A microfluidic chip for three-dimensional cell screening sorting

Abstract

The utility model provides a kind of microfluidic chip for three-dimensional cell screening sorting, including chip body and be set to chip body's flow channel, sink sorting area and three-dimensional cell recovery or buffer storage area, the flow channel, the sink sorting area and the three-dimensional cell recovery or buffer storage area are sequentially connected;The sink sorting area is in whole and presents the character of entering, including the first sorting branch and the second sorting branch with width difference and height difference, the first sorting branch and the second sorting branch are separated by width difference and height difference and realize the automatic separation of three-dimensional cell of different diameters;The three-dimensional cell recovery or buffer storage area includes first storage area and second storage area respectively corresponding the outlet of first sorting branch and second sorting branch connection.The utility model, by microfluidic continuous flow, realizes the quick, continuous separation, screening processing of three-dimensional cell;Realize low cost, low complexity three-dimensional cell efficient sorting.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of microfluidic chip design and microfabrication, toxicology, pharmacology, and cell biology, and relates to the design and manufacture of cell culture devices and cell separation and screening devices. Specifically, it is a microfluidic chip for three-dimensional cell screening and sorting, capable of rapidly separating and screening three-dimensional cells (such as organoids, cell clusters, etc.). Background Technology

[0002] Three-dimensional cell screening and sorting is based on the biological characteristics and shape (such as size, density, stiffness, surface markers, metabolic activity, and motility) of cells in a three-dimensional microenvironment (such as organoids, hydrogels, and microspheres) to achieve efficient separation, purification, or functional screening of target cell populations.

[0003] In existing technologies, three-dimensional cell sorting mainly includes: morphology-based automated sorting, which utilizes microscopic imaging and artificial intelligence (such as deep learning) to analyze the size, shape, and other morphological characteristics of three-dimensional cell aggregates, and then selects and transfers them using precision robotic arms or spraying technology; microfluidic sorting, which achieves the separation of three-dimensional cells within micrometer-scale chip channels by precisely controlling physical fields such as fluid dynamics and magnetic fields; inertial / deterministic lateral displacement sorting, which relies on the size-dependent inertial effect or label-free lateral displacement of cells / particles in microchannels; magnetically activated cell sorting, where target cells are pre-bound to magnetically labeled antibodies and separated from the mixture in a microfluidic chip using magnetic force; and label-free sorting based on physical properties, which utilizes the inherent physical properties of cells / aggregates such as density, size, and adhesion for separation without the need for labeling.

[0004] Currently, existing three-dimensional cell screening and sorting methods still have significant limitations in the following aspects: (1) High cost and complexity: Many advanced technologies (such as high-speed imaging combined with AI, flow cytometry) rely on expensive core equipment (precision optics, robotic arms, detectors) and consumables (specific fluorescent antibodies, magnetic labels), and require complex algorithms and support systems, resulting in high overall purchase, operation and maintenance costs. (2) Throughput and efficiency bottlenecks: Imaging-based methods are time-consuming, and the processing speed is much lower than that of two-dimensional cell analysis. (3) Potential damage to cell structural integrity: Many technologies pose the risk of damaging fragile three-dimensional cell clusters. This includes the physical contact stress of robotic arms, the impact and shear force of high-speed droplets in flow cytometry, and the fluid stress in microfluidic sorting technologies that rely on high flow rates. (4) High difficulty in technology integration and operation: The design and fabrication of chips that achieve multifunctional integration (such as culture and sorting) are complex, requiring professional interdisciplinary knowledge. When using multiple technology platforms for separation and screening operations, the steps are cumbersome, the requirements for users are high, and the system stability is easily affected by flow rate fluctuations, making it difficult to popularize in rapid applications in conventional biological laboratories. (5) The artificial sedimentation-selection method has a large subjective operational deviation. Utility Model Content

[0005] The purpose of this invention is to provide a microfluidic chip for three-dimensional cell screening and sorting through functional unit design. By designing fluid channels with height differences that can accommodate spheres, rapid separation without damaging cell cluster activity is achieved, eliminating the need for additional pretreatment or spiking operations.

