3D Hydrodynamic Flow Lithography

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results 3D Hydrodynamic Flow Lithography Yiying Zou , Mehmet Akif Sahin , Muhammad Zia Ullah Khan , Muhammad Usman Akhtar , View ORCID Profile Ghulam Destgeer doi: https://doi.org/10.1101/2025.10.27.684743 Yiying Zou 1 Control and Manipulation of Microscale Living Objects, Center for Translational Cancer Research (TranslaTUM), Munich Institute of Biomedical Engineering (MIBE), Department of Electrical Engineering, School of Computation, Information and Technology (CIT), Technical University of Munich , Einsteinstraße 25, Munich 81675, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mehmet Akif Sahin 1 Control and Manipulation of Microscale Living Objects, Center for Translational Cancer Research (TranslaTUM), Munich Institute of Biomedical Engineering (MIBE), Department of Electrical Engineering, School of Computation, Information and Technology (CIT), Technical University of Munich , Einsteinstraße 25, Munich 81675, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Muhammad Zia Ullah Khan 1 Control and Manipulation of Microscale Living Objects, Center for Translational Cancer Research (TranslaTUM), Munich Institute of Biomedical Engineering (MIBE), Department of Electrical Engineering, School of Computation, Information and Technology (CIT), Technical University of Munich , Einsteinstraße 25, Munich 81675, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Muhammad Usman Akhtar 1 Control and Manipulation of Microscale Living Objects, Center for Translational Cancer Research (TranslaTUM), Munich Institute of Biomedical Engineering (MIBE), Department of Electrical Engineering, School of Computation, Information and Technology (CIT), Technical University of Munich , Einsteinstraße 25, Munich 81675, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ghulam Destgeer 1 Control and Manipulation of Microscale Living Objects, Center for Translational Cancer Research (TranslaTUM), Munich Institute of Biomedical Engineering (MIBE), Department of Electrical Engineering, School of Computation, Information and Technology (CIT), Technical University of Munich , Einsteinstraße 25, Munich 81675, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ghulam Destgeer For correspondence: ghulam.destgeer{at}tum.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Continuous- and stop-flow lithography has been widely used for the fabrication of ingenious multi-dimensional microstructures, including fibers and microparticles. The flow profile of multiple co-flowing streams during the process, as one of the dimensions, dictates the cross-sectional morphology of the microstructures. Here, we introduce a three-dimensional hydrodynamic flow lithography (3D HFL) approach that enables rapid and programmable generation of desired flow profiles and their conversion into solid microstructures. By strategically combining multiple flow sculpting strategies, including variable inlet configurations, intra-channel pillar configurations, and outlet configurations, we achieved precise and versatile control over flow sculpting, as validated by computational fluid dynamics simulations and experimental production of microparticles and fibers, offering unparalleled design flexibility. Importantly, the flow sculpting device is fabricated using low-cost 3D-printed molds to cast PDMS channels with precisely aligned inlets and complex geometries that are difficult or impossible to achieve using conventional soft lithography. Our 3D HFL approach effectively overcomes the limitations of existing microfabrication techniques in device complexity, structural diversity, multi-material integration, and production throughput. Furthermore, we demonstrated the capability of this platform to produce anisotropic multi-material microparticles and fibers tailored for specific applications, such as amphiphilic particles for uniform microdroplet capture, biocompatible patterned hydrogels for controlled cell adhesion, and dual-layer fibers for temperature sensing. The simplicity of device fabrication, combined with the broad design flexibility, establishes this platform as a scalable, high-throughput, and versatile solution for engineering customized anisotropic microparticles and fibers, opening new avenues in various applications. 1. Introduction The functionalities of microstructures, e.g., microparticles and fibers, are fundamentally governed by their size, structure, and composition 1 – 4 . These physical attributes enable a wide range of specialized uses. For example, shape- or color-barcoded microparticles have been successfully employed as multiplexed bioassay platforms for allowing simultaneous detection of multiple analytes 5 – 7 . In parallel, biocompatible spherical or crescent-shaped particles 8 – 10 , as well as microfibers 11 , have emerged as ideal scaffolds for culture and analysis, providing well-controlled microenvironments for cell-level studies. Beyond static applications, microparticles also serve dynamic roles in micro-robotics. The magnetic, optical, and acoustic actuation capabilities of micro-robots typically stem from either responsive material compositions 12 , 13 or tailored geometries 14 , 15 , which facilitate deformation, rotation, or translational motion. These design strategies have demonstrated significant promise in biomedical applications requiring in vivo navigation, such as targeted drug delivery and minimally invasive microsurgery 16 , 17 . These diverse applications underscore the critical need for fabrication techniques capable of reliably producing functional microparticles and fibers with precisely tunable sizes, shapes, and compositions. Comprising continuous-flow lithography (CFL) 18 and stop-flow lithography (SFL) 19 , flow lithography (FL) 20 has emerged as a particularly versatile technique for generating these microstructures with a wide range of functionalities. Early implementations of SFL allowed the creation of 2D microparticles through the modulation of 2D photomask patterns, yielding geometrically simple but functionally robust architectures 21 , 22 . Innovations such as phase-mask interference 23 , two-step exposure 24 , non-uniform UV exposure or absorption 25 – 27 , and intra-channel flow restrictions 28 expanded this capability to 2.5D structures with controlled depth-wise heterogeneity. Additional approaches, including soft membrane 29 and vertical 30 flow lithography, have pushed the boundaries toward true 3D structuring. However, these methods often depend on precise, multi-step photomask alignment and are typically limited by moderate throughput and compositional flexibility. A promising alternative for fabricating 3D structured microparticles emerges in intersecting the 2D UV pattern projected from the photomask with an engineered multi-layered flow profile within microfluidic channels. Pioneering work by the Doyle group established hydrodynamic focusing lithography 31 , which employed bilayer polydimethylsiloxane (PDMS) microchannels to generate a dual-axis, multi-layered flow profile. However, despite its strengths, the technique is constrained by its reliance on relatively basic channel geometries and intricate alignment steps for multilayered microchannel fabrication, making it challenging to produce more sophisticated flow profile designs. Subsequently, the Di Carlo group developed an inertial flow sculpting method 32 , which employs single-layered PDMS microchannels embedded with pillar structures to generate secondary Dean flows at high Reynolds ( Re ) numbers, thereby dramatically expanding the diversity of achievable flow profiles 33 – 38 . However, the need