CN118813534B

CN118813534B - A culture system and method and application thereof for preparing cord blood NK cell preparation by removing trophoblast cells and producing floccules

Info

Publication number: CN118813534B
Application number: CN202411312661.8A
Authority: CN
Inventors: 吴姣琦; 田烁
Original assignee: Guangzhou Doublink Biological Products Co
Current assignee: Guangzhou Doublink Biological Products Co
Priority date: 2024-09-20
Filing date: 2024-09-20
Publication date: 2025-02-14
Anticipated expiration: 2044-09-20
Also published as: CN118813534A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a culture system for removing floccules in an umbilical cord blood NK cell preparation prepared by a trophoblast cell removal method, and a method and application thereof. The culture system A comprises a serum-free culture medium, a cell amplification reagent, IL-2 and a serum substitute, wherein the serum substitute does not contain fibrinogen. The culture system and the method thereof provided by the invention can eliminate floccules occurring when the cord blood NK cell preparation is prepared by a trophoblast cell method, improve the stability and the safety of the cell preparation, and solve the clinical use problem. The NK cells prepared by the method have higher amplification factor and purity and stronger biological activity.

Description

Culture system for removing floccules in umbilical blood NK cell preparation prepared by trophoblast cell method, and method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a culture system for removing floccules in an umbilical cord blood NK cell preparation prepared by a trophoblast cell removal method, and a method and application thereof.

Background

In recent years, cell therapy techniques have been rapidly developed, and in particular NK cell immunotherapy has been widely used in clinic. NK cells are effector cells of the natural immune system of a human body, are main functional cells for monitoring malignant pathological cells in vivo, and can directly kill target cells without antigen pre-sensitization, so that the NK cells play an important role in immune response of a human body against tumors, early viruses or intracellular bacterial infection. The proportion of NK cells in peripheral blood mononuclear cells is only 10-20%, and when diseases occur, the NK cells are affected by the conditions of patients, and some patients are not suitable for extracting peripheral blood of the patients for in-vitro expansion of NK cells. At this time, a new NK cell source needs to be selected, and compared with an adult peripheral blood NK cell, the NK cell from the umbilical cord blood source has lower immunogenicity, higher purity, stronger interferon expression capability and stronger bone marrow homing function.

There are several requirements for the in vitro preparation of NK cells, cell expansion number, NK cell purity and NK cell killing. How to obtain sufficient quality effector NK cells is one of the most central difficulties. In vitro culture of primary NK cells is largely classified into a cytokine method and a feeder cell method. The trophoblast method can realize the rapid expansion of NK cells, obtain high-purity high-quality NK cells and have good stability, but after the method is used for preparing the cord blood NK cell preparation, macroscopic floccules are easy to appear in a cell preparation bag in a short time, and the clinical use is influenced.

The reason why the NK cell preparation bag has floc in a short time is not clear, since this phenomenon may involve a variety of complicated factors. Such as (1) cell morphology and behavior, in some cases NK cells may proliferate rapidly and aggregate together during the culture to form a pellet. These clumps may appear visually as flocs, especially when the cell density is high or the culture conditions change. In addition, NK cells may undergo apoptosis or death if they are affected by adverse factors (e.g., temperature fluctuations, malnutrition, contaminants, etc.) during preparation or storage. Dead cells and fragments thereof may form flocs, affecting the purity and quality of the formulation. (2) Culture system factors certain components of the culture system (e.g., fibrinogen and other proteins, polysaccharides, etc. in autologous fire-fighting plasma) may combine with cells or cell debris under certain conditions (e.g., temperature, pH, ionic strength, etc.) to form very strong cross-linked flocs. (3) Solution factors NK cell formulations may also cause floc generation during storage or transport if stability of the solution is impaired (e.g. temperature fluctuation, light, vibration, etc.).

The appearance of floc is related to quality problems of the preparation and may be caused by various factors such as apoptosis, components of the culture system, transportation conditions, etc. Solving the problem can obviously improve the purity, stability and safety of NK cell preparations and ensure the effectiveness and reliability of the NK cell preparations in clinical application.

Disclosure of Invention

In order to solve the problems, the invention provides a method for removing floccules in an umbilical cord blood NK cell preparation prepared by a trophoblast cell method, so that the problem that clinical use is affected by the floccules in the cell preparation is solved.

In one aspect, the invention provides a culture system for NK cells, said culture system comprising a serum replacement.

In particular, the serum replacement does not contain fibrinogen.

Preferably, the serum replacement is added in an amount of 1% -5% of the volume of the rehydration solution, such as 1%, 2%, 4%, 5%, and any point in the range thereof.

