CN113604433A

CN113604433A - Kit for activating CTL (cytotoxic T lymphocyte) cells and activation method thereof

Info

Publication number: CN113604433A
Application number: CN202110961201.8A
Authority: CN
Inventors: 杨罕闻
Original assignee: Lanlian Hangzhou Biotechnology Co Ltd
Current assignee: Lanlian Hangzhou Biotechnology Co Ltd
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-11-05
Anticipated expiration: 2041-08-20
Also published as: CN113604433B

Abstract

The invention discloses a kit for activating CTL cells, which comprises: the syringe, set up the first liquid bag in the syringe, set up in the syringe and locate at the second liquid bag on the first liquid bag, set up in the syringe and locate at the third liquid bag on the second liquid bag, set up in the syringe and locate at the fourth liquid bag on the third liquid bag, set up in the syringe and locate at the fifth liquid bag on the fourth liquid bag, set up in the inner bottom of syringe and sting the department; the liquid adding device can complete liquid adding by only pressing the syringes in sequence and puncturing the liquid bag, is simple to operate and can comprehensively solve the problem of misoperation.

Description

Kit for activating CTL (cytotoxic T lymphocyte) cells and activation method thereof

Technical Field

The invention relates to the field of biological cell culture, in particular to a kit for activating CTL cells and an activation method thereof.

Background

Cytotoxic T Lymphocytes (CTLs), which are a subset of leukocytes, are specific T cells that secrete a variety of cytokines specifically for immune function. It has killing effect on some virus, tumor cell and other antigen matter, and forms important defense line with natural killer cell for resisting virus and tumor immunity.

The CTL cell prepared by the existing method has low yield and low purity; although the patent reported in the past overcomes the problems of low yield and high purity, the operation is complicated, the operation errors are easy to be confused by operators, and the Lymacin-T anti-CD 3 monoclonal antibody is frequently missed to be subjected to static culture for 2 hours, so that the CTL cell activation kit and the CTL cell activation method which are simple in instruction and operation are required in the market.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a kit for activating CTL cells and an activation method thereof.

In order to achieve the above object, the present invention adopts the following technical solutions:

a kit for CTL cell activation, comprising: the syringe, set up the first liquid package in the syringe, set up in the syringe and lie in the second liquid package on the first liquid package, set up in the syringe and lie in the third liquid package on the second liquid package, set up in the syringe and lie in the fourth liquid package on the third liquid package, set up in the syringe and lie in the fifth liquid package on the fourth liquid package, set up in the syringe the portion of spurting of bottom.

In the kit for activating CTL cells, the first liquid bag, the second liquid bag, the third liquid bag, and the fourth liquid bag are PE rubber membranes.

In the kit for activating CTL cells, the spur portion is a serration ring.

The CTL cell activation kit comprises a first liquid bag containing PBMCs culture solution, a second liquid bag containing 10-15ml of 5ug/ml Lymacin-T anti-CD 3 monoclonal antibody, and a third liquid bag containing 1-2ml of ALyS505N-0 and 60-80ul of 5x10⁵IU/ml IL-2, the fourth pack contained 1-2ml of ALyS505N-0 and 10-20ul of 5x10⁵IU/ml IL-6, the fifth pack contained 1-2ml of ALyS505N-0 and 10-20ul of 5x10⁵IU/ml IL-12。

The kit for activating CTL cells comprises the following PBMCs culture solution: 10ml-20ml ALyS 505N-0;

the kit for activating CTL cells comprises the PBMCs culture solution which further comprises: 30-60ul of 200mM amino acids.

The kit for activating the CTL cells comprises the following amino acids: one or more of L-glutamine, L-arginine, L-aspartic acid or L-phenylalanine.

As a kit for CTL cell activation, 5ug/ml of Lymacin-T anti-CD 3 monoclonal antibody was prepared by adding 50ul of 1mg/ml of Lymacin-T to 10ml of hanks buffer.

The activation method of the kit for CTL cell activation includes the following steps:

step one, treating plasma and collecting leucocytes;

pressing the injector to release the PBMCs culture solution in the first liquid bag, culturing the leucocytes for 2 hours by using the PBMCs culture solution, and washing to obtain cell suspension;

step three, after 3-5ml of autoserum is added into the cell suspension, the injector is continuously pressed, the second liquid bag, the third liquid bag, the fourth liquid bag and the fifth liquid bag are punctured in sequence, the cell suspension is transferred into a CTL culture bottle and placed at 37 ℃ and 5% CO₂CTL cells are harvested after 7-13 days of culture in the environment and frozen.

