# Stereolithography 3D printing method for multi-material hydrogel 2D photo-patterning in a microfluidic chip

Sandrine Assié-Souleille, Lionel Séguier, David Gauchard, I. Drobecq, Bernard

Franc, Laurent Malaquin, Julie Foncy

► To cite this version:

Sandrine Assié-Souleille, Lionel Séguier, David Gauchard, I. Drobecq, Bernard Franc, et al., Stereolithography 3D printing method for multi-material hydrogel 2D photo-patterning in a microfluidic chip. Micro and Nano Engineering, 2025, 27, pp.100301. 10.1016/j.mne.2025.100301. hal-05268436

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# Stereolithography 3D printing method for multi-material hydrogel 2D photo-patterning in a microfluidic chip☆

S. Assie-Souleille, L. Seguier, D. Gauchard, I. Drobecq, B. Franc, L. Malaquin, J. Foncy*

LAAS-CNRS, 7 Avenue du Colonel Roche, Toulouse, FR 31400, France

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

- Hydrogel photo-patterning

- 3D printer

- Microfluidic chip

- Stereolithography

- Syringe pump

## ABSTRACT

We present a novel and straightforward method using a standard stereolithography (SLA) 3D printer for high-resolution (20 µm x-y resolution), multi-material 2D hydrogel photo-patterning directly within a microfluidic chip. The process involves sequential injections of photosensitive hydrogel into a transparent microfluidic chip coupled with sequential direct laser writing by the printer through point-by-point photopolymerization. Our approach integrates a custom miniaturized syringe pump system into the SLA printer, thereby enabling fluid management and sequential injection of different photosensitive hydrogels directly into the microfluidic environment between each laser writing sequence. This technique enables the fabrication of intricate, multi-material hydrogel patterns (e.g., PEGDA and HAMA) with high spatial resolution over areas spanning several square millimeters. Future developments will focus on expanding the range of biomaterials and incorporating cell-laden hydrogels to facilitate the creation of biologically relevant microenvironments on chip.

This study opens new possibilities for high-resolution, multi-material hydrogel patterning in microfluidics and offers a valuable platform for advancing research in microsystems engineering.

## 1. Introduction

The integration of biomaterials [1] into microfluidic systems has attracted great interest in recent years, because of the ability to provide physiologically relevant environments for biological studies and diagnostics [2]. Among these materials, hydrogels—highly hydrated polymer networks—are notable for their ability to replicate the biochemical and mechanical properties of the extracellular matrix, and consequently support essential cellular functions such as proliferation, differentiation, and migration. These materials are relevant candidates for creating reactive biosensors [3] in 2D or 3D configurations [4], biomimetic tissue models [5,6], and organ-on-a-chip platforms [7–10], and may enable researchers to study cell behavior [11], screening materials [12], molecular diffusion [13], and tissue morphogenesis under controlled conditions.

Traditional approaches such as extrusion-based printing [14,15] and inkjet printing [16,17] offer the possibility of creating multi-material models but have limited resolution [18]. Laser-assisted techniques such as stereolithography [19,20], digital light processing [21,22], and two-photon lithography [23–26] provide superior resolutions ranging

from 3 µm to 200 nm [18]. In most cases, these techniques are restricted to single-material photopolymerization within a single resin tank, thus making multi-material printing complex and labor-intensive. Current strategies to achieve multi-material patterning often involve sequential immersion of samples in different material baths, and consequently require repetitive development and cleaning steps [27–29]. Recent innovations have provided interesting solutions to automating both the multimaterial injection and rinsing steps in SLA bioprinters [28,30]. For instance, Miller et al. [31] have developed a semi-automated multi-material layer by layer stereolithography printing approach (MMSLA) in which stereolithography is applied to photopolymerize liquid bioinks by using projected light patterns. This method’s multi-material capability relies on an automated bio-ink selection process and a manual rinsing step to avoid unwanted mixing of materials. This approach enables the creation of heterogeneous structures with precise spatial control (50 µm). However, the manufacturing process requires an open phase during the various printing stages.

