CN106085955B - Method that is a kind of while separating peripheral blood T, bone-marrow-derived lymphocyte - Google Patents

Abstract

The invention discloses a method for simultaneously separating peripheral blood T and B lymphocytes. This method is an improved nylon hair column separation method, which can achieve simultaneous separation of T lymphocytes and B lymphocytes at one time. The yield of cells is high, the cross-contamination is less, and there is no effect on the biological activity of cells, which is convenient for the next step. experimental research.

Description

A method for simultaneously separating peripheral blood T and B lymphocytes

technical field

The invention belongs to the technical field of cell culture, and relates to a high-efficiency separation and culture method for simultaneously obtaining peripheral blood T and B lymphocytes.

Background technique

Isolation of lymphocytes from peripheral blood is an important technique in modern molecular biology and immunology research. In recent years, cell separation technology has made great progress. At present, there are four main lymphocyte separation technologies: nylon hair column method, immunomagnetic bead method, gradient centrifugation method, and flow sorting method. The relative sorting cost of flow cytometry is relatively high, requiring large-scale flow sorting instruments, and the operation is laborious. At the same time, the sorting and screening process of flow cytometry may sometimes affect the activity and proliferation of cells. Although the purity of the cells obtained by the immunomagnetic bead method is high, the cell acquisition rate is very low, and the lymphocytes are easily activated by the immunomagnetic bead positive sorting antibody to activate downstream signaling pathways, which affects subsequent research. Although the gradient centrifugation method obtains more cells, the purity of the cells is poor. The yield of lymphocytes obtained by the traditional nylon hair column method is relatively low, and the interaction between T and B lymphocytes cannot be distinguished. Moreover, for the separation of lymphocytes in peripheral blood, the usual separation technology has low cell separation efficiency, and its activity is easily affected by magnetic bead antibodies and lost antibodies, and it cannot effectively separate and distinguish T lymphocytes and B lymphocytes, which may easily cause T and B lymphocytes. Interaction and cross-contamination between two types of lymphocytes.

Contents of the invention

The purpose of the present invention is to address the shortcomings of the existing lymphocyte separation technology, and to provide a method for simultaneously separating peripheral blood T and B lymphocytes, that is, an improved nylon hair column separation method, to achieve one-time simultaneous separation of T lymphocytes and B lymphocytes. There are two types of cells, the yield of cells is high, the cross-contamination is less, and the biological activity of cells is not affected, which is convenient for the next step of experimental research.

In order to achieve the above object, the technical solution provided by the invention is:

The method for simultaneously separating peripheral blood T and B lymphocytes comprises the following steps:

(1) After shredding the nylon wool, boil it with water, then cool it, repeat the boiling and cooling several times, then soak the nylon wool that has been repeatedly treated by boiling and cooling in acid, dry the nylon wool soaked in acid, and then soak the nylon wool again. Shredded to a single fiber state, put the shredded nylon wool into a 5 ml syringe barrel to make a nylon hair column tube, and fill each syringe barrel with 0.3-0.7 grams, preferably 0.5 grams of shredded nylon wool ; Soak at least three 10 ml syringes in acid and then wash them with water, dry and sterilize them as nylon hair column outer tubes for later use; preferably, the acid soaking is soaking with 0.2mol/L HCL;

(2) Prepare mixed lymphocyte suspension in vitro for future use (this step is a conventional method in the art);

(3) Inject the 1640 complete medium preheated at 37°C into the nylon wool column of the nylon wool column tube to ensure that all the nylon wool is completely wet; draw the mixed lymphocyte suspension preheated to 37°C and add it to the nylon wool column, and then add it to the nylon wool column. After adding a few drops of 1640 complete medium at 37°C to the nylon wool column, place the nylon wool column tube in a 37°C incubator to incubate;

(4) Place the nylon wool column tube in the nylon wool column outer sleeve, the lower end of the nylon wool column outer sleeve is connected to the infusion set tube, and the end of the infusion set tube is connected to the centrifuge tube; the infusion set tube is provided with a flow switch and a flow regulating clip; preferably Ground, an air regulating valve is provided between the nylon wool column outer sleeve and the infusion set tube;

