3D microfluidic high-throughput platform for simulating in vivo microenvironments and drug screening applications

Abstract

The invention discloses an automated microgels dispensing and micro-bioreactor system integrating air piezo pumps (1–4), liquid piezo pumps (5–6), solenoid valves (7–8), and dispensing chip docker (9) controlled by an edge computer (16) The system dispenses bioactive microgels from vials (10–13) through a microgels dispensing chip (21) with sheath media inlet (20) and nozzle (22), monitored by an active camera (23) at microfluidic intersection (24). A flow sensor (25) and temperature controller (26) regulate precise flow and uniform heating. Dispensed microgels are cultured in a micro-bioreactor (33) maintained by X–Y–Z translation stage (37) with selective perfusion and optical analysis via light source (30), beam splitter (31), objective lens (32), and monochrome camera (27). The system provides uniform thermal distribution, real-time imaging, and programmable microfluidic control for scalable, sterile, and automated biological assays.

Description

[0001] 3D MICROFLUIDIC HIGH THROUGHPUT PLATFORM FOR SIMULATING IN

[0002] VIVO MICROENVIRONMENTS AND DRUG SCREENING- APPLICATIONS

[0003] FIELD OF INVENTION

[0004] This present invention relates to a microfluidic Microspheroid Generator, dispensing System and an integrated Well Plate Reader for high-throughput drug discovery research.

[0005] BACKGROUND OF THE INVENTION

[0006] Current high-throughput drug discovery methods often rely on 2D cell cultures, which lack the physiological relevance of 3D tissue environments. This limited relevance can lead to misleading results and hinder the development of effective drugs.

[0007] The Microspheroid Generator employs microfluidic channels to manipulate fluids and cells with precise control. The channels can be fabricated from various materials, such as PDMS (polydimethylsiloxane), injection molding or 3d printing. The size of the generated microspheroids is precisely controlled by the flow focussing. The Microspheroid Generator can be equipped with interchangeable or modular extrusion heads, allowing researchers to tailor the system to their specific needs. Each head can be designed to handle a specific cell type.

[0008] There are prior-arts disclosing microarray compositions suitable for analysis by one or several spectrographic methods are disclosed, wherein the microarray composition includes a three- dimensional solid support and a plurality of reactive microbeads positioned on the solid support in spatially distinct and addressable locations (US20200011877A1).

[0009] The patent US 9464271B2 discloses a method that has been developed to produce stable cell-matrix microspheres with up to 100% encapsulation efficiency and high cell viability, using matrix or biomaterial systems with poor shape and mechanical stability for applications including cell therapeutics via microinjection or surgical implantation, 3D culture for in vitro expansion without repeated cell splitting using enzymatic digestion or mechanical dissociation and for enhanced production of therapeutic biomolecules, and in vitro modeling for morphogenesis studies. The modified droplet generation method is simple and scalable and enables the production of cell- matrix microspheres when the matrix or biomaterial system used has low concentration, with slow phase transition, with poor shape and mechanical stability.

[0010] The present invention addresses the limitations of traditional methods by providing a system for generating and analyzing multi-cell type 3D microspheroids under conditions that mimic blood flow dynamics.

[0011] SUMMARY OF THE INVENTION

[0012] The following summary is provided to facilitate a clear understanding of the new features in the disclosed embodiment and it is not intended to be a full, detailed description. A detailed description of all the aspects of the disclosed invention can be understood by reviewing the full specification, the drawing and the claims and the abstract, as a whole.

[0013] The system comprises two key components:

[0014] Multi Cell Type Microspheroid Generator: This component utilizes microfluidic technology to generate microspheres containing a mixture of cells from different types. The system allows for customization of the number of cell types used through interchangeable or modular extrusion heads. A precision dispensing mechanism enables the controlled delivery of microspheroids into microwell plates.

[0015] Real-Time Multi-Well Plate Reader Integrated with Flow Dynamics Simulator: This component features advanced imaging capabilities, including high-resolution bright field and fluorescence microscopy, for comprehensive analysis of microspheroids. The system is compatible with standard microwell plates and a specialized microfluidic chip designed to simulate flow dynamics around the microspheroids with interconnected wells enables the in-depth analysis of interrelated biological functions between distinct cell populations

[0016] BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 : Automated microgels dispensing system with control system, illustrating the arrangement of the components.

[0018] Figure 2: Microfluidic well-plate reader for biological studies and high-throughput drug screening. Figure 3: Micro-bioreactor microfluidic chip thermal study showing uniform heat distribution across the interconnected wells of the micro-bioreactor.

