CN115449483A - Cell sorting device and immune cell sorting method - Google Patents

Abstract

The invention relates to the technical field of cell separation, in particular to a cell sorting device and an immune cell sorting method. It includes a substrate structure, a cell filter membrane and an upper cover; at least one pit is arranged on the substrate structure, the opening area of the pit is larger than the area of the bottom of the pit, and the projection of the bottom of the pit is within the projected area of the opening; the pit is provided with The upper cover, the upper cover completely fills the space in the pit; the cell filter membrane is arranged between the substrate structure and the upper cover; the inner wall of the pit is provided with a first protrusion, and the outer wall of the upper cover is provided with a second protrusion . Utilizing the device of the embodiment of the present invention and the preparation of single-cell samples of immune cells that can be easily repeated has practical application capabilities of high efficiency, high throughput, high removal rate and low irritation.

Description

A cell sorting device and immune cell sorting method

technical field

The invention relates to the technical field of cell separation, in particular to a cell sorting device and an immune cell sorting method.

Background technique

The content of immune cells in human blood is very low, but it contains rich information on body immunity and disease diagnosis. The successful isolation and enrichment of immune cells is the first step in subsequent single-cell sequencing and disease screening. High throughput, high activity, high purity, and as low cell stimulation as possible are crucial. With the promotion of single-cell sequencing technology, single-cell sequencing applied to immune cells has expanded the understanding of traditional immune cell typing, and rapidly promoted the understanding of the pathological process of diseases and the discovery of therapeutic targets. However, for the preparation of peripheral blood cells and bone marrow-derived immune cells such as neutrophils and mononuclear cells, centrifugation and lysis steps are unavoidable in order to remove red blood cells and platelets. Due to the abnormal sensitivity of immune cells in disease states, existing studies have shown that centrifugation or chemical methods may activate or damage immune cells, which may cause deviations in sequencing results, thereby affecting the accuracy of understanding of immune cell typing. In addition, when the immune cells are isolated, the contamination of platelets and the RNA carried by them will also affect the sequencing results. However, there is no very effective method for the removal of platelets.

The current cell separation methods generally have problems such as low throughput and complicated process, and there is still a big gap between the expectations of clinical analysis in terms of platelet removal and low stimulation of immune cells.

At present, some methods have been used for clinical detection, and the more extensive traditional methods mainly include lysis, density gradient centrifugation and flow cytometry fluorescence-activated cell sorting (FACS). Although these traditional methods have advantages in terms of throughput and application, they all suffer from their inherent disadvantages. Fluorescence-activated cell sorting by flow cytometry has a complex system, high cost, and needs to stain the cells before operation, which will cause irreversible damage to the cells. The operation process of centrifugation and lysing usually takes a long time and needs to be operated by professionals and equipment. Moreover, the lysate uses the principle of osmotic pressure to rupture the red blood cells, and previous studies have shown that the lysate can damage the surface of the white blood cells. In addition, it has been reported that the high acceleration and impact force generated by repeated high-speed centrifugation can damage leukocytes, activate neutrophils, and even induce cell death. This also limits subsequent medical testing and bioanalysis.

With the development of clinical medicine, a series of microfluidic chips have been proposed and applied to cell sorting. Sorting methods can be divided into active sorting and passive sorting. Among them, active sorting includes sorting methods based on electricity, sound, light, magnetism and mechanical force, and has achieved great results in the field of enrichment and manipulation of rare cells. Passive sorting is mainly based on the difference in fluid force related to cell shape and size, including the use of inertial force, compressive flow, determination of lateral displacement, and microfiltration. However, microfluidic sorting usually requires special labeling of target cells, and the sorting equipment is complex and expensive; complex and delicate structures need to be designed and integrated, and the processing technology is complicated; in addition, microchips based on blood cell sorting will face cell clogging, sample Issues with low throughput and impaired cell viability. Therefore, the size-based two-dimensional screening method has the main advantages of being label-free, simple, and fast, and is more suitable for the sorting and enrichment of blood cells.

However, for a sieve membrane with a micron pore size, due to the existence of surface tension, there is a pressure threshold for the liquid to pass through the pores, that is, a higher pressure needs to be applied on one side of the membrane to make the liquid break through and pass through the membrane pores to achieve filtration. Effect. Especially for low-porosity (such as 25% and below porosity) micron pore filter membranes with small pore size (such as 3 micron pore size suitable for red blood cell removal), this threshold often reaches the kPa level, that is, tens of centimeters of water column equivalent water pressure. The filtration of microporous membrane must rely on external drive (such as pressure source or centrifuge, etc.) to complete the filtration effect. Using an external drive to apply pressure, on the one hand, increases the complexity and cost of the system, and on the other hand, high pressure and high flow rate are likely to cause damage to cells and the like. There is an urgent need to establish a method that can simply and conveniently reduce the membrane passing threshold in the filtration process, and realize the filtration effect only by water pressure without external drive.