Han et al. have proposed a multi-material 3D printing approach using projection micro-stereolithography integrated with dynamic fluidic control. Their system enables automated switching between

<footnote>* This article is part of a Special issue entitled: 'NanoBio 2024' published in Micro and Nano Engineering.</footnote>

<footnote>* Corresponding author.
E-mail address: jfoncy@laas.fr (J. Foncy).</footnote>
multiple photocurable resins, thus allowing for high-resolution patterning without manual intervention. This method markedly enhances fabrication speed and material versatility for microscale structures, but is impeded by switching time, residual cross-contamination, and sterility issues in the preparation of subsequent samples [32].

To overcome these limitations, the integration of photopatterning processes directly within microfluidic devices offers substantial advantages. The ability to pattern hydrogels with high resolution and multi-material configurations directly inside a microfluidic chip is a promising but challenging task in the field of biomanufacturing. In most approaches, the hydrogel is printed onto a glass substrate, and the microfluidic chamber is subsequently added on top [33]. However, working in a completely enclosed microfluidic chip offers a major advantage for bioprinting applications requiring sterility. Unlike open-phase printing methods, in which post-print fixation steps can expose samples to contamination, printing directly into a closed microfluidic chip ensures that the entire process can be performed in a sealed, sterile environment. This aspect is particularly important in printing cell-laden hydrogels or handling pathological or sensitive biological samples, because it markedly decreases the risk of contamination while enhancing experimental control and reproducibility. Mayer et al. [34] have developed a multi-material 3D laser printing technique that integrates a microfluidic chamber for the sequential injection of different liquids. This integration enables manufacturing of complex 3D structures composed of multiple materials without interruption for manual changes. However, the reliance of these methods on custom-made or specialized equipment limits their accessibility to researchers with standard tools.

Herein, we present a simple and straightforward approach to high-resolution (20 µm x-y resolution), multi-material 2D hydrogel photopatterning directly within a microfluidic chip, by using a standard stereolithography 3D printer. In contrast to existing methods, our approach does not require specialized equipment but instead uses a commercially available SLA printer enhanced with simple modifications. We integrated the stop-flow lithography technique [35] into a stereolithography 3D printer to achieve sequential injection and photopolymerization of photosensitive hydrogels directly within a transparent microfluidic chip positioned at the bottom of the 3D printer's empty resin tank. We developed and integrated a custom system of three independently controlled syringe pumps within the SLA printer. This compact fluidic system enables precise sequential injection of different photosensitive hydrogels into the chip. This setup, working in tandem with the printer's motorized platform and laser, enables accurate sequential photopolymerization of hydrogel patterns directly within the microfluidic environment. In summary, this process enables the fabrication of multi-material hydrogel 2D structures directly within a microfluidic chip, which can serve as both an observation platform and a cell culture chamber after printing, thus opening new avenues for multi-material bioprinting. By leveraging a standard SLA printer, our method democratizes access to high-resolution, multi-material hydrogel patterning, thereby ensuring the accessibility of this technique to a wide range of researchers.

## 2. Materials and methods

### 2.1. Materials and chemicals

Poly(ethylene glycol) diacrylate (PEGDA) with a molecular weight of 700 g/mol, hyaluronic acid methacrylate (HAMA), lithium-2,4,6-trimethylbenzoylphosphinate (LAP), rhodamine B, 3-methacryloxypropyltrimethoxysilane (MAPTMS), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. Fluorescein dimethacrylate was purchased from Polysciences. Double-sided adhesive tape (85 µm thick) was purchased from Misumi Company (5302 A-50), and the Flexdym™ layer (2 mm thick) was purchased from Eden Tech Company. Glass coverslips measuring 24 × 32 mm were purchased from VWR, and round

slides with a diameter of 15 mm were purchased from Knittel Glass.

