Variable high microfluidic cell automatic smearing and culturing system

Abstract

The application discloses a variable high microfluidic cell automatic smearing and culturing system. The system comprises three functionally independent and physically cooperating modules, namely, an automatic driving module, a microfluidic chip module and a living cell culturing module. By combining mechanical automation, micro-nano manufacturing and fluid mechanics principles, and by adopting a modular system design, the system successfully solves the problem that the three factors, namely, sample preparation standardization, high-quality data acquisition and long-term culturing, cannot be achieved simultaneously, which has been plaguing the field of high-content screening. The system is particularly suitable for application scenarios such as rare cell sample analysis, early development observation of organoids and drug toxicity testing requiring high-resolution morphological quantification, and has high scientific research value and commercial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering sorting technology, and particularly relates to the field of cell spreading technology, especially a variable high microfluidic automated cell spreading and culture system. Background Technology

[0002] With the rapid development of precision medicine, new drug development, and basic cell biology, the demand for precise quantitative analysis of cell samples is becoming increasingly urgent. To obtain reliable data such as morphological parameters and protein expression levels, the front-end cell sample preparation process is crucial. Ideal sample preparation requires achieving a high-quality, uniformly distributed monolayer on a substrate, while maintaining the long-term physiological activity of cells for continuous observation. However, current technologies often compromise on one aspect while striving to achieve this goal, failing to simultaneously meet the two core requirements of standardized monolayer preparation and long-term in-situ culture. This severely limits the data quality and efficiency of downstream analyses such as high-content imaging. Existing technologies and products mainly suffer from the following defects and shortcomings:

[0003] 1. To meet the oxygen exchange requirements of cells and simplify sample loading, microplates or open microfluidic chips are often used for cell culture. The channel height of a microfluidic chip is a key parameter for achieving a monolayer arrangement of cells and providing a controllable microenvironment. A review article (Meyvantsson I, Beebe D J. Cell culture models in microfluidic systems[J]. Annu. Rev. Anal. Chem., 2008, 1(1): 423-449.) discusses in detail the advantages and challenges of microfluidic cell culture models. If designed with high channels (e.g., >100 μm) to provide sufficient nutrient space and reduce fluid shear stress, cells tend to stack in multiple layers along the Z-axis under gravity, failing to meet the requirements for monolayer cell imaging. If designed with ultra-low channels (e.g., <20 μm) to physically restrict cell monolayer formation, a high driving pressure needs to be applied during cell loading, which generates enormous fluid shear stress, easily leading to cell damage or phenotypic changes. In addition, the confined space is prone to blockage during long-term culture, and the diffusion of nutrients and metabolic waste is restricted, which is not conducive to the long-term survival of cells.

[0004] 2. To address the limitations of fixed flow channel height, some studies have introduced dynamic elements. For example, pneumatic microvalves fabricated using multilayer soft lithography deform and compress a thin film by applying air pressure, thereby capturing cells or altering the height of the chamber. For instance, the literature (Booth R, Kim H. Characterization of a microfluidic invitro model of the blood-brain barrier (μBBB)[J]. Lab on a Chip, 2012, 12(10): 1784-1792.) demonstrates a device that uses pneumatic membrane compression to restrict cell movement for high-resolution imaging. These techniques typically rely on the deformation of flexible polydimethylsiloxane (PDMS) films to achieve compression. PDMS films often deform into an arc shape under air pressure rather than being perfectly parallel planes, and the PDMS material itself may have autofluorescence or surface defects. Its optical performance cannot compare with that of precision-polished optical glass, making it difficult to meet the extreme requirements of optical path uniformity for high-content imaging.

[0005] 3. When using microfluidic chips for large-area cell culture, the open liquid surface poses a significant challenge for subsequent cell analyses such as high-content imaging. Due to the interaction between the surface tension of the liquid and the container walls, a non-planar meniscus forms on the culture medium surface. In optical imaging, this meniscus acts as an irregular lens, causing optical path deflection. As some studies have pointed out, this effect leads to defocusing and uneven brightness in image edge areas, significantly reducing the effective imaging area and increasing the complexity and error rate of image analysis algorithms.

[0006] To address this issue, existing technologies attempt to employ specialized optical modulation devices. For example, the literature (Nienhaus F, Piotrowski T, Nießing B, et al. Adaptive phase contrast microscopy to compensate for the meniscus effect[J]. Scientific Reports,2023, 13(1): 5785.) compensates for imaging bias by placing an adaptive liquid prism in the optical path. While such methods improve optical quality to some extent, the process requires capturing and stitching multiple images, resulting in a lengthy process that is not conducive to the automation requirements of high-throughput laboratories. Furthermore, the system's correction capability is limited when the curvature angle of the liquid surface is too large.

[0007] In summary, existing technologies lack a solution that can dynamically integrate the aforementioned requirements. There is an urgent need for a novel system that can utilize automation to achieve low-damage, highly standardized, uniform spreading of monolayer cells, constructing a flat sample plane to meet the demands of subsequent high-precision analysis, and seamlessly switch within the device to an in-situ culture environment suitable for long-term cell growth, laying a solid foundation for downstream cell analysis. Summary of the Invention

[0008] The purpose of this invention is to address the common technical bottlenecks in the industry regarding high-quality cell sample preparation and culture by proposing a variable microfluidic automated cell spreading and culture system. This system combines the manipulability of open microfluidic chips with the precision of automated mechanical control, aiming to solve the problem that existing technologies struggle to simultaneously meet the two core requirements of standardized monolayer spreading and long-term in-situ culture in cell sample preparation.

