Compelling standardized and high-throughput micro-/millifluidic plates for biomedical research: from laboratory to market

2.1 Micro-/millifluidic plate design based on individual microwell

In micro-/millifluidic plates where microwells are treated as individual units, the microwells serve as containers that provide space for integrating functional inserts, including customized components,^{[24–25–26]} modified transwells,^{[27–28–29]} biomimetic substrates,^{[30–31–32]} and biosensors.^[33,34] Each microwell is thus endowed with specific functions, allowing it to act as an independent micro-/millifluidic device for various biomedical applications. Typically, customized components divide the microwells into multiple horizontal regions for cell culture, such as left or right regions,^[35,36] inner or outer regions,^[37,38] and self-defined regions.^[39] Different regions are either separated from each other or interconnected by microchannels,^[40,41] micropillars,^[42] and liquid rails.^[43,44] A notable example is the microcavity array, a design that consists of hundreds of independently and regularly arranged microcavities within a single microwell.^[45] It provides a promising strategy to overcome the limitations of the conventional organoid culture system based on the Matrigel dome, which lacks robustness in morphogenesis and compatibility with high-content analysis.^[46] Both our group and Decembrini et al.^[47] employed hydrogel-based microcavities as artificial niches, providing consistent physical and biological microenvironments to direct the generation of the liver,^[48,49] retinal,^[47] cerebral,^[50] and colorectal cancer^[51] organoids with uniform sizes and morphologies. In addition to the microcavity array, a 3D-printed inhibiting insert^[52] and hanging drop dripper^{[53–54–55]} integrated into the micro-/millifluidic plate have also been demonstrated to improve the cell spheroid and organoid homogeneity by unifying the spatial location and cell concentration. However, both inserts are limited by the throughput capacity predetermined by the microtiter plate format. In contrast to the microcavity array, the component with connected regions aims to construct histotypic and organotypic models with spatially-defined heterocellular architectures such as neurovascular units,^[56] neuromuscular junctions,^[57] and vascularized tumor tissues.^[58,59] In this kind of customized component, heterogeneous cells are precisely positioned in different regions with connecting microstructures promoting cell–cell interactions and guiding some special physiological processes like angiogenesis,^[60] axon elongation,^[57] and immune cell directional migration.^[61]

Transwell is another type of insert that provides 2 distinct vertical regions for cell culture^[62] and it can be modified to perform a wider range of tasks in reconstituting the microenvironment in vivo, such as dynamic culture^[63,64] and chemical gradient generation.^[27,65,66] Compared with customized components, the porous membranes on transwells provide a more adequate exchange of nutrients and gas, minimize hypoxia injury, and improve the maturity of models.^{[67–68–69–70–71]} However, the limited optical transparency of these membranes is a potential stumbling block for in situ observation. Moreover, transwells restrict microwell organization to up-down regions, limiting the potential for more complex cell arrangements.

Unlike customized components and modified transwells, which prefer to compartmentalize the microwells using physical boundaries to achieve the differential arrangement of heterogeneous cells, the micropatterned substrates employ invisible boundaries to construct heterocellular architectures with micron-level resolution analogous to that found in native tissues and organs. Different types of cells are precisely localized using removable micropatterned stencils^[72] and further coating the substrates with extracellular matrix to provide selective binding sites for specific cells.^{[73–74–75]} In addition to chemical modification of the substrate to accomplish the heterocellular arrangement, substrates can also be granted topographical cues that are crucial for regulating cell fate.^{[76–77–78]} For instance, integrating biomimetic polydimethylsiloxane (PDMS) substrate that mimicked the curvature of glomerular capillaries improved podocyte differentiation efficiency.^[79]

The final category, biosensor, represents a versatile class of inserts capable of monitoring multiple physiologically relevant tissue properties in a real-time, continuous, and noninvasive manner.^[80,81] Currently, the main transduction principles applied to micro-/millifluidic plates are based on electrical and mechanical signal propagation.^{[82–83–84]} The microelectrode array (MEA) is a common biosensor that is widely used for recording electrical activities in tissues, such as neural and cardiac tissues.^{[85–86–87]} Traditional MEAs are often based on opaque printed circuit board (PCB) substrates, which pose significant challenges for microscopic observation. To address this challenge, transparent glass or thermoplastic-based substrates are used as alternatives that offer additional assay flexibility, including bright-field imaging and immunofluorescence.^[88] However, these MEAs still face other limitations, such as reduced capability to monitor 3D tissues,^[89] and the metal electrodes often have short operational lifespans due to saturation or degradation.^[81] Except for electrical activity, mechanical forces generated by tissues such as cardiac and skeletal muscle are also essential to their function.^[90,91] Mechanical biosensors, such as microcantilevers or microwires, are commonly used in this context. These sensors bend in response to tissue contraction, and the resulting contraction forces can be measured by electronically or optically detecting the deflection of the sensors.^{[92–93–94]}