[0006] Therefore, the present invention provides the following technical solution:

[0007] A microfluidic chip for three-dimensional cell screening and sorting includes a chip body and a flow channel, a sinking sorting region, and a three-dimensional cell recovery or buffer storage region disposed on the chip body. The flow channel, the sinking sorting region, and the three-dimensional cell recovery or buffer storage region are connected sequentially. The sinking sorting region is generally U-shaped and includes a first sorting branch and a second sorting branch with a width difference and a height difference. The first sorting branch and the second sorting branch achieve automatic separation of three-dimensional cells with different diameters through the width difference and height difference. The three-dimensional cell recovery or buffer storage region includes a first storage region and a second storage region, which are respectively connected to the outlets of the first sorting branch and the second sorting branch.

[0008] Preferably, the flow channel includes a liquid inlet and a U-shaped channel. The liquid inlet is connected to the inlet of the U-shaped channel for injecting culture medium containing three-dimensional cells. The outlet of the U-shaped channel is connected to the sinking sorting area. When the culture medium flows through the U-shaped channel, the three-dimensional cells are arranged and buffered along with the culture medium.

[0009] Preferably, the liquid inlet is cylindrical, and the diameter of the liquid inlet is larger than the width of the U-shaped flow channel.

[0010] Preferably, the depths of the flow channel, the sinking sorting area, and the three-dimensional cell recovery or buffer storage area increase in sequence.

[0011] Preferably, the first sorting branch extends to the right, and the second sorting branch extends to the left; both the width and depth of the first sorting branch are larger than those of the second sorting branch.

[0012] Preferably, both the first storage area and the second storage area are cylindrical, and the radii of the first storage area and the second storage area are larger than the widths of the first sorting branch and the second sorting branch.

[0013] Preferably, it further includes a flow outlet, which is connected to the three-dimensional cell recovery or buffer storage area and is used to drain the culture medium stored in the three-dimensional cell recovery or buffer storage area as needed; the flow outlet includes a first flow outlet and a second flow outlet respectively connected to the first storage area and the second storage area.

[0014] Preferably, both the first flow outlet and the second flow outlet are cylindrical, and the diameters of the first flow outlet and the second flow outlet are larger than the widths of the first sorting branch and the second sorting branch.

[0015] The microfluidic chip for three-dimensional cell screening and sorting of the present utility model, compared with the prior art, has at least the following beneficial effects:

[0016] For example, the microfluidic chip for three-dimensional cell screening and sorting provided by the present utility model is provided with a sinking sorting area in a "Y" shape. Through the first sorting branch and the second sorting branch with a width difference and a height difference, sorting is achieved only by using the self-power of the fluid, without the need for external precision instrument drive, nor any markers or reagents, and the operation is simple and fast; it adopts a pure physical and low-shear force sorting mode, and the whole process is gentle, only affected by gentle laminar flow, avoiding mechanical extrusion, high-speed impact, and toxicity caused by adding exogenous markers, protecting the cell activity and integrity; rapid and continuous separation and screening of three-dimensional cells are achieved through microfluidic continuous flow; there is no need for complex labeling steps and strict dispersion requirements for cell clusters; high-efficiency sorting of three-dimensional cells with low cost and low complexity is achieved.

[0017] For another example, the microfluidic chip for three-dimensional cell screening and sorting provided by the present utility model realizes real-time separation and real-time recovery, can be compatible with a variety of laboratory consumables, and adapts to a variety of experimental operation requirements. Description of the Drawings

[0018] Figure 1This is a schematic diagram of the structure of a microfluidic chip for three-dimensional cell screening and sorting provided in an embodiment of the present invention;

[0019] Figure 2 This is a microscopic observation image of a three-dimensional cell cluster to be sorted provided in an embodiment of this utility model;

[0020] Figure 3 This is a large-diameter three-dimensional cell microscope observation image after sorting using the microfluidic chip for three-dimensional cell screening and sorting provided in this embodiment of the utility model;

[0021] Figure 4 This is a microscopic observation of small-diameter three-dimensional cells after sorting using the microfluidic chip for three-dimensional cell screening and sorting provided in this embodiment of the utility model. Detailed Implementation

[0022] This invention provides a microfluidic chip for three-dimensional cell screening and sorting.