[0009] The technical solution to achieve the purpose of this invention is: a variable high microfluidic cell automatic spreading and culture system, the system comprising three functionally independent but physically coordinated modules: an automatic drive module, a microfluidic chip module, and a live cell culture module;

[0010] The microfluidic chip module is provided with a culture chamber for containing cells and culture medium, and a limiting structure at the bottom of the culture chamber for defining the compression space;

[0011] The automatic drive module includes at least a pressure application component for pushing and spreading the cells in the culture chamber.

[0012] The live cell culture module includes at least a relatively enclosed environmental control chamber for housing the microfluidic chip module and providing a controlled physiological culture environment.

[0013] The automatic drive module drives the pressure application component to switch between a first position and a second position to achieve the conversion between cell culture state and monolayer board state: in the first position, the pressure application component is removed from the culture medium surface for open culture; in the second position, the pressure application component descends to touch the limiting structure, forming a monolayer board space of a preset height between the bottom of the pressure application component and the microfluidic chip module.

[0014] Furthermore, the system presents a modular, layered architecture:

[0015] The automatic drive module is located at the top layer;

[0016] The microfluidic chip module and the live cell culture module are located in the middle layer;

[0017] It also includes a cell post-analysis module located at the bottom layer, used to realize cell optical imaging or optical analysis; this module is located outside the culture environment, i.e. outside the live cell culture module, and corresponds to the optical window set at the bottom of the live cell culture module, that is, its optical path is aligned with the culture area at the bottom of the microfluidic chip module.

[0018] Furthermore, the automatic drive module also includes a sensing unit for real-time monitoring of changes in the mechanical signals of the pressure-applying component during its movement; it includes at least:

[0019] The sensing unit is used to identify the first signal of the pressure application component contacting the culture medium surface to determine the position reference point;

[0020] The sensing unit is also used to identify the sudden change in rigid resistance generated by the contact limiting structure of the pressure component as a second signal to trigger a stop motion command for the pressure component.

[0021] Furthermore, the microfluidic chip module includes a chip substrate, and the culture chamber and limiting structure are disposed on the chip substrate; the limiting structure is a micron-level rigid support fixed on the chip substrate, and its height is preset according to the cell type. It is used to precisely define the minimum distance between the lower surface of the pressure application component and the chip substrate when the pressure application component is in the second position, so that the pressure application component forms a physical limit when it is pressed down, ensuring that the cells are confined within the single-layer spread space.

[0022] Furthermore, during the movement of the pressure application component from the first position to the second position, that is, when the pressure application component performs the pushing and spreading operation, a radial extrusion flow is generated between its lower surface and the chip substrate, and the cells are evenly distributed on the horizontal plane through the shearing or dragging effect of the fluid.

[0023] Furthermore, the environmental control chamber is provided with a sealed interface through which the pressure application component passes, while the culture chamber is an open chamber. The two work together to enable the automatic drive module to enter and exit the environmental control chamber and the culture chamber.

[0024] Furthermore, the horizontal cross-sectional dimension of the pressure-applying component is smaller than the size of the top opening of the open chamber, so as to form an overflow gap between them for temporary storage of culture medium for backflow; and the side wall height of the culture chamber is higher than the highest liquid level of culture medium overflow when the pressure-applying component is in the second position; the surface of the pressure-applying component that contacts the cells has an anti-bioadhesion layer.

[0025] Furthermore, the automatic drive module has multiple adjustable speed modes, supporting switching between at least a high-speed mode for positioning the pressure component and a low-speed mode for pushing and spreading the sheet.

[0026] Furthermore, the environmental control chamber includes at least a temperature control component and a humidification control component;

[0027] The temperature control component is used to maintain the physiological temperature stability of the area where the microfluidic chip module is located;

[0028] The humidification gas control component is used to provide a preset composition of humidifying gas into the environmental control chamber and maintain a slightly positive pressure environment.

[0029] On the other hand, a method for operating the aforementioned variable high-microfluidic automated cell spreading and culture system is provided, the method comprising the following cyclical steps:

[0030] Open culture phase: The pressure application component is in the first position, allowing cells to undergo long-term growth and unrestricted gas exchange;

[0031] Compression analysis stage: The pressure application component moves to the second position, locking the cells in a thin layer of uniform height for cell optical imaging or optical analysis;

[0032] Recovery phase: The pressure application component returns to the first position, the overflowed culture medium flows back to cover the cells again, and the system returns to the large-volume open culture state.

[0033] Compared with the prior art, the significant advantages of this invention are:

[0034] (1) Balancing standardized sample preparation with long-term culture

[0035] This system achieves free switching between "compressed spreading" and "open culture" modes through the Z-axis movement of a movable pressure component (piston head). This solves the contradiction in existing technologies where fixed low-flow-channel chips are prone to clogging and have limited nutrient exchange, while high-flow-channel chips are difficult to use for single-layer cell imaging.

[0036] (2) Achieve high-quality, low-damage single-layer slab laying

[0037] Precise physical positioning: Utilizing the micron-level rigid positioning structure within the chip as an "absolute reference plane," the wafer height is precisely defined, ensuring that cells are confined within a single layer and are not mechanically damaged.

[0038] Fluid dynamics assistance: The radial extrusion flow generated by the pressure application component can effectively break up cell clusters, promote uniform cell distribution, and improve the standardization of the sample.

[0039] (3) Significantly improves optical imaging quality

[0040] Eliminating the meniscus effect: Under compression, the flat lower surface of the pressure-applying component replaces the irregular liquid meniscus, eliminating optical path deflection and defocusing, and expanding the effective imaging area.

[0041] The substrate is made of high-quality optical glass, avoiding the autofluorescence or surface deformation problems that may exist in flexible materials such as PDMS, thus meeting the requirements of high-content imaging.