(5) Use a pipette to draw 5 ml of 1640 complete medium at 37°C, slowly inject it into the nylon capillary tube, and control the drop rate of the filtered liquid entering the centrifuge tube to 1 drop/min by adjusting the flow switch and the flow adjustment clamp; After 5 ml of 1640 complete medium is completely filtered, centrifuge the filtrate collected in the centrifuge tube, and then resuspend the cells with 1640 complete medium at 37°C to obtain T lymphocytes;

(6) Put the nylon capillary tube treated in step (5) into a new nylon capillary outer tube, draw 10 ml of 1640 complete medium at 37°C with a pipette, slowly inject into the nylon capillary tube, pass Adjust the flow switch and the flow adjustment clamp to control the drop rate of the filtrate entering the centrifuge tube to be 1 drop/min; after 10 ml of 1640 complete medium is completely filtered, centrifuge the filtrate collected in the centrifuge tube, discard the supernatant, and obtain T, B lymphocyte mixture;

(7) Put the nylon capillary tube treated in step (6) into a new nylon capillary outer tube, draw 5 milliliters of 1640 complete medium at 4°C with a pipette, inject it into the nylon capillary tube, and adjust The flow switch and the flow adjustment clip control the drop rate of the filtrate entering the centrifuge tube to be 100 drops/min; after 5 ml of 1640 complete medium is completely filtered, centrifuge the filtrate collected in the centrifuge tube, discard the supernatant, and use The cells were resuspended in 1640 complete medium at 37°C to obtain B lymphocytes.

The method of the present invention can harvest 5*10 ^{6 T lymphocytes and 2*10 6 B} lymphocytes per milliliter, and subsequent cell culture and activation experiments show that the cells proliferate quickly and have high activity.

The present invention will be further described below:

Steps of the present invention specifically include:

1. Handle nylon wool and install nylon wool column tube

1 pack of 2 grams of nylon wool is taken out and shredded as much as possible. Then use deionized water to boil the nylon wool for 20 minutes, then let it cool for 20 minutes, repeat 6 times in total, and then soak it in 0.2mol/l HCL for 24 hours. The best extension effect can better absorb B lymphocytes. Then put the boiled nylon wool into the oven to dry for about 4 hours. After the nylon hair is completely dry, tear it carefully into a single fiber state, the finer the better. Then carefully fill these nylon hairs into a 5ml disposable syringe barrel (here is a plastic disposable syringe barrel, not including the push column inside), about 0.5 grams of nylon hair can hold 1 The column, with a volume of about 4 ml, should be moderately tight, and the packed nylon capillary tube should be autoclaved.

2. Prepare the nylon capillary casing

Three 10ml syringes were soaked with 0.2mol/l HCL for 24 hours, then rinsed with a large amount of distilled water, and placed in

In the sterile lunch box, rinse with 75% alcohol, dry naturally, and sterilize by autoclaving.

3. Preparation of mixed lymphocyte suspension in vitro

Take 10ml of peripheral blood, divide it into centrifuge tubes and dilute it with an equal volume of PBS solution, and then slowly add it to an equal volume of lymphocyte separation medium to form a layered interface. After centrifugation at 2200 rpm for 20 minutes, a layered interface can be seen. Aspirate the milky white liquid in the middle of the centrifuge tube, add 0.85% ammonium chloride to break the red blood cells and let stand at room temperature for 2 minutes, then stop the reaction with PBS. Centrifuge at 1800 rpm for 10 minutes at room temperature, and remove the supernatant. Continue to wash twice with PBS diluent, and centrifuge at 1600 rpm for 15 minutes each time; after removing the supernatant, adjust the precipitate to 1ml with RPMI-1640 culture medium containing 10% fetal bovine serum, gently blow and mix, and place at 37°C , incubate for 1 hour in a 5% CO2 incubator;

4. Preheating and incubation of nylon wool column

Use 5ML of 1640 complete medium preheated at 37 degrees and inject it into the nylon wool column tube to ensure that all the nylon wool is completely wet. Use a syringe to draw 1ml of the preheated to 37°C mixed lymphocyte suspension and add it vertically to the hair column, then add a few drops of 37°C 1640 complete medium, and place the nylon capillary tube in a 37°C incubator for 1 hour.

