CN209872948U - Cell separation tube for separating and extracting PBMC - Google Patents

Abstract

The utility model discloses a cell separation tube for separating and extracting PBMC, include: the blood plasma separating device comprises a pipe body, a pipe cover, an isolating sieve plate and isolating glue, wherein the isolating sieve plate is arranged in the pipe body and used for separating a blood plasma layer from a liquid separating layer; the diameter of the bottom of the isolation sieve plate is gradually reduced from top to bottom, and a liquid injection hole is formed in the bottom end of the isolation sieve plate; the structural design and the material characteristics of the isolation sieve plate can quickly add the blood sample to the liquid level of the separation liquid and can prevent the blood liquid layer from being mixed with the separation liquid; the physical property of the isolation glue can form a second layer of partition board; thereby improving the separation quality, improving the purity of PBMC, simplifying the operation steps and reducing the time for separation and extraction.

Description

Cell separation tube for separating and extracting PBMC

Technical Field

The utility model relates to an experimental apparatus field, especially a cell separation tube of PBMC is drawed in separation.

Background

Peripheral Blood Mononuclear Cells (PBMC) are primarily meant to include lymphocytes, monocytes, dendritic cells and other small numbers of cells. Monocytes are a more commonly used cell population in basic and clinically relevant in vitro studies. How to separate high-purity mononuclear cells from peripheral blood quickly and efficiently is a problem which is continuously discussed in the technical field of medicine. The traditional method for separating the mononuclear cells is a Ficoll density gradient separation method, and the mononuclear cells are mainly separated according to the specific gravity difference of each component in blood. After density gradient centrifugation, the uppermost layer is a plasma layer without cells, the lowermost layer is a separation liquid layer containing granular cells and platelets, PBMC is densely arranged at the interface of the plasma layer and the separation liquid and is in a white membrane shape, and the white membrane layer is sucked out to obtain PBMC. The density gradient method has the difficulties that in the sample adding process, if the sample adding speed of an operator is too high, a centrifugal tube is unbalanced or a brake is opened, the layering of a blood sample and a separating solution is not clear, the separating effect is influenced, the separating process is complicated, and the whole step needs to be 1.5 hours long; meanwhile, the white membrane layer aspiration process affects the key of cell yield and purity, and because each layer is easily scattered, dirty PBMC aspirated by the white membrane layer between the plasma layer and the separation liquid layer are more remained, or because too much separation liquid layer is aspirated to reduce the purity of PBMC, the remained platelets affect the cell count and the number of background spots of subsequent experiments, such as enzyme-linked immunosorbent assay (Elispot).

There are commercially available PBMC centrifuge tubes on the market, such as the SepMate tube from STEMCELL, which have a typical concave configuration with at least two openings, one of which is closer to the bottom end of the tube when the insert is in place and acts to pass liquid through the opening to the bottom; another function of the openings is to let air escape to equalize the pressure. The baffle plate also plays a role in physical isolation, and the concave configuration enables liquid to flow in a single direction and is not easy to flow back. However, the dosage of the ficoll separation solution needs to be well controlled and is slightly higher than the height of the concave configuration, partial platelet residues still remain in the supernatant after centrifugation, and the single cost of the SepMate tube is high; the market needs a cell separation tube that can solve the separation and extraction PBMC purity low, the separation and extraction time long and the inconvenient scheduling problem of operation, the utility model provides a such problem.

SUMMERY OF THE UTILITY MODEL

In order to solve the defects of the prior art, the utility model aims to provide a cell separation tube for separating and extracting PBMC, the structural design and the material characteristics of an isolation sieve plate can quickly add a blood sample on the liquid level of a separation liquid, and can prevent the mixing of a blood liquid layer and the separation liquid; the physical property of the isolation glue can form a second layer of partition board; thereby improving the separation quality, improving the purity of PBMC, simplifying the operation steps and reducing the time for separation and extraction.

In order to achieve the above object, the utility model adopts the following technical scheme:

a cell separation tube for separating and extracting PBMCs, comprising: the blood plasma separating device comprises a pipe body, a pipe cover, an isolating sieve plate and isolating glue, wherein the isolating sieve plate is arranged in the pipe body and used for separating a blood plasma layer from a liquid separating layer; the diameter of the bottom of the isolation sieve plate gradually decreases from top to bottom, and a liquid injection hole is formed in the bottom end of the isolation sieve plate.

The bottom of the isolation sieve plate forms a conical bottom.

The angle range of the conical bottom of the cell separation tube for separating and extracting the PBMC is more than or equal to 150 degrees and less than 180 degrees.

In the cell separation tube for separating and extracting PBMCs, the length ratio of the tube body above the isolation sieve plate to the tube body below the isolation sieve plate is 2: 1.

In the cell separation tube for separating and extracting PBMCs, the isolation sieve plate is an elastic isolation sieve plate.

In the cell separation tube for separating and extracting PBMCs, the isolation sieve plate is a polyvinyl chloride film or a polyethylene terephthalate film.

