A microfluidic chip for centrifugal generation of oil-free droplets

Abstract

This invention belongs to the field of microfluidics and discloses a microfluidic chip for centrifugal generation of oil-free droplets, comprising: a substrate; an intermediate layer including a droplet generation unit, the droplet generation unit comprising: a first aqueous phase storage cavity, a microchannel chip reserved area, a second aqueous phase storage cavity, and a droplet collection area arranged radially from the inside to the outside, and waste liquid cavities disposed on both sides of the second aqueous phase storage cavity; the microchannel chip is bonded to the substrate, and a rectangular microchannel communicating with the first and second aqueous phase storage cavities is provided at the bottom; the top cover includes: a sealing area connected to the microchannel chip, a first aqueous phase sample inlet and a first aqueous phase vent communicating with the first aqueous phase storage cavity, and a second aqueous phase sample inlet and a second aqueous phase vent communicating with the waste liquid cavities; the positions of the second aqueous phase sample inlet and the second aqueous phase vent are further away from the rotation center than the outlet position of the rectangular microchannel. Based on this device, the composition, size, uniformity, and morphology of droplets can be improved.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic technology applications, and more specifically, relates to a microfluidic chip for generating oil-free droplets by centrifugation. Background Technology

[0002] Organoids are the most representative models in 3D cell culture, composed of organ-specific cell types capable of self-organization. Matrigel is commonly used as the extracellular matrix for organoid in vitro culture, but its complexity and compositional variability lead to problems such as high cost, unclear definition, and batch-to-batch variability. To simulate the stem cell microenvironment, hydrogels, as a 3D matrix that can provide physical support, mechanical cues, and binding sites for cells, have attracted widespread attention. Alginate hydrogels are among the earliest studied and most widely reported hydrogel materials used in the synthesis of microgels in droplet microfluidic systems and for 3D cell loading and culture. Using droplet microfluidics to prepare microgel carriers allows for the confinement of fluids within a small area; this small volume control enables microfluidics to exhibit low sample consumption and microdomain effects, facilitating observation and analysis.

[0003] Commonly used methods for generating alginate droplets are mostly based on complex microfluidic systems, requiring specialized equipment and personnel. Traditional microfluidics generally uses flow focusing to generate droplets. Although the average flow rate when using a syringe pump can be very precise, the transient flow rate of the microfluidic chip is pulsating, and the pressure fluctuates over time, affecting the uniformity of the droplets. Existing centrifugal microfluidics use centrifugal pumps to generate centrifugal force. When the centrifugal force exceeds the interfacial tension of the dispersed phase, droplets are generated. Centrifugal microfluidics are highly scalable, requiring only a centrifuge for parallel processing of multiple samples. Centrifugal systems tend to suppress high-frequency pressure fluctuations through their angular momentum, thereby generating pulse-free flow and enhancing the repeatability of the cutoff process.

[0004] High-throughput generation and analysis of organoids help reduce randomness and variability, which is crucial for biological experiments and clinical trials involving numerous parameters and processing steps. Currently, gel microspheres used for cell culture are often obtained by washing with water-in-oil droplets. Oil and demulsifiers are cytotoxic, and the washing process is cumbersome and prone to loss. Centrifugal microfluidic chips utilize air as the mobile phase, shearing the first aqueous phase under centrifugal drive to generate droplets, which then solidify in a second aqueous phase, leveraging the cross-linking properties of the first and second aqueous phases. However, these "water-in-water" droplets exhibit unstable shapes, fusion, and low uniformity during generation.

[0005] Based on research into the homogeneity of organoids, a stable and efficient droplet generation method is needed. A simple and rapid method for generating oil-free droplet-loaded organoids holds promise for providing a more efficient culture foundation for subsequent drug screening, personalized medicine, and disease models. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a microfluidic chip for centrifugally generating oil-free droplets. The purpose is to adjust the morphology of the microchannels by designing the microchannel chip shape, and to adjust the air distance between the microchannel outlet and the second aqueous phase interface by designing the positions of the microchannel outlet, the second aqueous phase sample inlet, and the vent, or by setting the second aqueous phase chamber as multiple gradient-connected chambers. This solves the technical problems of droplet composition, size, uniformity, and morphology generated by centrifugation. It can be used for the culture and analysis of organoid loads to meet the needs of 3D cell culture scaffolds.

[0007] To achieve the above objectives, according to one aspect of the present invention, a microfluidic chip for generating oil-free droplets by centrifugation is provided, comprising: a substrate;

[0008] An intermediate layer bonded to the substrate has at least one droplet generating unit disposed around the rotation center on the intermediate layer. The droplet generating unit includes: a first aqueous phase storage cavity, a microchannel chip reserved area, a second aqueous phase storage cavity and a droplet collection area arranged radially from the inside to the outside, and waste liquid cavities disposed on both sides of the second aqueous phase storage cavity.

[0009] The microchannel chip placed in the reserved area of ​​the microchannel chip is bonded to the substrate and has a rectangular microchannel at the bottom for connecting the first aqueous storage cavity and the second aqueous storage cavity.

[0010] A top cover covering the intermediate layer and the microchannel chip; the top cover includes: a sealing area connected to the microchannel chip, a first aqueous phase sample inlet and a first aqueous phase vent communicating with the first aqueous phase storage cavity, a second aqueous phase sample inlet communicating with one side of the waste liquid cavity, and a second aqueous phase vent communicating with the other side of the waste liquid cavity; wherein the second aqueous phase sample inlet and the second aqueous phase vent are both located near the proximal end of the corresponding waste liquid cavity, and are further away from the rotation center than the outlet position of the rectangular microchannel;

[0011] And sealant filling the gaps between the substrate, the intermediate layer, the microchannel chip and the top cover.

[0012] Furthermore, the second aqueous phase sample inlet and the second aqueous phase gas outlet are located on the same arc, and the center of the circle where the second aqueous phase sample inlet and the second aqueous phase gas outlet are located, as well as the center of the circle where the rectangular microchannel outlet is located, coincide with the rotation center.