In general, the advantage of individual microwell-based micro-/millifluidic plates is that they can inherit and even increase the throughput of the original microtiter plate. The functional inserts can be designed independently from the microwells, allowing them to be easily installed in users’ standardized microtiter plates and detached for subsequent analysis. However, the fluid flow, which serves as a key hallmark in microfluidic devices, has not been widely demonstrated in this kind of micro-/millifluidic plate, probably due to lacking sufficient integration space and unified design for the fluid control system. In most cases, the fluid is driven manually, either by connecting to a syringe pump^[63] or employing an orbital shaker,^[66] which potentially reduces the compatibility and usability of the micro-/millifluidic plate.

2.2 Micro-/millifluidic plate design based on integrated microwell

For micro-/millifluidic plates where microwells are defined as integrated units, the microwells are regularly connected by microchannels, forming multiple parallel microfluidic chips, and the final micro-/millifluidic plates.^[95,96] Microwells serve diverse purposes, including liquid handling,^[97,98] cell culture,^[99] biochemical reaction,^[100,101] and sample detection.^[102,103] In most cases, the original microwell format is preserved, with microchannels integrated among or within the microwells. For example, a typical micro-/millifluidic plate may feature each chip containing 3 microwells, with up to 128 independent chips formed within a 384-well micro-/millifluidic plate. The microwells at either end function work as inlets and outlets for culture medium perfusion, while the middle microwells serve as the core, embedded with scaffolds designed to mimic native tissues.^{[104–105–106–107]} Specifically, a perfusable tubular scaffold termed “AngioTube” was suspended across the tissue culture microwell and connected to both inlet and outlet.^[108] It provides mechanical stability for a vessel lumen and supports the self-assembly of various parenchymal tissues, such as the liver,^[109] cardiac,^[108] kidney,^[110] lung,^[111] and solid tumors.^[112] The scaffold’s walls featured nanopores and microholes to enhance cell–cell interactions and improve biomolecular exchange between endothelial and parenchymal cells.^[113] Moreover, microcantilevers were strategically integrated into the scaffold to noninvasively probe the contraction of the tissues. PREDICT-96 micro-/millifluidic plate is another example that adapts and extends organ-on-a-chip (OoC) systems to a 384-well plate format.^[114] Unlike the AngioTube, PREDICT-96 utilizes bilayer chips with 2 overlapping microchannels positioned between 2 sets of inlets and outlets. Different cell types were cultured on opposing sides of a basement membrane, while the micropores on the membrane promoted cell–cell interactions. Furthermore, a recirculatory flow has been demonstrated by employing a programmable pneumatic pump positioned in the plate lid that was fluidically coupled to the micro-/millifluidic plate via a set of vertical tubes that extend into the microwells.^{[115–116–117]}

In addition to retaining the original microwells, they also can be removed or redefined to accommodate specific design requirements for microfluidic chips with well-defined configurations, such as double-T junction,^[23] Christmas tree-shaped network,^[118] parallel microwell array,^{[119–120–121]} and culture chamber with three assembly ridges.^[122] Essentially, this kind of micro-/millifluidic plate can be regarded as an array of conventional microfluidic chips. However, optimizing the number and arrangement of these chips is crucial to ensure compatibility with standard microtiter plates. For instance, in a micro-/millifluidic plate, 96 microwells were replaced by filtration devices of the same number, while each device was positioned at a 45-degree angle to accommodate a long filtration channel. Cell suspensions were loaded into the upper right inlets and driven through the micropillar arrays using air pressure. Cells that were poorly deformable could not pass through the micropillar gaps, resulting in the occlusion of these gaps. The micropillar arrays were strategically placed in the positions of the original microwells, allowing for high-throughput cellular imaging and quantification of the occluded gap percentage.^[123]