[0019] Figure 4: Selective flow control in each well of the micro-bioreactor (33), demonstrating software-controlled channels and fluidic pathways that can be selectively opened or closed to direct perfusion flow between specific wells, simulating physiological interconnections and tissue-specific microenvironments.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.

[0022] The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are constructed such that it provides a complete and a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.

[0023] Throughout this specification various indications have been given as to preferred and alternative embodiments of the invention. It should be understood that it is the appended claims, including all equivalents, which are intended to define the spirit and scope of this invention.

[0024] The present invention discloses a system that consists of two key components: a multi cell type Microspheroid Generator with a precision dispensing mechanism, and a Real-Time Multi-Well Plate Reader Integration that is compatible with both standard multi-well plates and our specialized microfluidic chip. This chip allows for the culture of different cell types in multiple wells with interconnected flow, incorporating flow dynamics to closely model blood flow, thereby providing an advanced platform for comprehensive drug discovery research.

[0025] The system incorporates a mechanism for accurate dispensing of microspheroids into microwells. This may involve piezoelectric elements or pressure-based control systems. The Plate Reader is equipped with high-resolution optics for bright field and fluorescence imaging of microspheroids within the wells. The system allows for control of illumination, focusing, and image acquisition through dedicated software.

[0026] The microfluidic chip features interconnected microchannels that mimic physiological flow patterns. This allows researchers to study how drugs interact with cells under conditions that resemble those found in the human body.

[0027] The present invention discloses an engineered microfluidic platform designed to generate, dispense, and analyze micro-scale tissue constructs or microgels containing one or more types of living cells. The platform integrates a Micro- Spheroid Generator, an Automated Dispensing System, and a Real-Time Multi -Well Plate Reader, enabling seamless generation, manipulation, and high-throughput biological analysis of three-dimensional (3D) microgels for use in drug screening, toxicity studies, and tissue modeling.

[0028] In one embodiment, the system comprises a microfluidic microspheroid generator that employs micro-scale flow channels fabricated using materials such as polydimethylsiloxane (PDMS), thermoplastics, or by additive manufacturing methods including injection molding or 3D printing. The microchannels are configured for flow focusing, allowing the precise control of droplet size and encapsulation parameters, resulting in uniform microgels or tissue spheroids containing one or more cell types.

[0029] The Microspheroid Generator may include modular or interchangeable extrusion heads configured to handle different viscosities, flow rates, or cell types. Each extrusion head can be customized to produce microgels containing specific combinations of cells, extracellular matrices, or hydrogels. The extrusion heads may be equipped with controlled pressure or piezoelectric actuation mechanisms for precise droplet generation. The produced microspheroids are maintained in an optimized fluid environment to ensure cell viability and physiological integrity.

[0030] The generated microspheroids are then directed to an Automated Dispensing System that precisely transfers them into wells of a micro-well plate or an integrated microfluidic chip. The dispensing system operates through a programmable liquid handling mechanism that provides spatial accuracy and minimizes mechanical stress during transfer. The dispensing process can be controlled through dedicated software that coordinates flow rates, timing, and droplet placement. The dispensing module is compatible with standard 96, 384, or 1536 well formats as well as custom-designed microfluidic plates.

[0031] In another embodiment, the invention includes a Real-Time Multi-Well Plate Reader integrated with an optical and imaging unit capable of detecting absorbance, fluorescence, and luminescence signals. The reader incorporates high-resolution optics, including bright field and fluorescence imaging modules, enabling both endpoint and time-lapse analyses of microgel growth, cellular morphology, and response to test compounds. The reader may also include a temperature- controlled stage and integrated fluidic flow control for simulating physiological shear conditions.

[0032] The present invention discloses an integrated 3D microfluidic high-throughput platform that combines micro-spheroid generation, precise dispensing, perfusion culture, and multi-modal optical readout in a single system. The device allows in-vitro simulation of in-vivo microenvironments such as organ-specific flow, temperature, and nutrient conditions, enabling real-time drug screening and cell-interaction studies with improved physiological relevance. The invention comprises three primary subsystems: a micro-gel or microspheroid dispensing module, a microfluidic perfusion and environmental control module, and a real-time optical sensing and imaging module. All subsystems are controlled by an edge computer (16) with feedback from optical and flow sensors to maintain precise control of droplet size, flow rates, temperature, and imaging parameters.