[0013] The difference between the radius of the arc corresponding to the second aqueous phase sample inlet and the second aqueous phase air outlet and the radius of the arc corresponding to the outlet of the rectangular microchannel is the air distance from the outlet of the rectangular microchannel to the second aqueous phase interface in the second aqueous phase storage chamber when the microfluidic chip generates droplets.

[0014] Furthermore, the microchannel chip includes PDMS and a rectangular glass sheet; the rectangular microchannel is a space formed by bonding the PDMS and the rectangular glass sheet, and the PDMS has a three-dimensional pattern facing the inner surface of the rectangular microchannel.

[0015] Furthermore, the bottom of the microchannel chip is provided with multiple parallel, straight rectangular microchannels.

[0016] Furthermore, the rectangular microchannel is Y-shaped, and the two branches of the Y-shape are configured as two unconnected first aqueous phase storage cavities; or the rectangular microchannel is multi-branched, and the multiple branches of the multi-branched microchannel are configured as multiple unconnected first aqueous phase storage cavities, wherein the multiple branches are more than two branches.

[0017] Furthermore, the inner diameter of the rectangular microchannel is smaller than the inner diameter of the second aqueous storage cavity; wherein, the thickness of the rectangular glass sheet is smaller than the inner diameter of the second aqueous storage cavity, and the bottom of the outlet of the rectangular microchannel is higher than the lower surface of the second aqueous storage cavity.

[0018] Furthermore, the thickness of the rectangular glass sheet is greater than the diameter of the largest cross-section of the generated droplet.

[0019] Furthermore, the substrate is a glass substrate, and the intermediate layer is made of PMMA; the substrate is a glass substrate, and the intermediate layer is made of PMMA.

[0020] The base, the intermediate layer, and the top cover are all disc-shaped, with mutually aligned centrifugal positioning holes in the center, and the intermediate layer and the top cover are uniformly provided with positioning holes.

[0021] According to another aspect of the present invention, a microfluidic chip for generating oil-free droplets by centrifugation is provided, comprising: a substrate;

[0022] An intermediate layer bonded to the substrate has at least one droplet generating unit disposed around the rotation center on the intermediate layer. The droplet generating unit includes: a first aqueous phase storage cavity, a microchannel chip reserved area, a second aqueous phase storage cavity, and a waste liquid cavity arranged radially from the inside to the outside, and a buffer cavity connected to the rightmost side of the second aqueous phase storage cavity; wherein the second aqueous phase storage cavity is configured as multiple interconnected chambers, the rightmost chamber is connected to the buffer cavity, and the bottom of the buffer cavity is connected to the waste liquid cavity;

[0023] The microchannel chip placed in the reserved area of ​​the microchannel chip is reversibly bonded to the substrate, and a rectangular microchannel is provided at the bottom for connecting the first aqueous storage cavity and the second aqueous storage cavity.

[0024] A top cover covering the intermediate layer and the microchannel chip; the top cover includes: a sealing area connected to the microchannel chip, a first aqueous phase sample inlet and a first aqueous phase vent communicating with the first aqueous phase storage chamber, a second aqueous phase sample inlet and a second aqueous phase vent communicating with the second aqueous phase storage chamber, and a vent communicating with the waste liquid chamber.

[0025] And sealant filling the gaps between the substrate, the intermediate layer, the microchannel chip and the top cover.

[0026] Furthermore, the geometric center of the chamber and the rectangular microchannel connected above are on the same straight line; the chamber is spatially connected near the outlet of the rectangular microchannel, and the distance from the proximal end of the right sidewall of the chamber to the outlet of the rectangular microchannel increases sequentially from left to right.

[0027] Furthermore, the microchannel chip includes PDMS and a rectangular glass sheet; the rectangular microchannel is a space formed by bonding the PDMS and the rectangular glass sheet, and the PDMS has a three-dimensional pattern facing the inner surface of the rectangular microchannel.

[0028] Furthermore, the bottom of the microchannel chip is provided with multiple parallel, straight rectangular microchannels.

[0029] Furthermore, the inner diameter of the rectangular microchannel is smaller than the inner diameter of the second aqueous storage cavity; wherein, the thickness of the rectangular glass sheet is smaller than the inner diameter of the second aqueous storage cavity, and the bottom of the outlet of the rectangular microchannel is higher than the lower surface of the second aqueous storage cavity.

[0030] Furthermore, the thickness of the rectangular glass sheet is greater than the diameter of the largest cross-section of the generated droplet.

[0031] Furthermore, the substrate is a glass substrate, and the intermediate layer is made of PMMA; the substrate, the intermediate layer and the top cover are all disc-shaped, and have mutually aligned centrifugal positioning holes in the center, and the intermediate layer and the top cover are uniformly provided with positioning holes.

[0032] In summary, compared with the prior art, the technical solutions conceived in this invention have the following main advantages:

[0033] (1) The centrifugal microfluidic chip provided by this invention utilizes air as the mobile phase in a centrifugal drive, and leverages the cross-linking or immiscibility of the first and second aqueous phases to generate oil-free, independent droplets. This simplifies the washing process and reduces cytotoxicity compared to water-in-oil droplets. Based on the device of this invention, the structure of the first aspect improves the uniformity of the composition and size of the generated droplets, while also adjusting their morphological uniformity or roundness; and the structure of the first aspect improves the uniformity of the composition and size of the generated droplets, while also regulating and controlling the formation of droplets of various different shapes under the same conditions; wherein the uniformity of droplets based on the same microchannel can be improved.