Specifically, the culture system further comprises one or more of serum-free culture medium, cell amplification reagent and cytokine.

Further specifically, the serum-free medium may be KBM581, AIM-V, GT-T551H 3 or SCGM.

Further specifically, the cytokines include, but are not limited to, one or more of IL-2, IL-7, IL-12, or IL-15.

Preferably, the cytokine may be IL-2.

Further preferably, the IL-2 may be added in an amount of 100-300IU/mL, such as 100IU/mL, 200IU/mL, 300IU/mL, and any point in the range thereof.

In yet another aspect, the present invention provides a method of NK cell culture, the method comprising the steps of:

(1) Cell inoculation, namely adding mononuclear cells into a culture system A for culture;

(2) Changing the liquid, namely replacing the culture system A in the step (1) with a culture system B, and continuing culturing;

(3) Adding a culture system B into the step (2) for the first time, and continuing culturing;

(4) Adding the culture system A into the step (3) for secondary fluid infusion, and continuing culturing;

(5) Adding a culture system C into the step (4) for the third fluid infusion, and continuing culturing;

(6) Harvesting the cells;

The culture system A comprises a serum-free culture medium, a cell amplification reagent, IL-2 and a serum substitute;

the culture system B comprises a serum-free culture medium, IL-2 and a serum substitute;

the culture system C comprises a serum-free culture medium and IL-2;

The serum replacement does not contain fibrinogen.

Specifically, the conditions for the culture in the steps (1) to (5) may be 2% to 5% CO2,35-37 ℃.

Preferably, the conditions for the culture in steps (1) - (5) may be 4% -5% CO2,36-37 ℃.

Further preferably, the conditions for the culture in the steps (1) to (5) may be 5% CO2,37 ℃.

Specifically, the addition amount of the serum replacement in the culture system A can be 1% -5% v/v, and the addition amount of the IL-2 is 100-300IU/mL.

Further specifically, in some embodiments, the serum replacement addition in culture system A may be 5% v/v and IL-2 addition 200IU/mL;

in other embodiments, the serum replacement addition amount in the culture system A can be 1% v/v, and the IL-2 addition amount is 200IU/mL;

or in still other embodiments, the two adding modes are added successively.

Or in still other embodiments, plasma may be added first and then either way.

Specifically, the addition amount of the serum replacement in the culture system B is 3% -5% v/v, and the addition amount of the IL-2 is 100-300IU/mL.

Further specifically, in some embodiments, the serum replacement addition in culture system A may be 3% v/v and IL-2 addition 200IU/mL;

in other embodiments, the serum replacement addition amount in the culture system A can be 5% v/v, and the IL-2 addition amount is 200IU/mL;

or in other embodiments, the two adding modes can be added successively.

Or in still other embodiments, plasma may be added first and then either way.

Specifically, the addition amount of IL-2 in the culture system C is 100-300IU/mL.

Preferably, the IL-2 addition amount in the culture system C is 200IU/mL.

Further specifically, the serum replacement is Helios UItraGROTM-Advanced serum replacement, purchased from Helios Bioscience company under the trade designation HPCFDCGL.

In yet another aspect, the invention provides the use of the foregoing culture system or method for the removal of trophoblast cells to produce floc in cord blood NK cell preparations.

The invention has the technical effects that:

The invention provides a method for preparing an umbilical cord blood NK cell preparation by a method for removing trophoblast k562 so as to solve the problem that the clinical use is influenced by the floccule appearing in the final cell preparation, and provides a novel use method of serum substitutes, namely the method for preparing the umbilical cord blood NK cell preparation by the method for preparing the trophoblast k562, wherein the serum substitutes do not contain fibrinogen, so that the problem that the clinical use is influenced by the floccule appearing in the umbilical cord blood NK cell preparation cultured by the method for preparing the trophoblast k562 is fundamentally solved, the serum substitutes are used for replacing umbilical cord blood autologous plasma for culturing NK cells by the method for preparing the trophoblast k562, the NK cells grow fast and stably, have high purity and biological activity, and the serum substitutes are clinical grades and can be used for clinical research.

Drawings

FIG. 1 is a microscope photograph, wherein A is example 1, B is comparative example 1;C is comparative example 2;D is comparative example 3;E is comparative example 4.

FIG. 2 is the total number of NK cells prepared in example 1 and comparative examples 1-2.

FIG. 3 shows NK cell expansion ratios prepared in example 1 and comparative examples 1-2.

FIG. 4 shows the apoptosis rate of NK cells prepared in example 1 and comparative examples 1-2.