In summary, this kind of micro-/millifluidic plates possess the ability to perform complex tasks. On the one hand, the available space for chip design extends beyond the microwells to encompass the entire plate, allowing for the integration of more functional structures into the chip. On the other hand, each microwell within the chip serves a unique function, and together, they collaborate to perform complex tasks. Despite these significant advantages, these derivative micro-/millifluidic plates still face challenges in fully achieving both functional complexity and multiplexing simultaneously due to the fixed dimensions of the microwell plate prototype. Depending on the application, an arbitrary number of chips (ranging from 4 to 192) are arranged in parallel on a single micro-/millifluidic plate.^[118,124] Looking forward, multilayer structure design may offer a promising solution to balance the throughput and the functional complexity of these micro-/millifluidic plates.^[125]

3.1 Active pump

Conventional syringe pumps,^{[130–131–132–133]} peristaltic pumps,^[134,135] pneumatic pumps,^[136] and vacuum pumps^[137,138] are all active pumps with prominent advantages in accurate fluid control in micro-/millifluidic plates.^[129,139] These active pumps are also amenable to generating complex flow patterns, such as pulsatile and recirculatory flow.^[140,141] For instance, Satoh et al.^[142] developed an OoC system using a pneumatic pump to control medium circulation across multiple culture chambers in a unidirectional manner. This medium circulation mechanism relied on the synergistic effects of the pneumatic pump, the elevated inlet, and the Laplace valve. Among them, the pneumatic pump was programmed to periodically apply pressure to tandem culture chambers for medium actuation. The elevated inlet and the Laplace valve were designed to prevent the medium from traveling back to the upstream chamber and stop the introduction of the gas into the microchannel, respectively. Although active pumps are still used as mainstream liquid-driven systems in micro-/millifluidic plates, these pumps are bulky and difficult to massively scale up.^[143] Moreover, active pumps typically contain multiple auxiliary components, such as power sources, fluid connections, and tubing, which require fluid handling expertise. These cumbersome components may ultimately hinder the widespread adoption of such systems by biomedical end-users.^[18] Therefore, enhancing usability should be a key consideration in the design of liquid-driven systems for micro-/millifluidic plates.

3.2 Passive pump

As an alternative to active pumps, passive pumps provide a solution for the limitations mentioned. Based on their advantages, including high level of integration, simplicity of operation, and portability, passive pumps have been widely adopted and applied to a large extent in biomedical applications.^[144] In current micro-/millifluidic plates, passive pumping methods such as gravity,^{[99,145–146–147–148]} surface tension,^[149,150] and capillary force^{[151–152–153]} have been successfully demonstrated for fluid actuation.

Gravity-driven pumps typically employ reservoirs filled with liquid at different heights to achieve fluid actuation. During the working process, the fluid flows continuously from the higher inlet to the lower outlet until the liquid levels in the reservoirs equalize. A continuous flow can be maintained over long experimental periods by regularly refilling the open inlet or placing the micro-/millifluidic plate on a perfusion rocker to create a hydraulic head difference between the inlet and outlet.^[154] However, as the hydraulic head difference decreases, the flow rate gradually diminishes.^[129] To maintain a constant flow rate, autonomous droplet dispensers are ingeniously integrated into the inlets to balance the volumes of the replenished and drained fluid.^[154,155] The prominent advantages of gravity-driven pumps include simplified system design, the elimination of complex auxiliary components, and improved usability. Additionally, air bubbles are easily removed in micro-/millifluidic plates with open microwell configurations.^[156] Despite these benefits, the drawbacks of gravity-driven pumps include a limited range of flow rates and the inability to generate pulsatile flow.

The surface tension effect is another strategy utilized for fluid actuation, which is generated by the internal pressure difference between 2 liquid drops with unequal volume. According to the Young–Laplace equation, smaller liquid drops have higher internal pressure than larger ones, and this pressure difference drives the fluid and dictates the flow direction.^[157] Similar to gravity-driven pumps, surface tension-driven pumps require only simple components (an inlet, an outlet, and a connection microchannel) for system operation, rendering them easy to integrate into micro-/millifluidic plates with a high degree of parallelism. Surface tension-driven pumps also function as an open system with standardized interfaces, allowing automatic liquid filling at the inlet via robotic liquid handlers. Surface tension-driven pumps have already been used to support dynamic cell cultures in micro-/millifluidic plates for applications such as drug screening and disease diagnosis.^{[158–159–160]} While they allow for the refilling of liquid to maintain continuous flow, surface tension-driven pumps are not suitable for long-term perfusion culture due to the limited volume of the liquid drops, which is typically in the microliter range.^[161,162]