[0034] (2) In the centrifugal microfluidic chip provided in the first aspect of the present invention, the position of the outlet of the rectangular microchannel is closer to the proximal end than the positions of the sample loading port and the vent in the second aqueous phase storage chamber. This ensures that during centrifugation, the second aqueous phase interface and the vent are on the same arc, and there is an air distance between them and the channel outlet. During droplet formation, the droplet expands due to the Laplace pressure at the air-water interface and necks at the channel outlet. The droplet is broken off due to Rayleigh-Taylor instability. At this time, the role of air is similar to that of the oil phase in a traditional water-in-oil droplet. During centrifugation, as the first aqueous phase droplet enters the second aqueous phase, the liquid volume in the second aqueous phase storage chamber increases. However, since the second aqueous phase interface and the vent are always on the same arc, the excess second aqueous phase will enter the waste liquid chamber under atmospheric pressure, and the air distance remains unchanged. A longer air distance will cause the droplet to deform into an elliptical shape under the shear force of the air, while a shorter air distance will cause the droplet to enter the second aqueous phase and solidify before being completely broken off at the channel outlet, forming a water droplet shape. An appropriate air distance can improve the sphericity of droplets, thereby achieving higher uniformity.

[0035] (3) The centrifugal microfluidic chip provided in the second aspect of the present invention uses a series of stepped second aqueous phase storage chambers within the same droplet generation unit. By using a buffer chamber on the far right of the second aqueous phase chamber, multiple different air distances are maintained between the outlet of the rectangular microchannel and the second aqueous phase interface during centrifugation, thereby controlling the droplet morphology and generating droplets of different shapes in a single operation. During centrifugation, the distance between the second aqueous phase interface and the outlet of the rectangular microchannel differs in each chamber. Therefore, the same sample can generate fibrous, teardrop-shaped, and elliptical droplets under the same conditions and rotation speed to explore the effects of symmetrical and asymmetrical microenvironments on organoids.

[0036] (4) The present invention provides a centrifugal microfluidic chip that allows for the alteration of droplet size and channel size. The droplet size can be adjusted by changing the rotation speed; the higher the rotation speed, the smaller the droplet size. Preferably, changing the PDMS pattern width can alter the channel size. Channels smaller than 80 micrometers, at the same rotation speed, produce droplets with little difference in size, providing a sorting mechanism for single cells or cell clusters of different sizes.

[0037] (5) Based on the centrifugal microfluidic chip provided in the first aspect of the present invention, it is preferable to use the regional spacing of the droplets, that is, to set two first aqueous phase storage chambers, and these two first aqueous phase storage chambers are connected to the second aqueous phase storage chamber through a "Y"-shaped channel pattern. During centrifugation, the two first aqueous phases will form laminar flow after passing through the "Y" intersection, generating droplets with two regions. Changing the channel pattern can also achieve more regional spacing. Given the importance of reconstructing the microenvironment for studying normal tissue function and disease progression, a suitable co-culture system is urgently needed when establishing physiologically relevant models. The chip designed in this invention can enable two or more cell types to form different compartments in the droplets to explore the influence between different cells.

[0038] (6) The microfluidic chip for generating oil-free droplets by centrifugation constructed in this invention preferably achieves droplet generation in six units simultaneously. These six units can be centrifuged at the same speed under the same conditions to achieve high-throughput and rapid droplet generation; alternatively, six different parameters can be designed simultaneously for experiments, greatly reducing the error between different groups. In traditional microfluidics, multiple pumps are required for different concentrations or different fluids, while this chip only requires a microfluidic centrifuge.

[0039] (7) The microfluidic chip for centrifugal generation of oil-free droplets constructed in this invention preferably uses soft photolithography to design channels with different patterns, and the number of parallel channels can be set simultaneously from 5 to 20. Compared with some centrifugal chips that use pinholes as channels, and only one pinhole can be placed in a unit (if the pinhole is blocked, the unit will be unusable), the microfluidic chip of this invention can achieve high-throughput generation of oil-free droplets. A chip can generate tens of thousands of droplets simultaneously in one minute at a rotation speed of 3000 rpm.

[0040] (8) This invention mixes cells with a first aqueous phase and then solidifies them with a second aqueous phase using a chip device to generate oil-free droplets, thus achieving droplet encapsulation of cells. Organoids are 3D cell clusters formed in vitro by specific stem cells through self-organization. Current organoid culture still suffers from problems such as heterogeneity, untunable microenvironment, and unclear cell-matrix interactions. This invention can provide high-throughput generation of oil-free droplets for loading organoids, enabling independent culture of organoids. This allows for the exploration of cell behavior under this model in a local and controlled environment, satisfying applications such as drug screening, personalized medicine, and organoid homogeneity. For example, confining organoids to micrometer-sized spheres maximizes their specific surface area, facilitating efficient biomolecule transport. The microenvironment of each hydrogel bead is precisely controlled, resulting in uniform distribution of oxygen, nutrients, etc., allowing for the exploration of cell behavior under this model in a local and controlled environment. Attached Figure Description

[0041] Figure 1 This is an exploded view of a microfluidic chip for culturing organoids, which is an example of the present invention for generating oil-free droplets by centrifugation.

[0042] Figure 2 This is a schematic diagram of the planar structure of a microfluidic chip for culturing organoids, which generates oil-free droplets by centrifugation, according to the first aspect of the present invention.

[0043] Figure 3 This is a three-dimensional schematic diagram of the droplet generation unit of the microfluidic chip of the first aspect of the present invention;

[0044] Figure 4 This is a planar schematic diagram of the droplet generation unit of the microfluidic chip of the first aspect of the present invention; R1 is the radius corresponding to the arc where the second aqueous phase sample inlet 82 and the second aqueous phase vent 81 are located, R2 is the radius corresponding to the arc at the tangent position of the channel outlet, and L is the air distance from the rectangular microchannel outlet to the second aqueous phase interface when centrifugally generating droplets.

[0045] Figure 5 This is a schematic cross-sectional view illustrating the droplet generation process of a microchannel component based on a microfluidic chip, as exemplified by this invention.

[0046] Figure 6The first aspect of this invention illustrates the centrifugation generation of oil-free droplets for culturing organoids using a microfluidic chip at different air distances and rotation speeds, with a scale bar of 200 μm.

[0047] Figure 7 This is a schematic diagram of the structure of a microfluidic chip for culturing organoids, provided by the second aspect of the present invention, which generates oil-free droplets of different shapes by centrifugation.