### 2.2. Syringe pump fabrication

The syringe pump consisted of three parts that were 3D printed with PLA-CF (0.16 mm slicing, 0.4 mm nozzle, Bambulab) by using fused deposition modeling (BambulabX1C, Bambulab): [1] the main body of the syringe pump, [2] the movable component that secures and controls the movement of the syringe piston, and [3] the syringe holder that keeps the syringe fixed to the pump body. The movable part of the syringe pump moved in a linear motion with a worm screw (M4 with a 0.7 mm pitch), which slid within two parallel stainless-steel rods (5 mm diameter). The rotation of the worm screw was driven by a stepper motor (Joy-it Stepper Motor NEMA11-02 Joy-IT 0.12 Nm 0.67 A, 5 mm shaft diameter (Conrad)) controlled by a microcontroller (Adafruit Feather HUZZAH ESP8266) and associated drivers (A4983 Stepper Motor Driver Carrier (Pololu Robotics & Electronics)), thus enabling communication with the pump control software. All 3D models (STL files), as well as the plans and software required to create and control the syringe pump, are available on Mendeley Data (DOI: 10.17632/tk3m4rvjsp.1).

The syringe pump had dimensions of 28 cm × 5 cm × 4.5 cm and weighed 450 g. This pump was compatible with 1 mL and 5 mL syringe models, and offered a minimum flow rate of 1 µL/min and a maximum flow rate of 100 µL/min for a 1 mL syringe, and a minimum flow rate of 10 µL/min and a maximum flow rate of 3000 µL/min for a 5 mL syringe.

The performance of the syringe pump was characterized with a flow rate sensor and PBS as the reference fluid. The sensor used for this purpose was a Flow Unit Size M (provided by Fluigent SA), which is designed for flow rate measurements in the 0.25 µL/min to 2 mL/min range. This setup allowed us to evaluate the pump's accuracy and stability. The flow rates were recorded and compared with the values for a commercial syringe pump (KDS syringe pump), thereby demonstrating the pump's ability to deliver consistent and precise flow rates under experimental conditions (Supplementary Data SII).

### 2.3. Syringe control software

The custom-built syringe pumps were operated through a Lab-Windows/CVI interface (National Instruments) (Supplementary Data SIII). The software allows users to calibrate the syringe models by defining parameters such as internal diameter (mm), volume (µL), and stroke length (mm), or by selecting a syringe model from the built-in syringe library. In the settings window, users can initialize syringes independently, set the flow rate (µL/min), adjust the acceleration (mm/s²), and define the dispensed volume (µL). The interface includes start and stop buttons to control the sequence. Additionally, users can design automated sequences for sequential injections across the three syringe pumps. All scripts used in this study are available on Mendeley Data (DOI: 10.17632/tk3m4rvjsp.1).

### 2.4. Microfluidic chip fabrication

The microfluidic chip was fabricated with thermal bonding and featured a sandwich-like structure composed of two MAPTMS-treated cover slides (Sigma-Aldrich) separated by double-sided tape (Misumi Company, 5302 A-50, 85 µm thick), to form an enclosed channel. A Flexdym™ layer (2 mm thick) was mounted on top of the structure to facilitate the connection to the microfluidic chip. The inlet and outlet, and the central windows were created with a manual puncher with a 3 mm or 11 mm diameter, respectively. All layers were thermally bonded to create the chip at a temperature of 50 °C for 45 s under a pressure of 0.01 MPa with a Sublym machine from Eden Tech Company. The shape and thickness of the channel (85 µm in our case) were determined by the double-sided tape's shape and thickness. The microfluidic chamber (11 mm diameter) was connected to the inlet and the outlet with
microfluidic channels with a width of 2 mm. The patterning of the double-sided adhesive tape was performed with a xurography Graph-tech Cutting plotter (FC8600-60).