[0033] The invention discloses an engineered platform that integrates microgel generation, microfluidic culture, and real-time analysis for high-throughput biological and drug screening applications. The system comprises a microfluidic microspheroid generator with an automated dispensing unit that deposits cell-laden microgels into a temperature-controlled microfluidic chip or standard multiwell plate. The custom-designed chip includes interconnected channels that enable continuous perfusion and simulation of in-vivo flow dynamics for studying cell behavior and drug response. A built-in optical plate reader performs high-resolution imaging and readouts of absorbance, fluorescence, and luminescence. The invention provides enhanced physiological relevance, scalability, and analytical efficiency for next-generation drug discovery research.

[0034] The dispensing system includes a micro-gel dispensing chip (21) with multiple on-chip reservoirs (17-19) for housing different cell suspensions or hydrogel precursors. Each reservoir is connected to a piezo-driven pneumatic actuator (1-4) that regulates air pressure through micro-channels terminating at the nozzle outlet (22). The cell mixtures and sheath media converge at a microfluidic intersection zone (24), where uniform micro-spheroids are formed through controlled co-flow and pressure-regulated droplet generation. The size of each micro-gel droplet is actively monitored by a camera (23) focused on the intersection zone. Real-time image feedback is processed by the edge computer (16), which adjusts the piezo driver array (15) to maintain a constant droplet volume and frequency. A flow sensor (25) provides additional feedback for sheath fluid stability. The generated micro-spheroids are dispensed into either a standard microwell plate or a custom microbioreactor chip (33) mounted on an X-Y-Z translation stage (37). The dispensing head can position droplets with micrometer precision across multiple wells, allowing high-throughput operation. The entire dispensing head and microscope assembly operate within a sterile environment filtered by an air filter (38).

[0035] The perfusion module maintains continuous media circulation between fresh media (10), used media (11), and a circulating media reservoir (12) via piezo pumps (5, 6) and three-way solenoid valves (7, 8). The solenoids are controlled by the solenoid driver (14) under software control to automate feed and waste exchange cycles. A PID temperature controller (26) maintains the chip environment at desired physiological temperatures, typically 37 ± 0.2 °C, using a transparent conductive heater integrated in the chip base. The temperature sensor on the chip acts as feedback to regulate the temperature. Each channel in the dispensing chip (21) includes a pressure sensor coupled with each piezo pump to enable closed-loop pressure regulation. The interconnected channels mimic physiological flow profiles and enable co-culture of different cell types under shear conditions comparable to capillary networks. The microfluidic micro-bioreactor (33) with interconnected wells incorporates transparent and conductive glass for regulating chip temperature and is software selectable for controlling the channels through which the fluid should flow.

[0036] The optical subsystem performs multi-modal imaging absorbance, fluorescence, and luminescence using shared optical paths. A light source and monochromator (30) provide excitation, which is reflected by a beam splitter (31) into the objective lens (32). Emitted light passes through an emission filter disk (29) that includes neutral-density and pass-through positions driven by a stepper motor (28). The filtered emission is captured by a monochrome camera (27). For absorbance measurements, an independent fiber-optic light source (36) couples through a collimator (35) and fiber optic cable (34) to illuminate the wells in the chip vertically. The transmitted intensity is detected and quantified to compute real-time absorbance for each culture well sample. The imaging and measurement sequence is automated, running on the edge computer (16), which synchronizes light source switching, filter positioning, and data capture. The data generated can be used for live-cell viability, morphology, and drug-response analytics. The microscope setup and dispenser are mounted on the X, Y, and Z translation stage (37), enabling automated scanning across wells and focus adjustment for imaging.

[0037] During operation, sterile media and cell suspensions are loaded into respective reservoirs (10-13, 17-19). The system is initialized through the edge computer interface, setting parameters for droplet size, temperature, and imaging schedule. The piezo-actuated dispensing chip generates uniform micro-gels that are automatically deposited into culture wells or interconnected chambers within the bioreactor chip (33). The perfusion module continuously refreshes culture media while maintaining controlled flow to simulate in-vivo shear forces. The optical system performs periodic imaging and absorbance scans without manual intervention. The entire process is managed through the edge computer (16), which controls all solenoid and piezo drivers, processes the live video feed from the camera (23) to control the sphere size for active feedback, manages filter and light source settings, and coordinates data processing and translation stage movements.