In the cell separation tube for separating and extracting PBMC, the thickness of the isolation sieve plate is in the range of 2-3.0 mm.

The density of the isolation gel of the cell separation tube for separating and extracting the PBMC is 1.065-1.077 g/ml.

The diameter of the injection hole of the cell separation tube for separating and extracting PBMC is 1-1.5 mm.

The utility model discloses an useful part lies in:

due to the design of the isolation sieve plate, the cell separation tube can quickly add a blood sample to the liquid level of the separation liquid, can prevent the mixing of a blood liquid layer and the separation liquid, and does not need to close a centrifugal machine to brake during centrifugation; meanwhile, the diameter of a liquid injection hole at the conical bottom formed by the isolation sieve plate is 1-1.5mm, so that ficoll separating medium and isolation glue can be conveniently added, the centrifuge tube is integrated, liquid can flow in a single direction, and the cell separating medium at the lower part of the isolation sieve plate compresses the funnel hole to close the opening and is not easy to flow back because the polyvinyl chloride film or the polyethylene terephthalate film has certain elasticity;

the isolation glue introduced into the cell separation tube is a mixture of polybutyl acrylate and silicon dioxide with the density of 1.065-1.077g/ml, the density of the isolation glue is close to that of PBMC, the isolation glue is solidified into a layer of film, the layer of film is blocked by an isolation sieve plate, and a 2-layer partition plate is formed;

keep apart sieve and isolation gum and can avoid injecting into the separating fluid of keeping apart the sieve below invert at the separator tube effectively, under jolting vibrations or the collision condition of transportation, "palirrhea" to the top of the isolation sieve of separator tube, guarantee that cell separating fluid is in the separator tube of keeping apart the below of sieve and isolation gum always before this separator tube is launched to make the cell extract operation more reliable, convenient, cell separator tube transportation is also more convenient.

Drawings

Fig. 1 is a schematic structural diagram of an embodiment of the present invention.

The meaning of the reference symbols in the figures:

the body 1, 2 pipe lids, 3 keep apart the sieve, 301 keep apart the bottom of sieve, 302 annotate the liquid hole, 4 keep apart glues, 5 separation liquid layers.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and specific embodiments.

A cell separation tube for separating and extracting PBMCs, comprising: the blood plasma separating tube comprises a tube body 1, a tube cover 2, an isolating sieve plate 3 and isolating glue 4, wherein the isolating sieve plate 3 is arranged in the tube body 1 and is used for separating a blood plasma layer from a separation liquid layer 5, and the isolating glue 4 is filled between the bottom 301 of the isolating sieve plate and the tube body 1; the diameter of the bottom 301 of the isolation sieve plate is gradually reduced from top to bottom, and the bottom end of the isolation sieve plate 3 is provided with a liquid injection hole 302. Preferably, the pipe body 1 is cylindrical, the pipe cover 2 is in threaded engagement and is hermetically mounted at the top of the pipe body 1 in a sealing manner, and the isolation sieve plate 3 is coaxially and hermetically mounted at the middle lower part in the pipe body 1.

As shown in fig. 1, the bottom 301 of the insulating screen deck preferably forms a conical bottom with an angular extent of 150 ° or more and less than 180 °. The isolation sieve plate 3 is an elastic isolation sieve plate 3; as an example, the isolation screen plate 3 is a polyvinyl chloride film or a polyethylene terephthalate film. Keep apart the toper bottom that sieve 3 formed and can make liquid one-way flow, because polyvinyl chloride membrane or polyethylene terephthalate membrane have certain elasticity, be difficult to "backward flow" for the centrifuging tube is a whole, has guaranteed the fastness of connection, makes things convenient for the sterilization operation simultaneously.

The length ratio of the pipe body 1 above the isolation sieve plate 3 to the pipe body 1 below the isolation sieve plate 3 is 2: 1. As an example, if the volume of the separation tube is 50ml of scale loading, an isolation sieve plate 3 is arranged at 15ml of the inner wall of the separation tube; if the volume of the separation tube is 15ml of scale loading, 4.5ml of the inner wall of the separation tube is provided with an isolation sieve plate 3.

The isolation glue 4 is a mixture of polybutyl acrylate and silicon dioxide (solvent dissolution is not needed), the mass final concentration of the polybutyl acrylate in the mixture is 71.8-75.0%, the density range of the isolation glue 4 is 1.065-1.077g/ml, the isolation glue 4 is close to PBMC, the PBMC can be solidified into a layer of film, the layer of film can be blocked by the isolation sieve plate 3, and a 2-layer partition plate is formed. The thickness range of the isolation sieve plate 3 is 2-3.0 mm. It should be noted that: the material of isolation glue 4 is unrestricted, as long as density is close PBMC, and can realize keeping apart, can both be applied to the utility model discloses.