[0023] Specifically, the microfluidic chip for three-dimensional cell screening and sorting includes a chip body and a flow channel, a sinking sorting region, and a three-dimensional cell recovery or buffer storage region disposed on the chip body. The flow channel, the sinking sorting region, and the three-dimensional cell recovery or buffer storage region are connected in sequence. The sinking sorting region is generally U-shaped and includes a first sorting branch and a second sorting branch with width and height differences. The first sorting branch and the second sorting branch achieve automatic separation of three-dimensional cells with different diameters through the width and height differences. The three-dimensional cell recovery or buffer storage region includes a first storage area and a second storage area, which are respectively connected to the outlets of the first sorting branch and the second sorting branch.

[0024] In some embodiments, the flow channel includes a liquid inlet and a U-shaped channel. The liquid inlet is connected to the inlet of the U-shaped channel for injecting culture medium containing three-dimensional cells. The outlet of the U-shaped channel is connected to a sinking sorting area. When the culture medium flows through the U-shaped channel, the three-dimensional cells are arranged and buffered along with the culture medium.

[0025] In some embodiments, the liquid inlet is cylindrical, and the diameter of the liquid inlet is larger than the width of the U-shaped channel.

[0026] In some embodiments, the depths of the flow channel, the sinking sorting area, and the three-dimensional cell recovery or buffer storage area increase sequentially.

[0027] In some embodiments, the first sorting branch extends to the right and the second sorting branch extends to the left; the width and depth of the first sorting branch are both greater than the width and depth of the second sorting branch.

[0028] In some embodiments, both the first storage area and the second storage area are cylindrical, and the radii of both the first storage area and the second storage area are greater than the widths of the first sorting branch and the second sorting branch.

[0029] In some embodiments, an outlet is further included, which is connected to a three-dimensional cell recovery or buffer storage area for draining culture medium stored in the three-dimensional cell recovery or buffer storage area as needed; the outlet includes a first outlet and a second outlet respectively connected to a first storage area and a second storage area.

[0030] In some embodiments, both the first flow outlet and the second flow outlet are cylindrical, and the diameters of both the first flow outlet and the second flow outlet are greater than the widths of the first sorting branch and the second sorting branch.

[0031] To make the objectives, features, and beneficial effects of this utility model more apparent and understandable, the specific embodiments of this utility model will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described below are merely for explaining this utility model and are not intended to limit it. Furthermore, the same or similar reference numerals may be used in the drawings to refer to the same or similar elements in different embodiments, and descriptions of the same or similar elements in different embodiments, as well as descriptions of prior art elements, features, effects, etc., may be omitted.

[0032] Reference Figures 1 to 4 This utility model provides a microfluidic chip for three-dimensional cell screening and sorting.

[0033] Reference Figure 1 The microfluidic chip for three-dimensional cell screening and sorting includes a chip body 1 and a flow channel A, a sinking sorting region B, and a three-dimensional cell recovery or buffer storage region C disposed on the chip body 1. The flow channel A, the sinking sorting region B, and the three-dimensional cell recovery or buffer storage region C are connected in sequence. The sinking sorting region B is generally U-shaped and includes a first sorting branch B1 and a second sorting branch B2 with width and height differences. The first sorting branch B1 and the second sorting branch B2 achieve automatic separation of three-dimensional cells with different diameters through the width and height differences. The three-dimensional cell recovery or buffer storage region C includes a first storage region C1 and a second storage region C2, which are respectively connected to the outlets of the first sorting branch B1 and the second sorting branch B2.

[0034] In some embodiments, the flow channel A includes a liquid inlet A1 and a U-shaped flow channel A2. The liquid inlet A1 is connected to the inlet of the U-shaped flow channel A2 for injecting culture medium containing three-dimensional cells. The outlet of the U-shaped flow channel A2 is connected to the sinking sorting area B. When the culture medium flows through the U-shaped flow channel A2, the three-dimensional cells are arranged and buffered along with the culture medium.

[0035] Specifically, the width, length, internal depth, and slope of the U-shaped flow channel A2 can be designed according to specific needs.

[0036] In some embodiments, the liquid inlet A1 is cylindrical, and the diameter of the liquid inlet A1 is greater than the width of the U-shaped channel A2.

[0037] In some embodiments, the depths of the flow channel A, the sinking sorting area B, and the three-dimensional cell recovery or buffer storage area C increase sequentially.

[0038] In some embodiments, the first sorting branch B1 extends to the right and the second sorting branch B2 extends to the left; the width and depth of the first sorting branch B1 are both greater than the width and depth of the second sorting branch B2.

