CN110713920A - A kind of filter centrifuge tube type lymphocyte culture tube and culture method - Google Patents

Abstract

The invention discloses a filtration centrifugal tube type lymphocyte culture tube and a culture method. The culture tube comprises a centrifugal tube, a filter and a tube cover. The upper part of the centrifugal tube is a cylindrical tube, the filter is a detachable cylindrical tube, and the diameter of the bottom of the filter is a cylindrical tube. It is smaller than the diameter of the filter tube body, the bottom of the filter is a mesh bottom surface, a filter membrane and a pressure ring are arranged on the top of the bottom in sequence, an external hanging edge is arranged at the mouth of the filter, a round hole is arranged in the center of the tube cover, and a rubber plug is arranged inside the tube cover. The cell culture method uses the above-mentioned culture tube to culture lymphocytes. This culture tube has a simple structure and an ingenious design. It integrates the functions of culturing, centrifuging and filtering cells. It uses an innovative centrifugal culture tube with a filter to provide human peripheral blood chromosome karyotype analysis. It is convenient, greatly simplifies the operation steps, shortens the experimental time, and can obtain stable experimental results at the same time.

Description

A kind of filter centrifuge tube type lymphocyte culture tube and culture method

technical field

The invention belongs to the field of cell culture containers, in particular to a filter centrifuge tube type lymphocyte culture tube and a culture method.

Background technique

In 1960, Nowell and Morhead verified that phytohaemagglutinin (PHA), which agglutinates red blood cells to separate white blood cells, is a stimulator of mitosis in human and other animal lymphocytes. Under the action of PHA, the lymphocytes originally in the G1 phase are converted into lymphoblasts, and then undergo mitosis. In this way, after short-term culture and treatment with colchicine or its derivative colchicamide, after hypotonicity and fixation, a large number of cells in the mitotic stage can be obtained, because colchicine (or colchicamide) can interfere with microtubules by interfering with microtubules. Assembled and inhibited the formation of spindle filaments, cell division could not smoothly enter anaphase and stopped in metaphase, so that a large number of metaphase divisions that were most suitable for chromosome analysis could be accumulated in a short period of time. In addition, colchicine can shorten and separate the chromatids, making the chromosomes appear distinct and easy to identify. After the lymphocytes are cultured, a cell population that is actively growing in vitro is formed, and is prepared by air-drying. The so-called air-drying method is actually a technique for obtaining chromosome preparations on a glass slide after the cells are subjected to the steps of colchicine-hypotonic treatment-sufficient fixation-dropping. With the continuous development of medical detection technology, more and more diseases need to be diagnosed by analyzing chromosomes, and karyotype analysis has become a very common genetic diagnosis method.

In the current common culture of peripheral blood or suspension cells, it is necessary to inoculate the peripheral blood or suspension cells into a sterile, airtight culture bottle, put them in an incubator for a period of time, and then transfer them to a centrifuge tube for operation. When the number of samples is large, we need to perform a lot of tedious operations, and the amount of experimental consumables such as centrifuge tubes is large. Multiple centrifugal transfers not only increase the experimental cost, but also greatly increase the probability of cell damage. Existing centrifugal culture tubes do not come with a filter unit.

The application number is: CN201620423268.0, the application date is: 20160510, and the title is: a patent for a sterile airtight culture tube with a fixed plug, a sterile airtight culture tube with a fixed plug is disclosed, which mainly includes a tube body, rubber stopper and tube cover, the rubber stopper is sealed at the mouth of the tube body, the tube cover includes an annular cover top and a cover ring hanging down from the annular outer edge, the cover top covers the top of the rubber stopper, The cover ring is covered and connected with the outer edge of the nozzle, and at least one fixing bolt is horizontally or vertically arranged on the outer side surface of the cover ring. This culture tube does not come with a filter unit.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a product suitable for culturing human peripheral blood lymphocytes in view of the deficiencies and actual needs of the prior art, which can simplify the process of culturing and preparing peripheral blood, ensure high experimental efficiency and save experimental costs.

