WO2015014291A1

WO2015014291A1 - Lymph cell amplification and activation method via serum-free cultivation

Info

Publication number: WO2015014291A1
Application number: PCT/CN2014/083368
Authority: WO
Inventors: 邵谊
Original assignee: 北京赛诺泰生物科技有限公司
Priority date: 2013-08-02
Filing date: 2014-07-31
Publication date: 2015-02-05
Also published as: CN103436492A; CN103436492B; HK1192282A1

Abstract

Provided is a lymph cell amplification and activation method via serum-free cultivation. The method comprises: a) the lymph cell activation step, in which a biological sample containing lymph cells is put in contact with a lymph cell activation agent coated on a cultivation container and is cultivated for 3 to 6 days in a serum-free medium; b) a first amplification step, in which the culture obtained from step (a) is added with a serum-free medium having a volume 0.8 to 1.2 times of the culture, and is further cultivated for 1 to 3 days; c) a second amplification step, the culture obtained from step (b) is added with a serum-free medium having a volume 1 to 1.5 times of the culture, and is further cultivated for 1 to 3 days; d) a third amplification step, the culture obtained from step (c) is added with a serum-free medium having a volume 2 to 4 times of the culture, and is further cultivated for 4 to 6 days; e) a fourth amplification step, the culture obtained from step (d) is added with a serum-free medium having a volume 0.5 to 1.5 times of the culture, and is further cultivated for 4 to 6 days; f) the activated and amplified lymph cells from step (e) are obtained.

Description

Method for amplifying activated lymphocytes by serum-free culture

The present invention relates to the field of immunotherapy, and in particular to a method for activating lymphocytes activated by serum-free culture. Background technique

Adoptive cellular immunotherapy is a method of tumor treatment after in vitro stimulation of lymphocytes by stimulator culture. Since the 1980s, the therapeutic effect of cytokine IL-2 on tumors has been discovered. The clinical application of cellular immunotherapy has rapidly expanded. Lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes have been cultured. TIL), cytokine-activated killer cells (CIK). In 2000, Yamazaki T et al. reported in the Lancet a randomized controlled clinical trial of in vitro expansion of autologous lymphocytes to prevent tumor recurrence after liver cancer surgery. The results showed that this non-specific immunotherapy greatly prolonged the survival time of patients with liver cancer, and there was a certain correlation between the amount of transfused cells and the killing performance and efficacy. It is a promising method for tumor treatment.

In the in vitro expansion culture of lymphocytes, different amplification steps, different media, and whether or not serum is added have different effects on the amplification effect, and whether or not serum is added to enhance lymphocyte expansion ability and eliminate the potential for clinical use. Security risks are critical.

There is a need in the art for a method for amplifying activated lymphocytes by serum-free culture that is safe, efficient, and has good product stability and is suitable for large-scale production. Summary of the invention

The present invention provides a method for activating activated lymphocytes by serum-free culture, the method comprising:

(a) a lymphocyte activation step in which a biological sample containing lymphocytes is contacted with a lymphocyte activating agent coated on a culture container and cultured in a serum-free medium for 3-6 days;

(b) a first amplification step, wherein the culture obtained in the step (a) is added to a serum-free medium having a volume of 0.8 to 1.2 times, and further cultured for 1-3 days; (c) a second amplification step, wherein the culture obtained in the step (b) is added to a serum-free medium having a volume of 1-1.5 times, and cultured for one to three days;

(d) a third amplification step, wherein the culture obtained in the step c) is added with a serum-free medium having a volume of 2-4 times, and cultured for 4-6 days;

(e) a fourth amplification step, wherein a culture medium obtained in the step d) is added with a serum-free medium having a volume of 0.5 to 1.5 times, and cultured for 4-6 days;

(f) Harvesting the amplified activated lymphocytes obtained in step (e).

In some embodiments, the lymphocyte activator used in the methods of the invention is an anti-CD3 antibody.

In some embodiments, the same serum-free medium is used in the method of the invention. In some embodiments, the serum-free medium used in the methods of the invention contains cytokines. The cytokines can include IL-2, IL-7, and INF-Y. The concentration of IL-2 in the medium used in the method of the present invention may be 750-1500 U/ml, for example 1000 U/ml _; the concentration of IL-7 may be 1-30 U/ml, for example 20 U/ml; INF- The concentration of γ can be 750-1500 U/ml, for example 1000 U/ml. Preferably, the serum-free medium used in the method of the invention is X-VIV015, TexMACS or IMSF100 medium. More preferably, the serum-free medium used is IMSF100 medium.