[0039] Specifically, the width, length, internal depth, and slope of the first sorting branch B1 and the second sorting branch B2 can be designed according to specific needs.

[0040] In some embodiments, both the first storage area C1 and the second storage area C2 are cylindrical, and the radii of both the first storage area C1 and the second storage area C2 are greater than the widths of the first sorting branch B1 and the second sorting branch B2.

[0041] Specifically, the diameter and internal depth of the first storage area C1 and the second storage area C2 can be designed according to specific needs.

[0042] In some embodiments, the system further includes an outlet D connected to a three-dimensional cell recovery or buffer storage area C for draining culture medium stored in the three-dimensional cell recovery or buffer storage area C as needed; the outlet D includes a first outlet D1 and a second outlet D2 respectively connected to a first storage area C1 and a second storage area C2.

[0043] In some embodiments, both the first outlet D1 and the second outlet D2 are cylindrical, and the diameters of both the first outlet D1 and the second outlet D2 are greater than the widths of the first sorting branch B1 and the second sorting branch B2.

[0044] The following uses the microfluidic chip for three-dimensional cell screening and sorting provided in this embodiment to illustrate the three-dimensional cell sorting method.

[0045] Culture medium containing spherical three-dimensional cells is injected into the liquid inlet A1. The cells flow through the U-shaped channel A2, creating a buffered flow effect. They then enter the U-shaped sinking sorting area B, which has varying depths and widths. A portion of the spherical three-dimensional cells enters the first storage area C1 via the first sorting branch B1, while the other portion enters the second storage area C2 via the second sorting branch B2.

[0046] The width and depth of the first sorting branch B1 are both greater than the width and depth of the second sorting branch B2. Through the first sorting branch B1, the first storage area C1 recovers three-dimensional cells with larger diameters, and through the second sorting branch B2, the second storage area C2 recovers three-dimensional cells with smaller diameters.

[0047] When the separated three-dimensional cells are needed for other experimental analysis and research, they can be drained to the first outlet D1 and the second outlet D2 as needed, or used for subsequent experimental steps through the next stage capillary.

[0048] Specifically, the first storage area C1, the second storage area C2, the first outlet D1, and the second outlet D2 are configured with a bottom structure; after sorting, the three-dimensional cells are retained in the first storage area C1 and the second storage area C2, and are then drained to the first outlet D1 and the second outlet D2 for removal or enter the next stage capillary for subsequent experimental analysis and research.

[0049] In some embodiments, the first storage area C1, the second storage area C2, the first outlet D1, and the second outlet D2 can also be configured as a through-hole structure without a bottom, which can be placed horizontally above commonly used laboratory liquid handling consumables such as EP tubes and centrifuge tubes, allowing the sorted three-dimensional cells to be directly recycled into the tubes.

[0050] The microfluidic chip for three-dimensional cell screening and sorting provided in this embodiment can be used independently. Figure 1 The structure shown can be modified by adding another chip. The added chip has through holes designed in the corresponding areas of the inlet A1 and outlet D1, D2 to achieve a local vertical connection while other areas are closed, which is convenient for the transfer and retrieval of three-dimensional cells by capillary tubes or disposable pipette tips. Alternatively, another chip can be added with a symmetrical sunken structure designed on the added chip, and through holes designed in the corresponding areas of the inlet A1 and outlet D1, D2 to achieve a local vertical connection while other areas are closed, which is convenient for the transfer and retrieval of three-dimensional cells by capillary tubes or disposable pipette tips.

[0051] Figure 2 This is a microscopic observation image of a three-dimensional cell cluster to be sorted provided in an embodiment of this utility model; Figure 3 This is a large-diameter three-dimensional cell microscope observation image after sorting using the microfluidic chip for three-dimensional cell screening and sorting provided in this embodiment of the utility model; Figure 4 This is a microscopic observation of small-diameter three-dimensional cells after sorting using the microfluidic chip for three-dimensional cell screening and sorting provided in this embodiment of the utility model.

[0052] The following uses a microfluidic chip of one specification for three-dimensional cell screening and sorting to illustrate the effect of three-dimensional cell sorting.

[0053] The microfluidic chip used has a first sorting branch B1 with a width of 200 μm and a depth of 500 μm; it is expected to obtain three-dimensional cells that are closer to spherical and have a diameter of less than 200 μm after separation. The second sorting branch B2 has a width of 150 μm and a depth of 300 μm; it is expected to obtain three-dimensional cells with a diameter of less than 150 μm, which are more irregular and more fragmented after separation.