[0042] (4) Possesses a highly intelligent feedback and protection mechanism

[0043] The system integrates a highly sensitive sensing unit capable of detecting liquid surface forces at the micro-Newton level and rigid contact forces at the Newton level. By capturing sudden changes in force signals to trigger a "soft landing" mechanism, it can accurately establish the Z-axis zero point and immediately stop movement to protect cells from mechanical pulverization damage.

[0044] (5) Modular and environmentally stable design

[0045] Physical isolation protection: The layered design effectively isolates the precision optical components from the high-temperature and high-humidity culture environment, ensuring the long-term stability of the detection system.

[0046] Adaptive environmental control: The live cell culture module provides a stable temperature control, high humidity saturated gas and micro-positive pressure environment, which prevents the evaporation of culture medium and maintains the physiological activity of cells.

[0047] (6) Supports dynamic tracking and in-situ analysis

[0048] The system supports repeated cycles of "sample addition / culturing" and "compression / analysis", allowing researchers to dynamically track the growth, differentiation, or drug response of the same group of cells at different time points with high spatiotemporal resolution without moving the samples.

[0049] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the system's slide preparation and culture process in one embodiment.

[0051] Figure 2 This is a schematic diagram of the system-integrated optical microscopy imaging process in one embodiment.

[0052] Figure 3 This is a schematic diagram of the system structure in one embodiment.

[0053] Figure 4 This is a cross-sectional view of the piston head and microfluidic structure in one embodiment.

[0054] Figure 5 This is a partial view of the piston head and the micropillar array of the microfluidic chip in one embodiment.

[0055] Figure 6 This is a schematic diagram of the piston head extrusion and ply-laying principle in one embodiment, wherein... Figure 6(a) in the diagram represents the cell loading and open culture stage. Figure 6 (b) in the text represents the compression lock state, i.e., the compression / analysis stage.

[0056] Figure reference numerals: 1-Automatic drive module, 2-Live cell culture module, 3-Microfluidic chip module, 4-Micro gas interface, 5-Force sensor, 6-Piston head, 7-Micron-level rigid limiting microcolumn, 8-Robotic arm, 9-Open chamber, 10-Concave liquid surface, 11-Extrusion flow. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0058] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indicators will also change accordingly.

[0059] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. If the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0060] In one embodiment, a variable high microfluidic cell automated spreading and culture system is provided, the system comprising three functionally independent but physically coordinated modules: an automated drive module 1, a microfluidic chip module 3, and a live cell culture module 2.

[0061] The microfluidic chip module is provided with a culture chamber for containing cells and culture medium, and a limiting structure at the bottom of the culture chamber for defining the compression space;

[0062] The automatic drive module includes at least a pressure application component for pushing and spreading the cells in the culture chamber to form a uniform single-layer spread.

[0063] The live cell culture module includes at least one independent, small, relatively enclosed environmental control chamber for housing the microfluidic chip module and providing a controlled physiological culture environment (capable of providing the required stable temperature, humidity and gas environment).

[0064] The automatic drive module drives the pressure application component to switch between a first position and a second position to achieve the conversion between cell culture state and monolayer board state: in the first position, the pressure application component is removed from the culture medium surface for open culture; in the second position, the pressure application component descends to touch the limiting structure, forming a monolayer board space of a preset height between the bottom of the pressure application component and the microfluidic chip module.

[0065] Furthermore, in one embodiment, the system presents a modular, layered architecture:

[0066] The automatic drive module is located at the top layer and is responsible for providing precise driving force.

[0067] The microfluidic chip module and the live cell culture module are located in the middle layer, which is the core functional area;

[0068] It also includes a cell post-analysis module located at the bottom layer, used to realize cell optical imaging or optical analysis; this module is located outside the culture environment, i.e. outside the live cell culture module, and corresponds to the optical window set at the bottom of the live cell culture module, that is, its optical path is aligned with the culture area at the bottom of the microfluidic chip module.

[0069] Here, the design of the cell subsequent analysis module position can physically isolate the environmentally sensitive optical components from the high temperature and high humidity culture environment, ensuring the long-term stability of the system and the imaging quality.

[0070] Preferably, in some embodiments, the optical window is located at the center of the bottom of the environmental control chamber, and it is a recessed opening. The microfluidic chip module is embedded in the optical window to form a relatively closed environment.

[0071] Furthermore, in one embodiment, the automatic drive module further includes a sensing unit for real-time monitoring of changes in the mechanical signals of the pressure-applying component during movement; it includes at least:

[0072] The sensing unit is used to identify the first signal of the pressure application component contacting the culture medium surface to determine the position reference point;

[0073] The sensing unit is also used to identify a second signal from the contact limit structure of the pressure component to trigger a command to stop the movement of the pressure component.

[0074] Preferably, in some embodiments, the sensing unit is a high-sensitivity sensing unit capable of detecting a range from microNewton-level liquid surface tension fracturing force to Newton-level rigid contact force.

[0075] Preferably, in some embodiments, the automatic drive module has a multi-level adjustable speed mode, supporting switching between at least a high-speed mode for positioning the pressure component and a low-speed mode for pushing the sheet.

[0076] Preferably, in some embodiments, the pressure-applying assembly includes a drive device capable of Z-axis lifting motion and a piston head; the Z-axis is vertical.

[0077] The Z-axis travel of the drive device needs to cover the range from the height of complete detachment from the liquid surface of the microfluidic chip module to the height of contact with the bottom limiting structure of the microfluidic chip module.

[0078] The piston head can extend into the environmental control chamber and the culture chamber to push and flatten the cells.

[0079] More preferably, in some embodiments, the movement speed of the drive device is adjustable, enabling switching from a high-speed mode for rapid positioning to an extremely low-speed mode for precision tablet pressing. Here, the drive device employs, but is not limited to, a robotic arm. The robotic arm employs, but is not limited to, a linear module driven by a stepper motor or servo motor equipped with a high-resolution optical encoder and closed-loop feedback control.