Using the method of amplifying activated lymphocytes of the present invention, lymphocytes in the biological sample can be amplified 100 to 1000 times. Preferably, wherein the expanded cells are predominantly CD8+ T cells, for example, CD8+ T cells can be >50% of the activated activated lymphocytes. Preferably, the activity of the expanded activated lymphocytes is maintained for 12 hours. DRAWINGS

Figure 1 shows a flow chart of a method for amplifying activated lymphocytes.

Figure 2 is a graph showing the time course of activated activated lymphocyte viability.

Figure 3 shows a comparison of the amplification performance and statistical analysis of the five media.

Figure 4 shows the percentage of cell phenotype and statistical analysis after amplification of different media. detailed description

The present invention provides a method for efficiently amplifying activated lymphocytes, which is cultured using a serum-free medium, which is safe, efficient, and has good product stability and is suitable for mass production. Optimized culture/amplification

The present invention provides a method of amplifying activated lymphocytes, the method comprising the following steps:

(b) a first amplification step, wherein the culture obtained in the step (a) is added to a serum-free medium having a volume of 0.8-1.2 times, and further cultured for 1-3 days;

(c) a second amplification step, wherein the culture obtained in the step (b) is added to a serum-free medium having a volume of 1-1.5 times, and cultured for one to three days;

(f) Harvesting the amplified activated lymphocytes obtained in step (e).

In the above method of amplifying activated lymphocytes, the number and volume of medium additions and the culture time of each step are optimized. The method of the invention allows amplification of samples of different patient origins using substantially uniform conditions and procedures. For large numbers of samples, amplification can be performed simultaneously to achieve scale production.

In addition, the starting cell raw material used for amplification is generally derived from the peripheral blood of the patient, and patients of different genders, ages, and disease states cause a large difference in cells before culture. After amplification by the method of the present invention, cells of relatively uniform quantity and quality can be obtained, conforming to uniform quality standards, and being advantageous for commercial applications.

As described in Example 1, lymphocytes of 143 patients were amplified using the method of the present invention, and all of them were efficiently amplified, and the amplification factor was about 100 to about 1000 times, and the number of cells and cell phenotype after culture were compared. The coefficient of variation before culture was significantly reduced by 62 to 85% (Table 5), which means that the cell preparation with higher quality can be obtained by the method of the present invention. Lymphocyte

Lymphocytes that can be cultured and expanded using the methods of the invention can be derived from peripheral blood lymphocytes. The lymphocytes to be expanded may also be lymphocytes present in other biological samples, such as epithelial lymphocytes, intratumoral infiltrating lymphocytes, cancerous ascites, or infiltrating lymphocytes in pleural fluid. Due to the simple separation of lymphocytes in peripheral blood, the high survival rate of lymphocytes after isolation, etc. Therefore, it is preferred to use lymphocytes isolated from fresh peripheral blood. Preferably, the "biological sample containing lymphocytes" in the method of the present invention refers to isolated peripheral blood mononuclear cells

(PBMC) o Methods for isolating PBMC from peripheral blood are well known to those skilled in the art. The amount of initial peripheral blood mononuclear cells used in the method of the present invention can be as low as 1 X 10 ⁷ ~ 4 X 10 ⁷ cells, which is equivalent to only about 20 ml of peripheral blood in normal humans, and 1 X 10 can be obtained after amplification. ⁹ ~ 12 X 10 ⁹ lymphocytes mainly composed of killer T cells. A lower initial cell volume avoids a large amount of blood sampling to the patient. Activated lymphocyte

The term "activated lymphocytes" refers to lymphocytes stimulated by specific antigens or non-specific lymphocyte stimulants/molecules which are capable of synthesizing macromolecular substances (RNA, protein, DNA) and cytokines. After activation, lymphocytes undergo cell proliferation and differentiate into progeny effector cells and memory cells of different functions.