The purpose of the present invention can be achieved through the following technical solutions:

A filtration centrifuge tube type lymphocyte culture tube, comprising a centrifuge tube 1, a filter 2, and a tube cover 3, and the upper part of the centrifuge tube 1 is a cylindrical tube;

The filter 2 is a detachable cylindrical tube, the bottom 21 of the filter 2 is a cylindrical tube, the diameter of the bottom 21 of the filter 2 is smaller than the diameter of the body of the filter 2, and the bottom 21 is connected to the body of the filter 2 through the inclined plane. , the outer diameter of the filter 2 is smaller than the inner diameter of the centrifuge tube 1;

The bottom 21 of the filter 2 is a mesh bottom surface, a filter membrane 22 and a pressure ring 23 are arranged above the bottom 21 in sequence, the pressure ring 23 is arranged on the filter membrane 22, and the pressure ring 23 is an annular pressure ring, An external hanging edge 24 is provided at the nozzle of the filter 2, the external hanging edge 24 extends out of the tube along the edge of the nozzle, and the outer diameter of the external hanging edge 24 is not greater than the outer diameter of the centrifuge tube 1;

The pipe cover 3 is provided with a circular hole in the center, and a rubber plug 31 is provided inside the pipe cover 3 , and the diameter of the rubber plug 31 is the same as the inner diameter of the pipe cover 3 .

Preferably, the inner side of the pipe cover 3 is provided with threads, the nozzle of the centrifuge tube 1 is provided with threads, and the threads of the pipe cover 3 are matched with the threads of the centrifuge tube 1 .

Preferably, the outer diameter of the outer hanging side 24 is the same as the outer diameter of the centrifuge tube 1 .

Preferably, the centrifuge tube 1 is a plastic transparent tube.

Preferably, the lower part of the centrifuge tube 1 has a conical structure.

Preferably, the outer wall of the centrifuge tube 1 is provided with a scale.

Preferably, the volume of the centrifuge tube 1 is 50 ml, and the volume of the filter 2 is 20 ml.

A method for culturing lymphocytes, the culturing method using the culture tube comprising any one of claims 1 to 7, and the culturing step is as follows:

S1: After disinfection with a sterile cotton sheet, use a heparin anticoagulation tube and a blood collection needle to collect peripheral venous blood, invert and mix;

S2: the centrifuge tube 1 of the culture tube described in any one of claims 1 to 8 contains culture medium, and is placed with a cover at room temperature;

S3: Use a disposable sterile syringe to draw the peripheral blood, insert the needle into the rubber stopper of the centrifuge tube 1, inject the peripheral blood into the culture medium, pull out the needle, discard the needle, and mix gently;

S4: Place the culture tube on the culture rack and place it in a 37°C constant temperature incubator for 72 hours. Shake the culture tube once a day to disperse the cells. Before terminating the culture for 2 hours, use a syringe to extract 20 mg/ml of colchicine and inject it into the Incubate the tube and continue to incubate to the end.

S5: Put the hypotonic solution in a water bath at 37°C, take out the centrifuge tube 1, unscrew the cap 3, pour the culture into the filter 2, then put the filter 2 into the centrifuge tube 1 and screw the cap 3 again , centrifuge at 1500 rpm for 1 min on a centrifuge, pour out the filtered medium, pour the hypotonic fluid into filter 2, gently shake to suspend the cells, gently pipette with a dropper to suspend the cells, and pour the obtained suspension into a centrifuge tube 1. Cover the tube cap 3, and place the centrifuge tube 1 in a water bath at 37°C for 25 minutes;

S6: Mix methanol and glacial acetic acid at a volume ratio of 3:1 to prepare a fixative solution. After 1 water bath in the centrifuge tube, open the cap and add 1 ml of the prepared fixative solution to the suspension, mix gently, and pour the suspension. Put it into filter 2, put it into a culture tube, let it stand for 5 min, then centrifuge at 1500 rpm for 1 min, discard the filtrate, add fixative to filter 2, gently blow with a dropper, let it stand for 20 min, then centrifuge at 1500 rpm for 1 min, discard Remove the filtrate, add fixative solution to filter 2, gently blow with a dropper, then let stand for 20 min, centrifuge at 1500 rpm for 1 min, discard the filtrate, suck the fixative solution with a syringe and inject it into the filter, blow up the cells, and then use Syringe to draw cell suspension;

S7: Pre-chilled ice box, place the glass slide on the ice box, after cooling, use a syringe to drop 5-10 drops of cell suspension on the glass slide, cover the glass slide, and bake the glass slide on the flame of an alcohol lamp Dry, the prepared slides can be directly stained with Giemsa staining solution or G-banding analysis, and the final chromosome slides are treated with G-banding and observed under a microscope.