Therefore, it is urgent to establish a new high-throughput physical method to prepare single-cell samples of immune cells with high purity, high activity and low activation rate.

Contents of the invention

In order to solve the above technical problems, the present invention provides a cell sorting device and method.

The invention provides a cell sorting device, comprising a substrate structure, a cell filter membrane and an upper cover; at least one pit is arranged on the substrate structure, the opening area of the pit is larger than the area of the bottom of the pit, and the projection of the bottom of the pit is Within the projected area of the opening; the pit is provided with an upper cover, and the upper cover completely fills the space in the pit; the cell filter membrane is arranged between the substrate structure and the upper cover; the inner wall of the pit is provided with a plurality of first protrusions A plurality of second protrusions are arranged on the outer wall of the upper cover; outlets are arranged in the bottom of the pit; the gaps between the upper cover and the cell filter membrane are all inlets.

Optionally, the pits are inverted cones or pyramids.

Optionally, the pyramid is selected from a regular triangular pyramid, a regular quadrangular pyramid, a regular pentagonal pyramid, a regular hexagonal pyramid, a regular heptagonal pyramid, a regular octagonal pyramid, a regular nine-pyramid, a regular ten-pyramid, a regular eleven-pyramid and a regular twelve-pyramid; vertex.

Optionally, the first protrusion is a hemisphere or a combination of a hemisphere and a base; the base is selected from a cylinder, a truncated cone or a polygonal prism, and the upper surface of the base and the hemisphere are in a smooth transition.

Optionally, the bottom diameter or side length of the first protrusions is 0.1 mm to 10 mm; the height of the first protrusions is 0.1 mm to 10 mm; at least one row of the first protrusions is arranged along the inner wall of the pit.

Optionally, the porosity of the cell filter membrane is 11% to 14%, the thickness is 12 μm to 27 μm, and the pore size is 3 μm to 8 μm; the pore size is preferably 3 μm to 5 μm; % BSA solution for cell filter membranes after surface protein modification.

Optionally, a plurality of second protrusions are used to prop up the cell filter membrane and at the same time form passages for liquid circulation; the height of the second protrusions is 1 μm˜5000 μm.

Optionally, the substrate structure and the upper cover are made of transparent resin materials; the cell filter membrane is selected from polyethylene terephthalate materials.

The present invention proposes a method for sorting immune cells, using the above-mentioned cell sorting device to separate the target cells in the sample to be tested, at least including the steps:

S1. Rinse the cell filter membrane with DPBS solution without calcium and magnesium ions:

S2. Add the sample to be tested from the entrance:

S3. After the filtration is completed, collect the target cells sorted on the cell filter: rinse the surface of the upper cover and the surface of the cell filter with DPBS solution without calcium and magnesium ions to obtain a suspension containing the target cells.

Optionally, in S2, the blood sample is diluted and added, and the dilution factor is 1 to 3 times;

In S2, after adding the sample to be tested, the container containing the sample to be tested is cleaned with DPBS solution, and the cleaning solution is added to the cell sorting device from the inlet; the volume of the DPBS solution used for cleaning is 2 to 5 times that of the blood sample;

In S3, the DPBS solution used for washing is 8-15 times the volume of the blood sample.

Compared with the prior art, the technical solution provided by the embodiments of the present invention has the following advantages:

The embodiment of the present invention utilizes self-driven capillary force and three-dimensional filtration, and successfully realizes whole blood filtration and screening of immune cells using a 3 μm cell filter membrane, and can achieve high-throughput enrichment of 40 mL/min.

Compared with the lysis method, the cell sample obtained by the device and method of the embodiment of the present invention can achieve the purity of immune cells (about 25%) similar to that of lysis; in the non-target cell removal rate, the red blood cell removal rate is as high as 99.9%, while in In terms of platelet removal performance, the platelet removal rates were 99.48%±0.26% (3 μm) and 99.61%±0.54% (5 μm), which were significantly higher than the lysis operation (92.5%±2.24%).

The device and method of the embodiments of the present invention significantly reduce the stimulation rate (at least 7 times) to immune cells, and greatly reduce the damage to immune cells.

The device and method of the embodiments of the present invention also improve the RNA quality of immune cells and greatly reduce the degradation of RNA.

In summary, using the device of the embodiment of the present invention and the preparation of single-cell samples of immune cells that can be easily repeated, has the practical application capabilities of high efficiency, high throughput, high removal rate and low irritation, and is expected to replace lysis as a clinically based Practical single-cell preparation techniques for immune cell disease diagnosis.

Description of drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description serve to explain the principles of the invention.