## 2.5. Substrate preparation

The glass cover slips used in the microfluidic chip (24 × 32 mm from VWR) and 15 mm diameter round slides from Knittel glass were treated with MAPTMS to promote hydrogel adhesion during the printing process. The glass cover slips were first cleaned with acetone, deionized water, and ethanol, then dried under a nitrogen stream. The slides were subsequently activated with air plasma treatment for 5 min (Diener PICO, 0.5 mBar, 40 kHz, 50 W). A solution of 1 % MAPTMS, 0.3 % acetic acid in deionized water, and 98.7 % water was prepared. The glass slides were immersed in this solution of MAPTMS for 1 h, rinsed with ethanol, and dried under nitrogen. The glass cover slips were subsequently stored under vacuum until use for microfluidic fabrication.

## 2.6. Hydrogel preparation

Hydrogel solutions were prepared with 60 v/v% PEGDA in PBS and 3 w/w% HAMA in PBS. In both cases, hydrogels were mixed with LAP photo-initiator (0.1 w/w%) diluted in PBS.

## 2.7. Printing workflow /process

The structures were printed with a Dilase 3D Bio (Kloé SA) high-resolution, multi-resolution stereolithography 3D printer equipped with a temperature-controlled tank to maintain a constant temperature during the printing process. The printer was operated with a 405 nm laser (7 mW) and offered a range of laser speeds from 0.1 mm/s to 100 mm/s. The system was equipped with two objectives (10× and 65×) allowing for X and Y resolutions of 10–65 µm, and a Z resolution of 10–100 µm.

Three miniaturized syringe pumps were directly integrated into the printer chamber. The three independent syringes were filled with either hydrogel or PBS, and connected to the microfluidic chip through a microfluidic manifold (Manifold 4 PortsCross 1/4"-28 ETFE, Darwin Microfluidics). The fluidic control software allowed for the sequential injection of different photosensitive hydrogels or rinsing solution (PBS) into the microfluidic chip (at rates of 1 µL/min to 1000 µL/min). Simultaneously, the printer’s motorized platform, in combination with its laser, sequentially photopolymerized the hydrogel patterns inside the microfluidic chip. Sequential injections and the printing phase were coordinated manually.

The printing parameters were set as follows: for PEGDA, 2 % laser power and speed of 2 mm/s, corresponding to a dose of 7.6 mJ/mm²; for HAMA, 100 % laser power and a speed of 1 mm/s, corresponding to a dose of 755 mJ/mm².

## 2.8. Imaging

Bright-field images were taken with an Olympus microscope, and fluorescence images were taken with a confocal Leica SP8 microscope. Images were analyzed in ImageJ software.

# 3. Results and discussion

## 3.1. Description of the setup for multi-material hydrogel 2D photo-patterning in a microfluidic chip

We developed an integrated system compatible with a standard SLA 3D printer to achieve high-resolution, multi-material hydrogel patterning. The setup includes a transparent microfluidic chip placed at the bottom of the printer’s empty resin tank and a how-made compact module with three independently controlled syringe pumps for

sequential injection of photosensitive hydrogels.

This approach leveraged the printer’s motorized platform and laser for precise photopolymerization directly within the microfluidic environment, and required only minor modifications to the printer. Below, we present the system design and demonstrate its ability to create intricate multi-material hydrogel patterns. We developed and integrated three custom-made miniaturized homemade syringe pump systems into the printer, equipped with fluidic monitoring (Fig. 1). The original substrate holder of the printer was removed during these experiments, and the syringe pumps were vertically mounted on the printing stage. The system integrated three independent syringes (red, blue, and green in Fig. 1) containing either hydrogel or PBS, connected to a microfluidic chip via four microfluidic manifold ports. The syringe pumps were controlled by dedicated software enabling precise fluid management and the sequential injection of various photosensitive hydrogels into the chip. The custom-made microfluidic chip was fully transparent and was placed on the printer stage above the printer’s optical objectives (Fig. 1). The fluidic system allowed for the sequential injection of different photosensitive hydrogels into the chip, whereas the printer’s motorized platform, in combination with the laser, photopolymerized the hydrogel patterns within the microfluidic chip (Fig. 3).