Beneficial effects of the present invention:

This culture tube has a simple structure and ingenious design. It integrates the functions of culturing, centrifuging and filtering cells. It uses an innovative centrifugal culture tube with filter, which provides convenience for the analysis of human peripheral blood chromosome karyotype, greatly simplifies the operation steps, and shortens the experiment. time, and stable experimental results can be obtained.

Description of drawings

FIG. 1 is a schematic structural diagram of a filter centrifuge tube type lymphocyte culture tube.

In the figure: 1-centrifuge tube; 2-filter; 21-bottom; 3-tube cover; 31-plastic stopper.

Figure 2 is a schematic diagram of the structure of the filter.

In the picture: 2-filter; 21-bottom; 22-filter membrane; 23-pressing ring; 24-external hanging edge.

FIG. 3 is an enlarged schematic view of the structure of the bottom of the filter.

In the figure: 21-bottom; 22-filter membrane; 23-press ring.

FIG. 4 is a structural diagram of placing a culture medium in a centrifuge tube according to Example 2. FIG.

In the figure: 1-centrifuge tube; 3-tube cap; 31-plastic stopper; 4-culture medium.

FIG. 5 is a schematic diagram of the lymphocytes cultured in Example 2 observed under a low magnification microscope.

Fig. 6 is the chromosome observed under the oil microscope of the cultured lymphocytes described in Example 2.

Detailed ways

As shown in conjunction with the accompanying drawings, the technical scheme of the present invention is further described:

Example 1:

As shown in the accompanying drawings 1-3: The present invention develops a pH-stable lymphocyte culture medium for the characteristics of human peripheral blood lymphocyte culture and chromosome preparation, which can be used for the in vitro culture of peripheral blood lymphocyte culture medium. For closed cultivation, it can be cultivated in a water-proof constant temperature incubator or a water bath. Centrifugal culture tubes can simplify the post-production process and facilitate operation.

A filtration centrifugal tube type lymphocyte culture tube, comprising a centrifugal tube 1, a filter 2, a tube cover 3, the upper part of the centrifugal tube 1 is a cylindrical tube, the lower part is conical, and the inner side of the tube cover 3 is provided with threads , the nozzle of the centrifuge tube 1 is provided with a thread, and the thread of the tube cover 3 matches the thread of the centrifuge tube 1 .

The filter 2 is a detachable cylindrical tube, the bottom 21 of the filter 2 is a cylindrical tube, the diameter of the bottom 21 of the filter 2 is smaller than the diameter of the body of the filter 2, and the bottom 21 is connected to the body of the filter 2 through the inclined plane. , the outer diameter of the filter 2 is smaller than the inner diameter of the centrifuge tube 1 .

The bottom 21 of the filter 2 is a mesh bottom surface, a filter membrane 22 and a pressure ring 23 are arranged above the bottom 21 in sequence, the pressure ring 23 is arranged on the filter membrane 22, and the pressure ring 23 is an annular pressure ring, An external hanging edge 24 is provided at the nozzle of the filter 2 , the external hanging edge 24 extends out of the tube along the edge of the nozzle, and the outer diameter of the external hanging edge 24 is not larger than the external diameter of the centrifuge tube 1 .

The pipe cover 3 is provided with a circular hole in the center, and a rubber plug 31 is provided inside the pipe cover 3 , and the diameter of the rubber plug 31 is the same as the inner diameter of the pipe cover 3 .

In this embodiment, the volume of the centrifuge tube is 50 milliliters, the volume of the filter 2 is 20 milliliters, and the outer diameter of the centrifuge tube 1 is 29 mm and the inner diameter is 27 mm. The outer diameter of the filter 2 is 25 mm, the inner diameter is 23 mm, and the outer diameter of the outer hanging side 24 is the same as the outer diameter of the centrifuge tube 1, which is 29 mm. The bottom 21 of the filter 2 has a diameter of 10 mm and a depth of 5 mm. The outer wall of the centrifuge tube 1 is provided with graduations in units of 5 ml.