The invention has the advantages that:

the liquid adding can be completed by only pressing the syringes in sequence and puncturing the liquid bag, and the operation is simple;

the Lymacin-T anti-CD 3 monoclonal antibody does not need to be subjected to static culture in advance, and can be directly subjected to culture after the static culture process is finished in a liquid bag;

the invention can comprehensively solve the problem of misoperation.

Drawings

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

the meaning of the reference symbols in the figures:

1 injector, 2 first liquid bags, 3 second liquid bags, 4 third liquid bags, 5 fourth liquid bags, 6 fifth liquid bags and 7 stabbing parts.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

A kit for CTL cell activation, comprising: the syringe 1, set up the first liquid package 2 in the syringe 1, set up in the syringe 1 and lie in the second liquid package 3 on the first liquid package 2, set up in the syringe 1 and lie in the third liquid package 4 on the second liquid package 3, set up in the syringe 1 and lie in the fourth liquid package 5 on the third liquid package 4, set up in the syringe 1 and lie in the fifth liquid package 6 on the fourth liquid package 5, set up the spur portion 7 of bottom in the syringe 1, as a preferred choice, first liquid package 2, second liquid package 3, third liquid package 4, fourth liquid package 5 are PE rubber membrane, spur portion 7 is the saw ring gear.

The first pack 2 contains PBMCs culture solution, the second pack 3 contains 10-15ml of 5ug/ml Lymacein-T anti-CD 3 monoclonal antibody, and the third pack 4 contains 1-2ml of ALyS505N-0 and 60-80ul of 5x10⁵IU/ml IL-2, the fourth pack 5 contains 1-2ml of ALyS505N-0 and 10-20ul of 5x10⁵IU/ml IL-6, the fifth pack 6 contains 1-2ml of ALyS505N-0 and 10-20ul of 5x10⁵IU/ml IL-12。

PBMCs culture solution includes: 10ml-20ml ALyS 505N-0; preferably, the culture solution of PBMCs further comprises: 30-60ul of 200mM amino acid; the amino acids include: one or more of L-glutamine, L-arginine, L-aspartic acid or L-phenylalanine.

The 5ug/ml Lymacin-T anti-CD 3 monoclonal antibody was prepared by adding 50ul of 1mg/ml Lymacin-T to 10ml hanks buffer.

A method for activating a kit for CTL cell activation, comprising the steps of:

step one, treating plasma and collecting leucocytes;

step two, pressing the injector 1, releasing the PBMCs culture solution of the first liquid bag 2, culturing the leucocytes for 2 hours by using the PBMCs culture solution, and washing to obtain cell suspension;

step three, after 3-5ml of autoserum is added into the cell suspension, the injector 1 is continuously pressed, the second liquid bag 3, the third liquid bag 4, the fourth liquid bag 5 and the fifth liquid bag 6 are punctured in sequence, the cell suspension is transferred into a CTL culture bottle and placed at 37 ℃ and 5% CO₂CTL cells are harvested after 7-13 days of culture in the environment and frozen.

The following experiments verify that the activation effect of the present invention is consistent with the conventional operation:

the method comprises the following steps: processing the plasma and collecting white blood cells;

1. confirming the sealing of the aspiration tube of the plasma bag. The aspiration tube and scissors were wiped with two different clean sheets dedicated to the sterile room soaked in 70% ethanol.

2. The pipette was grasped with forceps, the front end of the tubing was cut open and the flow rate was controlled with forceps to transfer plasma to a 50ml tube. The plasma volume transferred was recorded.

3. The tubes were centrifuged (400g, 4 ℃) for 30 minutes to remove platelets.

4. The supernatant was poured into a new 50ml tube.

5. The tube was placed in a constant temperature water bath (56 ℃ C.), and incubated for 30 minutes.

6. The tube was centrifuged (400g, 4 ℃) for 60 minutes to remove precipitated fibrin.

7. The supernatant (autologous serum) was poured into a new 50ml tube. The patient name was marked on the tube, the date of preparation, and refrigerated (4 ℃).

8. Confirm the aspiration tube seal of the PBMC bag. The PBMC bags were suspended. The aspiration tube and scissors were wiped with two different strips of clean paper specially soaked in 70% ethanol sterile room.