Fig. 1 is a schematic structural diagram of a cell sorting device provided by an embodiment of the present invention;

Fig. 2 is a schematic structural diagram of another cell sorting device provided by an embodiment of the present invention;

Fig. 3 is a schematic structural diagram of another cell sorting device provided by an embodiment of the present invention;

4 is a schematic diagram of the principle of screening of the cell sorting device of the embodiment of the present invention;

Fig. 5 is a schematic diagram of the self-driving process in which the first protrusion of the cell sorting device of the embodiment of the invention gradually eliminates the gas-liquid interface under the action of capillary force;

Fig. 6 is a photo of the cell sorting device of the embodiment of the present invention, wherein, Fig. e is the sorting device after assembly, Fig. f is the upper cover, Fig. g is the substrate structure, and Fig. h is the SEM of the cell filter membrane with a pore size of 3 μm Image, Figure i is a SEM image of a cell filter with a pore size of 5 μm;

Figure 7 The performance results of the immune cell preparation method, wherein, Figure a is the lysis method, Figure b is the pore size of 3 μm and Figure c is the cell sample composition diagram of the blood routine analyzer after filtration by the cell filter membrane with the pore size of 5 μm; Figure d and Figure e are the removal rates of red blood cells and platelets in the immune cell samples after preparation, respectively;

Figure 8 is a comparison of the damage stimulation of immune cell samples by the preparation method: after lysis, 3 μm and 5 μm cell filter membrane filtration, the results of flow cytometry analysis, in which the activation ratio of CD62L and CD11b in the cell sample represents the immune cells stimulation rate.

Figure 9 is the RNA analysis of single-cell samples of immune cells after preparation, showing the peak diagram of RNA samples of immune cell samples after lysis repeated three times and filtered through 3 μm and 5 μm cell filters;

Figure 10 is the performance verification result of the three-dimensional sieving method;

Figure 11 is the characterization method and principle verification results of the three-dimensional sieving preparation method;

Figure 12 is a photo of the use of the two-dimensional filtration and screening device.

in:

10 - substrate structure;

11 - pit;

12 - the first protrusion;

13 - export;

2 - cell filter;

3- cover;

31 - the second protrusion;

4- Entrance.

detailed description

In order to understand the above-mentioned purpose, features and advantages of the present invention more clearly, the solutions of the present invention will be further described below. It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

In the following description, many specific details have been set forth in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described here; obviously, the embodiments in the description are only some embodiments of the present invention, and Not all examples.

The embodiment of the present invention proposes an immune cell sorting device, which uses capillary force to self-drive to three-dimensionally screen immune cells, and realizes high-throughput, high-activity, and low-stimulation immune cell enrichment. The immune cell sorting device of the present invention cleverly realizes cascade triggering. By adding a bump structure under the inclined membrane surface, a small amount of liquid is induced to flow through, and spread on the lower surface of the membrane through surface tension, infiltrating the nearby membrane pores and Around, reduce the penetration threshold of the infiltrated pores, further induce more liquid, form a cascade extraction effect, and finally realize the pressure threshold of the liquid through the micropores on the entire membrane surface.

Specifically, the structure schematic diagram of the immune cell sorting device according to the embodiment of the present invention is shown in Fig. Pit 11, wherein FIG. 1 is a case of multiple pits 11, and FIG. 2 is a case of only one pit 11. The opening area of the pit 11 is greater than the area of the bottom of the pit, and the projection of the bottom of the pit is within the projected area of the opening; a loam cake 3 is arranged in the pit 11, and the loam cake 3 completely fills the space in the pit 11; the cell filter membrane 2. Set between the substrate structure 10 and the upper cover 3; the inner wall of the pit is provided with a first protrusion 12, and the outer wall of the upper cover is provided with a second protrusion 31; the bottom of the pit is provided with an outlet 13; the upper cover The gap between the cell filter and the cell filter is the inlet 4. Similar to the filter paper used for filtration, the cell filter membrane 2 needs to be prepared in the same or similar shape as the pit 11 .

Through the contact between the first protrusion 12 on the inner wall of the pit 11 and the cell filter 2, the liquid is partially drawn out under the cell filter 2, and the gas-liquid interface under the cell filter 2 is gradually eliminated under the action of capillary force and gravity , greatly reducing the difficulty of liquid passing through the holes, increasing the utilization rate and filtration efficiency of the micropores on the cell filter membrane.

In FIG. 1, the substrate structure is a platform with multiple pits, so that multiple samples can be prepared at the same time. In FIG. 2 , when the substrate structure 10 is also designed in a tapered shape, in order to place it stably, it needs to be used together with a bracket. In FIG. 2 , a row of first protrusions 12 is arranged along the inner wall of the substrate structure 10 near the outer edge, forming a circle. In FIG. 3 , the inner wall of the substrate structure 10 is provided with multiple rows of first protrusions 12 .

In order to further increase the flow of liquid, the pits are designed as inverted cones or pyramids. The pyramids are selected from regular triangular pyramids, regular quadrangular pyramids, regular pentagonal pyramids, regular hexagonal pyramids, regular heptagonal pyramids, regular octagonal pyramids, regular nine-pyramids, regular ten-pyramids, and regular eleven-pyramids. Pyramids and regular twelve-pyramids, the more variables there are in a pyramid, the closer it is to a cone. The outlet is arranged at the apex of the cone or pyramid.