[0048] Figure 8 This is a schematic diagram of the droplet generation unit of the microfluidic chip provided in the second aspect of the present invention;

[0049] Figure 9 This invention illustrates the generation of oil-free droplets from a microfluidic chip for culturing organoids using centrifugation at different channel sizes and rotation speeds. Figure a shows the droplet sizes generated at 2000-5000 rpm for channels of 20, 40, 60, and 80 μm. Figure b shows the frequency of droplet sizes generated at 2000-5000 rpm for an 80 μm channel. Figure c shows the fluorescence of droplets generated at 2000-5000 rpm for an 80 μm channel. The scale bar is 200 μm.

[0050] Figure 10 This is a schematic diagram of the droplet generation unit of a microfluidic chip for centrifugation to generate Janus oil-free droplets for organoid co-culture, as exemplified by the present invention.

[0051] Figure 11 This is an example of an improvement of the microfluidic chip for centrifugation to generate oil-free droplets for culturing organoids, with an added rectangular glass plate below the PDMS channel assembly. Figure a shows the droplets formed before the improvement, Figure b shows the droplets formed after the improvement, and Figure c shows the improvement schematic diagram.

[0052] Figure 12 This is a cell viability diagram of droplet-loaded MCF-7 cells during the culture process, as exemplified in Embodiment 1 of the present invention.

[0053] Figure 13 This is a comparison diagram of droplet-loaded CRC organoids and ordinary cultured organoids as exemplified in Embodiment 2 of the present invention; wherein Figure a is a cell viability staining fluorescence image of droplet-encapsulated cultured organoids and ordinary cultured organoids, green represents live cells stained with calcein, and red represents dead cells stained with propidium iodide; Figure b is a cell viability histogram of the two; and Figure c is a histogram of organoid size of the two.

[0054] In all the accompanying drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0055] 1-Substrate, 2-Droplet generation unit, 3-Microchannel chip, 31-PDMS, 32-Rectangular glass plate, 4-Top cover, 5-Connection channel between waste liquid chamber and second aqueous phase storage chamber, 6-First aqueous phase storage chamber, 71-First aqueous phase vent, 72-First aqueous phase sample inlet, 81-Second aqueous phase vent, 82-Second aqueous phase sample inlet, 9-Waste liquid chamber, 10-Second aqueous phase storage chamber, 11-Rectangular microchannel, 12-Droplet collection area, 13-Positioning hole, 14-Centrifugation positioning hole, 15-Buffer chamber. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0057] like Figure 1-5 As shown, the microfluidic chip of the present invention has a rotation center set in a centrifugal manner. Its basic hierarchical structure from bottom to top includes a substrate 1; an intermediate layer with multiple droplet generating units 2 arranged around the rotation center; and a top cover 4. The gaps between the substrate 1, the intermediate layer and the top cover 4 are filled with sealant.

[0058] In a first aspect of the invention, the microfluidic chip of the present invention, wherein the droplet generation unit 2, as... Figure 2-4 As shown, it specifically includes: a first aqueous phase storage cavity 6, a microchannel chip reserved area, a second aqueous phase storage cavity 10 and a droplet collection area 12 arranged radially from the inside to the outside, and waste liquid cavities 9 arranged on both sides of the second aqueous phase storage cavity 10.

[0059] The top cover 4 includes various inlet and outlet components, specifically: a sealing area connected to the microchannel chip 3, a first aqueous phase sample inlet 72 and a first aqueous phase vent 71 connected to the first aqueous phase storage chamber 6, a second aqueous phase sample inlet 82 connected to one side of the waste liquid chamber 9, and a second aqueous phase vent 81 connected to the other side of the waste liquid chamber 9; wherein, the second aqueous phase sample inlet 82 and the second aqueous phase vent 81 are both located near the proximal end of the corresponding waste liquid chamber 9, and are further away from the rotation center than the outlet position of the rectangular microchannel 11.

[0060] When the microfluidic chip of the present invention produces droplets using centrifugation, the first aqueous phase and the second aqueous phase are placed in the first aqueous phase storage chamber 6 and the second aqueous phase storage chamber 10 respectively, and the second aqueous phase is a curing agent for the first aqueous phase, or the second aqueous phase and the first aqueous phase are incompatible with each other, so as to generate oil-free droplets.

[0061] The microchannel chip reserved area is used to place the microchannel chip 3, which is composed of a PDMS component 31 and a rectangular glass plate 32. The two are plasma-bonded, and the spatial structure formed at the bonding point is a rectangular microchannel 11. This rectangular microchannel 11 is used to connect the first aqueous phase storage chamber 6 and the second aqueous phase storage chamber 10, such as... Figure 5 As shown.

[0062] When the microfluidic chip of the present invention produces droplets using centrifugation, it adds samples and allows airflow to the first aqueous phase storage chamber 6 and the second aqueous phase storage chamber 10 through inlet and outlet components provided on the top cover. Specifically, on both sides of the first aqueous phase storage chamber 6, one side can be selected as the first aqueous phase sample inlet 72, and the other side as the first aqueous phase air outlet 71, and the first aqueous phase sample inlet 72 and the first aqueous phase air outlet 71 are on the same arc with the centrifugation positioning hole or rotation center as the center. The second aqueous phase sample inlet 82 and the second aqueous phase air outlet 81 are respectively provided on both sides of the waste liquid chamber 9, and the waste liquid chamber 9 is symmetrically arranged on both sides of the second aqueous phase storage chamber 10 to place excess solution of the second aqueous phase during centrifugation.