The term "lymphocyte activator" refers to a reagent/molecule capable of stimulating lymphocyte activation. For example, an anti-CD3 antibody can be used as a lymphocyte activating agent which stimulates CD3 molecules on the surface of T cells and efficiently activates T lymphocytes through T cell receptors. Lymphocyte mitogens are another class of potent lymphocyte activators. These molecules rapidly activate lymphocytes and do not pass through T lymphocyte receptors.

An example of a suitable lymphocyte activator is an anti-CD3 antibody. In a specific embodiment of the lymphocyte expansion method of the present invention, the lymphocyte activating agent is an anti-CD3 antibody coated (: immobilized:) on the surface of the culture vessel. The use of an immobilized anti-CD3 antibody is preferred because it mainly activates CD8+ T cells having a cell killing function, which is particularly advantageous for increasing the antitumor activity of the expanded lymphocytes. A method of immobilizing an anti-CD3 antibody is known, and for example, immobilization can be carried out as follows.

When the anti-CD3 antibody is immobilized in a culture container, the anti-CD3 antibody can be diluted with a sterile sodium phosphate buffer (PBS) to a concentration of 1 to 50 μg/ml. When the concentration is less than 1 μg/ml, the proliferation of T cells tends to be insufficient. When the proliferation of T cells exceeds 50 μg/ml, the proliferation of T cells is sufficient, but the free anti-CD3 antibody which is not immobilized on the insoluble carrier tends to increase. . In addition, the dilution liquid is not particularly limited as long as it is a physiological solution such as a sterilized sodium phosphate buffer solution, and albumin, agarose or the like may be added as a protective agent.

The diluted anti-CD3 antibody was aseptically plated on the bottom surface of the culture flask. The reaction temperature at the time of fixation is 4 to 40 ° C, more preferably 4 to 25 ° C, and most preferably 4 to 10 ° C. Anti-CD3 antibody at less than 4 ° C The immobilization efficiency tends to decrease, and when it exceeds 40 ° C, the activity of the anti-CD3 antibody tends to decrease. The reaction time at the time of immobilization is from 30 minutes to 24 hours, preferably from 1 hour to 24 hours. At less than 30 minutes, the immobilization amount of the anti-CD3 antibody tends to decrease, and when it exceeds 24 hours, the fixation time is too long, and the production efficiency tends to decrease.

Immediately following the immobilization of the above anti-CD3 antibody, the residual anti-CD3 antibody dilution which was not immobilized was removed. The anti-CD3 antibody which is not fixed is washed with an appropriate washing solution such as sodium phosphate buffer (PBS) and dried. The drying method can be carried out by natural drying in a cold storage at 4-10 ° C, heating drying at 10-40 ° C, lyophilization, etc. after removing the cleaning liquid. In view of the activity and stability of the anti-CD3 antibody, lyophilization is preferred, and natural drying in a cold storage at 4-10 °C is most preferred. As described above, the anti-CD3 antibody is immobilized in a culture container.

Those skilled in the art will appreciate that there is no need to use a lymphocyte activator in the amplification step following lymphocyte activation. For example, optionally, the culture can be transferred to a culture vessel containing no lymphocyte activating agent after the incubation step (a:), (b), W or d) for subsequent incubation. Optimized cytokines

During cell expansion and culture, it is usually necessary to add cytokines to the medium to promote cell proliferation. The cytokine used in the amplification of lymphocytes is optimized in the method of the present invention.

In the present invention, the cytokine uses a combination of IL-2, IL-7 and INF-Y. Among them, IL-2 can amplify CD8+ T cells with high efficiency and specificity, and CD8+ T cells are the most important cellular components of the immune system to exert anti-tumor effects. IL-7 and INF- y additionally promote lymphocyte proliferation, for example, to maintain the viability of memory cells and reduce cell death after cell activation, thereby achieving the best cell expansion and cell activity. In the medium used in the method of the present invention, the concentration of IL-2 may be 250-3000 U/ml, 500-2000 U/ml, 750-1500 U/ml, for example 1000 U/ml; the concentration of IL-7 may be 1-100 U/ml, 5-50 U/ml, 10-30 U/ml, eg 20 U/ml; INF- γ concentration can be 250-3000 U/ml, 500-2000 U/ml, 750-1500 U /ml, for example 1000U/ml. Preferably, the cells expanded using the method of the present invention are mainly CD8+ T cells. For example, the proportion of CD8+ T cells in the activated activated lymphocytes can be >50%. Optimized serum-free medium