5. Assemble the nylon wool column casing

In the ultra-clean bench, place the nylon wool column tube in the nylon wool column outer sleeve (10Ml syringe syringe), place the syringe on a shelf 80 cm high and fix it, the lower end is connected to the air regulating valve, and the air regulating valve The lower end is connected to a 50 cm infusion tube equipped with a flow switch, and two additional flow adjustment clips are installed on the infusion tube. The end of the infusion tube was connected to a 50ML centrifuge tube.

Sixth, pass the column

Use a pipette to draw 5ML of 1640 complete medium preheated at 37 degrees, and slowly inject it into the hair column under manual control. By adjusting the flow switch and flow adjustment clip on the infusion tube, it is basically guaranteed to collect and filter at a speed of 1 drop/minute. liquid. After the 5Ml medium was completely filtered, the eluate was collected, and after centrifugation, the cells were resuspended with 37°C preheated 1640 complete medium to obtain T lymphocytes.

Then put the hair column into the new nylon hair column outer casing, and manually control and slowly inject the hair column with 10ml of preheated 37°C 1640 complete medium. By adjusting the flow switch and flow adjustment clamp on the infusion tube, basically at 1 The filter was washed at a rate of drops/min, the eluate was collected, and the supernatant was discarded by centrifugation. Can be obtained, T, B lymphocytes mixed with two kinds of cells.

Then put the hair column into the new nylon hair column outer sleeve, draw 5ml of 4 degree 1640 complete medium into the syringe quickly (100 drops/minute) into the hair column, completely release the flow switch, and rinse and filter at the fastest speed, Collect the eluate, centrifuge and discard the supernatant to obtain B lymphocytes

Seven, cell content and activity detection

The isolated cells were counted. This modified nylon hair column method can harvest 5*10 ⁶ T lymphocytes and 2*10 ⁶ B lymphocytes per milliliter of peripheral blood.

In the lymphocyte population after passing through the column, the results of flow cytometry showed that the purity of T cells can reach 91.59%, and the purity of B cells can reach 89.36%, which is significantly higher than that of the traditional nylon hair column method.

Compared with prior art, the beneficial effect of the present invention is:

1. Nylon hair treatment: the traditional method is to directly tear the nylon wool by hand and then disinfect it directly after cleaning. Our improved method is to combine nylon wool with acid soaking and repeated boiling and cooling to ensure that the nylon wool is as fluffy and stretchable as possible to achieve the fluffiest state, which can better absorb B cells.

2. Improved design of the nylon hair column: the traditional nylon hair column is to put the nylon hair directly in the syringe tube, but our nylon hair column is to first put the nylon hair in the 5ml syringe tube, and then put the 5ML Put the syringe tube into a 10ml syringe tube to form a sleeve of nylon wool column, and use multiple nylon wool column outer sleeves and a nylon wool column tube to achieve repeated filtration and separation of nylon wool column, effectively avoiding the use of Interaction and cross-contamination between T and B lymphocytes that exist when an infusion tube is collected.

3. The sequence and flow control of passing through the column: the traditional nylon wool column passing through the column is to let the medium naturally pass through the nylon wool and then collect the separation liquid, but we control the filtration speed at 1 through the combination of the infusion tube switch and the flow controller. Drops per minute, to ensure that the lymphocyte suspension is fully combined with the nylon wool column and then filtered. This filtration speed almost completely binds the B lymphocytes to the nylon wool, and collects more pure T lymphocytes. At the same time, when collecting B lymphocytes, the maximum flow rate is used to ensure that the B lymphocytes can be washed and filtered by the automatic liquid flow rate.

4. Temperature control when passing through the column: the traditional nylon wool column passing through the column does not strictly control the temperature of the filtrate, but our improved method of passing through the column uses 37 degrees preheated 1640 to fully culture when filtering T lymphocytes B lymphocytes are very sensitive to temperature changes, and low temperature significantly inhibits the adhesion of B lymphocytes to nylon wool, which can be controlled by temperature Effective separation of T lymphocytes and B lymphocytes.

Description of drawings

Figure 1: The isolated T lymphocytes and B lymphocytes were labeled with CD4 and CD19 respectively. The purity of T lymphocytes can reach 91.59% and the purity of B cells can reach 89.36% according to flow cytometry;

Figure 2: Observation of the proliferation ability of T lymphocytes after in vitro culture. The isolated T lymphocytes and B lymphocytes were labeled with CFSE fluorescent dyes, and the results showed that the isolated T lymphocytes and B lymphocytes proliferated significantly after being cultured in vitro for 3 days, respectively. 28.6% and 13.1% of the cells were proliferating and dividing.