As an improved technical solution of the embodiment of the present invention, the first protrusion is a hemisphere or a combination of a hemisphere and a base; the base is selected from a cylinder, a truncated cone or a polygonal prism, and the upper surface of the base and the For a smooth transition. The bottom diameter or side length of the first protrusion is 0.1mm-10mm; the height is 0.1mm-10mm, preferably 0.5mm-5mm, most preferably 1mm.

As an improved technical solution in the embodiment of the present invention, as shown in Figures 1 to 3, on the slope of the upper cover 3, a series of second protrusions 31 are designed as a diversion structure according to the bridge water diversion body, and multiple first The two protrusions 31 are used to prop up the cell filter membrane, and at the same time form a passage for liquid circulation. As a specific implementation manner, the plurality of second protrusions are regularly arranged. In order to further reduce the filtration dead zone, multiple second protrusions 31 are regularly arranged parallelogram prisms, which can improve the utilization rate of the micropores on the cell filter membrane, effectively increase the liquid flow rate, and minimize the upper cover 3. Residual cells on the top, improve the effect of filtration. When processing a blood sample, it is only necessary to add the sample along the top of the upper cover 3, and directly recover the white blood cells from the tip of the cell filter 2 after washing. As shown in FIG. 3 , the second protrusion 31 is preferably a rhomboid prism. The height of the second protrusion 31 is 1 μm˜5000 μm.

Among them, the substrate structure and the upper cover can be manufactured by 3D printing technology, and can also be prepared in batches, and it also has the advantage of low cost and price. As an improved technical solution in the embodiment of the present invention, the cell filter membrane has a porosity of 11% to 14%, a thickness of 12 μm to 27 μm, and a pore diameter of 3 μm to 8 μm. And preferably 3 μm, 5 μm, specifically, the size of the pore size can be determined according to the type of cells required, it has been found that the cell filter membrane with a pore size of 3 μm has a better retention effect on the target cells, and when the pore size of the cell filter membrane is 5 μm, the filter Faster, less cell activation.

As an improved technical solution in the embodiment of the present invention, the cell filter membrane is selected from polyethylene terephthalate materials, and soaked in a BSA solution containing 0.5-1.5% by mass percentage before use to carry out surface protein purification. Modification, thereby reducing the damage and residue on the membrane surface to cells.

As an improved technical solution in the embodiment of the present invention, the substrate structure and the upper cover are made of transparent resin material, which can effectively reduce cell residues and visually observe blood filtration conditions.

As an improved technical solution in the embodiment of the present invention, the first protrusion is arranged at least one circle along the upper edge of the inner wall of the pit. To increase its effect, multiple turns can also be set. When multiple turns are provided, the first protrusions of two adjacent turns are arranged alternately.

The schematic diagrams of the immune cell sorting device of the embodiment of the present invention are shown in Fig. 4 and Fig. 5 . It can be seen from FIG. 4 that the capillary force effect brought by the first protrusion 12 greatly reduces the membrane rupture pressure of the cell filter 2 and the liquid at the first protrusion 12. Driven by the capillary force, the first The liquid at the bulge 12 continuously passes through the cell filter membrane 2 to condense into water droplets, and then is driven by gravity and capillary force to form a liquid flow. During the flow of the liquid, the gas-liquid interface at the flow-through area is gradually eliminated, so that the nuclear pores at the gas-liquid interface are eliminated, and the liquid flows out continuously. This is like a positive feedback, which greatly reduces the liquid through-hole Difficulty, improve the filtration area and filtration efficiency of the effective cell filter membrane.

As shown in Fig. a to Fig. c in Fig. 5, through the contact of the first protrusion 12 on the substrate structure 10 and the cell filter membrane 2, Fig. a shows that the design of the bleeding point structure helps the liquid to penetrate the membrane, Fig. Figure b shows that the liquid is partially drawn out under the cell filter membrane 2 and wets the bottom surface of the membrane; Figure c shows that under the action of capillary force and gravity, the gas-liquid interface under the membrane is gradually eliminated, more liquid is guided through the membrane, and the liquid is greatly reduced The difficulty of passing through holes increases the utilization rate and filtration efficiency of micropores.

In the embodiment of the present invention, filtration experiments were carried out through filter membranes of 3 μm and 5 μm. The performance verification experiment and the quality verification of immune cells were carried out with the lysis operation. While the operation is simple, fast and efficient, it can achieve similar immune cell purity and non-target cell removal rate as lysis, and at the same time significantly reduce the stimulation rate of immune cells.