A capillary-driven pump operates on the principle of capillary action that allows fluid to move through narrow channels or porous materials. Based on the substrate material, capillary-driven pumps based on solid material and porous material have been widely used in micro-/millifluidic plates.^[163] The capillary-driven pump was initially proposed in the context of a Si microfluidic chip.^[164] In microfluidic systems, the spontaneous liquid transport through microchannels is governed by capillary forces arising from the interaction between liquid surface tension and solid interface geometry. Flow cessation occurs upon complete channel filling.^[165] To address this limitation, absorbent matrices have been strategically implemented at channel termini to sustain continuous flow cycles. For example, when liquid is introduced into microwells of 96-well ELISA-optimized micro-/millifluidic plates, it propagates through spiral microchannels via capillarity. The terminal absorbent pads, exhibiting enhanced capillary potential compared with the microchannel network, continuously extract liquid from the system. This configuration enables sequential introduction of samples and reagents through the microchannels, thereby completing the analytical workflow.^[166] In addition to solid materials, capillary-driven pumps have also been closely associated with the use of porous materials. Paper is a common-used porous material on which hydrophobic boundaries can be created by using techniques such as photolithography^[167] and wax printing,^{[168–169–170]} guiding fluid through patterned hydrophilic regions.^[171] Except for paper, functionalized powders have also been demonstrated as ideal materials to form porous microzones on hydrophobic polymer plates.^[172] The porous capillary-driven pump eliminates the need for narrow channels and prevents bubble entrapment.^[165] However, it primarily relies on stochastic capillary flow within pore spaces, indicating that the inherent properties of porous materials, such as the porosity, will determine the fluid behaviors and cannot be regulated once the pumps are fabricated.^[163,173]

Simplifying fluid handling challenges in micro-/millifluidic plate design through passive pumping strategies is an inevitable trend that will accelerate adoption. Advances in passive pumps will eliminate the need for auxiliary components and improve the integration of micro-/millifluidic plates. Passive pumps effectively lower the barrier to entry for the end-users, as they only require an automated liquid handler to operate. A prime example is the paper micro-/millifluidic plate used in POCT, where the liquid-driven mechanism is hidden, allowing users to focus solely on interpreting test results.^[174,175] Another advantage of passive pump-based micro-/millifluidic plate is that they are easy to integrate, which helps establish standards for plate design, manufacturing, and testing within the industrial supply chain. This ultimately reduces costs and makes micro-/millifluidic plates more accessible.^[1,176] While current active pumps offer superior fluid control and the ability to generate complex flow patterns, passive pumps gradually match these capabilities through the introduction of functional microstructures. For instance, continuous unidirectional perfusion with recirculation has been achieved in a gravity-driven flow system by incorporating supporting microchannels and passive valves.^[177,178]

4.1 OoC

A preclinical study is a necessary stage in the drug development pipeline, where the primary goal is to evaluate the efficacy and safety of drug candidates to eliminate ineffective and highly toxic candidates as soon as possible. To do so, the drug candidates undergo a series of stringent tests in 2D static cell cultures and animal models.^[183] However, more than 80% of drug candidates fail to demonstrate efficacy and safety in clinical trials,^[184] indicating the current models often cannot faithfully predict human responses to these drug candidates.^[185] The limitations of these models have prompted the urgent need to develop models with better predictive ability. To meet this need, OoC systems were hatched through the convergence of microtechnology and biology.^{[186–187–188]} On the one hand, the OoC systems recapitulate key tissue- and organ-level functions by mimicking physiologically relevant microenvironments in microfluidic devices. On the other hand, the species difference can be eliminated by using cells of human origin in OoC systems. OoC is a promising in vitro model bridging the gap between traditional 2D cell cultures and animal models. Despite their promise, most OoC systems currently operate at very low throughput, allowing only a limited number of replicates at a time.^[189] Consequently, it is difficult to meet the requirements of practical pharmaceutical applications that need to screen thousands of drug candidates in a short time.^[190] Developing novel OoC systems based on microtiter plate format can effectively increase the number of replicates per plate, promote the faster transition from personalized to standardized design, and enhance the repeatability and reproducibility of the system.