Detailed ways

1. Handle nylon wool and install nylon wool column tube

1 pack of 2 grams of nylon wool is taken out and shredded as much as possible. Then use deionized water to boil the nylon wool for 20 minutes, then let it cool for 20 minutes, repeat 6 times in total, and then soak it in 0.2mol/l HCL for 24 hours. The purpose is to make the nylon wool as fluffy and stretchable as possible after repeated boiling and soaking in acid. To achieve the best extension effect, it can better absorb B lymphocytes. Then put the boiled nylon wool into the oven to dry for about 4 hours. After the nylon hair is completely dry, tear it carefully into a single fiber state, the finer the better. Then these nylon hairs are carefully filled into a 5ml disposable syringe barrel (the syringe barrel used here does not include the push column inside), about 0.5 grams of nylon hair can hold a column, and the volume is about 4 Milliliters, pay attention to the moderate tightness, and autoclave the packed nylon capillary tube.

2. Prepare the nylon capillary casing

Three 10ml syringes were soaked in 0.2mol/l HCL for 24 hours, then rinsed with a large amount of distilled water, placed in a sterile lunch box, rinsed with 75% alcohol, dried naturally, and sterilized under high pressure.

3. Preparation of mixed lymphocyte suspension in vitro

Take 10ml of peripheral blood, divide it into centrifuge tubes and dilute it with an equal volume of PBS solution, and then slowly add it to an equal volume of lymphocyte separation medium to form a layered interface. After centrifugation at 2200 rpm for 20 minutes, a layered interface can be seen. Aspirate the milky white liquid in the middle of the centrifuge tube, add 0.85% ammonium chloride to break the red blood cells and let stand at room temperature for 2 minutes, then stop the reaction with PBS. Centrifuge at 1800 rpm for 10 minutes at room temperature, and remove the supernatant. Continue to wash twice with PBS diluent, and centrifuge at 1600 rpm for 15 minutes each time; after removing the supernatant, adjust the precipitate to 1ml with RPMI-1640 culture medium containing 10% fetal bovine serum, gently blow and mix, and place at 37°C , incubate for 1 hour in a 5% CO2 incubator;

4. Preheating and incubation of nylon wool column

Use 5ML of 1640 complete medium preheated at 37 degrees and inject it into the nylon wool column tube to ensure that all the nylon wool is completely wet. Use a syringe to draw 1ml of the preheated to 37°C mixed lymphocyte suspension and add it vertically to the hair column, then add a few drops of 37°C 1640 complete medium, and place the nylon capillary tube in a 37°C incubator for 1 hour.

5. Assemble the nylon wool column casing

In the ultra-clean bench, place the nylon wool column tube in the nylon wool column outer sleeve (10Ml syringe syringe), place the syringe on a shelf 80 cm high and fix it, the lower end is connected to the air regulating valve, and the air regulating valve The lower end is connected to a 50 cm infusion tube equipped with a flow switch, and two additional flow adjustment clips are installed on the infusion tube. The end of the infusion tube was connected to a 50ML centrifuge tube.

Sixth, pass the column

Use a pipette to draw 5ML of 1640 complete medium preheated at 37 degrees, and slowly inject it into the hair column under manual control. By adjusting the flow switch and flow adjustment clip on the infusion tube, it is basically guaranteed to collect and filter at a speed of 1 drop/minute. liquid. After the 5Ml medium was completely filtered, the eluate was collected, and after centrifugation, the cells were resuspended with 37°C preheated 1640 complete medium to obtain T lymphocytes.

Then put the hair column into the new nylon hair column outer casing, and manually control and slowly inject the hair column with 10ml of preheated 37-degree 1640 complete medium. By adjusting the flow switch and flow adjustment clamp on the infusion tube, basically ensure the Wash and filter at a speed of 1 drop/min, collect the eluate, and discard the supernatant by centrifugation. Can be obtained, T, B lymphocytes mixed with two kinds of cells.