The second aspect of the embodiment of the present invention also proposes an immune cell sorting method, using the aforementioned cell sorting device to separate the target cells in the sample to be tested, at least including the steps of:

S1. Use DPBS solution without calcium and magnesium ions to rinse the cell filter membrane, which can effectively prevent blood from reacting with reagents and cause blood coagulation;

S2. Add the sample to be tested from the entrance:

S3. After the filtration is completed, collect the target cells sorted on the cell filter: wash the surface of the upper cover and the surface of the cell filter with DPBS solution to obtain a suspension containing the target cells.

The DPBS solution without calcium and magnesium ions is Duchenne's phosphate buffered saline, which is a balanced salt solution for short-term maintenance of mammalian cell activity, and can maintain the structural and physiological integrity of isolated cells within a limited period of time. Before separating the target cells, first use the DPBS solution without calcium and magnesium ions to rinse the cell filter membrane. The specific operation method can be to add the DPBS solution from the entrance of the device. And the gap between the cell filter membrane is added in circles.

As an improved technical solution in the embodiment of the present invention, the sample to be tested is a diluted blood sample, and the dilution factor is 1 to 3 times; the purpose of dilution is to reduce the concentration and improve the filtering effect.

As an improved technical solution in the embodiment of the present invention, in the process of adding the sample to be tested, it is necessary to control the flow rate and the height of the dripping liquid level at the same time, so as to prevent the cells from being damaged by the shear stress caused by the excessive flow rate.

As an improved technical solution in the embodiment of the present invention, in the sorting process, various conventional experimental means, such as washing, etc. are used, so as to reduce the loss of blood cells and improve the sorting effect. Specifically include: In S2, after adding the sample to be tested, in order to reduce the loss of blood cells; use DPBS solution without calcium and magnesium ions to clean the container holding the sample to be tested, and add the cleaning solution from the inlet to the cell sorting device for filtration and sorting; the volume of DPBS solution used for cleaning is 8 to 15 times that of blood. In S2, in order to further reduce the loss of blood cells, after lifting the upper cover, use DPBS solution without calcium and magnesium ions to rinse the upper cover and filter membrane again.

As an improved technical solution in the embodiment of the present invention, in S3, after the filtration is completed, the cell suspension in the enrichment area is washed and filtered, and the DPBS solution used for washing is 8 to 15 times the blood volume, and preferably 10 times. Reduce the residue of blood cells attached to the surface of the device, increase the probability of red blood cells and platelets passing through the hole, and improve the purity of white blood cells in the enrichment area of the cone tip of the cell filter membrane.

As an improved technical solution in the embodiment of the present invention, the specific preparation process can be:

1. During the experiment, first draw 2 mL of blood into a test tube, add 1 to 3 times, preferably 2 times (based on the volume of the blood sample) DPBS to dilute, and at the same time use DPBS to wet and rinse the cell filter membrane.

2. Use a pipette gun to add the diluted blood sample into the filter device along the inlet of the tapered edge, while controlling the flow rate and the height of the dripping liquid surface;

3. After the blood sample is added dropwise, wash the test tube with 1 to 3 times, preferably 1.5 times (based on the volume of the blood sample) DPBS solution and add it to the filter device

4. Use 2 to 5 times, preferably 10 times (based on the blood sample volume) of DPBS to directly wash the surface of the upper cover and the surface of the cell filter,

5. Lift the upper cover, and use 2 to 5 times, preferably 2.5 times (based on the blood sample volume) of DPBS solution to rinse the upper cover channel and filter membrane;

6. Pipette and filter the cell suspension in the enrichment area to recover the enriched cell suspension;

7. Rinse the filter membrane with 0.2-1 times, preferably 0.5 times (based on the volume of the blood sample) DPBS solution, and recover the cell suspension.

Example

As shown in FIG. 6 , the embodiment of the present invention proposes a photo of a cell sorting device for screening immune cells. The photo of the device is shown in Figure e in Figure 6, the photo of the upper cover is shown in Figure f, and the photo of the substrate structure is shown in Figure g, all of which are manufactured by 3D additive printing technology. Experiments were carried out using cell filter membranes with pore diameters of 3 μm (as shown in Figure h) and 5 μm (as shown in Figure i).

As shown in Figure e, the device is mainly composed of a substrate, an upper cover, and a cell filter membrane, and is used in conjunction with a recovery device. The recovery device can use commonly used instruments and consumables. As shown in Figure f, it is the upper cover designed and manufactured, and the upper cover and the substrate are made of transparent resin. The second protrusion is a series of rhombic prisms with a height of 2 mm. The arrangement of the rhomboid prisms can support the cell-forming filter membrane and form a passage for liquid to flow. As shown in Figure g, it is a substrate structure that matches the upper cover. There are multiple first protrusions on the surface. The first protrusions are arranged in multiple circles along the upper edge of the inner wall of the pit. staggered arrangement. It can reduce the rupture pressure of the liquid, increase the fluid flux, and provide support with the minimum contact area at the same time, increasing the effective use area of the filter membrane. The upper cover and substrate structure of the device are manufactured using 3D printing technology. The middle interlayer of the filter device is a conical cell filter membrane, and the cell filter membrane with a pore size of 3 μm and 5 μm is selected for experimental operation. The specific parameters are shown in Table 1. When making a cone-shaped cell filter, only simple heat sealing and cutting are required to obtain a stable three-dimensional cone structure. Finally, soak the cell filter membrane in 1% BSA to modify the surface protein to reduce the damage and residue on the membrane surface to the cells.