for high-pressure inertial flow at Re ∼O (10) or higher, along with extended pillar sequences within long microfluidic channels to achieve the desired flow profiles, can pose significant challenges. More recently, we have demonstrated 3D-printed microfluidic devices with customizable nozzles to sculpt a coaxial flow profile at low Re ∼O (1) or smaller to produce a wide variety of shape-encoded, multi-material particles 6 , 39 – 42 . This approach expands the design space through the flexibility of 3D printing but relies on oxygen-impermeable channels, necessitating inert sheath flows to prevent clogging, which is not an issue with conventional PDMS-based microchannels used in hydrodynamic and inertial flow sculpting methods. We summarize the features of conventional SFL-based particle fabrication techniques for comparison in the supporting information ( Table S1 ). To address these trade-offs, we envision a hybrid strategy that combines the oxygen permeability of PDMS microchannels with the design flexibility of 3D printing, aiming to enable scalable, sheath-free fabrication of complex microparticles and fibers 43 with enhanced structural tunability and functional versatility. View this table: View inline View popup Table S1. Summary of representative SFL approaches for anisotropic microparticle fabrication. In this work, we present a 3D hydrodynamic flow lithography (3D HFL) method ( Figure 1 ) for producing multi-material, anisotropic microparticles and fibers with engineered shapes and tunable sizes. We use an oxygen-permeable 3D PDMS microfluidic channel with multiple inlets (up to five) converging into a single outlet (∼100–500 μm) to photo-cure particles under a cyclic UV exposure through a photomask (SFL, >10 4 particles/hour) or long fibers under a continuous bulk UV exposure (CFL), without the need for an inert sheath flow stream ( Figure 1A ). For a precise positioning of the inlet ports within the main channel, we devised a new cleanroom-free microchannel fabrication workflow. We 3D-printed the low-cost channel molds with integrated pillars that form the inlet ports within the PDMS channel after the mold replica step. This hybrid device fabrication method, combining 3D printing and soft lithography, enables us to produce microfluidic devices with various inlets, pillars, and outlet configurations to shape multi-layered co-flowing streams into >10 distinct flow profiles, with potential for further expansion. For each microfluidic device, we performed computational fluid dynamics simulations to numerically analyze the sculpted flow profiles before fabricating the desired particles or fibers ( Figure 1B ). The fabricated structured particles and fibers clearly show multiple precursor streams cured into a desired cross-sectional shape and composition ( Figure 1C ). Download figure Open in new tab Figure 1. 3D hydrodynamic flow lithography for producing structured microparticles and fibers. (A) A 3D hydrodynamic flow sculpting device producing multi-layered structured microparticles and fibers upon cyclic and continuous UV exposure of curable polymer precursors, respectively. Square PDMS microchannels with nominal outer diameters of 100, 250, and 500 µm, defined the outer dimensional limit of the cured particles and fibers. (B) Numerical modeling of the sculpted flow profile with five layers (1-5) controlled by the flow rate through the five independent inlets. (C) Brightfield and fluorescent microscopy images of the multilayered structured microparticles and fibers. Top: Five curable streams, transparent (1), red (2), blue (3), red (4), and transparent (5), led to multilayered particles with outer trapezoidal shape matching the channel cross-section. Middle: Three inert (1, 3, 5) and two curable blue (2) and red (4) streams resulted in bi-layered structured particles shaped like the alphabet “A”. Bottom: A continuous UV exposure of a similar flow profile, shaped like the alphabet “A”, produces a structure fiber with a matching cross-section (inset). A bubble trapped within the fiber confirms a hollow cavity. 2. Results and discussion 2.1 Flow sculpting strategies To create structured flow profiles inside the microchannels, we have demonstrated three flow sculpting strategies using variable inlet, intra-channel pillar, and outlet configurations. First, the inlets introduce multiple streams simultaneously or sequentially within the channel at desired locations to form an initial multi-layered flow profile, which then would be further reshaped by the different indented pillar structures inside the main channel. Lastly, the cross-sectional shape and size of the outlet would determine the final cross-sectional outline of the sculpted flow before UV curing. Contrary to the conventional methods that require complex multi-layered PDMS microchannels with manually punched inlet ports and limited flow sculpting possibilities 31 , 44 , we leverage 3D-printed molds methodology to enable highly flexible design and easy fabrication of PDMS microchannels with precisely aligned inlet ports and pillars within the main channel as well as with variable outlet channel cross-sections, allowing multiple streams to be freely stacked and sculpted under these three flow sculpting strategies. 2.1.1 Flow sculpting using variable inlet configurations We have investigated three different inlet configurations to sculpt multiple co-flowing streams ( Figure 2A ). In the first inlet configuration, the diameter ( D i ) of a single circular inlet is equal to the channel width ( W c ), however, for multiple juxtaposed inlets placed at a given YZ-plane, the sum of inlet diameters is still equal to W c , i.e., Σ D i = W c . In the second inlet configuration, an inlet with a relatively smaller diameter with respect to the channel width is placed symmetrically in the middle of the channel or asymmetrically closer to one of the side walls, such that Σ D i W c , is placed symmetrically or asymmetrically over the microchannel width. Multiple inlets, each with any of the three inlet configurations, can be further arranged in series or in parallel formation within the main channel along the X- or Y-axis, respectively. Download figure Open in new tab Figure 2. Flow sculpting using variable inlet configurations. (A) Three different inlet configurations are realized such that the sum of inlet diameters ( Di ) at a given YZ-plane is equal to, smaller than, or larger than the channel width ( Wc ), i.e., Σ Di = Wc , Σ Di Wc , respectively. The inlets can be further arranged along the X- or Y-axis in series, or in parallel configuration, respectively, within the main channel. (B) After the first inlet, any subsequent inlet can be positioned in a symmetric or asymmetric configuration with respect to the main channel. The relative diameter Di of the second inlet, i.e., 25-200% of Wc , and its position with respect to the channel center lead to different sculpted cross-sectional flow profiles. The second inlet is tangential to one of the side walls of the channel. (C) The series and parallel inlet configurations produce vertical and horizontal stacking of the flow streams within the channel cross section. An array configuration leads to a hybrid checkered flow profile. The thicknesses of individual layers are defined by the flow rates and Poiseuille velocity profile. (D, E) The multi-layered flow streams are converged into a smaller outlet channel (250 μm × 250 μm) where structured microparticles are UV-cured. (D) The multi-layered particles are cured with vertical, horizontal, and hybrid layer stacking for Σ Di = Wc . (E) Particle shapes resembling “latte art” are cured using symmetric and asymmetric configurations