Up to now, micro-/millifluidic plates have been utilized in the development of both single-organ and multiorgan systems, depending on the target applications.^{[191–192–193]} Single-organ micro-/millifluidic plates are commonly used to study specific tissue and organ functions without involving the complexity of other organ systems. For example, the liver is the largest internal organ in the human body. It performs various essential biological functions, such as the synthesis of proteins and lipids, secretion of bile, and metabolism of drugs.^[194] However, hepatocytes cultured as monolayers typically exhibit nonpolarized and rapidly lose their liver-specific functions.^[195] The PhysioMimix micro-/millifluidic plate integrates 12 chips in parallel, enabling the dynamic culture of hepatocytes to maintain their phenotypes and functions by providing continuous oxygen and nutrients as well as biomechanical stimuli through the fluid shear stress.^[196] Additionally, by replacing the laminar flow with vertical flow, the high flow resistance of porous membranes will induce pressure inside culture chambers, which exerts compaction forces on hepatocytes. This mechanical compaction simulates the pressure experienced by hepatocytes inside the abdominal cavity. As a result, the hepatocytes exhibit accelerated repolarization, in vivo-like cuboidal morphology, and better liver metabolic and synthetic functions.^[197] Moreover, biophysical cues like arterial strain waveform^[198] and oxygen tension^[199] have also been proven to play an important role in regulating the phenotypes and functions of vascular smooth muscle cells and alveolar basal epithelial cells, respectively. Except for biochemical and biophysical cues, the spatial arrangement of the cellular structure also has a significant impact on regulating the microenvironment of the embedded cells.^[200,201] By converging OoC and dielectrophoretic bioassembly, cells can be spatially arranged to form liver lobules and then transferred and cultured in 96-well micro-/millifluidic plates.^[202] Similarly, advanced extrusion-based bioprinting^[203,204] and acoustic bioassembly^{[205–206–207]} techniques have also been demonstrated to construct functional tissues with predetermined cellular structures in micro-/millifluidic plates, such as cerebral cortex, liver lobule, cardiac, and tumor.

Different from single-organ micro-/millifluidic plates, multiorgan micro-/millifluidic plates are suitable for investigating the interactions between different tissues and organs.^[208] To investigate the potential toxicity linked to metabolism by the liver, at least one other OoC is connected to the liver-on-a-chip. For example, a lung–liver micro-/millifluidic plate has been developed to assess both acute and chronic toxicity of pathogens, where lung tissues are cocultured with liver tissues. The micro-/millifluidic plate had 4 multiorgan chips, and each chip comprised 2 adjacent compartments to house the lung and liver tissues. Peristaltic pumps were employed to drive the culture medium in a circulatory manner. The liver’s detoxification ability was validated using aflatoxin B1 (AFB1), demonstrating that cocultured liver tissues reduced the AFB1 toxicity in lung tissues, indicating liver-mediated detoxification decreased lung tissue cytotoxicity^[209] In addition to toxicity screening, liver-target organ micro-/millifluidic plate also opens possibilities for testing prodrugs that only become biologically active after liver metabolism. The bioactivation process for prodrugs named capecitabine and cyclophosphamide targeting colorectal cancer has been recapitulated in liver-colorectal cancer micro-/millifluidic plates.^[41,110] In these 2 micro-/millifluidic plates, capecitabine and cyclophosphamide were transformed into metabolites 5-fluorouracil and 4-hydroxycyclophosphamide respectively, and then acted on colorectal cancer located at the compartments downstream.