In this embodiment, the centrifuge tube 1 and the tube cover 3 can be screwed tightly. There is a round hole in the center of the tube cover 3, and a rubber stopper is attached to the inner side, which not only has a sealing function, but also facilitates the injection of peripheral blood or cell suspension into the tube with a syringe. The filter 2 is separable from the centrifuge tube 1 , and the center of the bottom of the filter 2 is a circular mesh hollow structure, which supports the filter membrane 22 , and an annular pressure ring 23 fixes the filter membrane 22 . When the cell culture needs to be centrifuged, first pour the cell suspension into the filter 2, then put it into the tube, screw on the cap for centrifugation.

Embodiment 2:

As shown in Figures 1-6:

A method for culturing lymphocytes comprising the above-mentioned culture tube.

After disinfection with sterile cotton sheets, 2 ml of peripheral venous blood was collected using a heparin anticoagulant tube and a blood collection needle, and mixed by inversion. Take a tube of peripheral blood lymphocyte culture medium (as shown in Figure 4) of the present invention and place it at room temperature to thaw. In the ultra-clean workbench, use a 2 ml disposable sterile syringe to draw about 0.3 ml of peripheral blood, and insert the needle into it. The rubber stopper of the centrifuge tube 1, the peripheral blood was poured into the medium, the needle was pulled out (discarded), and the mixture was gently mixed. Place the culture tube on the medium rack and place it in a 37°C constant temperature incubator for 72 hours. Shake the culture tube once a day to disperse the cells. Before terminating the culture for 2 hours, use a 2 ml syringe (do not discard it and continue to use it in subsequent operations) to draw 0.1 ml. 20 mg/ml colchicine was injected into the culture tube, and the culture was continued until the end.

Put the hypotonic solution in a water bath at 37°C, take out the centrifuge tube 1, unscrew the tube cap 3, pour the culture into filter 2, then put the filter 2 into the centrifuge tube 1, screw the cap back on, and run the centrifuge at 1500 rpm Centrifuge for 1 min, pour out the filtered medium, pour 8 ml of hypotonic solution into the filter, gently shake to suspend the cells (gently pipette with a dropper), pour the suspension into centrifuge tube 1, and cover the tube cap 3. Place in a 37°C water bath for 25 minutes, mix methanol and glacial acetic acid at a volume ratio of 3:1 to prepare a fixative solution. After the water bath, add 1 ml of the prepared fixative solution to the suspension, mix gently, and mix the suspension. Pour the liquid into filter 2, put it into centrifuge tube 1, let it stand for 5 minutes, then centrifuge at 1500 rpm for 1 minute, discard the filtrate, add 9 ml of fixative to filter 2, blow gently with a dropper and let it stand for 20 minutes, then Centrifuge at 1500 rpm for 1 min and discard the filtrate. Add 9 ml of fixative to filter 2, blow gently with a dropper, let it stand for 20 min, centrifuge at 1500 rpm for 1 min, and discard the filtrate. Pipette 0.5 ml of fixative solution into the filter with a 2 ml syringe, blow up the cells, and then use a syringe to aspirate the cell suspension.

Take a pre-frozen ice box, place the glass slide on the ice box, use a syringe to drop about 5-10 drops of cell suspension 20cm above the glass slide after cooling, cover the glass slide, and then place the glass slide in an alcohol lamp. After drying on flame, the prepared slides can be directly stained with Giemsa staining solution or analyzed by G banding. The finally prepared chromosome slides were treated with G banding and observed under a microscope, and the results are shown in FIG. 5 and FIG. 6 .

Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the present invention can still be modified or Equivalents are substituted without departing from the spirit and scope of the invention, and the appended claims are intended to cover such modifications or equivalents as fall within the spirit and scope of the invention.

Claims (8)