[0080] More preferably, in some embodiments, the sensing unit is disposed between the drive device (the drive end of the robotic arm) and the piston head, specifically to: provide a feedback signal at the instant the piston head contacts the liquid surface to establish the Z-axis zero point or the liquid surface reference surface; monitor the resistance in real time during the piston head's downward pressing process; and simultaneously, generate a steep force signal change when the piston head contacts the limiting structure, and the system uses this signal as a "soft landing" termination command to immediately control the drive device to stop moving.

[0081] Furthermore, in one embodiment, the microfluidic chip module includes a chip substrate, and the culture chamber and limiting structure are disposed on the chip substrate; the limiting structure is a micron-level rigid support fixed on the chip substrate, and its height is preset according to the cell type. It is used to precisely define the minimum distance between the lower surface of the pressure application component and the chip substrate when the pressure application component is in the second position, so that the pressure application component forms a physical limit when it is pressed down, ensuring that the cells are confined within the single-layer spread space.

[0082] Preferably, in some embodiments, the chip substrate is made of high-quality optical glass substrate, and the upper surface of the optical glass substrate is pre-coated with extracellular matrix proteins to promote cell adhesion and growth.

[0083] Preferably, in some embodiments, the micron-level rigid support is, but is not limited to, an array of micron-level rigid limiting micropillars, which serve as a physical "absolute reference plane" and "safety limiter".

[0084] Furthermore, in some embodiments, the sidewalls of the culture chamber are formed by, but not limited to, injection molding. The volume of the culture chamber should be designed to be large enough to support cell culture in an open state for several hours to several days.

[0085] Furthermore, in some embodiments, the height of the micron-level rigid limiting micropillar is a key parameter that determines the quality of cell spread. It should be set according to the type of target cells. The principle is that the height should be slightly greater than the average height of the cells after they spread on the wall (the inner wall of the culture chamber), or slightly greater than the average diameter of the suspended cells, so as to ensure that the cells are confined in a monolayer but are not subjected to mechanical compression.

[0086] Furthermore, in some embodiments, the micron-level rigid limiting micropillars are uniformly distributed, and the spacing between the micron-level rigid limiting micropillars is designed to meet the following conditions: cells exceeding a preset proportional threshold (most cells) fall in the gap area between the micron-level rigid limiting micropillars, rather than on top of the micron-level rigid limiting micropillars; at the same time, the duty cycle of the micron-level rigid limiting micropillars should be as low as possible to maximize the effective spread area.

[0087] Furthermore, in some embodiments, the micron-scale rigid limiting micropillars can be directly manufactured on a glass substrate by, but not limited to, photolithography and wet / dry etching techniques, or by spin-coating a layer of photoresist on a glass substrate and manufacturing it by photolithography microfabrication techniques, or by integrally forming it on an injection-molded plastic substrate. The specific formation method is not further specifically limited.

[0088] Preferably, in some embodiments, during the movement of the pressure application component from the first position to the second position, that is, when the pressure application component performs the pushing and spreading operation, a radial extrusion flow is generated between its lower surface (lower surface of the piston head) and the chip substrate, and the cells are evenly distributed on the horizontal plane by the shearing or dragging effect of the fluid.

[0089] Preferably, in some embodiments, the horizontal cross-sectional dimension of the pressure-applying component is smaller than the size of the top opening of the open chamber, so as to form an overflow gap between them for temporary storage of culture medium for backflow. Specifically, exemplarily, the horizontal cross-sectional dimension of the piston head is slightly smaller than the size of the top opening of the open culture chamber to ensure smooth insertion and leave narrow slits around it for liquid overflow. At the same time, the sidewall height of the open culture chamber ensures that when the piston head is pressed down to its lowest position, i.e., the second position, the squeezed-out culture medium is completely contained within the overflow gap and does not overflow outside the microfluidic chip module, thereby ensuring the sterility of cell culture and the conservation of culture medium volume.

[0090] Preferably, in some embodiments, the surface of the pressure-applying component in contact with the cells has an anti-bioadhesion layer, achieved through material surface modification to prevent cell or protein adsorption. Specifically, the lower surface of the piston head undergoes a rigorous anti-bioadhesion surface treatment. This is achieved, but is not limited to, chemical modification methods, such as covalently grafting a hydrophilic polymer coating onto a precision-polished glass surface. These coatings form a stable hydration layer on the material surface, effectively resisting protein and cell adsorption, ensuring complete separation of the piston head from the cell layer upon lifting, and maintaining the long-term cleanliness of the optical interface.

[0091] Furthermore, in one embodiment, the environmental control chamber is provided with a sealed interface through which the pressure application component passes, while the culture chamber is an open chamber. The two work together to enable the automatic drive module to enter and exit the environmental control chamber and the culture chamber.

[0092] Furthermore, in one embodiment, the environmental control chamber includes at least a temperature control component and a humidification control component;

[0093] The temperature control component is used to maintain the physiological temperature stability of the area where the microfluidic chip module is located;

[0094] The humidification gas control component is used to provide humidifying gas of preset composition into the environmental control chamber and maintain a slightly positive pressure environment to form a stable airflow circulation.

[0095] Preferably, in some embodiments, the temperature control component achieves temperature control by means of, but not limited to: integrating precision resistance heating elements or conductive glass heating layers into the cavity wall and bottom optical window, and equipping them with high-precision temperature sensors and controllers to maintain a physiological temperature of 37°C, while ensuring that the temperature fluctuation in the area where the microfluidic chip module is located is minimal (less than a preset fluctuation range).