for Σ Di Wc . (F) Dimensional characterization shows high uniformity across particles and individual layers. The layer thicknesses match the numerical predictions in (C). (G) The particles fabricated within a single batch demonstrate high uniformity >90% for any given shape. Scale bars: 250 μm. The configuration of the first inlet in the fluidic circuit with i = 1 does not have any significance; however, the size and position of any subsequent inlet will have a significant effect on the sculpting of a multi-layered flow profile ( Figure 2B ). For example, the relative diameter D i of the second inlet varied as a fraction of the channel width W c , i.e, D i = 25-200% of W c , and its position with respect to the channel center, i.e., a symmetric or asymmetric configuration, can lead to different sculpted cross-sectional flow profiles. For configuration one with Σ D i = W c , a square microchannel having two inlets predicts a perfect vertical stacking of two flow streams. For Σ D i < W c , the flow stream from the second inlet forms an upward curved layer on top, where the curvature of the interface and the bi-layer cross-sectional flow profile are determined by the relative inlet diameter D i with respect to the channel width W c , i.e., 25, 50, or 75%, and the relative position of the inlet within the channel. For Σ D i > W c , the second flow stream forms an oppositely curved interface, further tuned by the symmetric or asymmetric positioning of the inlet. In a multi-layered sculpted flow profile, the thicknesses and spatial positionings of individual layers within the channel cross-section are governed by the flow rate ratio and the selected inlet configuration, respectively ( Figure 2C ). For example, an inlet configuration (Σ D i = W c ) with three inlets connected to the main channel in series produces a vertical stacking of three flow streams within the channel cross-section such that a flow rate ratio Q 1 : Q 2 : Q 3 = 1:1:1 results in the corresponding layer thicknesses with ratio of t 1 : t 2 : t 3 ≈ 1.5:1:1.5 defined by the Poiseuille velocity profile. The flow streams flowing closer to the side walls occupy a larger width of the channel, i.e., 37.5%, whereas the innermost stream flows faster with a smaller layer thickness, i.e., 25%, despite having the same flow rate. A flat stacking of streams results from a non-inertial laminar flow with Re ≲ 1, whereas an inertial flow with Re ≈ O (20) will bend the stacked streams downwards due to the Dean flow vortices ( Figure S1 and Note S1). For a parallel inlet configuration (Σ D i = W c ) with four inlets, a horizontal stacking of four flow streams is obtained with layer thicknesses, i.e., t 1,4 and t 2,3 equal 30.2% and 19.8% of W c , respectively, defined by the Poiseuille velocity profile such that t 1,4 : t 2,3 ≈ 1.5:1. An array configuration with four inlets, where two inlets are placed in parallel followed by another two in series, leads to a hybrid checkered flow profile with four uniformly distributed areas associated with each inlet flow. For an experimental demonstration of the flow sculpting capabilities of these inlet configurations, the multi-layered flow streams are converged from mm-scale channel cross-sections into an order-of-magnitude smaller outlet channel (250 μm ྾ 250 μm) where structured microparticles are cured upon UV exposure through a photomask ( Figures 2D and 2E ). The microchannel aspect ratio, i.e., channel width W c to channel height H c , at the point of inlet entry can influence the flow profile within the square channel outlet, in addition to the inlet configuration ( Figure S2 ). For consistency, we fixed the channel height, H c = 1.5 mm, for the experimental demonstrations unless otherwise mentioned, which results, for example, in W c : H c = 0.5, 2, and 1, respectively, for the inlet configuration (Σ D i = W c ) depicted in Figures 2C and 2D . We have numerically investigated that additional variations of the sculpted flow profiles are possible for significantly higher channels, up to H c = 9 mm, and higher aspect ratios W c : H c = 3, combined with variable W c : D i = 0.5 to 4 (Note S2). However, these larger microchannel designs lead to increased delay time in a stop-flow cycle due to higher capacitance in the fluidic circuit. Therefore, we did not investigate these microchannel designs experimentally. The shapes of fabricated microparticles match well with the numerically predicted cross-sectional flow profiles, owing to the meticulously crafted numerical models ( Figure S3 and Note S3). For the first inlet configuration with Σ D i = W c , we cured multi-layered particles with vertical, horizontal, and hybrid flat layers stacking ( Figure 2D ). Beyond flat flow profiles, we also demonstrated a controlled creation of multi-layered upward or downward curved flow profiles by using the second and third inlet configurations ( Figure 2E ). We cured particle shapes resembling ‘ latte art ’ using symmetric and asymmetric inlet configurations for Σ D i W c . We have performed dimensional characterization to confirm high uniformity across particles and individual layers ( Figure 2F ). Moreover, the layer thicknesses measured experimentally from the fabricated particles matched nicely with the numerical prediction in Figure 2C , with 90% by calculating the intersection-over-union scores ( Figure 2G ). 2.1.2 Flow sculpting using variable intra-channel pillar configurations We also investigated the effect of flow-obstructing pillar structures on flow sculpting within microfluidic channels ( Figure 3 ). Full-height pillars, commonly fabricated via single-layer soft lithography in PDMS channels, are widely used for inertial flow sculpting. However, their effectiveness emerges only at higher Reynolds numbers ( Re > 10), where Dean flow vortices form around them 33 – 38 , 45 , 46 . In contrast, non-inertial Stokes flows ( Re < 1) remain largely unaffected by such pillars 32 . Partial-height herringbone structures have been used to mix or manipulate Stokes flows 44 , 47 – 49 , but their fabrication requires multi-step soft lithography with precise layer alignment, which is laborious. Our hybrid fabrication approach, combining 3D-printed molds with soft lithography, enables precise, on-demand positioning of partial-height pillars, expanding the range of flow sculpting capabilities. Download figure Open in new tab Figure 3. Flow sculpting using variable pillar configurations. (A) A representative flow sculpting mode demonstrates that a given initial flow profile (input) is going to be transformed into a resulting flow profile (output) after passing through an indented pillar structure (unit operator). Full-height and partial-height pillars exhibit distinct flow sculpting behaviors in non-inertial ( Re = 0.2) and inertial ( Re = 20) flow regimes. For pillar structures symmetric about the YZ-plane, a net-zero flow sculpting effect is produced in non-inertial flow. (B) A non-net-zero flow sculpting effect is produced in non-inertial flow only when the pillar structures are partially indented and asymmetric about the YZ-plane, regardless of whether they maintain XZ-plane symmetry or exhibit complete asymmetry. (C) Effect of the interaction between adjacent pillars on flow sculpting. (D) Representative examples of flow sculpting microchannels incorporating composite partial-pillar designs in a non-inertial flow, and fabricated uniquely shaped particles. Scale bar: 250 μm. We examined how the symmetry of arbitrarily shaped pillars affects their ability to sculpt multilayered flow profiles ( Figure 3A ). A 2D shape (e.g., triangular) extruded along the Z-axis forms a full-height ( H p : H c = 1) or partial-height ( H p : H c < 1) pillar within the channel, where H p is the height of the pillar. At low Reynolds numbers ( Re ≲ 1), both full- and partial-height symmetric pillars (about the YZ plane) do not significantly disturb non-inertial flows with a checkered or quadrant-shaped profile. However, at Re > 10, both pillar types generate inertial effects and Dean vortices that modify the incoming flow, consistent with inertial flow sculpting methods. In contrast, our approach operates in the Stokes regime ( Re < 1), where flow manipulation relies on guiding co-flowing laminar streams using topological features that induce secondary flows in the YZ plane ( Figure 3B ). Symmetric pillars about the YZ plane cause reversible shaping of the flow 32 , 44 , resulting in a net-zero sculpting effect due to the complementary deformation and relaxation of streamlines in the counterclockwise (CCW) and clockwise (CW) directions, respectively ( Figure S4A and Note S4). To achieve irreversible flow sculpting in the Stokes regime, asymmetric structures about the YZ plane are necessary ( Figure S4B ). These include partial-height YZ-asymmetric pillars with symmetry or asymmetry about the XZ plane, which result in a pair of vortices or a single vortex, respectively, as depicted in the advection maps ( Figure 3B ). Each oblique side of the triangular pillar (green or red) contributes to a vortex in a CW or CCW direction. By mirroring an XZ-symmetric triangular pillar about the YZ plane, i.e., from the triangle pointing downstream to the triangle pointing upstream, the direction of the vortex-pair pointing downwards is reversed to pointing upwards ( Figure S4C ). Similarly, the direction of the single CW vortex can be reversed to a CCW vortex by mirroring the XZ-asymmetric pillar about the YZ plane. Adjacent pillars can become hydrodynamically connected, generating an effective slope that induces a vortex, which persists until the pillars are sufficiently spaced apart ( Figure 3C ). Pillar features, such as depth ( H p : H c ), width ( W p : W c ), aspect ratio ( L p : W p ), edge bevel, and orientation, can be tuned to produce diverse sculpting effects ( Figure S5 and Note S5). A composite channel design incorporating such partial-height ( H p : H c < 1) pillars with different inlet configurations and superimposed CW/CCW vortices produces uniquely shaped structured microparticles ( Figure 3D ). 2.1.3 Flow sculpting using variable outlet configurations The geometry of the outlet channel, including its cross-sectional shape and dimensions, is crucial in defining the final cross-sectional outline of the produced microparticles and fibers, since the UV illumination is applied in this region to rapidly solidify the confined structured flow. Although existing techniques can fabricate microchannels with various cross-sectional boundary shapes 50 , 51 , they remain limited in achieving complex channel designs that integrate both geometrically defined outlets and multiple inlets. The 3D-printed mold method effectively addresses this limitation. Multi-inlet microchannel molds with diverse outlet geometries, e.g., inverted trapezoidal, triangular, slit-shaped, and square outlines, can be readily fabricated via 3D printing ( Figure 4A ). Moreover, the size of the flow profile, and consequently that of the resulting microparticles, can be precisely tuned by adjusting the outlet channel dimensions. Using this strategy, structured microparticles with diameters ranging from approximately 100 μm to 500 μm were successfully fabricated ( Figure 4B ). Naturally, as in any flow sculpting method, controlling the flow rate ratio remains the most straightforward means of adjusting the layer proportions within the flow profile ( Figure 4C ). Download figure Open in new tab Figure 4. Flow sculpting using variable outlet channel configurations. Microchannel designs simulated flow profiles and experimental microparticle productions with (A) various outlet channel geometries: inverted trapezoidal, triangular, slit-shaped, and square outlines. (B) Square outlet channel geometry with various dimensions: ∼ 500 μm, 250 μm, and 100 μm side length. (C) 500 μm square-shaped multi-layered microparticles were produced under various pressure ratios between the three inlets. Scale bars: 250 μm. 2.2 Representative fabrications of tailored microparticles and fibers As one of the fundamental properties of microparticles or fibers, material composition plays a crucial role in defining their functionalities and thus guiding their potential applications. By integrating different material streams in 3D hydrodynamic flow lithography, a broad range of functional features can be achieved, e.g., solid or hollow interiors, functionalized surfaces, and localized expansion or contraction in response to specific stimuli, depending on the intrinsic material properties. For preliminary demonstrations, we fabricated several representative microparticles and fibers. Firstly, we fabricated biocompatible PEGDA - GelMA particles with a three-layered pattern that can direct cell growth and intercellular interaction. As shown in time-lapse images, cells gradually migrated towards the particle and adhered exclusively to the GelMA regions 52 , and finally glued the microparticles together ( Figure 5A , Figure S6 , Note S6, Movie S1). Secondly, we produced amphiphilic PEGDA - PPGDA particles with internal hydrophilic cavities that can facilitate the formation of uniform droplets without the need for conventional droplet microfluidics 6 , 39 , 40 ( Figure 5B ). Thirdly, we produce single-material PEGDA particles and fibers with structured cross sections and anisotropy along the fiber length, which is incorporated by tuning the UV exposure through the photomask ( Figure 5C ). Lastly, we realized a thermally-responsive PEGDA - PNIPAM bilayer fiber that exhibited reversible shrinkage and curling behavior as the temperature was increased from room temperature to 60 °C 53 and back ( Figure 5D ). Download figure Open in new tab Figure 5. Representative microparticles and fibers fabricated via 3D hydrodynamic flow lithography. (A) Time-lapse images showing targeted cell adhesion on patterned, biocompatible PEGDA - GelMA microparticles. (B) Microfluidics-free droplet formation using hollow, amphiphilic PEGDA - PPGDA microparticles. (C) Slit-shaped PEGDA microparticles and fibers with anisotropic features along the fiber length. (D) Thermal-responsive PEGDA-PNIPAAM microfibers. Scale bars: 250 μm for (A)–(C), 1 mm for (D). Download figure Open in new tab Figure 6. Workflow for 3D hydrodynamic flow lithography. 3. Materials and Methods 3.1 Workflow 3.1.1 Step 1: microchannel design The microchannel model was designed using 3ds Max ® computer-aided drafting software (Autodesk Inc.) and exported as the SAT file for numerical simulation or the STL file for 3D printing. The following general dimensions and guidelines were used for the microchannel design: Inlet pillars: The inlet diameter was typically set as 0.75 or 1.5 mm to match the outer diameter of the inlet tubing. For designs with parallel inlets, the inlet pillars were extended beyond the mold wall height with a smaller top (0.6 mm) to eliminate the need for hole punching in the PDMS replica and prevent liquid leakage at the inlet junctions. Main channel body: The main channel width was set as 0.75, 1.5, or 3 mm based on the inlet diameter, number, and configuration, with a fixed channel height of 1.5 mm. Transition section: A gradually converging section, with a length of 2.5 mm and an original height of 1.5 mm, was incorporated to connect the main channel and the outlet channel, ensuring a smooth hydrodynamic flow transition. Outlet channel: The outlet channel length was typically set as 2 mm in the numerical model and 35 mm for the 3D printing model. 