## 3.2. Description of the glass-glass microfluidic chip

The microfluidic chip, fabricated with thermal bonding custom, consisted of two glass cover slips separated by double-sided adhesive tape (Fig. 2I). The shape and thickness of the channel (8.5 µm herein) were defined by the geometry and thickness of the double-sided tape (Fig 2II and 2III). This design offers several advantages: injection tests demonstrated the absence of leaks up to at least 1 bar, thus confirming the reliability of the design under pressure conditions; moreover, the design was made from biocompatible materials and provided complete transparency. The glass-to-glass observation area’s accessibility for both upright and inverted microscopes facilitated imaging (Fig 2III).

## 3.3. Multi-material hydrogel photo-patterning in a microfluidic chip

The process began with the injection of the first material, a photosensitive hydrogel from syringe no. 1 (red in Fig. 3I), into the microfluidic chip. The laser from the 3D printer was then used to

<figure><img src="image_4.png" /><figcaption>Fig. 1. 3D illustration of integration of the microfluidic setup into the 3D printer. Three miniature syringe pumps (no. 1 with the red syringe, no. 2 with the blue syringe, and no. 3 with the green syringe) are positioned vertically in the printer, thus supplying a transparent microfluidic chip via a four-port manifold. The system enables sequential injection of various photosensitive hydrogels, while the printer’s motorized platform and laser (405 nm/7 mW) photopolymerize the patterns within the chip. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Glass-glass microfluidic development chip. I Thermal bonding process. II Photograph of the microfluidic chip (top view). III, Microfluidic chip cross-section showing the Flexdym (dark gray), double sided tape (brown), MAPTMS cover slip (light gray), and inlet and outlet ports. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

photopolymerize the desired pattern within the chip (Fig 3II). The chip was subsequently flushed and rinsed with PBS (blue in Fig 3III) from syringe no. 2. The rinsing step was crucial in ensuring complete removal of unpolymerized hydrogel from the microfluidic chip while also cleaning the microfluidic tubing and channels with PBS. To achieve these objectives, we flushed the system with 1 mL PBS, which corresponded to approximately five times the chamber volume, at a controlled flow rate of 500 µL/min. This controlled rinsing protocol administering 5 times the volume of the microfluidic chip minimized the risk of cross-contamination between materials. These conditions were chosen to prevent potential effects of shear stress on the adhesion and integrity of the polymerized structures. Proper orientation of printed hydrogel structures with the fluid flow direction is critical for achieving effective rinsing. When hollow or open structures are oriented perpendicularly to the flow, residual hydrogel can remain trapped, thus resulting in incomplete development, structural defects, or cross-contamination between materials. Controlling this mechanism is critical for optimizing the design of microfluidic chips to ensure high-quality, multi-material printing (simplified explanation in supplementary data SIV).

Subsequently, the second photosensitive hydrogel was delivered from syringe no. 3 (green in Fig 3IV) into the chip and photopolymerized in the next step (Fig. 3V). Finally, the chip was flushed and rinsed with PBS once more to remove excess material, thus resulting in the finalized multi-material hydrogel pattern (Fig 3VI). No additional post processing steps were necessary after the final rinsing in PBS. As illustrated in the cross-sectional view after printing (Fig. 3VII), the hydrogel was fully polymerized from the bottom to the top of the channel and covalently bonded to the cover slip, because of the MAPTMS treatment.