Table 1

Aperture Porosity film thickness cell filter 1 3μm 14% 27μm cell filter 2 5μm 11% 12μm

Based on the built prototype device, leukocytes were sorted from whole blood samples. After the experimental device is set up, the cell filter membrane soaked in 1% BSA solution by mass percentage is assembled into the filter element. The cell filter membrane and blood were washed with DPBS solution without calcium and magnesium ions.

1. During the experiment, first draw 2 mL of blood into a test tube, add 4 mL of DPBS to dilute to 2 times, and at the same time use DPBS to wet and rinse the cell filter membrane.

2. Use a pipette gun to add the diluted blood sample into the filter device along the inlet of the tapered edge, while controlling the flow rate and the height of the dripping liquid surface;

3. After the blood sample is added dropwise, wash the test tube with 3mL of DPBS solution and add it to the filter device

4. Use 20mL of DPBS to directly wash the surface of the upper cover and the surface of the cell filter,

5. Lift the upper cover and rinse the upper cover and filter membrane with 5mL DPBS solution;

6. Pipette and filter the cell suspension in the enrichment area, and recover 500 μL of enriched cell suspension;

7. Finally, in order to increase the cleaning effect, rinse the filter membrane with 1mL of DPBS solution, and recover 500μL of liquid, recovering a total of 1mL of cell suspension.

The recovered cell samples can be directly used for subsequent bioanalysis experiments. During the experiment, a timer was used to time the operation process. The time for filtration and recovery of 2mL whole blood samples was about 5 minutes for the 5 μm cell membrane, and about 35 minutes for the 3 μm membrane. After the operation is completed, the experimental samples will be placed in a constant temperature environment at 4°C or analyzed for verification at the first time.

Experimental example

This experimental example is used to illustrate the effect of using the device and method of the embodiment of the present invention to separate immune cells

In this experiment, unless otherwise stated, the blood used in the experiment was donated by myself or volunteers in the research group, and all volunteers gave informed consent. Ethylenediaminetetraacetic acid (EDTA) tubes were used to collect peripheral blood from healthy volunteers without any added anticoagulant. The commonly used lysis method was used as the control group to compare the removal of red blood cells, the enrichment and recovery of white blood cells, and the damage to white blood cells to verify the excellent performance of the three-dimensional filtration method. Before the experiment, the drawn blood was subpackaged to ensure that the initial blood volume was consistent at 2mL. In the blood filtration and enrichment operation, cell filter membranes with a pore size of 3 μm and 5 μm were used to test the effect of pore size on the enrichment of immune cells. In order to ensure the reliability of the results, all biological experiments were repeated three times with different populations.

1. Cracking method operation:

To ensure the effect of lysis, after screening, use Gibco's ACK lysate to remove red blood cells from whole blood. During the experimental use, strictly follow the instructions for operation.

Firstly, 2mL of whole blood was selected for operation each time to ensure the consistency of the experiment and facilitate comparison with the filter group. Add 5mL of ACK lysate to each 1mL sample; then incubate at room temperature for 10-15 minutes (no more than 15 minutes) , during which the turbidity was observed to evaluate the lysis of red blood cells. Once the sample became clear, the lysis was completed; after that, it was centrifuged at 500g for 5 minutes at room temperature, and the supernatant was decanted gently to obtain the cell pellet; BSA was washed with DPBS to clear the residual lysate and ensure the same reagent conditions in the two experiments; finally, 1 mL of DPBS buffer was used to resuspend the enriched cells for subsequent analysis experiments.

2. Machine testing

In order to further characterize and verify the preparation effect, a clinical blood cell analyzer was selected to analyze the prepared samples, and the removal rate and recovery purity of non-target cells (RBC&PLT) were calculated. Clinical blood collection and experiments were performed in accordance with the guidelines and protocols approved by the hospital ethics committee and scientific committee. The collected blood was divided into 4 parts, which were marked as the original blood control group, the lysed operation group, and the 3 μm and 5 μm nuclear pore membrane screening group. After the preparation operation is completed, resuspend with 1mL of DPBS, load the sample on the machine to analyze the original blood to obtain the initial value of each cell in the blood, and then load the sample to analyze the lysis recovery and filter membrane (3μm, 5μm) sieving and weight respectively Suspend the sample, calculate and analyze the recovery purity of cells and the removal rate of non-target cells by the preparation method.