[0038] The system integrates droplet generation, controlled perfusion, and multimodal analysis into a compact and automated platform suitable for a wide range of research and pharmaceutical applications. It enables high-throughput screening of drug libraries using physiologically relevant 3D cultures, simulates vascularized tissue microenvironments for oncology, neurobiology, and regenerative medicine research, reduces reagent use and assay variability through precise microfluidic control, and performs multi-parameter readouts including optical, absorbance, and fluorescence measurements within the same platform. By enabling real-time feedback and continuous perfusion, the invention bridges the gap between in-vitro and in-vivo models, delivering improved predictive accuracy for drug efficacy and toxicity testing.

[0039] Figure 1 illustrates an automated microgels dispensing system with control system, showing air piezo pumps (1,2, 3, 4) for microgels dispensing, liquid piezo pumps (5,6), three-way solenoid valves (7,8), microgels dispensing chip docker (9), liquid vials (10-13) for culture media and media circulation, solenoid driver array (14), piezo driver array (15), and the edge computer (16) controlling the system. It also includes on-chip vials (17-19), sheath media inlet (20), microgels dispensing chip (21), nozzle (22), active camera (23), microfluidic intersection location (24), flow sensor (25), temperature controller (26), and translation stage (37) for dispensing and positioning.

[0040] Figure 2 illustrates the microfluidic well-plate reader for biological studies and high-throughput drug screening, showing the light source and monochromator (30), beam splitter (31), objective lens (32), micro-bioreactor (33), fiber optic cable (34), collimator (35), light source for absorbance (36), monochrome camera (27), emission filter disk (29), and stepper motor (28) for filter selection.

[0041] Figure 3 illustrates the micro-bioreactor microfluidic chip thermal study showing uniform heat distribution across interconnected wells regulated by the PID temperature controller (26) and conductive glass substrate of the chip (33), ensuring consistent physiological temperature control across the wells.

[0042] Figure 4 illustrates selective flow control in each well of the micro-bioreactor (33), demonstrating software-selectable channels through which fluid can be directed to specific wells, enabling precise simulation of flow patterns and intercellular communication in connected culture environments.

[0043] Optionally, a custom microfluidic chip may replace the conventional well plate for applications requiring dynamic flow conditions. This chip features a network of interconnected microchannels linking multiple wells, thus allowing nutrient or drug diffusion across regions and enabling the study of intercellular communication and drug transport. The microchannels are dimensioned to mimic physiological blood flow dynamics, enabling analysis of drug behavior under quasi-in vivo flow regimes.

[0044] The entire system may be controlled by a unified software interface that synchronizes microgel formation, dispensing, and imaging operations. The software enables the selection of specific parameters such as flow rates, droplet diameters, imaging intervals, and data acquisition modes. Data collected from the plate reader can be processed for quantitative metrics including cell viability, proliferation, and drug response profiles. The present invention is highly inventive over existing manual or semi-automated systems because it integrates micro-spheroid generation, precise dispensing, continuous perfusion, and multi-modal optical analysis into a single automated platform. Unlike conventional manual methods that require repetitive handling, separate incubation, and independent imaging of 3D cell cultures, this system enables automated generation and deposition of uniform microgels or microspheroids from on- chip vials (17-19) through a microgels dispensing chip (21) using piezo-actuated pumps (1-6) and solenoid valves (7-8). Real-time feedback from the active camera (23), flow sensor (25), and temperature controller (26) allows precise control of droplet size, flow dynamics, and environmental conditions within the micro-bioreactor (33). The integration of an edge computer (16) ensures synchronized operation of all modules, which eliminates human variability, reduces reagent consumption, and enables high-throughput experimentation that is not feasible with existing manual systems.

[0045] Moreover, the invention provides significant advantages in biological research and drug discovery by closely mimicking physiological microenvironments. The interconnected wells of the microbioreactor (33) with software-selectable flow channels allow controlled perfusion and co-culture of multiple cell types, replicating in-vivo shear conditions and nutrient distribution. High- resolution optical imaging and absorbance measurement through the microfluidic well-plate reader (30-36) enable real-time monitoring of cell viability, morphology, and drug responses, all without manual intervention. By combining automation, environmental control, and multi-parameter readouts in a single compact system, the invention dramatically increases throughput, reproducibility, and efficiency, providing a transformative improvement over traditional manual 3D cell culture and drug screening methods.

[0046] The 3D Microfluidic Drug Discovery Platform is useful for various applications in drug discovery research, including:

[0047] Screening of bioactive molecules for potential therapeutic effects.