[0063] In some embodiments, the second aqueous phase injection port 82 and the second aqueous phase vent 81 are located on opposite sides of the waste liquid chamber 9, closer to the proximal end than the outlet position of the rectangular microchannel 11, and situated on the same arc. The centers of the arcs containing the second aqueous phase injection port 82 and the second aqueous phase vent 81, as well as the center of the arc containing the outlet of the rectangular microchannel 11, coincide with the centrifugation positioning hole or the rotation center. During centrifugation, because the density of air is lower than that of water, the air will be closer to the center. Therefore, by setting the difference between the arc radius of the second aqueous phase injection port 82 and the second aqueous phase vent 81 centered on the rotation center and the arc radius of the rectangular microchannel 11 centered on the rotation center, the air distance from the outlet of the rectangular microchannel 11 to the second aqueous phase interface during droplet generation is controlled, thereby controlling the shape of the generated droplets.

[0064] like Figure 4 As shown, the difference between the radius R1 of the arc corresponding to the second aqueous phase injection port 82 and the second aqueous phase vent 81 and the radius R2 corresponding to the outlet of the rectangular microchannel 11 is the horizontal distance L from the rectangular microchannel 11 to the second aqueous phase interface. That is, changing the position of the second aqueous phase injection port 82 and the second aqueous phase vent 81 can change the horizontal air distance L = R1 - R2 from the rectangular microchannel 11 to the second aqueous phase interface.

[0065] In this embodiment, the second aqueous phase injection port 82 is located at the top of the waste liquid chamber 9. For example, the waste liquid chamber is approximately a right-angled triangle with a circular arc on the hypotenuse. The waste liquid chambers of the second aqueous phase storage chamber are located on the left and right sides and are connected to the second aqueous phase storage chamber through PMMA channels with a width of 0.05 mm.

[0066] In this embodiment, different droplet shapes are achieved by adjusting the air distance.

[0067] By drawing the sample inlet 81 and the second aqueous phase vent 81 at different locations using CAD, the corresponding PMMA layer structure was obtained, with the air distance set to 1, 3, 5, and 7 mm; Figure 6 As shown, the droplet shapes generated at different air distances are different.

[0068] Among them, the droplets generated by the chip with an air distance of 1mm at a rotation speed of 2000rpm are tadpole-shaped, while the droplets generated by the chip with an air distance of 7mm at the same rotation speed are elliptical.

[0069] In a second aspect of the invention, as Figure 7-8 As shown, the microfluidic chip of the present invention is based on a centrifugal structure designed in a clockwise direction. It differs from the microfluidic chip of the first aspect in that it is a droplet generation unit 2, specifically including: a first aqueous phase storage cavity 6, a microchannel chip reserved area, a second aqueous phase storage cavity 10 and a waste liquid cavity 9 arranged radially from the inside to the outside, and a buffer cavity 15 connected to the rightmost side of the second aqueous phase storage cavity 10; wherein, the second aqueous phase storage cavity 10 is configured as multiple interconnected chambers, the rightmost chamber is connected to the buffer cavity 15, and the bottom of the buffer cavity 15 is connected to the waste liquid cavity 9;

[0070] The top cover 4 includes various inlet and outlet components, specifically: a sealing area connected to the microchannel chip, a first aqueous phase sample inlet 72 and a first aqueous phase vent 71 connected to the first aqueous phase storage chamber 6, a second aqueous phase sample inlet 82 and a second aqueous phase vent 81 connected to the second aqueous phase storage chamber 10, and a vent 83 connected to the waste liquid chamber 9.

[0071] During centrifugation, the distribution of the second aqueous phase within the buffer chamber of this microfluidic chip is as follows: Figure 8 As shown, excess second aqueous phase will enter buffer chamber 15 and then enter waste liquid chamber 9 through channel.

[0072] In some embodiments, the second aqueous phase storage chamber 10 is configured as multiple interconnected chambers, which are spatially connected near the outlet of the rectangular microchannel 11. The distance from the proximal end of the right sidewall of each chamber to the outlet of the rectangular microchannel 11 increases sequentially from left to right, thereby altering the interfacial air distance between the chamber and the rectangular microchannel 11 sequentially from left to right during centrifugation.

[0073] Furthermore, the geometric center of the chamber and the rectangular microchannel 11 connected above are on the same straight line.

[0074] Compared to the structure of the first aspect of the present invention, it mainly achieves the regulation of droplet morphology by setting a series of stepped second aqueous phase storage chambers in the same droplet generation unit, controlling the air distance from the outlet of the rectangular microchannel to the second aqueous phase interface in the same droplet generation unit, thereby generating droplets of different shapes at one time.

[0075] Therefore, by changing the distance between the second aqueous phase storage chamber and the PDMS channel in the microfluidic chip of the second aspect, it is possible to generate droplets of different shapes in the same unit. That is, a microfluidic chip that generates oil-free droplets of different shapes by centrifugation for culturing organoids can be used to explore the effect of different droplet shapes encapsulating organoids on cell polarity.

[0076] For example, such as Figure 8 As shown, a droplet generation unit comprises five second aqueous phase storage chambers 10, which are interconnected. The chambers are arranged in a stepped configuration. On the far right of the second aqueous phase storage chambers 10 is a buffer chamber 15, which connects to the second aqueous phase chambers on its left side and has a channel at its bottom connecting to the waste liquid chamber. During centrifugation, the distances from the horizontal interface of the second aqueous phase to the channel outlet from left to right are 0, 1, 2, 3, and 4 mm, respectively, which can meet the requirement of generating droplets of different shapes from the same sample.

[0077] In some implementations, the preferred embodiments applicable to the first and second aspects described above are used.

[0078] The rectangular microchannel 11 is formed by bonding a PDMS 31 containing a channel pattern to a rectangular glass sheet 32. The rectangular microchannel 11 is a micrometer-level horizontal channel with the same width and height, and a square vertical cross-section. The channel is bonded to the rectangular glass sheet at the bottom, and the channel outlet is on the same vertical plane as the edge of the rectangular glass sheet.

[0079] For example, the rectangular microchannel has the same height and width, a length of 1 cm, and a square vertical cross-section, ensuring that the pressure on the droplet is equal around its perimeter at the outlet. The rectangular glass plate has the same length and width as the PDMS assembly, for example, a height of 0.55 mm, and the outlet of the rectangular microchannel 11 is on the same vertical plane as the edge of the rectangular glass plate.