The composition of the prepared immune cell sample after verification by the routine blood analyzer is shown in Figure 7a-c. The results show that in the prepared samples, the proportion of immune cells obtained by the method of enrichment by filtration and sieving is basically the same as that obtained by the lysis method, accounting for about 25%. This shows that the filtration and screening method has the potential to replace lysis, and cell samples can be applied to subsequent clinical medical experiments. Pie chart analysis found that the proportion of filtered red blood cells was relatively high. This was because a large number of platelets and small-sized immune cells were removed during the immune cell enrichment process, resulting in a slightly lower total number of cells.

Calculated by machine sampling values, the red blood cell removal rate is shown in Figure 7d, and the red blood cell removal rates of the two preparation methods are both about 99.9%. The results show that the filtration method has a very high red blood cell removal rate, which is sufficient to replace the lysis method in terms of red blood cell removal. In terms of platelet removal performance, the filtration operation has obvious advantages. The pie chart results show that platelets account for about 54% of the lysed cell sample, while the platelets after filtration are only 12% (3 μm) and 0 (5 μm).

The platelet removal rate in Figure 7e was obtained by calculation. Filtration operation can get 99.48%±0.26% (3μm) and 99.61%±0.54% (5μm) platelet removal rate, significantly higher than lysis operation (92.5%±2.24%). This proves that lysis and centrifugation cannot effectively remove platelets, and filtration can greatly reduce the residual platelets by using the size of the cells to ensure the quality of subsequent tests.

3. Quality Characterization of Immune Cells

Based on the preparation method of immune cells, the quality characterization of immune cells is divided into the following two methods: the first is to use specific antibodies and flow cytometry to characterize and analyze the stimulation rate of immune cells; the second is to use RNA sequencing to analyze the immune cell Quality and degradability were characterized and verified. To ensure consistency, in this experiment, cells were counted after each sample preparation, and a uniform number of immune cells were extracted for quality characterization, and all experiments were repeated three times with different populations.

3.1 Flow representation

After the immune cell preparation operation, the obtained immune cell samples are processed according to the method of flow cytometry. The first step is to divide the cell samples obtained by different preparation methods into four cell suspensions, and centrifuge the samples. Then wash with 300μL Flow Cytometry Staining Buffer, centrifuge at 400xg for 5min at room temperature, and discard the supernatant. Next, mix the antibody and Flow Cytometry Staining Buffer according to the recommended amount, add it to the cells, vortex gently, and incubate at 4°C in the dark for 30 minutes. After incubation, centrifuge at 400 g for 5 min at room temperature and discard the supernatant. Afterwards, wash with 300 μL DPBS, centrifuge at 400 g for 5 minutes at room temperature, and finally resuspend with 300 μL DPBS for detection on the machine. CD11b and CD80 are used to characterize the stimulation rate of immune cells, among which CD11b is used to characterize the degree of overall immune cell stimulation, and CD80 can mark m1 macrophages, which can be used to characterize the degree of stimulation of macrophages in immune cells. The 4 groups of cell samples were double-negative, single-negative, single-negative and double-positive control groups. When the flow cytometer is on the machine, first test the double-negative group without antibody as the control group, draw a cross line according to the cell distribution as the limit of the stimulation rate, and then load the sample to analyze the stimulation of the cells, and use the proportion of antibody-labeled cells as the limit. Stimulation rate of immune cells.

The double-positive rate of CD45/CD62L is shown in Figure 8. The overall immune cell stimulation degree of lysis and membrane filtration with a pore size of 3 μm and a pore size of 5 μm were 78.70%±2.59%, 70.78%±3.61%, and 62.40%±11.20%, respectively .

Similarly, Figure 8 shows the CD45/CD11b double-positive rate of immune cells after preparation, which can characterize the degree of immune cell stimulation. It can be seen that the overall immune cell stimulation degree of lysis and 3μm and 5μm cell membrane filtration operations were respectively They are 61.03%±2.63%, 57.98%±6.04%, 38.20%±17.97%.

3.2 RNA quality characterization

Use RNA quality control analysis to characterize the damage and quality impact on immune cells during the preparation process. After the immune cells are prepared, the cell sample is first subjected to counting and analysis of immune cells. On the basis of ensuring the consistent number of cells and meeting the single-cell analysis conditions, the final number of cells in each group is guaranteed to be on the order of 10 ⁵ . Cell samples were resuspended with 1 mL of Trizol Reagent. In order to ensure the accuracy of the experiment, after the operation was completed, it was treated with dry ice to ensure that it was delivered to Lianchuan Biotechnology Co., Ltd. for RNA sequencing and quality analysis in a low temperature environment of -80 °C.

Figure 9 is a statistical graph of absorbance at 260/280, which is used to represent the quality of RNA. A value between 1.8 and 2.0 indicates that the RNA quality is good and no degradation occurs. It can be seen from the results that the RNA quality of the immune cells prepared by the three-dimensional filtration operation is in a good range, while the lysis shows a disadvantage in terms of RNA quality due to degradation.