FIG. 5 is the content of CD3-CD56+ which is a cell surface marker of NK cells prepared in example 1 and comparative examples 1-2.

FIG. 6 shows the biological activities of NK cells prepared in example 1 and comparative examples 1-2.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

The information of the main reagents and materials in the embodiment of the invention is as follows:

NK cell amplification reagent is purchased from Hangzhou winning biomedical technology Co., ltd, and has the product number ZY-NKZ-0104 for stimulating NK cell activation and amplification, wherein the NK cell amplification reagent comprises two specifications, A1 is 1mL, and A2 is 4mL.

NK cell serum-free medium KBM581, available from Corning Inc., cat# 88-581-CMF, was used for NK cell expansion culture.

IL-2 recombinant human interleukin 2, purchased from tetracyclic pharmaceutical company, was added during the whole culture process to stimulate cell expansion.

Helios UItraGROTM-Advanced serum replacement (GMP grade) purchased from HELIOS Bioscience company under the trade designation HPCFDCGL05 at an additive concentration of 1-5% for NK cell expansion to provide nutrients required for cell growth. Hereinafter, serum replacement will be abbreviated.

Annexin V-APC/PI double-dyeing apoptosis detection kit is purchased from Jiangsu Kaiki biological company, with the product number KGA1107.

Annexin V-FITC/PI double-stained apoptosis detection kit is purchased from Jiangsu Kaiki biological company under the product number KGA1102.

Calcium gluconate purchased from Shandong Midu pharmaceutical Co.

Autologous inactivated plasma (frozen) plasma was obtained after centrifugation of whole cord blood, after half an hour of inactivation at 56 ℃,2300g was centrifuged for 15min, and autologous inactivated plasma was harvested for comparison experiments.

Autologous inactivated blood plasma (8% calcium gluconate) is obtained by centrifuging whole blood of cord blood, adding 8% calcium gluconate, mixing, inactivating at 56 deg.C for half an hour, centrifuging for 15min, and collecting autologous inactivated blood plasma for comparison experiment.

Example 1

This example uses serum replacement for cord blood-derived NK cells.

The specific cell culture method generally comprises the following steps:

(1) Day 0 NK cell expansion reagent A1 was resuscitated by transferring 10 mL of the rewrited KBM581 medium into 115 mL centrifuge tubes in advance, rapidly withdrawing NK cell expansion reagent A1 from liquid nitrogen, placing it in a 37 ℃ water bath until complete dissolution (time no more than 3 min), rapidly transferring cells into the 15mL centrifuge tubes after dissolution, 300 g centrifuging 5min, discarding the supernatant, resuspensioning cells with 5mL of the rewrited KBM581 medium, and recording as A1 cell suspension.

(2) Day 0 cell inoculation, namely separating and counting mononuclear cells, taking 3×10 ⁷ cells, re-suspending the cells by using a re-warmed KBM581 medium, adding all the re-suspended 3×10 ⁷ cells and the A1 cell suspension in the step (1) into 1T 75 suspension culture bottles, adding 5% serum replacement of the total volume, adding 200IU/mL KBM581 medium of the final concentration of IL-2 to 30mL of the total volume, uniformly mixing, and culturing in a culture box of 5% CO2 and 37 ℃;

(3) Day 3 centrifugation and liquid exchange, namely placing the cells in a T75 suspension culture bottle into a 50mL centrifuge tube when the inoculated cells are cultured to the 3 rd Day, centrifuging the cells by 300 g for 5min, discarding the supernatant, re-suspending the cells by using a re-warmed KBM581 culture medium, re-seeding the cell suspension into an original T75 suspension culture bottle, supplementing a serum replacement with the total volume of 5% and a KBM581 culture medium with the final concentration of 200IU/mL of IL-2 into the total volume of 30mL, uniformly mixing, and culturing in a culture box with the temperature of 5% CO2 and 37 ℃;

(4) Day4-6, wherein KBM581 culture medium with the final concentration of IL-2 of 200IU/mL is supplemented every Day, the cell concentration is maintained to be 0.8-1.2X10 ⁶ cells/mL, 5% of serum replacement with the liquid supplementing amount is supplemented on days 4 and 5, 3% of serum replacement with the liquid supplementing amount is supplemented on Day 6, the volume exceeds 100mL, and the culture is transferred to a T182 suspension culture flask;