[0054] Microscopic observation and analysis using common image processing software such as ImageJ revealed three-dimensional cell clusters of varying diameters and shapes before sorting, such as... Figure 2 As shown, the diameter is 132.01±44.78μm, and the coefficient of variation is 33.93%.

[0055] After screening by the microfluidic chip, the three-dimensional cells with larger diameter and higher cell density that flowed through the U-shaped channel A2 to the wider first sorting branch B1 (width 200μm) and then to the first storage area C1 had a diameter of 156.78±25.32μm and a coefficient of variation of 16.13%. Figure 3 As shown.

[0056] The diameter of the smaller, more irregularly shaped, and more fragmented three-dimensional cells flowing through the U-shaped channel A2 to the narrower second sorting branch B2, and then through the second storage area C2 before entering the liquid outlet D2 is 93.67±33.75 μm, with a coefficient of variation of 36.03%. Figure 4 As shown.

[0057] The three-dimensional cell sorting effect via the first sorting branch B1 and the second sorting branch B2 can meet expectations.

[0058] Although specific embodiments have been described above, these embodiments are not intended to limit the scope of this utility model disclosure, even when only a single embodiment is described with respect to a particular feature. The feature examples provided in this utility model disclosure are intended to be illustrative and not limiting, unless otherwise stated. In practice, one or more technical features of the dependent claims may be combined with the technical features of the independent claims as needed and where technically feasible, and the technical features from the respective independent claims may be combined in any suitable manner rather than solely by the specific combinations listed in the claims.

[0059] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A microfluidic chip for three-dimensional cell screening and sorting, characterized in that, The device includes a chip body and a flow channel, a sinking sorting area, and a three-dimensional cell recovery or buffer storage area disposed on the chip body. The flow channel, the sinking sorting area, and the three-dimensional cell recovery or buffer storage area are connected in sequence. The sinking sorting area is generally U-shaped and includes a first sorting branch and a second sorting branch with a width difference and a height difference. The first sorting branch and the second sorting branch achieve automatic separation of three-dimensional cells with different diameters through the width difference and height difference. The three-dimensional cell recovery or buffer storage area includes a first storage area and a second storage area, which are respectively connected to the outlets of the first sorting branch and the second sorting branch.

2. The microfluidic chip for three-dimensional cell screening and sorting according to claim 1, characterized in that, The flow channel includes a liquid inlet and a U-shaped channel. The liquid inlet is connected to the inlet of the U-shaped channel for injecting culture medium containing three-dimensional cells. The outlet of the U-shaped channel is connected to the sinking sorting area. When the culture medium flows through the U-shaped channel, the three-dimensional cells are arranged and buffered along with the culture medium.

3. The microfluidic chip for three-dimensional cell screening and sorting according to claim 2, characterized in that, The liquid inlet is cylindrical, and the diameter of the liquid inlet is larger than the width of the U-shaped flow channel.

4. The microfluidic chip for three-dimensional cell screening and sorting according to claim 1, characterized in that, The depths of the flow channel, the sinking sorting area, and the three-dimensional cell recovery or buffer storage area increase sequentially.

5. The microfluidic chip for three-dimensional cell screening and sorting according to claim 1, characterized in that, The first sorting branch extends to the right, and the second sorting branch extends to the left; the width and depth of the first sorting branch are both greater than the width and depth of the second sorting branch.

6. The microfluidic chip for three-dimensional cell screening and sorting according to claim 1, characterized in that, Both the first storage area and the second storage area are cylindrical, and the radii of the first storage area and the second storage area are both greater than the widths of the first sorting branch and the second sorting branch.

7. The microfluidic chip for three-dimensional cell screening and sorting according to claim 1, characterized in that, It also includes an outlet connected to the three-dimensional cell recovery or buffer storage area for draining the culture medium stored in the three-dimensional cell recovery or buffer storage area as needed; the outlet includes a first outlet and a second outlet respectively connected to the first storage area and the second storage area.

8. The microfluidic chip for three-dimensional cell screening and sorting according to claim 7, characterized in that, Both the first and second outlets are cylindrical, and their diameters are greater than the widths of the first and second sorting branches.

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