Then put the hair column into the new nylon hair column casing, draw 5ml of 1640 complete medium pre-cooled at 4 degrees by the syringe, inject it into the hair column quickly (100 drops/min), completely release the flow switch, and rinse and filter at the fastest speed ,, collect the eluate, centrifuge and discard the supernatant to obtain B lymphocytes

7. Detection of cell content and activity (results shown in Figure 1 and Figure 2)

The isolated cells were counted. This modified nylon hair column method can harvest 5*10 ⁶ T lymphocytes and 2*10 ⁶ B lymphocytes per milliliter of peripheral blood.

In the lymphocyte population after passing through the column, the results of flow cytometry showed that the purity of T cells can reach 91.59%, and the purity of B cells can reach 89.36%, which is significantly higher than that of the traditional nylon hair column method.

After separation, the proliferative activity of T lymphocytes and B lymphocytes is very high, and they respond obviously to various in vitro stimuli, which meets the needs of further experimental observation in vitro.

Claims (3)

1. a kind of method for separating peripheral blood T, bone-marrow-derived lymphocyte simultaneously, described method includes following steps:

(1) it with boiling boiling, then cooling after tearing up nylon hair, repeats to boil and cool down repeatedly, then cold through boiling with acid soak Nylon hair after the nylon hair after drying acid soak, is torn up to single fiber state again, will be torn by but treated repeatedly nylon hair Broken nylon hair is fitted into 5 milliliters of injector syringe, and nylon hair column tube is made, is packed into 0.3-0.7 in every injector syringe Gram nylon hair torn up；It will be washed after at least 3 10 milliliters of syringe acid soaks, nylon hair column housing be used as after drying and sterilizing It manages spare；

(2) mixed lymphocytes suspension is prepared in vitro, it is spare；

(3) in the nylon hair with 37 DEG C of 1640 complete mediums preheated injection nylon hair column tubes, it is ensured that whole nylon hairs are complete It is complete wet；Nylon hair column is added in the mixed lymphocytes suspension that absorption is preheated to 37 DEG C, then is added few drops 37 to nylon Mao Zhuzhong DEG C 1640 complete mediums after nylon hair column tube be placed in 37 DEG C of incubators be incubated for；

(4) nylon hair column tube is placed in nylon hair column outer tube, nylon hair column outer tube lower end connects infusion apparatus pipe, infusion apparatus Pipe end connects centrifuge tube；Infusion apparatus pipe is equipped with flow switch and flow adjusts folder；

(5) 1640 complete mediums that 5 milliliters 37 DEG C are drawn with pipettor, are slowly injected into nylon hair column tube, by adjusting flow It is 1 drop/minute that switch and flow, which adjust folder control to enter the filtration drop speed of centrifuge tube,；It is complete to 5 milliliter of 1640 complete medium After portion's filtration, the filtered solution that centrifuge tube is collected is centrifuged, then cell is resuspended with 37 DEG C of 1640 complete mediums, obtains T lymph Cell；

(6) will through step (5), treated that nylon hair column tube is put into new nylon hair column outer tube, draw 10 millis with pipettor Rise 37 DEG C of 1640 complete mediums, be slowly injected into nylon hair column tube, by adjust flow switch and flow adjust folder control into The filtration drop speed for entering centrifuge tube is 1 drop/minute；After 10 milliliter of 1640 complete medium all filtration, centrifuge tube is collected Filtered solution centrifugation, discard supernatant, obtain T, bone-marrow-derived lymphocyte mixture；

(7) will through step (6), treated that nylon hair column tube is put into new nylon hair column outer tube, draw 5 millis with pipettor 4 DEG C of 1640 complete mediums are risen, nylon hair column tube is injected, folder control is adjusted by adjusting flow switch and flow and enters centrifugation The filtration drop speed of pipe is 100 drops/minute；After 5 milliliter of 1640 complete medium all filtration, by the filtration of centrifuge tube collection Liquid centrifugation discards supernatant, then cell is resuspended with 37 DEG C of 1640 complete mediums, obtains bone-marrow-derived lymphocyte.

2. the method as described in claim 1, which is characterized in that step (1) described acid soak is soaked with the HCL of 0.2mol/L Bubble.

3. the method as described in claim 1, which is characterized in that be equipped with sky between the nylon hair column outer tube and infusion apparatus pipe Gas regulating valve.