Different from OoC, organoid follows intrinsic developmental programs and forms by self-organization and differentiation of stem cells.^{[210–211–212]} Organoids represent a complementary yet distinct approach to achieving the same goal as OoCs: replicating key aspects of native organ structures and functions in vitro.^{[213–214–215]} The convergence of OoC in organoid is leading to technological advances that allow proper biochemical and biophysical microenvironments and consequently overcome the limitations of this nascent technology such as lack of reproducibility, significant heterogeneity, and limited level of maturity and function.^[216,217] As a striking example, 3D-printed miniaturized spinning bioreactors, called SpinΩ, were developed to fit standard 12-well microtiter plates and used for generating forebrain organoids with high reproducibility as well as minimized heterogeneity and variability.^[218] Above the cover, a single electric motor drove 12 spinning shafts through a set of interconnected gears. The parameters of spinning shafts and leaves were systematically optimized to dynamically culture organoids in the suspension medium while preventing their aggregation at the center of each microwell. The forebrain organoids recapitulated critical features of human cortical development, including progenitor zone organization, neurogenesis, and especially a distinct human-specific outer radial glia cell layer. These organoids could also present pathologic features of microcephaly when infected with Zika virus. Together, SpinΩ provided a versatile system for modeling human brain development and disease and for drug screening. Furthermore, OoC also provides opportunities to mimic biological interactions at higher levels of organization, which remains a critical challenge in current organoids. In practice, liver, intestinal, and stomach organoids were cultured in 3 adjacent compartments connected via microchannels. Gravity-driven flow was generated by employing a perfusion rocker and further allows communication between different organoids. Bile acid homeostasis regulated by the interactions between liver organoid and intestine organoid is proved in such a multiorganoid micro-/millifluidic plate.^[219]

4.2 POCT

POCT plays a vital role in the improvement of clinical outcomes in medical healthcare management.^[220] In 2022, the global market of POCT has reached US$43.2 billion, with projections to grow to US$72.0 billion by 2027. Lab-on-a-chip (LOC) is considered one of the important technological drivers to transform the POCT industry. Especially the micro-/millifluidic plate with high throughput and sensitivity is now paving the way for the next generation of POCT platforms.^[221] For example, an ELISA micro-/millifluidic plate based on a 96-well format has been developed for immunological detection of Staphylococcal Enterotoxin B at concentrations as low as 0.1 ng mL⁻¹.^{[222–223–224]} The plate contained 3 layers of functional structures for sample loading, detection, and removal, and thus could provide a sample-to-answer process for immunological assays. Similarly, a droplet-based magnetic bead immunoassay was proposed to simplify washing steps and reduce sample or reagent consumption during the ELISA testing process. The immune reaction occurred within a small droplet in the reaction microwell and the antibody-conjugated magnetic beads were driven and manipulated by an XY-motorized magnet array stage to accomplish the following washing and detection step in the other 2 connected microwells, respectively. By using such a micro-/millifluidic plate, less than 5 pg/mL synthetic amyloid beta oligomers were detected as a model analyte for the early diagnosis of Alzheimer disease.^[102] To further increase the detection throughput of the device, Meso Scale Discovery launched a multiplexed electrochemiluminescent micro-/millifluidic plate that enabled to simultaneously measure up to 10 analytes in the same 96-well. The plate also enabled highly sensitive wash-free sandwich immunoassays in complex sample matrices by using SULFO-TAG labels on the antibody, which emitted light when electrochemically stimulated at the carbon electrode surface.^[225]

4.3 Other applications

In addition to the mainstream applications aforementioned, some micro-/millifluidic plates are also applied in cell biology for purposes such as cell transfection,^[226] cell proliferation,^[134] and cell agglomeration.^[227] Cell transfection involves the deliberate introduction of exogenous molecules into living eukaryotic cells.^[228] Compared with classical chemical transfection strategies, electroporation offers a nontoxic, cost-effective, and efficient alternative. Despite its advantages, traditional electroporation systems are not well-suited for high-throughput experiments due to their cumbersome manual operations. To meet this challenge, a standard 96-well suspended-drop electroporation device was developed in which biomolecules were able to be introduced into difficult-to-transfect cell types, such as primary neurons and differentiated neutrophils.^[229] This device contained 2 machined gold-coated copper pieces and each of them had 96 electrodes. Vertical electrode pairs created by these plates function as suspended electroporation chambers, allowing for rapid and high-throughput cell transfection. However, the electroporation parameters for each chamber were not customizable. This issue could be mitigated by integrating a PCB into the suspended-drop electroporation system.^[226] The PCB incorporates 8-row electrodes and 12-column electrodes mounted on its top and bottom surfaces, which are connected to 2 electrodes within each electroporation chamber. This modification provides individually addressable capabilities, enabling the transfection of various cell types with a wide range of biomolecules.