[0096] Preferably, in some embodiments, the humidification gas control component achieves humidification gas control in the following ways, but not limited to: the environmental control chamber is also provided with a micro gas interface for connecting to an external premixed gas source, and the gas passes through a built-in heated water tank humidifier before entering the environmental control chamber to reach near-saturation humidity, so as to prevent rapid evaporation of the culture medium in the open chamber.

[0097] Furthermore, in one embodiment, the live cell culture module allows access to other external modules to adapt to or expand different scenario requirements.

[0098] In one embodiment, a method for operating the variable high-microfluidic automated cell spreading and culture system is provided, the method comprising the following cyclical steps:

[0099] Open culture phase: The pressure application component is in the first position, and the cells undergo long-term growth culture and unrestricted gas exchange;

[0100] Compression analysis stage: The pressure application component moves to the second position, locking the cells in a thin layer of uniform height for cell optical imaging or optical analysis;

[0101] Recovery phase: The pressure application component returns to the first position, the overflowed culture medium flows back to cover the cells again, and the system returns to the large-volume open culture state.

[0102] Specifically, the system executes the following work cycle: repeatedly switching between "sample addition / culturing" and "compression / analysis";

[0103] First, during the system initialization and environment setup phase, the pre-sterilized microfluidic chip module is placed inside the live cell culture module and fixed on the bottom optical window.

[0104] Start the temperature control and gas path system of the live cell culture module, set the target temperature and gas environment, and wait for the system to preheat and reach equilibrium; at this time, the piston head of the automatic drive module is in the raised state, which does not obstruct gas exchange.

[0105] Next, we proceed to the cell loading and open culture stage. We open the loading port of the live cell culture module and inject the cell suspension into the open culture chamber of the microfluidic chip module. The cells settle to the chip substrate surface under gravity. At this time, the system is in an "open state" and the piston head is not in contact with the liquid surface. The liquid layer in the open culture chamber is relatively thick, and the liquid surface is naturally concave due to surface tension. In this state, the cells enjoy sufficient culture medium and unrestricted gas exchange, which is suitable for initial adhesion and long-term growth.

[0106] When the predetermined time point is reached for high-content optical imaging, the system executes an automatic wafer placement and compression locking procedure, entering a "compression state." The automatic drive module drives the piston head through a sealed through-hole at the upper end of the adaptive environmental control chamber into the controlled environment, and rapidly descends into the open culture chamber. When the sensing unit detects the instantaneous change in resistance when the piston head contacts the liquid surface, the system switches to an extremely low-speed mode. The piston head continues to slowly press into the liquid. As the distance between the lower surface of the piston head and the chip substrate decreases, the liquid between them is forced out radially, forming a radial extrusion flow that exerts pressure on cells in a suspended or semi-adherent state. Shearing and dragging forces break up the aggregated cell clusters and promote the cells to spread evenly on the horizontal plane. The piston head continues to descend until its lower surface contacts the top of the micron-level rigid limiting micropillar. The sensing unit detects a sharp increase in resistance exceeding the preset standard, triggering a "soft landing" mechanism. The drive device stops working, keeping the piston head in its current position. At this point, the system enters a "compression-locked state," where a thin liquid space with extremely uniform height, precisely defined by the height of the micron-level rigid limiting micropillar, is formed between the lower surface of the piston head and the upper surface of the chip substrate. In the "compression-locked state," the system performs subsequent high-content optical imaging analysis.

[0107] After the imaging task is completed, the automatic drive module slowly and steadily raises the piston head, causing it to detach from the liquid surface and retreat to the top or outside of the live cell culture module. As the piston head is raised, the culture medium that previously overflowed into the side slit formed between the piston head and the open culture chamber flows back to the central area under the action of gravity and surface tension, re-covering the cells, and the microfluidic chip module returns to its large-volume open state. The humidifying gas in the live cell culture module is once again in full contact with the liquid surface for exchange. In this state, the system maintains the microfluidic chip module in a constant temperature environment for long-term culture of several hours to several days.

[0108] Based on actual needs, time intervals are set to repeatedly execute the compression, imaging, and recovery steps, thereby achieving high spatiotemporal resolution dynamic tracking of the growth, differentiation, migration, or drug response processes of the same group of cells.

[0109] In summary, this invention combines mechanical automation control, micro / nano structure confinement, and cellular microenvironment construction, solving the problem of existing technologies struggling to balance standardized cell sample preparation, convenient long-term in-situ culture, and high-precision subsequent content analysis. This is mainly reflected in the following aspects:

[0110] 1. Traditional cell spreading techniques primarily rely on manual pipetting to add cell suspensions into culture dishes or well plates, allowing them to settle and adhere naturally due to gravity. This method is highly susceptible to human error, liquid disturbance, and container edge effects, leading to uneven cell distribution, uncontrollable density, and cell aggregation and overlap, severely limiting the accuracy and reproducibility of high-content quantitative analysis. This invention employs an automated drive module to precisely control the piston head's downward pressure, utilizing the principle of extrusion flow in fluid mechanics to efficiently push suspended or semi-adherent cells towards the bottom plane of the chip. More importantly, combined with a pre-fabricated array of micron-level rigid limiting micropillars at the bottom of the chip, the minimum distance between the piston head and the chip's bottom surface is precisely defined. This design forcibly confines cells within a highly uniform two-dimensional thin-layer space, effectively preventing longitudinal overlap and achieving highly standardized, uniform large-area monolayer cell spreading, significantly improving the statistical significance and reliability of subsequent image analysis.