9. Transfer cells to 50ml tubes. The capacity of the transfer is recorded.

10. The number of 50ml tubes required was counted and 15ml of lymphocyte isolate 1077 was added to all tubes.

11. Enough new 50ml tubes were prepared to dilute the sample 2-fold. 25ml hanks buffer was poured into all 50ml tubes prepared. Transfer 25ml of the cell suspension to each tube using a 10ml pipette. 30ml of diluted cell suspension was transferred to each of all tubes of step 11.

12. All tubes (400g, room temperature) were centrifuged for 40 min.

13. And sucking up and discarding the upper liquid until the distance from the liquid surface to the white blood cells at the bottom layer is 1 cm.

14. A new 50ml tube was prepared. The leukocytes were collected cautiously using a 10ml pipette.

Culturing the leucocytes for 2 hours by using PBMCs culture solution, and washing to obtain cell suspension;

1. the leukocytes were collected in every two tubes and transferred to a new 50ml tube.

2. T75 cell culture flasks were prepared. Labeling dates; the syringe 1 is pressed to puncture the first bag 2 and allow culture of PBMCs to be dispensed into each flask.

3. 5ml of the cell suspension was transferred to each T75 cell culture flask.

4. The cover of the culture flask is buckled. The bottle was gently shaken to homogenize the cell suspension. The cells were cultured in an incubator for 2 hours (37 ℃ C., 5% CO 2).

Culturing the cell suspension with an AT culture solution for 7 days, harvesting AT cells AT 37 ℃ and 5% CO 2, and freezing for storage;

1. t225 cell culture flasks were prepared.

2. The syringe 1 was pressed to puncture the second bag 3, and Lymacein-T was added to the T225 cell culture flask and the lid was closed.

Gently shake the T225 cell culture flask back and forth until the liquid covers the entire bottom, and allow to stand at room temperature for 2 hours.

3. After the two hour incubation of the PBMCs was completed, the cells were microscopically secured to the bottom of the flask.

4. Gently shake the T75 flask back and forth 10 times to suspend the non-adherent cells, and transfer the cell suspension to a 50ml tube for use.

5. The cell culture flasks were washed using the following method:

a. sucking up and discarding the solution in the culture flask.

b. To each flask was added 25ml of physiological saline and shaken back and forth.

c. The physiological saline was poured into a sterile container and discarded.

d. B and c were repeated twice.

e. Sucking and cleaning the residual solution in the culture bottle and at the bottle mouth.

7. The cells in the 50ml tube were resuspended with a pipette. On average, the cell suspension was transferred to each cell culture flask.

8. And continuously pressing the injector 1, puncturing the third liquid bag 4, the fourth liquid bag 5 and the fifth liquid bag 6, adding 4ml of autologous serum, mixing and transferring to a culture bottle.

9. The cover of the culture flask is buckled and the mixture is shaken up gently. The flask was placed in an incubator and cultured for 7 days (37 ℃ C., 5% CO 2).

Harvesting of CTL cells

1. The CTL cell culture flasks to be harvested were confirmed.

2. Ensure endotoxin detection as negative.

3. A new 50ml tube was prepared. Preparation of a culture solution A: to the tube was added 45ml of ALyS505N-0 medium (room temperature). 50ul of 2ug/ml ionomycin and 50ul of 200ng/ml PMA were added to the tube.

4. A new 250ml tube was prepared.

5. All CTL culture flasks were examined by microscopic observation. The endotoxin test result was confirmed to be negative again. All the culture broth was poured into a 250ml test tube.

6. Transfer approximately 25ml of broth A to an empty flask, and pour the flask.

7. Centrifuge tube 250ml tube for 5 minutes (1500rpm, room temperature)

8. After centrifugation, the supernatant was discarded into a clean container at room temperature.

9. Transferring 25ml of the culture solution A obtained in the step 6 to a test tube, resuspending the cells, and transferring the cells into a CTL cell culture flask.

10. The CTL cell culture flask was placed in an incubator and cultured for 3 hours (37 ℃, 5% CO 2).

After 11.3 hours, the cells were suspended by transferring the liquid up and down several times using a pipette. Transfer the cell suspension to a new 250ml tube. About 10ml of ALyS505N-0 was added to the flask and the cells were suspended by pipette. Transfer cell suspension to 250ml tubes. The adherent cells at the bottom of the tube were observed with the naked eye and a microscope.