Fig. 10 is the performance verification result of the three-dimensional sieving method of the present invention. Figure 10a and Figure 10b show the flow rate and flux as a function of time when sieving 20 mL of DPBS solution using 3 μm and 5 μm microporous membranes, respectively. Figure 10c shows the average flux results (means ± SEM) of 3 μm and 5 μm microporous membranes obtained after repeated experiments.

Fig. 11 shows the characterization method and principle verification results using the three-dimensional sieving method of the present invention.

Fig. 11a is a photo of the sieving using the basic fluid unit, the characterizing device used in the present invention, for detection. As shown in Fig. 11a, when the liquid level is 15mm higher than the highest water level, the filtration can be completed.

Figure 11b is a microscope verification picture of the working principle of the bleeding point structure. The results in Figure 11b show that at the bleeding point structure, after the liquid passes through the membrane (i) under the action of capillary force, it continuously gathers to form droplets (ii), and finally gradually eliminates the self-driven step diagram of the gas-liquid interface work (iii).

Figure 12 is a photo of the use of the two-dimensional filtration and screening device built with 3 μm and 5 μm microporous membranes. As shown in Figure 12, the two-dimensional filtration and screening device built with 3 μm and 5 μm microporous membranes has a specific structure: a microporous membrane is installed at the bottom of a vertically placed transparent conduit. Even if the liquid level difference reaches 30mm, the filtration cannot be completed.

The above descriptions are only specific embodiments of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. A cell sorting apparatus, comprising: a substrate structure, a cell filter membrane and an upper cover;

the substrate structure is provided with at least one pit, the opening area of the pit is larger than the area of the pit bottom, and the projection of the pit bottom is in the projection area of the opening;

the upper cover is arranged in the pit and completely fills the space in the pit;

the cell filter membrane is arranged between the substrate structure and the upper cover;

a plurality of first bulges are arranged on the inner wall of the pit, and a plurality of second bulges are arranged on the outer wall of the upper cover;

an outlet is arranged in the pit bottom; the gaps between the upper cover and the cell filter membrane are inlets.

2. The cell sorting device according to claim 1, wherein the pits are inverted cones or pyramids.

3. The cell sorting apparatus according to claim 2,

the pyramid is selected from regular triangular pyramid, regular rectangular pyramid, regular pentagonal pyramid, regular hexagonal pyramid, regular seven pyramid, regular eight pyramid, regular nine pyramid, regular decapyramid, regular undecalapyramid and regular dodecapyramid; the outlet is disposed at the apex of the cone or pyramid.

4. The cell sorting device according to claim 1, wherein the first protrusion is a hemisphere or a combination of a hemisphere and a base; the base is selected from a cylinder, a truncated cone or a polygonal prism, and the upper surface of the base and the hemispheroid are in smooth transition.

5. The cell sorting apparatus according to claim 1, wherein the bottom diameter or side length of the first projection is 0.1mm to 10mm; the height of the first bulge is 0.1 mm-10 mm; the first bulges are at least arranged in one row along the inner wall of the concave pit.

6. The cell sorting device according to claim 1, wherein the porosity of the cell filtration membrane is 11 to 14%, the thickness is 12 to 27 μm, and the pore diameter is 3 to 8 μm; the pore diameter is preferably 3-5 μm;

the cell filter membrane is soaked in BSA solution with the mass percentage concentration of 0.5-1.5% for surface protein modification.

7. The immune cell sorting device according to claim 1, wherein a plurality of the second protrusions are used for supporting the cell filtering membrane while forming a passage for liquid to flow through;

the height of the second protrusion is 1-5000 μm.

8. The cell sorting apparatus according to claim 1, wherein the substrate structure and the upper cover are of a transparent resin material; the cell filter membrane is made of polyethylene terephthalate material.

9. An immune cell sorting method for separating a target cell in a sample to be tested by using the cell sorting device according to any one of claims 1 to 8, comprising at least the steps of:

s1, rinsing the cell filter membrane by using a DPBS solution without calcium and magnesium ions:

s2, adding the sample to be detected from the inlet:

s3, after filtration is completed, collecting target cells obtained by sorting on the cell filter membrane: and (3) washing the surface of the upper cover and the surface of the cell filter membrane by using a DPBS solution without calcium and magnesium ions to obtain a suspension containing target cells.

10. The method of claim 9,

in S2, diluting the blood sample and then adding the diluted blood sample, wherein the dilution multiple is 1-3 times;

in S2, after the sample to be detected is added, a container containing the sample to be detected is cleaned by adopting a DPBS solution, and a cleaning solution is added into the cell sorting device from the inlet; the volume of the DPBS solution for cleaning is 2-5 times of that of the blood sample;

in S3, the DPBS solution used for the rinsing is 8 to 15 times the volume of the blood sample.

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