[0111] 2. In conventional open culture containers, liquid culture inevitably results in a meniscus at the gas-liquid interface due to surface tension. This curved liquid surface acts as an irregular lens during large-field-of-view, high-resolution microscopic imaging, leading to severe imaging deviations, image field curvature, and inconsistent optical path lengths across different regions, significantly limiting the effective imaging field of view and image quality. The piston head designed in this invention presses down to contact and smooth the liquid surface, forming an ideal parallel optical system together with the chip substrate at the bottom. This allows the system to fully utilize the performance of high numerical aperture objectives, rapidly acquiring large-field-of-view, high-resolution image data.

[0112] 3. Traditional live-cell analysis workflows typically require frequent sample transfers between a CO2 incubator and a separate data acquisition platform, such as optical imaging, which is cumbersome and increases the risk of sample contamination and environmental stress. While some existing closed microfluidic chips can integrate culture and imaging, they often face problems such as insufficient gas exchange, difficulties in long-term culture, and bubble blockage. This invention employs a unique operating mode: in the open culture state, the piston head is raised, and the chip returns to a large-volume open micropore that facilitates gas exchange and long-term culture; when detection is needed, it quickly switches to a compressed state for automated slide preparation and instantaneous imaging. This design cleverly combines the advantages of open culture systems with the advantages of closed imaging systems, achieving end-to-end integration from cell seeding and long-term culture to automated and standardized sample preparation. This greatly simplifies experimental operations, improves detection efficiency, and better maintains cell physiological activity, laying the foundation for subsequent cell analysis.

[0113] As a specific example, in one embodiment, the invention will be further described in detail with reference to the accompanying drawings.

[0114] This embodiment provides a variable high-performance microfluidic cell spreading and culture system driven by an automated mechanical device, along with its supporting technical solutions. This system combines the manipulability of an open microfluidic chip with the precision of automated mechanical control, aiming to solve the problem of existing technologies struggling to simultaneously meet the two core requirements of standardized monolayer spreading and long-term in-situ culture during cell sample preparation. This embodiment utilizes a controllable piston head 6 to perform precise Z-axis movement within an open culture chamber, thereby dynamically changing the effective working height of the chamber and reversibly switching between "compressed spreading" and "open culture," as shown below. Figure 1 As shown. This design utilizes the hydrodynamic principles of squeeze flow to achieve automated monolayer cell spreading, eliminates imaging biases caused by open liquid surfaces, and retains the ability to conduct long-term culture in an open environment. The entire system mainly consists of three functionally independent but physically coordinated modules: an automatic drive module 1, a microfluidic chip module 3, and a live cell culture module 2. It is suitable for various downstream analytical experiments, such as high-content imaging. Figure 2 As shown.

[0115] The overall architecture of the system presents a modular and layered design, such as Figure 3 As shown. The top layer is the automatic drive module, which is responsible for providing precise mechanical driving force; the middle layer is the core functional area, containing the live cell culture module and the microfluidic chip module—the carrier for cell culture—placed inside it; the bottom layer can hold modules for subsequent cell analysis, such as microscopic imaging. This module is located outside the culture environment. This design physically isolates the environment-sensitive optical components from the high-temperature and high-humidity culture environment, ensuring the long-term stability of the system and the imaging quality.

[0116] The microfluidic chip module, as the core consumable of this system, employs a unique design to work in conjunction with the automatic drive module, such as... Figure 4 As shown. Its bottom is made of high-quality optical glass substrate, and its upper surface is pre-coated with extracellular matrix proteins to promote cell adhesion and growth. A fully open-top culture chamber is constructed above the chip substrate to contain cells and culture medium. The sidewalls of the chamber can be formed using injection molding. The chamber's volume should be large enough to support cell culture in an open state for several hours to several days. The most crucial feature of this chip is the array of micron-sized rigid confinement micropillars 7 fabricated within its chamber, such as... Figure 5As shown. These micropillars function as physical "absolute reference planes" and "safety limiters." When the piston head of the automatic drive module depresses, the top surface of the micropillars is the only solid plane that the piston head can contact, precisely defining the minimum distance between the lower surface of the piston head and the chip substrate under compression. The height of the micropillars is a key parameter determining the quality of cell spread and should be set according to the type of target cells. The principle is to slightly exceed the average height of cells after adhesion and spread, or slightly exceed the average diameter of suspended cells, to ensure that cells are confined within a monolayer without being mechanically compressed. For example, for most mammalian cells, the micropillar height can be set between 15 μm and 25 μm; for larger cells or cell clusters, the height can be appropriately increased. The micropillars are uniformly arrayed on the substrate, with sufficient spacing to ensure that the vast majority of cells fall in the gaps between the micropillars, rather than on top of the micropillars. The duty cycle of the micropillars should be as low as possible to maximize the effective spread area. These micropillars can be manufactured directly on a glass substrate using photolithography and wet / dry etching techniques, or by spin-coating a layer of photoresist onto a glass substrate and then manufacturing them using photolithography microfabrication techniques. They can also be integrally molded onto an injection-molded plastic substrate.

[0117] The live cell culture module is a self-contained, small, environmentally controlled chamber, similar to a miniature incubator on a microscope stage, used to house microfluidic chip modules and provide them with a stable temperature, humidity, and gas environment. This module is a relatively enclosed space, but allows other modules to be connected. Its main body is a temperature-controlled frame with an internal space perfectly suited to accommodate the microfluidic chip modules. A large light-transmitting aperture is located at the center of the bottom of the chamber, allowing the chip to be placed directly on this window, ensuring unobstructed observation by the microscope objective from below. To maintain a physiological temperature of 37°C, precision resistance heating elements or conductive glass heating layers are integrated into the chamber walls and bottom window, along with a high-precision temperature sensor and controller, ensuring minimal temperature fluctuations in the chip area. The module also features a miniature gas interface 4, connecting to an external premixed gas source (e.g., 5% CO2 + 95% air). Before entering the chamber, the gas passes through a built-in heated water tank-type humidifier to achieve near-saturation humidity, preventing rapid evaporation of the culture medium within the open chamber. A slight positive pressure is maintained within the chamber, creating a stable airflow circulation. The top cover of the culture module has a sealed flexible interface or precision fitting hole at its center, which is specifically designed to allow the piston head of the automatic drive module to extend into the chamber for operation, while minimizing environmental leakage.