[0080] The microchannel chip 3 is fabricated by soft photolithography, and the PDMS 31 is sealed to the rectangular glass sheet 32 ​​by plasma bonding.

[0081] In this embodiment, the size of the PDMS channel can be changed by altering the pattern of the soft lithography mask, i.e., designing different 3D patterns of PDMS.

[0082] Simply replacing the PDMS31 component allows you to set different channel sizes, such as setting the PDMS channel width to 20, 40, 60, or 80 μm, with the height being the same as the width.

[0083] Chips with different channel sizes produce droplet sizes that do not differ significantly at the same rotation speed. Figure 9 As shown. Therefore, the droplet size generated by the microfluidic chip involved in this invention is not affected by the channel size (below 80 μm), and can provide a sorting mechanism for single cells or cell clusters of different sizes.

[0084] In some embodiments, preferred solutions applicable to the first and second aspects described above are used.

[0085] The bottom of the microchannel chip 3 has multiple parallel rectangular microchannels 11. By setting up parallel channels, high-throughput droplet generation can be achieved, and setting up parallel droplet generation units can enable simultaneous testing of different parameters.

[0086] The intermediate layer can contain six droplet generation units, all of which can be centrifuged at the same speed and under the same conditions to achieve high-throughput and rapid droplet generation.

[0087] The intermediate layer can contain 6 droplet generation units, each of which is different, enabling high-throughput exploration of droplet generation of different shapes under the same experimental conditions.

[0088] In some embodiments, based on the preferred microfluidic chip of the first aspect described above,

[0089] The rectangular microchannel 11 is Y-shaped, and the two branches corresponding to the Y shape are set as two unconnected first aqueous phase storage cavities 6. Each first aqueous phase storage cavity includes a first aqueous phase inlet 72 and a first aqueous phase vent 71.

[0090] In this embodiment, droplet region separation can be achieved by changing the channel shape, which is a microfluidic chip for centrifugation to generate Janus oil-free droplets for organoid co-culture.

[0091] like Figure 10 As shown, the PDMS channel pattern is designed in a Y shape, and the first aqueous phase storage chamber is divided into two regions, which are respectively connected to the forks of the Y shape.

[0092] When the microfluidic chip uses centrifugal drive to produce droplets, two first aqueous phase storage chambers can be injected with different cell suspensions respectively. Through the centrifugally driven chip, the two cell suspensions will converge at the intersection of the Y-shaped channel of the PDMS and reach the channel outlet in a laminar flow form, forming Janus droplets at the channel outlet.

[0093] For example, the first aqueous phase of the cell suspension contains sodium alginate, and the second aqueous phase is a calcium chloride solution. Sodium alginate and calcium chloride will cross-link, so the Janus droplet precursor will enter the calcium chloride solution and solidify, resulting in a droplet with two separated regions.

[0094] Since traditional organoid culture lacks vascularization and immunomodulation, Janus droplets can be used to explore the effects of vascular endothelial cells or immune cells on organoid formation and function.

[0095] Based on the above, in some real-time methods, the PDMS channel pattern can be changed to a multi-branch confluence form, where "multi-branch" refers to more than two branches. This allows for the adaptation and expansion by introducing multiple first aqueous phases in conjunction with a second aqueous phase to utilize centrifugal drive for droplet production.

[0096] In some implementations, the preferred embodiments applicable to the first and second aspects described above are used.

[0097] The inner diameter height is smaller than the inner diameter height of the second aqueous phase storage chamber 10. The bottom of the outlet of the rectangular microchannel 11 is higher than the lower surface of the second aqueous phase storage chamber 10.

[0098] Furthermore, the rectangular glass plate has a certain thickness, which is greater than the diameter of the generated droplet but less than the height of the second aqueous phase storage chamber. The purpose is to prevent the generated droplet from contacting the glass substrate and the upper surface of the second aqueous phase storage chamber.

[0099] For example, the bottom of the outlet of the rectangular microchannel 11 is 0.65 mm higher than the surface of the second aqueous phase storage chamber 10, which is the thickness of the rectangular glass sheet. Figure 11 As shown, the first aqueous phase in the rectangular microchannel 11 forms dispersed droplets at the channel outlet. Under the action of centrifugal force, the droplets do not contact the bottom glass substrate or the upper surface of the second aqueous phase storage chamber and directly enter the second aqueous phase.

[0100] Within the rectangular microchannel 11, the first aqueous phase forms dispersed droplets at the channel outlet. Under centrifugal force, these droplets do not contact the bottom glass substrate or the upper surface of the second aqueous phase storage chamber, directly entering the second aqueous phase. Without the rectangular glass plate, the bottom of the channel outlet and the lower surface of the second aqueous phase chamber are on the same plane. When the droplets are generated at the channel outlet, they directly react with the glass substrate, resulting in a reduced velocity as they enter the second aqueous phase, making it difficult for them to cross the gas-liquid interface and causing droplet deformation.

[0101] In some embodiments, the droplet collection area 12 connected to the second aqueous phase storage chamber 10 in the first aspect is located at the distal end of the entire device; while in the second aspect, the droplet collection area 12 is not provided.

[0102] In the first aspect, for example, the opening size of the droplet collection area 12 occupies 1 / 6 of the planar area of ​​the second aqueous phase storage chamber 10, the thickness of the droplet collection area 12 is 3mm, and the sealing method of the collection port is single-sided adhesive bonding, which can be easily sealed and opened.

[0103] In some embodiments, the preferred solutions applicable to the first and second aspects described above are used.

[0104] The lower substrate is a glass substrate, which is a disc-shaped structure with a centrifugal positioning hole at the geometric center.

[0105] Except for the glass substrate, rectangular glass sheet, and PDMS, all other layers are drawn using CAD and then cut from PMMA using a laser engraving machine.

[0106] The base, intermediate layer and top cover 4 are all disc-shaped, and each has a centrifugal positioning hole 14 aligned with the others in the center.