The diameter of the liquid injection hole 302 is 1-1.5mm, so that a ficoll separating medium and an isolating glue 4 can be conveniently added, wherein the isolating glue 4 needs to be formed by centrifuging the ficoll separating medium and the isolating glue 4 in advance (2000g, 4min), so that a separation system with the ficoll separating medium positioned at the lower layer and the isolating glue 4 positioned at the upper layer is formed. Then, 30ml of the blood sample can be quickly added to the liquid level of the bottom sieve plate and the isolating glue 4, and the bottom sieve plate and the isolating glue 4 prevent the blood sample layer from being mixed with the separation liquid layer 5; the centrifugal force of 1000g is used for 10 minutes, and the brake of the centrifugal machine is not required to be closed during centrifugation. After centrifugal separation, red blood cell, platelet and granular cell etc. in separation liquid layer 5 and the blood sample are kept apart in bottom sieve and isolation gum 4 below, the required most PBMC cell is for being located the white membrane layer between blood plasma layer and the separation liquid layer 5, because the setting of isolation gum 4, can directly pour the supernatant into new container easily after the centrifugation, take out easily, convenient operation, swiftly, still save time, the quality of separation cell has been improved, the operating procedure has been simplified, PBMC is extracted high-efficiently.

The use method of the cell separation tube of the utility model is as follows:

1) preparing an isolation adhesive 4: 71.8% by weight of polybutylacrylate (molecular weight 9.59X 10)³) And 28.2 wt% of a silica mixture having a density of 1.065 g/ml. The isolation glue 4 is filled in a medical disposable syringe and is preserved by irradiation sterilization (25kGray cobalt-60 gamma ray irradiation), and the endotoxin content is less than or equal to 0.5 EU/ml.

2) 6g of the isolation glue 4 is pushed into a 50ml sterile centrifuge tube through a liquid injection hole 302 by using a syringe, 7.5ml of ficoll separating medium is added for centrifugation (2000g, 4min), so that a separation system with the ficoll separating medium positioned at the lower layer and the isolation glue 4 positioned at the upper layer is formed, and the interface of the separation system is flush with the isolation sieve plate 3.

3) The whole blood sample is diluted with 1 × PBS in a ratio of 1:1, transferred to a 50ml sterile centrifuge tube of step 2), and centrifuged for 10 minutes at a centrifugal force of 1000g without turning off the centrifuge brake during centrifugation.

4) After centrifugation, the supernatant was directly poured into a new 15ml sterile centrifuge tube in a new container and centrifuged (300g, 6min) to obtain supernatant a and pellet a.

5) And (3) cleaning the precipitate a twice by using a diluent, precipitating the PBMC to be extracted at the bottommost part of the test tube, and taking out the PBMC to ensure that the recovery rate is more than or equal to 85 percent and the cell survival rate is more than or equal to 95 percent.

In order to further prove the effect achieved by the structure, the effect of separating the mononuclear cells by the device for separating the mononuclear cells from the whole blood is compared with that of separating the mononuclear cells by the traditional Ficoll; the comparison results are shown in table 1:

TABLE 1

It can be seen from the table that the utility model discloses a centrifuging tube has improved the purity that the separation drawed PBMC, reduces the length of time that the separation drawed.

The utility model relates to a cell separation tube for separating and extracting PBMC, the structural design and the material characteristics of an isolation sieve plate 3 can quickly add a blood sample on the liquid level of a separation liquid and can prevent the mixing of a blood liquid layer and the separation liquid; the physical properties of the isolating glue 4 will form a second layer of spacers; thereby improving the separation quality, improving the purity of PBMC, simplifying the operation steps and reducing the time for separation and extraction.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by adopting equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims (9)

1. A cell separation tube for separating and extracting PBMCs, comprising: the blood plasma separating device comprises a pipe body, a pipe cover, an isolating sieve plate and isolating glue, wherein the isolating sieve plate is arranged in the pipe body and used for separating a blood plasma layer from a liquid separating layer; the diameter of the bottom of the isolation sieve plate is gradually reduced from top to bottom, and a liquid injection hole is formed in the bottom end of the isolation sieve plate.

2. The cell separation tube for separating PBMCs according to claim 1, wherein the bottom of the partition sieve plate forms a conical bottom.

3. The cell separation tube for separating and extracting PBMCs according to claim 2, wherein the angle range of the tapered bottom is greater than or equal to 150 ° and less than 180 °.

4. The cell separation tube for separating and extracting PBMCs according to claim 1, wherein the length ratio of the tube body above the isolation sieve plate to the tube body below the isolation sieve plate is 2: 1.

5. The cell separation tube for separating and extracting PBMCs according to claim 1, wherein the isolation sieve plate is a resilient isolation sieve plate.

6. The cell separation tube for separating and extracting PBMCs according to claim 5, wherein the isolation sieve plate is a polyvinyl chloride film or a polyethylene terephthalate film.

7. The cell separation tube for separating and extracting PBMCs according to claim 1, wherein the isolating screen has a thickness ranging from 2 to 3.0 mm.

8. The cell separation tube for separating and extracting PBMCs according to claim 1, wherein the density of the isolation gel is in the range of 1.065-1.077 g/ml.

9. The cell separation tube for separating and extracting PBMCs according to claim 1, wherein the diameter of the injection hole is 1-1.5 mm.