[0118] An automated drive module is mounted above the live cell culture module, responsible for driving the piston head through the top interface of the culture module to push and flatten the cells. Its core drive component is a robotic arm 8 capable of precise Z-axis lifting and lowering motion, preferably a linear module driven by a stepper motor or servo motor equipped with a high-resolution optical encoder and closed-loop feedback control. Its Z-axis travel should cover a range from the height completely detached from the chip liquid surface to the height contacting the bottom limiting structure of the chip. Crucially, its motion resolution and repeatability should reach the micrometer level to ensure precise control of the compressed liquid layer thickness. The robotic arm's movement speed should be adjustable, enabling switching from a high-speed mode for rapid positioning to an extremely low-speed mode for precision tableting. Between the drive end of the robotic arm and the piston head, a highly sensitive micro-force sensor 5 is integrated, with a range designed to detect a range from microNewton-level surface tension fracturing forces to Newton-level rigid contact forces. The sensor plays a crucial role: firstly, it provides a feedback signal the instant the piston head contacts the liquid surface, establishing the Z-axis zero point or the liquid surface reference plane; secondly, it monitors resistance in real time during the piston head's downward press, especially upon contact with the rigid limiting micropillars at the bottom of the chip, where a steep force signal change occurs. The system uses this signal as a "soft landing" termination command, immediately stopping the motor movement to prevent further pressure from the piston head causing mechanical damage to the cells. The horizontal cross-sectional dimensions of the piston head are designed to be slightly smaller than the opening size of the microfluidic chip culture chamber, ensuring smooth insertion and leaving narrow slits around the perimeter for liquid overflow. To prevent cells from non-specifically adhering to the lower surface of the piston head during pressing, it must undergo rigorous anti-bioadhesion surface treatment. Chemical modification methods are typically used, such as covalently grafting a hydrophilic polymer coating onto a precision-polished glass surface. These coatings form a stable hydration layer on the material surface, effectively resisting protein and cell adsorption, ensuring complete separation of the piston head from the cell layer upon lifting, and maintaining the long-term cleanliness of the optical interface.

[0119] The system provided by this invention can perform an innovative work cycle, repeatedly switching between "sample loading / culturing" and "compression / analysis". A typical workflow of a system combining high-content imaging is as follows: First, in the system initialization and environment setup phase, a pre-sterilized microfluidic chip module is placed inside the live cell culture module and fixed to the bottom window. The temperature control and gas path system of the live cell culture module is activated, the target temperature and gas environment are set, and the system is allowed to preheat and reach equilibrium. At this time, the piston head of the automatic drive module is in the raised state, not obstructing gas exchange. Next, the cell sample loading and open culture phase begins (e.g., Figure 6As shown in (a)), the sample loading port of the culture module is temporarily opened using a pipette, and an appropriate amount of cell suspension is injected into the open chamber 9 of the chip. The cells settle to the chip substrate surface under gravity. At this time, the system is in an "open state," the piston head is not in contact with the liquid surface, the liquid layer in the chamber is relatively thick, and the liquid surface is naturally concave due to surface tension (concave meniscus 10). In this state, the cells enjoy sufficient culture medium and unrestricted gas exchange, suitable for initial adhesion and long-term growth. When high-content imaging is required at the predetermined time point, the system executes an automatic slide preparation and compression locking procedure, entering a "compression locking state" (as shown in (a)). Figure 6 (As shown in (b)). The automatic drive module drives the piston head through the interface at the top of the culture module, into the controlled environment, and rapidly descends into the chip chamber. The force sensor detects the instantaneous change in resistance when the piston head contacts the liquid surface, and the control system switches the speed to a precise slow mode. The piston head continues to slowly press the liquid in, and as the distance between the bottom surface of the piston and the chip substrate decreases, the liquid between them is forced to be squeezed out radially in all directions, forming a radial extrusion flow 11. This flow generates shearing and dragging effects on cells in a suspended or semi-adherent state, effectively breaking up aggregated cell clusters and promoting the uniform distribution and spread of cells on the horizontal plane. The piston head continues to descend until its lower surface gently contacts the top of the rigid limiting micropillar at the bottom of the chip. The force sensor detects a sharp increase in resistance, triggering a soft landing mechanism, and the motor immediately stops and maintains the current position. At this point, the system enters a "compression state," and a thin liquid space with an extremely uniform height, precisely defined by the height of the micropillar, is formed between the lower surface of the piston head and the upper surface of the chip substrate. The physical confinement effect forcibly restricts the cells within this two-dimensional thin layer, achieving monolayer deployment and preventing cell overlap along the Z-axis. Simultaneously, the micropillar support ensures the piston head does not directly press on the cell bodies, avoiding mechanical damage. Excess culture medium squeezed out during compression overflows into the gap between the piston head and the chamber sidewall for temporary storage, preventing leakage outside the chip. In the compressed and locked state, the system performs high-content optical imaging and other analytical tasks.