12. Centrifuge tube for 5 minutes (1500rpm, 4 ℃ C.)

13. After centrifugation, the supernatant was aspirated and discarded. Cells were resuspended with a small amount of ALyS 505N-0. Transfer all cell suspensions to one of the tubes. ALyS505N-0 was added up to 50 ml.

14. The number of cells was counted using Trypan Blue stain to calculate the survival rate.

15. Centrifugal pipe 5 minutes (1500rpm, 4 ℃ C.)

16. The supernatant was aspirated and 50ml of ALyS505N-0 was added to suspend the cells.

17. The tube was centrifuged for 5 minutes (1500rpm, 4 ℃) and sampled for FACS detection.

18. Cell cryo-solutions (AIM-V, 20% autologous serum, 10% DMSO) were prepared and buffered on ice.

19. Performing sterility testing

20. Cells were stored temporarily on ice.

21. Add the freezing fluid to the cell tube and mix well with a pipette.

22. And (5) freezing and storing.

Test one: the cell numbers detected in the above two bags of plasma from different patients are represented by plasma A and plasma B, respectively, and are shown in Table 1.

TABLE 1 cell number detection Table

	A plasma cell number	B plasma cell number
			Sample (I)	1.51x10^7/ml	1.48x10^7/ml

As can be seen from the results in Table 1, the number of plasma cells can be the same or even higher with the method of the invention compared to the same reagent formulation with the conventional activation method.

And (2) test II: the results of cell surface CD identification are shown in the plasmA of example N-A and example N-B, respectively, and are shown in Table 2.

TABLE 2 cell surface CD identification results

From the results of table 2, it can be seen that: the effect of the invention on activating T lymphocytes is similar to that of the prior patent CN110229789A, and even has better value.

Compared with the prior art, the liquid adding device can complete liquid adding only by sequentially pressing the syringes 1 and puncturing the liquid bag, and is simple to operate; the Lymacin-T anti-CD 3 monoclonal antibody does not need to be subjected to static culture in advance, and can be directly subjected to culture after the static culture process is finished in a liquid bag; the invention can comprehensively solve the problem of misoperation.

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 using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Claims

1. A kit for CTL cell activation, comprising: the syringe, set up the first liquid package in the syringe, set up in the syringe and lie in the second liquid package on the first liquid package, set up in the syringe and lie in the third liquid package on the second liquid package, set up in the syringe and lie in the fourth liquid package on the third liquid package, set up in the syringe and lie in the fifth liquid package on the fourth liquid package, set up in the syringe the portion of spurting of bottom.

2. A CTL cell activation kit according to claim 1, wherein said first, second, third and fourth liquid bags are PE rubber membranes.

3. A CTL cell activation kit according to claim 1, wherein said spike is a serration.

4. A kit for CTL cell activation according to claim 1, wherein the first liquid bag contains PBMCs culture solution, the second liquid bag contains 10-15ml of 5ug/ml Lymacin-T anti-CD 3 monoclonal antibody, and the third liquid bag contains 1-2ml of ALyS505N-0 and 60-80ul of 5x10⁵IU/ml IL-2, said fourth pack containing 1-2ml of ALyS505N-0 and 10-20ul of 5x10⁵IU/ml IL-6, said fifth pack containing 1-2ml of ALyS505N-0 and 10-20ul of 5x10⁵IU/ml IL-12。

5. A kit for CTL cell activation according to claim 4, wherein the PBMCs culture medium comprises: 10ml to 20ml of ALyS 505N-0.

6. A kit for CTL cell activation according to claim 5, wherein the PBMCs culture medium further comprises: 30-60ul of 200mM amino acids.

7. A kit for CTL cell activation according to claim 6, wherein the amino acids comprise: one or more of L-glutamine, L-arginine, L-aspartic acid or L-phenylalanine.

8. A kit for CTL cell activation according to claim 4, wherein the 5ug/ml Lymacin-T anti-CD 3 monoclonal antibody is prepared by adding 50ul of 1mg/ml Lymacin-T to 10ml hanks buffer.

9. The method for activating a CTL cell-activating kit according to claim 4, comprising the steps of:

step one, treating plasma and collecting leucocytes;

step threeAdding 3-5ml of autologous serum into the cell suspension, continuously pressing the injector, puncturing the second liquid bag, the third liquid bag, the fourth liquid bag and the fifth liquid bag in sequence, transferring the cell suspension into a CTL culture bottle, placing at 37 ℃ and 5% CO₂CTL cells are harvested after 7-13 days of culture in the environment and frozen.