3.1.2 Step 2: numerical modeling The SAT file containing the microchannel design was imported into COMSOL ® Multiphysics for numerical analysis. The Single-Phase Laminar Flow module was used to predict the flow profile and estimate pressure and velocity distributions. All simulations were performed using a working fluid composed of 60 vol.% PEGDA and 40 vol.% Ethanol (density: 987 kg·m 3 ; viscosity: 7.55 mPa.s). The colors and positions of the inlet pillars in the numerical model correspond to those of the streamlines, enabling clear visualization of the layered flow profile. The inlet boundary condition was usually set as a flow rate of 5.7e-10 m 3 /s per inlet for channels with a 250 µm × 250 µm square outlet, unless otherwise specified. Additional pillars (diameter: 0.05 mm, length: 3.54 mm) were added to mimic inlet tubings with an inner diameter of 0.18 mm and a length of 60 cm in the high-flow-resistance model ( Figure S3C ). Step 3: microchannel fabrication 3D printing of the microchannel mold: After obtaining a satisfactory flow profile from the numerical simulation, the microchannel design was finalized by adding mold walls and adjusting the outlet channel dimensions. The finalized mold design was exported as an STL file and converted to sliced CTB files using CHITUBOX ® software. The CTB file was then imported to the 3D printer (Sonic MINI 8K and Aqua-Gray 8K Resin, PHROZEN TECH CO., LTD) for microchannel mold printing. For the 500 μm × 500 μm square outlet channel, the design specified a channel width of 500 μm and a height of 530 μm to compensate for the offset in the Y-axis of the 3D printing. The printing parameters were set to a printing layer thickness of 50 μm and an exposure time of 2.1 s per layer. For the 250 μm × 250 μm outlet channel, the dimensions were adjusted to 250 μm × 325 μm, with a layer thickness of 25 μm and 1.0 s exposure time. For the 100 μm × 100 μm outlet channel, the design dimensions were 100 μm × 160 μm, printed with a layer thickness of 20 μm and 1.4 s exposure time. Surface treatment of the microchannel mold: The 3D-printed mold was then thoroughly washed with isopropanol, dried with an air flush, and subjected to a UV post-curing for 60 minutes (Anycubic Wash & Cure 2.0). After a 1-minute plasma activation, the mold was exposed to the vapor atmosphere of 50 μL Trichlor(1H,1H,2H,2H-perfluorooctyl)silane (448931, Sigma-Aldrich) for 20 minutes in a Petri dish, to complete the hydrophobic surface treatment. Microchannel fabrication: The PDMS microchannel was fabricated using soft lithography. Briefly, the PDMS base and curing agent (184 Silicone Elastomer Kit, Dow Inc.) were mixed at a 5:1 mass ratio and stirred until homogeneous. Then the mixture was poured over the microchannel mold, degassed, and cured at 65°C for 1 h (90 °C was used for the first curing step to clean the mold surface). After curing, the PDMS replica was removed from the mold, cut off both step ends, and the inlet holes were perfectly extended by vertical punching. For channel sealing, a 100-150 μm PDMS thin membrane was prepared by spin coating the PDMS mixture onto a silicon wafer at 500 rpm for 30 s, followed by curing at 65 °C for 15 min. The PDMS channel replica and thin membrane were then bonded using oxygen plasma treatment for 1 min, forming a sealed PDMS microchannel. After detaching the sealed channel from the silicon wafer, an additional cover glass (20 × 26 × 0.4 mm; Paul Marienfeld GmbH & Co. KG) was attached beneath the PDMS thin membrane at the inlet and converging section (excluding the outlet channel) using a very thin layer of uncured PDMS mixture as an adhesive. The assembled device was further cured at 90 °C for 24 h to enhance rigidity. Alternatively, a thin cover glass pre-cured with a thin PDMS membrane can also be used to seal the entire channel, though the additional glass layer at the outlet region would slightly affect the UV exposure. Finally, an open outlet for particle collection was created by cutting at the end of the outlet channel. 3.1.4 Step 4: flow lithography The FL setup consists of a laptop, a PDMS microchannel chip connected with PEEK inlet tubings, a fluid driving system (LineUp Flow EZ 7000, Fluigent Deutschland GmbH), and a UV curing unit (S2000 elite, Excelitas Canada Inc.). The precursor flow is first introduced and shaped at the sculpting channel and subsequently solidified as microparticles or fibers at the outlet region under the UV exposure. A home-made Python script was developed to automate the system, synchronizing the signals sent to both the UV light source and the pressure controllers. Specifically, SFL achieves particle fabrication through the cyclic coordination of flow stoppage and UV exposure 6 , 39 , 40 , 54 , whereas CFL enables fiber fabrication by keeping the flow continuous under UV exposure 43 . The UV light source was coupled with a collimator at an adjusted working distance to ensure a uniform exposure on the precursor flow across the entire photomask area. 100% UV light intensity was used in all experiments to maintain high UV exposure uniformity. A key consideration in the SFL configuration is the management of the high hydraulic flow resistance within the microfluidic system. As the flow was regulated using the pressure controller without additional mechanical valves, the hydrostatic pressure from the height difference between the pressure controllers’ reservoirs and the microfluidic chip can induce unwanted flow continuation even when the applied pressure is removed, which is detrimental to the SFL process 54 . So, tubings with different I.D.s (PEEK tubings with 1/32-inch O.D., 250 μm, 180 μm, and 130 μm I.D., Analytics-Shop) were intentionally chosen based on the size of the microchannel ( Table S2 ), to appropriately increase flow resistance so as to minimize the unwanted flow and sustain uniform particle production for a long time. The photomask was attached directly underneath the PDMS thin membrane at the outlet channel area for particle fabrication. Any bubbles trapped inside the channel are detrimental to flow sculpting, so before connecting the tubing with the channel, the air inside the channel should be expelled, and the channel should be preoccupied with ethanol or water. The environmental temperature for the GelMA experiment was controlled at around ∼ 30 °C. View this table: View inline View popup Download powerpoint Table S2. Microchannel and inlet tubing configurations. 3.1.5 Particles/fibers collection The produced particles/fibers were collected from the outlet channel, thoroughly washed three times with Ethanol, and stored in either ethanol or a 0.1% Pluronic ® F-127 (P2443, Sigma-Aldrich) aqueous solution to prevent aggregation. The particles for the cell experiment were washed and stored in cell culture medium. 3.2 Precursor solution preparation The precursor solution consisted of 60 vol.% Poly (ethylene glycol) diacrylate (PEGDA, M w 575, 437441, Sigma-Aldrich) and 40 vol.% Ethanol (1.00983, Sigma-Aldrich), additionally with 5 vol.% photoinitiator (2-hydroxy-2 methylpropiophenone, Darocur 1173, 405655, Sigma-Aldrich) and 50 μg/mL fluorescent dyes (red: Methacryloxyethyl thiocarbamoyl rhodamine B, 23591, Polysciences, Inc. Headquarters; blue: Methacrylate-modified blue-emitting dye, MA-P4VB: 2-(methacryloyloxy)propyl 6-(4-methoxy-2,5-bis((E)−2-(pyridin-4-yl)vinyl)phenoxy)hexanoate)), provided by a collaborating laboratory), or 5 vol.