[0120] In the compressed and locked state, the previously irregular meniscus gas-liquid interface is replaced by the smooth lower surface of the piston head. During imaging, because the cells are confined to an extremely narrow depth of focus, autofocus is rapid and accurate. The system utilizes a wide-field objective lens to quickly capture large-area, high-resolution images, acquiring massive amounts of single-cell data. After the imaging task is completed, to ensure long-term cell survival, the cells must be restored to a suitable culture environment. The automatic drive device slowly and smoothly raises the piston head, detaching it from the liquid surface and retracting it to the high-level storage area at the top of the culture module. This storage area is located below the sealed interface to ensure that the piston head remains in a sterile environment with controlled temperature and humidity, preventing contamination and maintaining temperature and humidity balance within the system. As the piston head is raised, the culture medium that previously overflowed into the side slits flows back to the central area under the influence of gravity and surface tension, re-covering the cells. The chip returns to a large-volume open state. The humidifying gas within the culture module once again comes into full contact with the liquid surface for exchange. In this state, the system can maintain the chip in a constant-temperature environment for long-term culture of several hours to several days. Depending on the experimental requirements, time intervals (e.g., every hour) can be set to repeatedly perform the compression, imaging, and recovery steps, thereby achieving high spatiotemporal resolution dynamic tracking of the growth, differentiation, migration, or drug response processes of the same group of cells.

[0121] The system proposed in this invention, by combining mechanical automation, micro / nano manufacturing, and fluid mechanics principles, and employing a modular system design, successfully solves the long-standing challenge of achieving a balance between standardized sample preparation, high-quality data acquisition, and long-term culture in the field of high-content screening. This system is particularly suitable for applications such as rare cell sample analysis, early organoid development observation, and drug toxicity testing requiring high-resolution morphological quantification, demonstrating significant scientific research value and promising commercial applications.

[0122] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention without departing from its spirit and scope should be included within the protection scope of the present invention.

Claims

1. A variable high-performance microfluidic automated cell spreading and culture system, characterized in that, The system comprises three functionally independent but physically coordinated modules: an automatic drive module, a microfluidic chip module, and a live cell culture module. The microfluidic chip module is provided with a culture chamber for containing cells and culture medium, and a limiting structure at the bottom of the culture chamber for defining the compression space; The automatic drive module includes at least a pressure application component for pushing and spreading the cells in the culture chamber. The live cell culture module includes at least a relatively enclosed environmental control chamber for housing the microfluidic chip module and providing a controlled physiological culture environment. The automatic drive module drives the pressure application component to switch between a first position and a second position to realize the conversion between cell culture state and monolayer spread state: in the first position, the pressure application component is removed from the culture medium surface for open culture; in the second position, the pressure application component descends to touch the limiting structure, forming a monolayer spread space of a preset height between the bottom of the pressure application component and the microfluidic chip module. The automatic drive module further includes a sensing unit for real-time monitoring of changes in the mechanical signals of the pressure-applying component during movement; it includes at least: The sensing unit is used to identify the first signal of the pressure application component contacting the culture medium surface to determine the position reference point; The sensing unit is also used to identify the sudden change in rigid resistance generated by the contact limiting structure of the pressure component as a second signal to trigger a stop motion command for the pressure component; The microfluidic chip module includes a chip substrate, and the culture chamber and limiting structure are disposed on the chip substrate. The limiting structure is a micron-level rigid support fixed on the chip substrate. Its height is preset according to the cell type. It is used to precisely define the minimum distance between the lower surface of the pressure application component and the chip substrate when the pressure application component is in the second position, so that the pressure application component forms a physical limit when it is pressed down, ensuring that the cells are confined within the single-layer spread space. During the movement of the pressure application component from the first position to the second position, that is, when the pressure application component performs the pushing and spreading operation, a radial extrusion flow is generated between its lower surface and the chip substrate, and the cells are evenly distributed on the horizontal plane through the shearing or dragging action of the fluid. The system presents a modular, layered architecture: The automatic drive module is located at the top layer; The microfluidic chip module and the live cell culture module are located in the middle layer; It also includes a cell post-analysis module located at the bottom layer, used to realize cell optical imaging or optical analysis; this module is located outside the culture environment, i.e. outside the live cell culture module, and corresponds to the optical window set at the bottom of the live cell culture module, that is, its optical path is aligned with the culture area at the bottom of the microfluidic chip module; The culture chamber is an open chamber, and the horizontal cross-sectional dimension of the pressure application component is smaller than the size of the top opening of the open chamber, so as to form an overflow gap between them for temporary storage of culture medium for backflow; and the side wall height of the culture chamber is higher than the highest liquid level of culture medium overflow when the pressure application component is in the second position; the surface of the pressure application component that contacts the cells has an anti-bioadhesion layer.

2. The variable high-microfluidic automated cell spreading and culture system according to claim 1, characterized in that, The environmental control chamber is provided with a sealed interface through which the pressure application component passes. This sealed interface cooperates with the open chamber to enable the automatic drive module to enter and exit the environmental control chamber and the culture chamber.

3. The variable high-microfluidic automated cell spreading and culture system according to claim 1, characterized in that, The automatic drive module has multiple adjustable speed modes, and supports switching between at least a high-speed mode for positioning the pressure component and a low-speed mode for pushing and laying the sheet.

4. The variable high-microfluidic automated cell spreading and culture system according to claim 1, characterized in that, The environmental control chamber includes at least a temperature control component and a humidification control component; The temperature control component is used to maintain the physiological temperature stability of the area where the microfluidic chip module is located; The humidification gas control component is used to provide a preset composition of humidifying gas into the environmental control chamber and maintain a slightly positive pressure environment.

5. A method for operating the system according to any one of claims 1 to 4, characterized in that, The working method includes the following cyclical steps: Open culture phase: The pressure application component is in the first position, and the cells undergo long-term growth culture and unrestricted gas exchange; Compression analysis stage: The pressure application component moves to the second position, locking the cells in a thin layer of uniform height for cell optical imaging or optical analysis; Recovery phase: The pressure application component returns to the first position, the overflowed culture medium flows back to cover the cells again, and the system returns to the large-volume open culture state.

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