6.1 Standardization of micro-/millifluidic plates

Existing standards, such as ANSI/SLAS, ISO, and ASTM standards, are loosely applied to micro-/millifluidic plates. These standards primarily address plate designs and general laboratory practices but do not fully address the unique challenges of micro-/millifluidic systems, such as fluid dynamics, surface chemistry, and integration with external systems. As a result, micro-/millifluidic plates are still in the early stages of standardization. Future efforts should focus on refining existing standards and developing new, comprehensive guidelines that cover the entire lifecycle of micro-/millifluidic plates.^[263] Establishing consistent performance evaluation criteria and quality control standards will be crucial to ensure the reproducibility, reliability, and scalability of microfluidic systems. A collaborative effort between researchers, industry leaders, and standardization bodies is necessary to address these needs.

6.2 Materials and manufacturing strategies

The materials and manufacturing strategies for micro-/millifluidic plates have not been standardized. During the research phase, efficiency and ease of fabrication are prioritized to facilitate rapid prototyping and experimentation. However, transitioning to large-scale production requires considerations such as market demand, cost efficiency, and manufacturing scalability. These differing priorities often result in discrepancies in material selection and fabrication techniques, making it challenging to bridge the gap between laboratory development and commercial viability.

Silicone rubber, particularly PDMS, is widely used in the fabrication of micro- and millifluidic plates due to its optical transparency, gas permeability, and biocompatibility^[264]. However, the tendency of PDMS to absorb small molecules can affect the temporal and spatial concentrations of chemicals, compromising the reliability of experimental results, particularly in clinical diagnosis and drug testing.^{[265–266–267]} Furthermore, the existing fabrication techniques for PDMS-based plates are unsuitable for large-scale production due to challenges in scaling-up manufacturing process.^[143]

Thermoplastics like polystyrene (PS) and cyclic olefin copolymer are promising alternatives for both laboratory experiments and commercial production. Fabrication techniques such as hot embossing,^[268] laser engraving,^[269] and micromilling^[270] can optimize microstructures during the laboratory stage. Once the final design is determined, injection molding can enable rapid prototyping for industrial-scale production.^[271] However, injection molding is unsuitable for plates with complex microstructures or functional features due to limitations in material flexibility and filling narrow, deep cavities.^[272] Advances in 3D printing technology offer solutions to these challenges by enabling the rapid and cost-effective fabrication of intricate structures, enhancing design flexibility, and accelerating prototyping.^[273,274] Future innovations in high-resolution printing and multimaterial integration will be key to bridging the gap between research prototypes and commercial products.

6.3 User accessibility and integration

The interaction between end-users and micro-/millifluidic platforms is critical for their adoption.^[275] Many current plates rely on multiple external devices, such as liquid-driven pumps, microscopes, and computers, which require professional skills and experience to operate. This reliance complicates automation and scalability.^[276] Future efforts should focus on integrating micro-/millifluidic plates with automated experimental platforms and intelligent analysis systems. By incorporating advanced sensors and real-time data analysis, these systems can continuously acquire, process, and interpret complex data, enabling intelligent adjustments during experiments without human intervention.^[277,278] Artificial intelligence (AI) can optimize data collection, analysis, and decision-making, significantly enhancing automation, accelerating workflows, and improving accuracy.^{[279–280–281–282]} For instance, Tebon et al. and Al Shihabi et al.^[283,284] developed a high-throughput drug screening platform combining bioprinting, high-speed live cell interferometry (HSLCI) imaging, and machine learning. This platform continuously monitors bioprinted tumor organoids via HSLCI imaging, while AI-driven segmentation and tracking enable precise, label-free mass measurements and automated analysis.^[283,284] Future AI-driven micro-/millifluidic plates will benefit from more intuitive tools and standardized algorithm selection guidelines. Advances in explainable AI and automated model training will further enhance AI’s reliability and accelerate its adoption in high-throughput screening and precision analysis.^[285] Additionally, replacing multiple external devices with smaller, integrated components within the micro-/millifluidic platform will streamline workflows, reduce the need for specialized skills, and make the technology more scalable and accessible.

6.4 Future directions

Significant progress remains to be made before micro-/millifluidic plates can be fully integrated into the biomedical research pipeline. Microfluidic engineers have already made strides in design strategies, materials, fabrication techniques, and liquid-driven systems, which are essential for expanding the adoption of this technology. These efforts aim to build a broad user base and facilitate academic advancements. Moreover, micro-/millifluidic plates have advanced beyond the proof-of-concept stage and are now positioned to demonstrate their value in key biomedical applications, such as drug development and clinical diagnostics.