% fluorescent nanoparticles dispersion solution, to visualize the particle structures ( Table S3 ). For multiple-material particles, the compositions of the precursor solution are listed in Table S4 , including polyethylene glycol (PEG, M w 200, P3015, Sigma Aldrich), polypropylene glycol diacrylate (PPGDA, M w 800, 455024, Sigma-Aldrich), Gelatine-Methacryloyl (GelMA, 900622, Sigma-Aldrich), N-Isopropylacrylamide (NIPAAM, H66262.22, Thermo scientific). GelMA precursor was prepared in the ∼ 50 °C water bath to melt and homogenize the solution, under the cover of aluminum foil. Density matching among different precursor streams is critical for maintaining stable flow patterns. A density mismatch between streams can lead to noticeable deviations between the simulated and experimentally obtained structures. To ensure flow stability and fidelity to theoretical designs, the densities of the precursor solutions should be carefully adjusted within ∼1% during preparation (PEGDA: 1.12 g/mL, PPGDA: 1.01 1.12 g/mL, PEG: 1.127 g/mL, Ethanol: 0.79 g/mL, 60 vol.% PEGDA & 40 vol.% Ethanol: 0.987 g/mL). View this table: View inline View popup Table S3. Fabrication parameters for compartmentalized microparticles. View this table: View inline View popup Download powerpoint Table S4 Fabrication of structured multi-material microparticles 3.3 Cell culture Human embryonic kidney cell line HEK293T was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The GFP-expressing U87 glioblastoma cell line was kindly provided by a collaborating laboratory. U87 and HEK293T cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, GlutaMAX™ Supplement; Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 1% (v/v) penicillin–streptomycin (10,000 IU/mL penicillin and 10 mg/mL streptomycin; Sigma-Aldrich). All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ and were routinely tested for mycoplasma contamination. For passaging, the culture medium was aspirated, and the adherent cells were washed once with phosphate-buffered saline (PBS). Cells were then detached by adding 5 mL of trypsin–EDTA solution (Gibco™) and incubating at 37 °C for 2–5 minutes, depending on the cell line, until detachment was observed. The enzymatic reaction was stopped by adding an equal volume of complete medium. The cell suspension was centrifuged, resuspended in fresh culture medium, and seeded into new culture flasks for further growth. 3.4 Cell-particle experiment protocol and imaging We seeded U87 (18,000 cells) and HEK293T (38,000 cells) onto composite GelMA-PEGDA–GelMA microparticles and monitored attachment and proliferation by time-lapse imaging. The plate was maintained under environmental control conditions (37 °C, 5% CO₂) on a Leica LAS X fluorescence microscope. Time-lapse imaging was performed over a period of 72 hours, with Z-stack images captured at 3-hour intervals to monitor cell behavior and interactions. 4. Conclusions In conclusion, 3D hydrodynamic flow lithography shows great potential in the on-demand design and continuous fabrication of multi-material, anisotropic microparticles and fibers. We have demonstrated that: 1) Precise flow profile sculpting of up to five co-flowing streams with tunable size (∼100–500 μm), diverse outline geometries (e.g., inverted trapezoidal, triangular, slit-shaped, and square), and theoretically unlimited internal structures, achieved through multiple flow sculpting strategies, including variable inlet configurations, intra-channel pillar configurations, and outlet configurations. These flow sculpting strategies are validated by numerical simulations and experimental production of anisotropic microparticles and fibers; 2) Simplified cleanroom-free microdevice manufacturing was achieved through low-cost 3D-printed molds combined with soft lithography to produce 3D PDMS microchannels, which enables simple and precise positioning of the inlet ports with respect to the main channel, distinct main channel topology for flow sculpting, and various cross-sectional shapes of the outlet channel; 3) Inertia-free and sheath-flow-free operation, which allows stable performance across a wide range of fluidic material velocities and fluid viscosities and improve material utilization efficiency; 4) Broad material compatibility, demonstrated with PEGDA, PNIPAM, GelMA, and PPGDA, enabling the fabrication of functional microparticles and fibers tailored for specific applications. Together, these advancements establish 3D hydrodynamic flow lithography as a versatile and high-throughput (SFL, more than 10 4 particles/hour) platform for engineering anisotropic microparticles with customizable size, structure, and composition. Nevertheless, several challenges remain. For instance, certain limitations in channel design, such as intra-channel pillars that expand from top to bottom, deep concave structures with higher aspect ratios, and structures at the bottom of the channels, are inherent to the current molding methods used for microchannel fabrication. Direct 3D printing of oxygen-permeable channels could potentially overcome these geometric limitations and broaden the design possibilities in 3D hydrodynamic lithography. Moreover, the resolution of the 3D-printed molds or microchannels has a significant influence on the sculpted flow structures. At present, the design of 3D flow sculpting channels relies largely on trial and error, requiring intuitive spatial reasoning from the designer, which becomes increasingly difficult for channels with complex topological structures. Developing an inverse computational model assisted by artificial intelligence 55 , 56 to derive 3D flow sculpting channel designs from desired flow profiles would greatly streamline the design process and reduce the effort required for future device optimization. Supplementary Material See the supplementary material for additional supporting movies, figures, and notes. Supplementary Information Download figure Open in new tab Figure S1. Non-inertial vs. inertial flow sculpting. (A) A representative flow sculpting microchannel with three inlets arranged in series following inlet configuration one (Σ Di = Wc ). (B) Numerical simulations model the multi-layered flow profile for non-inertial, Re ≲ O (1), and inertial, Re ≈ O (10), flow regimes. (C) Dean flow vortices produced downstream of the 3 rd inlet due to inertial flow. The vector plots are obtained from YZ cross-sectional planes at distances 0, 0.25, 0.5, 0.75, and 1mm downstream of the 3 rd inlet. (D) Circular, squared, and rectangular inlet geometries produce different flow profiles at varying Re . Note S1. Non-inertial vs. inertial flow sculpting. Three inlets ( D i = 0.75 mm) are arranged in series along a rectangular cross-sectional ( W c = 0.75 mm, H c = 1.5 mm) segment of a flow sculpting channel following inlet configuration one, i.e., Σ D i = W c , (Figure S1A). The rectangular channel converges into a square outlet channel (250 × 250 µm) to obtain the final sculpted flow profile. We perform numerical simulations to model the multi-layered flow profile for non-inertial, Re ≲ O (1), and inertial, Re ≈ O (10), flow regimes (Figure S1B). The non-inertial flow with Re c = 0.20 and Re d = 0.89 within the rectangular and squared channel cross-section, respectively, resulted in a vertical and flat stacking of the three layers at the outlet. However, the inertial flow with 100x higher flow rate, i.e., Re c = 20 and Re d = 89, produced a downward curvature in the three stacked layers, which is attributed to the Dean flow vortices produced by the high velocity flow from the inlet turning by 90 degrees into the main channel. A vector plot within the rectangular cross-section clearly indicates the presence of the Dean flow vortices