[0107] The rectangular microchannel component within the droplet generation unit is sealed to the surrounding components using a photocurable adhesive, while the other layers are bonded together using a transparent double-sided thermosensitive adhesive.

[0108] The microfluidic chip designed in this invention for centrifuging to generate oil-free droplets for culturing organoids has adjustable design parameters to meet different droplet requirements.

[0109] This invention discloses a simple and rapid oil-free droplet generation chip for encapsulating organoids. Traditional droplet microfluidics generates water-in-oil droplets using flow focusing, but both the oil and the demulsifier used in subsequent washing steps are cytotoxic, and the washing process is cumbersome and prone to loss. Organoids, as tissue analogs with ordered arrangements of multiple cells, often require a more biocompatible extracellular environment due to their complexity. This chip enables the rapid generation of oil-free droplets to encapsulate organoids, thereby facilitating the construction of a 3D culture platform.

[0110] In some embodiments, the chip mainly consists of a four-layer disk structure and several channel components. The first layer is a 0.5mm thick glass substrate, serving as the bottom of the chambers; the second to fourth layers are all PMMA; the second layer is a 2mm thick droplet generation unit layer, mainly consisting of a first aqueous phase and a second aqueous phase storage chamber, as well as channel components; the channel components consist of a rectangular glass sheet and a PDMS channel, the PDMS channel being a horizontal channel with the same width and height, plasma-bonded to a 0.55mm high rectangular glass sheet below the channel, with the channel outlet on the same vertical plane as the edge of the glass sheet; the third layer is a 1mm thick intermediate layer of the chambers, mainly functioning to separate the first and second aqueous phase storage chambers, allowing the first aqueous phase to enter the second aqueous phase only through the PDMS channel; the fourth layer is a 0.4mm thick top cover, mainly serving as the inlet and outlet for each storage chamber; each disk structure has centrifugal positioning holes and positioning holes; the channel components are sealed with photocurable adhesive; the collection port is reversibly sealed with single-sided adhesive; each PMMA layer is bonded with double-sided adhesive, and hot-pressing is performed after assembly.

[0111] Sodium alginate solution used in cell experiments is the solvent for cell culture medium, and CaCl2 solution is the solvent for sterile water. Chips used in cell experiments require ultrasonic water cleaning, drying, and ultraviolet sterilization.

[0112] The following will provide some specific embodiments, and the nickel-based alloys will be described in detail with reference to the accompanying drawings.

[0113] Example 1: A microfluidic chip for centrifugal droplet generation in multi-cell spheroid culture, comprising the following steps:

[0114] MCF-7 (human breast cancer cells) cells were cultured in DMEM medium supplemented with 1% penicillin-drug antibiotics and 10% fetal bovine serum. The culture environment was a 37°C incubator containing 5% CO2, and cells were passaged every other day at a 1:4 ratio. When the cells reached 80%-90% confluence in the culture flasks, they were digested with trypsin at 37°C for 2-5 minutes until the cells no longer came into contact with each other. The cells were resuspended in 400 μL of medium and mixed with 600 μL of 25 mg / mL sodium alginate solution, resulting in a final sodium alginate concentration of 20 mg / mL and a cell density of approximately 10⁶ cells / mL. 300 μL of 50 mM CaCl₂ (containing 0.1% v / v Tween-20) was added to the second aqueous phase reservoir of the chip, and 50 μL of the cell mixture was added to the first aqueous phase reservoir. The chip was then centrifuged in a microfluidic centrifuge at 3000 rpm for 100 s. After centrifugation, remove the chip. Remove the single-sided adhesive from the collection port and aspirate the second aqueous phase into a centrifuge tube. Centrifuge the CaCl2 solution containing droplets at 3000 rpm for 5 min. After centrifugation, aspirate the supernatant, resuspend the bottom precipitate in culture medium, and transfer it to a 24-well plate. Perform a partial medium change for the cells every 1-2 days.

[0115] MCF-7 cells were encapsulated in droplets, and cell proliferation and viability were observed on days 0, 2, 5, and 12. The control group consisted of gel clusters with a diameter of 2 mm. Figure 12 As shown, the cells retain high viability within the hydrogel droplets. Clearly, even when enclosed in a confined environment, the exchange of nutrients and metabolites is still permitted. This method enables 3D culture of multicellular spheroids to explore the interaction between the spheroids and the matrix.

[0116] Example 2: A centrifugal droplet generation microfluidic chip for organoid culture, the method comprising the following steps:

[0117] CRC (colorectal cancer) organoids were encapsulated in a matrix gel containing 60% Matrigel and 40% organoid culture medium and cultured in a 37°C incubator with 5% CO2, with medium changes every other day. After one week of culture, the cells were digested using Trypsin LE digestion solution to form cell spheres with a diameter of less than 50 μm. After centrifugation to remove the supernatant, the cell pellet was resuspended in 400 μL of organoid culture medium, and 600 μL of 25 mg / mL sodium alginate solution and 50 μL of Matrigel were added, resulting in a final sodium alginate concentration of 20 mg / mL and a Matrigel concentration of 5%. 300 μL of 50 mM CaCl2 (containing 0.1% v / v Tween-20) was added to the second aqueous phase storage chamber of the chip, and 50 μL of the above cell suspension was added to the first aqueous phase storage chamber. The chip was then placed in a microfluidic centrifuge at 3000 rpm for 100 s. After centrifugation, remove the chip, peel off the single-sided adhesive from the collection port, and aspirate the second aqueous phase into a centrifuge tube. Centrifuge the CaCl2 solution containing the droplets at 900g for 5 minutes. After centrifugation, aspirate the supernatant, resuspend the bottom precipitate in organoid culture medium, and transfer it to a 24-well plate. Perform a half-medium change of the medium for the droplet-loaded cells every 1-2 days.