(5) Day 7, transfer 40 mL of rewarmed KBM581 medium into 150 mL centrifuge tubes in advance, rapidly remove NK cell expansion reagent A2 from liquid nitrogen, place it in 37 ℃ water bath until it is completely dissolved (time not exceeding 3 min), rapidly transfer cells into the 50mL centrifuge tubes after dissolution, centrifuge 300 g for 5 min, discard supernatant, resuspend cells with 10mL of rewarmed KBM581 medium, and record as A2 cell suspension. Adding the A2 cell suspension into a culture bottle, supplementing KBM581 culture medium with the final concentration of IL-2 of 200IU/mL, maintaining the cell concentration to be 0.8-1.2X10 ⁶ cells/mL, supplementing 1% of serum replacement of the supplementing amount, and transferring the cell culture bag to a cell culture bag with the volume of more than 400 mL;

(6) Day 8-10 cell expansion, namely adding KBM581 culture medium with the final concentration of IL-2 of 200IU/mL every Day, and maintaining the cell concentration of 0.8-1.2X10 ⁶ cells/mL;

(7) Day 11 cells were harvested by collecting cells on Day 11-13 of culture, this time by collecting cells on Day 11, centrifuging to collect all cells obtained by culture, using 300g, centrifuging for 8min, removing the culture medium, washing with physiological saline for 2 times, finally resuspending with physiological saline to maintain the cell density at 2-3×10 ⁷/mL, taking 5mL of cells, placing in a T25 flask, observing under a microscope, photographing, placing the rest cell suspension in a 150mL cell preparation bag, storing in a 4 ℃ cell preparation bag, and observing at certain intervals.

(8) Sampling and detecting, namely counting 1mL of cell suspension every day from the 5 th day, calculating the total cell number and expansion times, sampling 5mL of the cell suspension at the 7 th, 9 th and 11 th days for NK cell apoptosis rate, and detecting the surface marker CD3-CD56+, and biological activity.

The detection method comprises the following steps:

(a) Fold expansion/total number of cells

When the liquid is replenished every day, 1mL is taken and counted by using a blood separator, and the total cell number of the day is calculated.

Total cell count formula total cell count = cell density x total product count.

Amplification factor formulaTotal number of cells per initial number of cells inoculated on the day.

(B) Apoptosis rate

Apoptosis was detected using the FITC/PI double-stain apoptosis kit. Taking NK cells of 2E6, uniformly placing in 2 1.5mL EP tubes, namely a control tube and an experimental tube, centrifuging 300g for 5min, removing a culture medium, adding 1mL of PBS for washing once, centrifuging 300g for 5min, removing a supernatant, adding 500 mu L of apoptosis kit Buffer and 5 mu L of FITC and 5 mu L of PI into an experimental group, adding 500 mu L of apoptosis kit Buffer into the control group, incubating for 10min at normal temperature and in a dark place, detecting by using a flow cytometer, and recording the double negative proportion of FITC and PI.

Apoptosis rate =-b。

Wherein, a is the proportion of FITC and PI dianion of the control group, and b is the proportion of FITC and PI dianion of the experimental group.

(C) Cell surface marker CD3-CD56+

Flow cytometry was performed to detect the CD3-CD56+ ratio of cultured cells, NK cells of 2E6 were taken and placed uniformly in 2 1.5mL EP tubes, respectively control tube and experimental tube, 300g was centrifuged for 5min, medium was removed, 1mL PBS was added for washing once, 300g was centrifuged for 5min, 100. Mu.L of physiological saline was added to resuspend cells, 5. Mu.L of CD3 flow antibody was added to the experimental group, 5. Mu.L of CD56 flow antibody was added, and CD3 negative CD56 positive ratio was recorded.

(D) Biological Activity assay

NK 562 (GFP) =5:1 incubation for 1h, K562 apoptosis rate was flow tested using APC/PI apoptosis kit to test NK cell killing activity. Spreading to 6-well plates, K562 (GFP) positive control well, K562 (GFP) negative control well, NK cell control well and NK: K562 (GFP) =5:1 well, 37 ℃ and CO ₂% incubation for 1h, collecting cells, centrifuging for 5min at 300g, removing the medium, washing once with 1mL PBS, centrifuging for 5min at 300g, removing the supernatant, adding 500 μl apoptosis kit Buffer and 5 μl APC and 5 μl PI to K562 (GFP) =5:1 tube, adding 500 μl apoptosis kit Buffer to K562 negative control and NK control, incubating for 10min at normal temperature in dark, detecting by using a flow cytometer, and recording the ratio of APC to PI double negative.

Killing Activity =%。

Where a is the ratio of K562 positive control to APC and PI double negative, and b is the ratio of NK: K562 (GFP) =5:1 pipe APC and PI double negative.

Comparative example

Comparative example was set forth with reference to example 1, and the differences between the comparative example and example 1 are shown in table 1, and the other examples are the same as example 1.