produced downstream of the 3 rd inlet due to inertial flow, which gradually disappears away from the inlet (Figure S1C). This inertial flow effect and subsequent Dean vortices could be minimized by altering the shape and size of the inlet channel (Figure S1D). As the inlet geometries are modified from circular to square and rectangular cross-sections, with longer dimension along the flow direction, the curvature effect is reduced gradually even at higher Re . A wider rectangular inlet port will produce a relatively lower flow velocity for the given flow rate and will allow a longer time for the two merging streams to interact, thereby reducing the inertial effects and Dean flow vortices that curve the interface between the layers. Download figure Open in new tab Figure S2. Sculpted flow profiles with variable inlet diameter ( Di ), channel width ( Wc ), height ( Hc ), and aspect ratio ( Wc / Hc ). (A) A representative flow sculpting microchannel with three inlets arranged in series using the inlet configuration one (Σ Di = Wc ). (B) Sculpted flow profiles at the outlet following different inlet configurations, i.e., Σ Di = Wc , and Σ Di < Wc , using a constant Di of 0.75 mm. Wc is varied from 0.75 mm to 3 mm. Hc is varied from 0.25 mm to 6 mm. (C) A representative flow sculpting microchannel with three inlets arranged in series using the inlet configuration three (Σ Di > Wc ). (D) Sculpted flow profiles at the outlet following different inlet configurations, i.e., Σ Di = Wc , Σ Di Wc , using a constant Di of 1.5 mm. Wc is varied from 0.75 mm to 4.5 mm. Hc is varied from 0.25 mm to 9 mm. Green boxes: unexpected deviation, red boxes: expected flow profiles. Note S2. Sculpted flow profiles with variable inlet diameter ( D i ), channel width ( W c ), height ( H c ), and aspect ratio ( W c / H c ). The shape of the sculpted flow profiles for the three inlet configurations (Σ D i = W c , Σ D i W c ) holds consistently for a broad range of the microchannel dimensions ( D i , W c , H c , and W c / H c ) (Figure S2). However, deviation from the expected flow profile shapes was observed for limited cases (green boxes). For example, a vertical and flat stacking of three flow streams, following inlet configuration one (Σ D i = W c , the fourth row of Figure S2B and the third row of Figure S2D), was as expected under W c / H c ≤ 1.5, whereas, a higher aspect ratio channel with W c / H c = 3 resulted in enhanced upward curvature (green boxes in the fourth row of Figure S2B and the third row of Figure S2D) mimicking the inlet configuration two. For the inlet configuration two (Σ D i < W c , the first-third rows of Figure S2B and the first-second rows of Figure S2D), the stacked layers are curved upwards as expected for W c / H c ≥ 1 (red boxes); however, for W c / H c = 0.5, we observed unexpectedly vertical stacking of the layers mimicking the inlet configuration one (green box). For the inlet configuration three (Σ D i > W c , the last row of Figure S2D), we obtained a sculpted flow profile with stacked layers curved downwards as expected for W c / H c ≤ 1.5, whereas, for W c / H c = 3, the flow profile with reduced curvature deviates slightly from the expected shape. Download figure Open in new tab Figure S3. Defining appropriate inlet boundary conditions in the numerical models: flow rate Q vs. pressure P . (A) A representative flow sculpting microchannel with inlet configuration one (Σ Di = Wc ), and numerical modeling of sculpted flow profile using inlet boundary conditions based on constant flow rate Q and pressure P across three inlets within non-inertial flow regime, i.e., Re ≲ O (1). (B) Comparison of sculpted flow profiles resulting from different channel designs and inlet connections under constant Q and P boundary conditions. (C) A microchannel model mimicking high flow resistances across four inlets results in similar sculpted flow profiles for constant Q and P boundary conditions. Note S3. Defining appropriate inlet boundary conditions in the numerical models: flow rate Q vs. pressure P . We conducted numerical simulations using a 3-inlet flow sculpting microchannel (Σ D i = W c ) to investigate the effects of inlet boundary conditions (BCs) with constant flow rate Q of 5.7×10 - 10 m 3 /s or pressure P of 2.2 mbar (corresponding to an outlet Re of 0.89) on the resulting tri-layered vertically stacked flow profiles and velocity distributions (Figure S3A). A constant Q boundary condition with flow rate ratio Q 1 : Q 2 : Q 3 = 1:1:1 resulted in layer thicknesses with a symmetric ratio of t 1 : t 2 : t 3 ≈ 1.5:1:1.5 (i.e., 37.5%, 25%, and 37.5% of the channel height) defined by the Poiseuille velocity profile. For a constant Q boundary condition, the first inlet converges numerically with the highest inlet pressure of 2.1767 mbar, followed by 2.1735 and 2.1670 mbar for the second and third inlets, respectively. The inlet pressures vary across the channel length; however, the relative proportions of different stream layers remained symmetric following the Poiseuille velocity profile and constant Q . In comparison, a constant P (= 2.2 mbar, similar to the converged pressure values from the last numerical model) boundary condition with pressure ratio P 1 :P 2 :P 3 = 1:1:1 resulted in layer thicknesses with an asymmetric ratio of t 1 : t 2 : t 3 ≈ 1.29:1:2.18 (i.e., 28.8 %, 22.4 %, and 48.8 % of the channel height). Different hydraulic flow resistances ( R h ) for a constant pressure drop ΔP = 2.2 mbar between the three inlets and the outlet translated into variable flow rates of 3.94, 5.13, and 8.26 ×10 -10 m 3 /s, respectively (following Hagen-Poiseuille equation: Q = Δ P / R h ). Hence, variable layer thicknesses within the sculpted flow profile were obtained, which were consistent with the inlet flow rates. We have further investigated various inlet designs with different flow resistances for inlets positioned along the channel length (Figure S3B). The connecting geometries between two juxtaposed inlets (parallel configuration) affect the interface between the co-flowing streams from these inlets and the final sculpted flow profile. Notably, shallower channels ( H c = 250 µm between the series inlets), with a greater difference in R h between inlets placed in series along the channel length, produce flow profiles having thinner bottom layers associated with the first two inlets for a constant P boundary condition. For a constant Q boundary condition, the layer thicknesses within the checkered flow profile are relatively consistent for different channel designs; however, the interface shape is influenced by the inter-inlets geometric feature. Once the channel height is increased ( H c = 1,500 µm between the series inlets), the difference in R h between inlets along the channel length is reduced. The bottom layer thickness for the constant P boundary condition starts to approach that for the constant Q boundary condition. We mitigate the inter-inlets difference in R h by introducing additional flow resistance into the system (mimicking connecting tubing with much smaller diameters), such that the sculpted flow profile is not affected by the minor flow resistance variation between the inlets (Figure S3C). The flow profile obtained for constant Q and P boundary conditions matched well. Therefore, we have adopted these findings in our experimental workflow by using inlet connecting tubing with significantly higher flow resistances compared to the channel itself. Download figure Open in new tab Figure S4. Flow sculpting principle of a partial-height pillar in non-inertial flow. (A) A representative flow sculpting mode showing the input (initial flow profile from a given