[0118] CRC organoids were encapsulated in droplets, and organoid size and cell viability were observed on days 1, 3, 5, and 7. The control group consisted of 30 μL of Matreigl hemispheres cultured in 3D. Figure 13 As shown, the organoids exhibit significant growth when encapsulated in droplets, with both organoid size and cell viability significantly exceeding those of the control group.

[0119] Example 3: A centrifugal droplet generation microfluidic chip for exploring the external microenvironment of organoids, the method comprising the following steps:

[0120] CRC (colorectal cancer) organoids were encapsulated in a matrix gel containing 60% Matrigel and 40% organoid culture medium and cultured at 37°C in an incubator with 5% CO2, with medium changes every other day. After one week of culture, the cells were digested using Trypsin LE digestion solution to form cell spheres with a diameter of less than 50 μm. After centrifugation to remove the supernatant, the cell pellet was resuspended in 400 μL of organoid culture medium, and 600 μL of 25 mg / mL sodium alginate solution and 50 μL of Matrigel were added. The final concentration of sodium alginate was 20 mg / mL, and the proportion of Matrigel was 5%. Add 300 μL of 50 mM CaCl2 (containing 0.1% v / v Tween-20) to the second aqueous phase storage chamber of the chip and perform a first centrifugation at 3000 rpm for 10 s. The purpose is to centrifuge the interfacial heights of the various second aqueous phase storage solutions to achieve a gradient distribution. Excess solution will enter the waste liquid chamber below through the rightmost buffer chamber. After centrifugation, add 50 μL of the above cell suspension to the first aqueous phase storage chamber and perform a second centrifugation at 4000 rpm for 100 s.

[0121] After centrifugation, droplets of different shapes will be generated in each chamber, and the organoids will be encased in an asymmetric cellular microenvironment to explore their effects on proliferation, differentiation, and migration.

[0122] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microfluidic chip for centrifugal generation of oil-free droplets, wherein a rotation center is disposed thereon, characterized in that, include: Base (1); An intermediate layer bonded to the substrate (1) has at least one droplet generating unit (2) arranged around the rotation center on the intermediate layer. The droplet generating unit (2) includes: a first aqueous phase storage cavity (6), a microchannel chip reservation area, a second aqueous phase storage cavity (10), and a droplet collection area (12) arranged radially from the inside to the outside; and waste liquid cavities (9) and buffer cavities (15) arranged on both sides of the second aqueous phase storage cavity (10). A microchannel chip (3) is placed in the microchannel chip reservation area, which is bonded to the substrate, and has a bottom for connecting the first aqueous phase storage cavity (10). The aqueous phase storage chamber (6) and the second aqueous phase storage chamber (10) are arranged in parallel with multiple straight rectangular microchannels (11); the second aqueous phase storage chamber (10) is configured as multiple interconnected chambers, which are connected in space near the outlet of the rectangular microchannel (11). The distance from the aqueous phase interface of the multiple interconnected chambers to the outlet of the rectangular microchannel (11) is the air distance. The multiple interconnected chambers are stepped so that the multiple interconnected chambers correspond to different air distances. The rightmost chamber is connected to the buffer chamber (15), and the bottom of the buffer chamber (15) is connected to the waste liquid chamber (9). A top cover (4) covering the intermediate layer and the microchannel chip (3); the top cover (4) includes: a sealing area connected to the microchannel chip (3), a first aqueous sample inlet (72) and a first aqueous gas vent (71) communicating with the first aqueous storage chamber (6), a second aqueous sample inlet (82) communicating with one side of the waste liquid chamber (9), and a second aqueous gas vent (81) communicating with the other side of the waste liquid chamber (9); wherein the second aqueous sample inlet (82) and the second aqueous gas vent (81) are both located near the proximal end of the corresponding waste liquid chamber (9), and are further away from the rotation center than the outlet position of the rectangular microchannel (11); And the sealant filled between the substrate (1), the intermediate layer, the microchannel chip (3) and the top cover (4).

2. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 1, characterized in that, The second aqueous phase sample inlet (82) and the second aqueous phase gas inlet (81) are located on the same arc, and the center of the circle where the second aqueous phase sample inlet (82) and the second aqueous phase gas inlet (81) are located, as well as the center of the circle where the rectangular microchannel (11) is located, coincides with the rotation center.

3. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 1, characterized in that, The geometric center of the chamber and the rectangular microchannel (11) connected above are on the same straight line; and the air distance from the plurality of connected chambers to the outlet of the rectangular microchannel (11) increases sequentially from left to right.

4. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 1, characterized in that, The microchannel chip (3) includes PDMS (31) and a rectangular glass plate (32). The rectangular microchannel (11) is a space formed by bonding the PDMS (31) and the rectangular glass sheet (32), and the PDMS (31) has a three-dimensional pattern on the inner surface facing the rectangular microchannel (11).

5. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 1, characterized in that, The rectangular microchannel (11) is Y-shaped, and the two branches of the Y-shape are set as two unconnected first aqueous phase storage cavities (6). Alternatively, the rectangular microchannel (11) may be in the form of a multi-branched tree, with multiple branches corresponding to the multi-branched tree as multiple unconnected first aqueous phase storage cavities (6), wherein the multi-branched structure consists of more than two branches.

6. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 4, characterized in that, The inner diameter of the rectangular microchannel (11) is smaller than the inner diameter of the second aqueous storage cavity (10); wherein the thickness of the rectangular glass plate (32) is smaller than the inner diameter of the second aqueous storage cavity (10), and the bottom of the outlet of the rectangular microchannel (11) is higher than the lower surface of the second aqueous storage cavity (10).

7. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 4, characterized in that, The thickness of the rectangular glass plate (32) is greater than the diameter of the largest cross-section of the generated droplet.

8. The microfluidic chip for generating oil-free droplets by centrifugation as described in claim 1, characterized in that, The substrate (1) is a glass substrate, and the intermediate layer is made of PMMA; The base, the intermediate layer and the top cover (4) are all disc-shaped, and the center is provided with centrifugal positioning holes (14) aligned with each other, and the intermediate layer and the top cover are uniformly provided with positioning holes (13).

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