1. a filter centrifuge tube type lymphocyte culture tube, comprising centrifuge tube (1), filter (2), tube cover (3), it is characterized in that: The upper part of the centrifuge tube (1) is a cylindrical tube; The filter (2) is a detachable cylindrical tube, the bottom (21) of the filter (2) is a cylindrical tube, and the diameter of the bottom (21) of the filter (2) is smaller than the diameter of the body of the filter (2), The bottom (21) is connected with the body of the filter (2) through the inclined plane, and the outer diameter of the filter (2) is smaller than the inner diameter of the centrifuge tube (1); The bottom (21) of the filter (2) is a mesh bottom surface, a filter membrane (22) and a pressure ring (23) are arranged on the top of the bottom (21) in sequence, and the pressure ring (23) is arranged on the filter membrane (22). ), the pressure ring (23) is an annular pressure ring, the filter (2) nozzle is provided with an external hanging edge (24), and the external hanging edge (24) extends out of the pipe along the edge of the nozzle, The outer diameter of the outer hanging side (24) is not greater than the outer diameter of the centrifuge tube (1); A circular hole is provided at the center of the tube cover (3), a rubber stopper (31) is arranged inside the tube cover (3), and the diameter of the rubber stopper (31) is the same as the inner diameter of the tube cover (3). 2. The centrifugal culture tube according to claim 1, characterized in that: the inner side of the tube cover (3) is provided with threads, the nozzle of the centrifugal tube (1) is provided with threads, and the tube cover (3) match the thread of the centrifuge tube (1). 3. The centrifugal culture tube according to claim 1, wherein the outer diameter of the outer hanging edge (24) is the same as the outer diameter of the centrifugal tube (1). 4. The centrifugal culture tube according to claim 1, wherein the centrifugal tube (1) is a plastic transparent tube. 5 . The centrifugal culture tube according to claim 1 , wherein the lower part of the centrifugal tube ( 1 ) has a conical structure. 6 . 6. The centrifugal culture tube according to claim 1, characterized in that: the outer wall of the centrifugal tube (1) is provided with a scale. 7. The centrifugal culture tube according to claim 1, wherein the volume of the centrifugal tube (1) is 50 ml, and the volume of the filter (2) is 20 ml. 8. A method for culturing lymphocytes, characterized in that: the method for culturing comprises the culturing tube described in any one of claims 1 to 7, and the culturing step is as follows: S1: After disinfection with a sterile cotton sheet, use a heparin anticoagulation tube and a blood collection needle to collect peripheral venous blood, invert and mix; S2: the centrifuge tube (1) of the culture tube described in any one of claims 1 to 8 contains culture medium, and is placed with a cover at room temperature; S3: Draw peripheral blood with a disposable sterile syringe, insert the needle into the rubber stopper of the centrifuge tube (1), inject the peripheral blood into the culture medium, pull out the needle, discard the needle, and mix gently; S4: Place the culture tube on the culture rack and place it in a 37°C constant temperature incubator for 72 hours. Shake the culture tube once a day to disperse the cells. Before terminating the culture for 2 hours, use a syringe to extract 20 mg/ml of colchicine and inject it into the Incubate the tube and continue to incubate to the end. S5: Put the hypotonic solution in a water bath at 37°C, take out the centrifuge tube 1, unscrew the tube cover 3, pour the culture into the filter (2), and then put the filter (2) into the centrifuge tube 1 and twist it again Put the cap on the tube (3), centrifuge at 1500 rpm for 1 min on the centrifuge, pour off the filtered medium, pour the hypotonic fluid into the filter (2), gently shake to suspend the cells, and gently pipette with a dropper to suspend the cells. The obtained suspension was poured into the centrifuge tube (1), the tube cap (3) was closed, and the centrifuge tube (1) was water bathed at 37°C for 25 minutes; S6: Mix methanol and glacial acetic acid at a volume ratio of 3:1 to prepare a fixative solution. After 1 water bath in the centrifuge tube, open the cap and add 1 ml of the prepared fixative solution to the suspension, mix gently, and pour the suspension. Put it into the filter (2), put it into the culture tube, let it stand for 5 minutes, then centrifuge at 1500rpm for 1min, discard the filtrate, add the fixative to the filter (2), gently blow with a dropper and let it stand for 20 minutes, then 1500rpm Centrifuge for 1 min, discard the filtrate, add the fixative solution to the filter (2), blow gently with a dropper, let it stand for 20 min, centrifuge at 1500 rpm for 1 min, discard the filtrate, suck the fixative solution with a syringe and inject it into the filter, Blow up the cells, then aspirate the cell suspension with a syringe; S7: Pre-chilled ice box, place the glass slide on the ice box, after cooling, use a syringe to drop 5-10 drops of cell suspension on the glass slide, cover the glass slide, and bake the glass slide on the flame of an alcohol lamp Dry, the prepared slides can be directly stained with Giemsa staining solution or G-banding analysis, and the final chromosome slides are treated with G-banding and observed under a microscope.

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