### 3.4. Multi-material 2D structures

We optimized the fabrication processes for different natural and synthetic hydrogel (PEGDA and HAMA) solutions associated with photoinitiators (e.g., LAP) (Fig. 4). Our first validations were performed by printing a double dot matrix with a diameter of 20 µm to demonstrate the technique's resolution capabilities. We sequentially printed 60 % PEGDA with fluorescein diacrylate and 3 % HAMA with rhodamine diacrylate, by using doses of 7.6 mJ/mm² for PEGDA and 755 mJ/mm² for HAMA. The optimal printing conditions were determined by balancing the printing speed and laser power to achieve adequate resolution, as established during a preliminary calibration step, in which a

test pattern was printed with a from 0.5 to 800 mJ/mm² dose range. The dose was adjusted by modification of specific printing parameters, particularly the speed (0.03 to 50 mm/s) and laser power (1 % to 100 %) at 405 nm (7.55 kW/cm²). Fluorescence confocal imaging was then used for observation (Fig. 4.I.a). Our results indicated that XY resolution was determined primarily by the laser spot size (10 µm) and the printability of the hydrogel.

We first demonstrated multi-material printing capabilities by producing intricate structures (Figs. 4.I.b and 4.I.c). Although the 3D printer lacked an alignment system, the **microfluidic chip remained fixed throughout the process**, thus enabling the integration of both materials within the same pattern through sequential printing, by ensuring that the **X and Y coordinates of both patterns precisely aligned to maintain contact**.

In this process, the thickness of the structure was defined by the distance between cover slips, which was determined by the thickness of the double-sided adhesive tape. The hydrogel fully polymerized from the bottom to the top of the channel and was covalently bonded to the cover slip, as a result of the MAPTMS treatment. As shown in Fig. 4.II, the pattern height was adjusted by modification of the channel height. For example, we successfully printed structures with heights of 85 µm (Fig. 4.II.a), 170 µm (Fig. 4.II.b), and 255 µm (Fig. 4.II.c) in a single print.

Moreover, the large surface printing capability of the printer (up to 4 cm²) enabled the creation of multi-material patterns over areas exceeding several mm² (Fig. 4III).

To evaluate the reproducibility of the printing process, we analyzed the dimensions of multiple printed patterns (N = 10) under identical experimental conditions (PEGDA dose: 7.6 mJ/mm², HAMA dose: 755 mJ/mm²). The results demonstrated high replicability, with minimal variation across different prints (Fig. 4.IV). The measured standard deviation of feature dimensions was 2 µm for the smallest feature (20 µm circular pattern diameter) and 20 µm for the largest feature (700 µm structure), corresponding to a variation of less than 10 %, thus highlighting the robustness of the process.

To assess the accuracy of the printed structures, we compared their dimensions in the X-Y plane to the original CAD design. The average deviation between the expected and actual printed dimensions was 2 µm for the smallest feature (20 µm circular pattern diameter) and 14 µm for the largest feature (700 µm structure). These findings confirmed that our approach ensured reliable patterning with precise control of feature size (less than 10 % variation). For the circular pattern, we observed better
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Multi-material printing workflow. I. Injection of the 1st material. II. 2D laser writing by SLA. III. Flushing and rinsing (PBS). IV. Injection of the 2nd material. V. 2D printing by SLA. VI. Flushing and rinsing (PBS). VII. Top view after printing. VIII. Cross-section view.</figcaption></figure>

accuracy in the printed structures compare to the CAD design when PEGDA (20 µm ± 1.7 µm) rather than HAMA hydrogel (22 µm ± 2.7 µm) was used. This variation was explained by the swelling properties of the HAMA hydrogel, as previously described in the literature [36].

### 3.5. Technical limitations and constraints

In this study, we demonstrated the ability of a stereolithography-based 3D printer to achieve high-resolution, multi-material 2D hydrogel photo-patterning within a microfluidic chip. We also outlined the primary technical limitations and constraints of this approach. One key limitation is hydrogel viscosity, which can be challenging because of the thickness of the microfluidic channel. High-viscosity hydrogels may hinder both chip filling and the formation of high-resolution patterns. In our experiments, we used hydrogels with a viscosity similar to that of water to mitigate these issues.

