iPS cell culture for embryoid body formation

4 Conclusions

Declarations

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

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