[0048] Evaluating drug efficacy and toxicity in a more relevant 3D cell culture model.

[0049] Studying cellular interactions and signaling pathways in a controlled microenvironment.

[0050] Investigating mechanisms of drug resistance. Advantages of the present invention are below:

[0051] Enhanced physiological relevance of drug discovery assays through the use of 3D microspheroids and blood flow simulation.

[0052] Increased throughput and scalability of drug screening processes.

[0053] Improved efficiency in generating and analyzing complex 3D cell cultures.

[0054] Facilitation of studies on intricate cellular interactions and mechanisms of action.

Claims

AMENDED CLAIMSreceived by the International Bureau on 05 May 2026 (05.05.2026).I / We Claim:

1. A 3d microfluidic high throughput platform comprising:(a) a microgel generation and dispensing subsystem comprising:a plurality of air piezo pumps (1-4) and liquid piezo pumps (5-6) operatively coupled to three-way solenoid valves (7-8),a microgels dispensing chip docker (9) receiving liquid from vials (10-13) through solenoid driver array (14) and piezo driver array (15),wherein an active camera (23) monitors droplet formation in real time at a microfluidic intersection (24) and provides image feedback to an edge computer (16), which dynamicallyadjusts the piezo driver array (15) to maintain a constant droplet volume and dispensing frequency, and a flow sensor (25) providing additional feedback for sheath fluid stability;on-chip vials (17-19) supply cell mixtures to a microgels dispensing chip (21) via a sheath media inlet (20), dispensing through a nozzle (22),wherein an active camera (23) monitors droplet formation in real time at a microfluidic intersection (24) and provides image feedback to an edge computer (16), which dynamically adjusts the piezo driver array (15) to maintain a constant droplet volume and dispensing frequency, and a flow sensor (25) providing additional feedback for sheath fluid stability;(b) a perfusion and environmental control subsystem comprising:a microfluidic micro-bioreactor (33) having a plurality of culture wells interconnected by microchannels dimensioned to simulate physiological blood flow dynamics, the interconnected wells being selectively perfusable through software-controlled solenoid valves operated by the edge computer (16), enabling directional flow between designated wells to simulate tissue-specific physiological interconnections, a temperature controller (26) maintaining the micro-bioreactor (33) at physiological temperature through a transparent conductive glass base providing uniform heat distribution across all wells,an X-Y-Z translation stage (37) for positioning the dispensing nozzle (22) over designated wells of the micro-bioreactor (33),and a disk air filter (38) maintaining sterile conditions throughout dispensing and incubation;(c) a real-time multi-modal optical readout subsystem comprising:a light source and monochromator (30), a beam splitter (31), an objective lens (32), an emission filter disk (29) driven by a stepper motor (28), and a monochrome camera (27) for fluorescence and luminescence imaging of each well, anda fiber optic cable (34), collimator (35), and absorbance light source (36) for independent absorbance measurement of each well of the micro-bioreactor (33); wherein all three subsystems are controlled and coordinated by the edge computer (16) through the solenoid driver array (14) and piezo driver array (15), and all operate within a sterile environment maintained by the disk air filter (38).

2. The platform as claimed in claim 1, wherein the edge computer (16) processes real-time image data from the active camera (23) to compute droplet diameter comparing the measured diameter to a target value, and adjusting drive signals to the piezo driver array (15) to correct deviations, thereby maintaining uniform microgel size during continuous high-throughput dispensing3. The platform as claimed in claim 1, wherein the temperature controller (26) operates as a PID controller maintaining the micro-bioreactor (33) at 37 ± 0.2°C using a temperature sensor on the chip as feedback input, and the conductive glass base distributes heat uniformly across all interconnected wells.

4. The platform as claimed in claim 1, wherein the micro-bioreactor (33) comprises interconnected wells allowing fluidic communication controlled by software-regulated valves for selective perfusion between wells.

5. The platform as claimed in claim 1, wherein the microgels dispensing chip (21) produces controlled microgels using a sheath media inlet (20) and microfluidic intersection (24) geometry to regulate droplet size and encapsulation rate.

6. The platform as claimed in claim 1, wherein the flow sensor (25) ensures precise sheath fluid control and droplet stability in coordination with the air piezo pumps (1-4).

7. The platform as claimed in claim 1, wherein the X-Y-Z translation stage (37) aligns the dispensing nozzle (22) with the micro-bioreactor (33) and optical setup for automated multipoint operation.

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