In addition, the orientation of the patterns in the chip was essential. Proper alignment with the flow direction is critical to facilitate the effective removal of unpolymerized material during pattern development.

Finally, printing fully enclosed structures was not possible, because some open space was required to allow the unpolymerized hydrogel to

flow out of the chip. For successful pattern development, the minimal spacing achieved between two adjacent patterns was 20 µm (Fig. 4.I.b). This minimum spacing was closely associated with the hydrogel's viscosity and consequently may vary. Finally, the height of the patterns within the microfluidic chip remained constant and was directly determined by the height of the microfluidic channel.

### 4. Conclusion

In summary, we developed a high-resolution, multi-material 2D hydrogel photo-patterning technique within a microfluidic chip, by using a stereolithography based 3D printer. By integrating custom miniaturized syringe pump systems with an SLA printer, we achieved fluid management and sequential injection of various photosensitive hydrogels into the microfluidic chip. Combined with the high-resolution point-by-point printing capability of SLA, this setup allows for the creation of complex multi-material patterns over large areas.

Despite certain limitations, such as hydrogel viscosity and the inability to print fully enclosed structures, our results demonstrate this technique's adaptability to a wide range of material combinations and patterns. Future work will be aimed at expanding the variety of compatible materials and exploring bioprinting applications by
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Multi-material hydrogel 2D patterning. I. Fluorescence images of 2D patterns composed of 60 % PEGDA labeled with rhodamine (red) and 3 % HAMA labeled with fluorescein (green). II. Fluorescence images (top view and horizontal cross-section) of patterns made with 60 % PEGDA labeled with rhodamine (red) and with varying thicknesses of 85 µm, 170 µm, or 255 µm (II.a, II.b, and II.c, respectively). III. Bright-field images of 60 % PEGDA and 3 % HAMA patterns over large surfaces. IV. Comparison of CAD file dimensions and experimental pattern measurements (N = 10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
incorporating live cells in the hydrogels, thereby creating relevant microbiological systems on chip. An important feature of our approach is that the printed patterns are covalently bonded to both the top and bottom surfaces of the chip through direct photopolymerization, thereby ensuring strong integration of the patterns into the chip, and preventing undesired fluid flow beneath or above the structures. This robust anchoring is particularly advantageous for applications such as circulating cell capture with hydrogel patches, or quantitative diffusion measurements (e.g., by FRAP), in which precise localization and structural stability are essential.

Furthermore, our closed-chip configuration offers a major advantage for bioprinting applications requiring sterility. Unlike open-phase printing methods, in which post-printing binding steps may expose samples to contamination, our method ensures that the entire process can be performed in a sealed, sterile environment. This aspect is particularly important in printing with cell-laden hydrogels or manipulating pathological or sensitive biological samples, because it substantially decreases contamination risk while enhancing experimental control and reproducibility.

Overall, this study provides new opportunities for high-resolution hydrogel patterning, by offering promising advancements in the development of multi-material systems within microfluidic environments and bioprinting with cell-laden hydrogels.

## Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, we used ChatGPT to check grammar and spelling, and improve the readability and language of the manuscript. After using this tool/service, we reviewed and edited the content as needed, and we take full responsibility for the content of the published article.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We acknowledge Julie Batut (Centre de Biologie Intégrative, Toulouse, FRANCE), Renaud Coste (Kloé company, Montpelier, FRANCE), and Pierre Blanchet (Kloé company, Montpelier, FRANCE) for their support in development. This work was partly supported by the LAAS-CNRS characterization platform and FabLAAS; the MultiFAB project funded by FEDER European Regional Funds and French Region Occitanie (16007407/MP0011594); the French National Agency for research (ANR Printiss, ANR21CE1941, ANR PRISM ANR22CE18002001); and Toulouse University (Projet Tremplin AO).

## Data availability

No data was used for the research described in the article.

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