inlet configuration), unit operator (partial-height pillar structure), and output (resulting flow profile). Any partial-height pillar or pillar pair structure symmetric about the YZ-plane produces a net-zero sculpting effect on the flow profile. Advection maps indicate the direct flow transformation (vortices) between specific planes. (B) Effective slopes in the partial-height pillars are responsible for inducing the sculpting effect. The direction (C) and the intensity (D) of the flow sculpting vortices are determined by the orientation and the superimposition of these effective slopes. Note S4. Flow sculpting principle of a partial-height pillar in non-inertial flow. Flow profile reshaping in the YZ plane is achieved by generating secondary flows in this plane, which are perpendicular to the direction of primary flow. Such flow reshaping can occur either through Dean flow in the inertial flow regime or through flow dislocation in the non-inertial flow regime. Since the flow must remain continuous, any local fluid displacement would be immediately compensated by the surrounding fluid. Consequently, changes in the flow profile usually manifest as vortical flow transformations within the plane. In non-inertial flow sculpting, this type of flow transformation, i.e., flow dislocation, is initially induced by the lateral offsets (y-direction) between flow layers obstructed by pillar structures and those that remain unobstructed as the fluid moves downstream the primary flow direction. The transformation is then completed by forming vortices under fluid advection. The oblique sides of partial-height pillar structures serve to generate these lateral offsets. Any given initial flow profile will be mapped to a corresponding output profile after passing through a partial-height pillar with an oblique side. Advection maps reveal the direct flow transformations produced by specific partial-pillars. For example, Map p2-p1 illustrates exactly how the fluid flows and is transformed through the first pillar, from the flow profile at plane 1 to the flow profile at plane 2. Comparing Map p2-p1 and Map p3-p2 shows that this YZ-plane-symmetric partial-height pillar pair generates pattern-matched, counter-rotating vortex pairs, resulting in a net-zero sculpting effect (Figure S4A,). In contrast, a YZ-plane-asymmetric partial-height pillar with effective slope can induce a non-net-zero flow sculpting effect (Figure S4B), where the direction and the intensity of the flow sculpting vortices are governed by the orientation (Figure S4C) and superposition (Figure S4D) of these effective slopes. Closer inspection reveals that the plane 2 flow profile of Figure S4A is not identical to the leftmost flow profile of Figure S4B, even though they share the same initial flow profile and pass through the same pillar structure. This difference arises from hydrodynamic interactions between adjacent pillars, which persist until the pillars are sufficiently spaced apart ( Figure 3C ). Download figure Open in new tab Figure S5. Effect of pillar dimensions on non-inertial flow sculpting. (A) Comparison of flow sculpting effects produced by straight vs. tapered pillars. The tapered pillar generates smaller lateral offsets in the y-direction compared to the straight pillar, thereby resulting in a gentler flow sculpting effect. (B) Influence of the straight pillar’s width ( Wp ), length ( Lp ), and height ( Hp ) on the flow dislocation (advection maps showing the vortices) and, consequently, on the final flow sculpting effect (flow profiles). Note S5. Effect of pillar dimensions on non-inertial flow sculpting. In flow reshaping, the flow dislocation (vortex) observed on the YZ-plane represents the projection of the 3D vortex within the channel onto this plane. Consequently, the geometric dimensions of the pillar that project onto the YZ-plane, i.e., width ( W p ) and height ( H p ), significantly affect the central position and region of the resulting vortex on this plane, while the pillar length ( Lₚ ) exerts only a minor influence on this. Download figure Open in new tab Figure S6. Time-lapse images of U87 cells cultured with three-layered GelMA-PEGDA-GelMA microparticles. GFP-expressing U87 glioblastoma cell line. From top to bottom: bright field, fluorescent, overlapped, and zoomed-in fluorescent and bright field images. From left to right: images captured after 0 h, 12 h, 24 h, 36 h, 48 h, and 72 h of culture. Cells migrate gradually towards the particles and adhere exclusively to the top and vertical surfaces of the GelMA region, avoiding the PEGDA area. Scale bars: 250 μm. Movie S1. U87 and HEK293T cells cultured with three-layered GelMA-PEGDA-GelMA microparticles and imaged for 48 hours. Note S6. Across replicates, both cell types rapidly adhered to and spread on GelMA domains, forming contiguous colonies that expanded over time (Figure S6; Movies S1). In contrast, PEGDA regions remained largely cell-free during the observation window, showing only transient contacts without sustained spreading, consistent with PEGDA’s non-adhesive character. Cells were also attached to the vertical walls of the particles and subsequently migrated onto the horizontal planes. 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Fabrication of 3D concentric amphiphilic microparticles to form uniform nanoliter reaction volumes for amplified affinity assays . Lab Chip 2020 , 20 ( 19 ), 3503 – 3514 . DOI: 10.1039/d0lc00698j . OpenUrl CrossRef PubMed (18). Akhtar , M. U. ; Sahin , M. A. ; Werner , H. ; Destgeer , G . Fabrication of Size-Coded Amphiphilic Particles with a Configurable 3D-Printed Microfluidic Device for the Formation of Particle-Templated Droplets . Advanced Materials Technologies 2024 . DOI: 10.1002/admt.202301967 . OpenUrl CrossRef (19). Helen Werner , E. T. , Mehmet Akif Sahin , Moritz Leuthner , Peer Erfle , Oliver Hayden , Andreas Dietzel , and Ghulam Destgeer . Stopping a Multilayered Co-Axial Flow in a 3D Printed Microchannel with Cascaded Nozzles. Adv . Mater. Technol . 2025 , 10 , 2500203 . (20). Yuan , R. ; Nagarajan , M. B. ; Lee , J. ; Voldman , J. ; Doyle , P. S. ; Fink , Y . Designable 3D Microshapes Fabricated at the Intersection of Structured Flow and Optical Fields . Small 2018 , 14 ( 50 ), e1803585 . DOI: 10.1002/smll.201803585 . OpenUrl CrossRef PubMed (21). Zhou , C. ; Liang , S. ; Li , Y. ; Chen , H. ; Li , J . Fabrication of sharp-edged 3D microparticles via folded PDMS microfluidic channels . Lab Chip 2021 , 22 ( 1 ), 148 – 155 . DOI: 10.1039/d1lc00807b . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted October 28, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following 3D Hydrodynamic Flow Lithography Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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Share 3D Hydrodynamic Flow Lithography Yiying Zou , Mehmet Akif Sahin , Muhammad Zia Ullah Khan , Muhammad Usman Akhtar , Ghulam Destgeer bioRxiv 2025.10.27.684743; doi: https://doi.org/10.1101/2025.10.27.684743 Share This Article: Copy Citation Tools 3D Hydrodynamic Flow Lithography Yiying Zou , Mehmet Akif Sahin , Muhammad Zia Ullah Khan , Muhammad Usman Akhtar , Ghulam Destgeer bioRxiv 2025.10.27.684743; doi: https://doi.org/10.1101/2025.10.27.684743 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Bioengineering Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17633) Bioengineering (13856) Bioinformatics (41841) Biophysics (21399) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24282) Genetics (15582) Genomics (22462) Immunology (17700) Microbiology (40295) Molecular Biology (17140) Neuroscience (88419) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)