TABLE 1

Results and analysis

1. Microscopic examination result

As a result, as shown in FIG. 1, the cell preparation prepared in example 1 had no floc and was left at 4℃for 24 hours without macroscopic floc, whereas comparative examples 1 to 4 had more flocs under the microscope and was left at 4℃for 1 hour with macroscopic small particles and at 2 hours with macroscopic floc. It was shown that clinical grade, i.e., floc-free, was only achieved with Helios UItraGROTM-Advanced fibrinogen-free serum substitutes.

2. Fold expansion/total number of cells

As shown in fig. 2-3, the serum replacement of example 1 produced a cell preparation that was not only free of floc, but also had a higher number of cells to be finally harvested and a higher expansion factor.

3. Apoptosis rate

As shown in FIG. 4, the whole apoptosis of the cell preparation prepared by the serum replacement of example 1 was lower than that of plasma, and the cell viability was higher.

4. Cell surface marker CD3-CD56+

As a result, as shown in FIG. 5, the serum replacement of example 1 produced a cell preparation having a higher purity, and reached 80% or more on day 7.

5. Biological Activity assay

The results are shown in FIG. 6, where the serum replacement of example 1 produced a cell preparation with a higher killing power, reaching 80% on day 7.

In conclusion, the embodiment 1 of the invention can fundamentally solve the problem that the clinical use is affected due to floccules in the preparation of the cord blood NK cell preparation by a trophoblast cell method, and the cord blood NK cells cultured by the method have better effects on the total cell quantity, the expansion times, the apoptosis rate, the NK cell purity and the biological activity. Therefore, the invention provides a new and good method for preparing the clinical cord blood NK cell preparation.

Claims

1. A method for culturing NK cells, characterized in that the method comprises the following steps:

(1) Cell inoculation: Add mononuclear cells into culture system A for culture;

(2) Medium change: replace the culture system A in step (1) with the culture system B and continue culturing;

(3) Rehydration on days 4-6: Add culture system B to step (2) and continue culturing;

(4) Rehydration on the 7th day: add culture system A to step (3) and continue culturing;

(5) Rehydration on days 8-10: Add culture system C to step (4) and continue culturing;

(6) Harvesting cells;

The culture system A comprises serum-free culture medium, cell expansion reagent, IL-2 and serum substitute; the serum substitute in the culture system A is added in an amount of 1%-5% v/v, and the IL-2 is added in an amount of 100-300 IU/mL;

The culture system B comprises serum-free culture medium, IL-2 and serum substitute; the serum substitute added in the culture system B is 3%-5% v/v, and the IL-2 added is 100-300 IU/mL;

The culture system C includes serum-free culture medium and IL-2; the amount of IL-2 added in the culture system C is 200 IU/mL;

The serum substitute does not contain fibrinogen, and the serum substitute is Helios UItraGROTM-Advanced serum substitute;

The rehydration on days 4-6 is as follows: KBM581 medium with a final IL-2 concentration of 200 IU/mL is added every day to maintain the cell concentration at 0.8-1.2×10 ⁶ cells/mL, 5% v/v of the serum replacement volume is added on days 4 and 5, and 3% v/v of the serum replacement volume is added on day 6, the volume exceeds 100 mL, and the cells are transferred to a T182 suspension culture flask;

The 7th day rehydration is as follows: 40 mL of rewarmed KBM581 medium is transferred to a 50 mL centrifuge tube in advance, NK cell expansion reagent A2 is quickly taken out from liquid nitrogen, and placed in a 37°C water bath until completely dissolved. After dissolution, the cells are quickly transferred to the above 50 mL centrifuge tube, centrifuged at 300 g for 5 min, the supernatant is discarded, and the cells are resuspended with 10 mL of rewarmed KBM581 medium, which is recorded as A2 cell suspension; the A2 cell suspension is added to the culture bottle, and KBM581 medium with a final IL-2 concentration of 200 IU/mL is added to maintain the cell concentration at 0.8-1.2× 10 ⁶ cells/mL, and 1% of the serum replacement amount is added to the rehydration volume. The volume exceeds 400 mL and the cell culture bag is transferred;

The fluid replenishment on days 8-10 is as follows: KBM581 culture medium with a final IL-2 concentration of 200 IU/mL is added every day to maintain the cell concentration at 0.8-1.2×10 ⁶ cells/mL.

2. The method according to claim 1, characterized in that the culture conditions in steps (1) to (5) are: 2%-5% CO ₂ , 35-37°C.

3. Use of the method according to any one of claims 1-2 in the preparation of cord blood NK cell preparation by removing trophoblast cells in the presence of floccules.