Cell culture media commonly used in the art, such as RPMI-1640 or DMEM, must be supplemented with fetal bovine serum or human serum when culturing cells. However, adding serum will cause some problems, such as the introduction of serum. Source pathogen contamination, possible differences between different batches of serum, risk of immune rejection, etc. Some serum-free cell culture systems have been developed in the art. However, these serum-free culture systems cannot achieve high-efficiency amplification during lymphocyte culture, fail to meet clinical requirements for the number and activity of cells for treatment, and the obtained cells lose activity in a short period of time.

The present invention is directed to an existing serum-free cell culture method, which screens a serum-free medium suitable for amplifying lymphocytes, and avoids the above problems in combination with the optimized cytokine combination described above and an optimized culture step. . Serum-free media suitable for use in the methods of the invention include X-VIV015 (Lonza), TexMACS (Miltenyi Biotech) or IMSF100 (LYMMU OTECH) media. The most preferred serum-free medium is IMSF100 medium.

An advantage of the method of the present invention is that the same medium (including the same optimized combination of cytokines) can be used during lymphocyte culture without changing in culture, which simplifies the culture step and facilitates large-scale operation, which is beneficial to Commercial application.

The method of the present invention has a stable cell culture efficiency as compared with other serum-free culture methods in the art, and can achieve or exceed the amplification efficiency of a culture system to which animal serum is added, and the obtained cell activity is stable and has a long expiration date. Using the method of amplifying activated lymphocytes of the present invention, lymphocytes in the biological sample can be amplified at least 10-fold, preferably at least 100-fold, more preferably at least 1000-fold. The viability of the activated lymphocytes expanded using the method of the invention can be maintained for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours. Immunotherapy method and pharmaceutical composition

The invention also provides an immunotherapeutic method comprising administering to an individual an activated lymphocyte obtained by the method of the invention.

In one embodiment of the immunotherapy method of the present invention, the lymphocytes obtained by the above amplification method are autologous lymphocytes, and the immunotherapy method comprises the following steps:

(1) obtaining a biological sample containing lymphocytes from an individual;

(11) amplifying the lymphocytes by the method of the present invention, thereby obtaining amplified activated lymphocytes;

(ill) returning the amplified activated lymphocytes to the individual.

In the immunotherapy method of the present invention, the lymphocytes used are derived from a small amount of peripheral blood of the patient himself. These lymphocytes can be activated and amplified up to 100-1000 times in a short period of time and then returned to the patient. The activated cells obtained by the method of the present invention are mostly CD8+ T lymphocytes (: >50%:). CD8+ T lymphocytes play an important role in killing virus-infected cells and cancer cells. Human T lymphocytes include many specific cells directed against different antigens. These cells are cultured and have a wide range of recognition and killing effects against different tumor antigens/viral antigens. The immunotherapeutic method of the present invention can be used to treat individuals suffering from, for example, tumors, infectious diseases, congenital or acquired immunodeficiency, and infectious diseases after infection and infection-induced tumors.

Tumors that can be treated using the immunotherapeutic methods of the invention include, but are not limited to, liver cancer, lung cancer, cardiac cancer, colon cancer, breast cancer, medulloblastoma, gastric cancer, renal cancer, and malignant melanoma, and individuals with tumors It can be an individual who is receiving radiation therapy and/or chemotherapy.

The invention also provides a pharmaceutical composition for immunotherapy comprising activated lymphocytes obtained by the method of the invention. Various advantages of the present invention

In summary, the method of amplifying activated lymphocytes provided by the present invention has the following advantages over the prior art:

1. Use serum-free medium for culture

The method of the present invention can be carried out using a serum-free medium which avoids pathogenic microbial contamination and other adverse factors affecting cell growth which may result from the use of serum, and also avoids possible safety factors.

2. High cell amplification efficiency, no need to use blood cell single mining machine

The patient's own T lymphocytes can be efficiently activated and amplified in vitro by the method of the present invention, and 1 X 10 ⁹ ~ 12 X 10 ⁹ activation amplification can be obtained from 1 X 10 ⁷ ~ 4 X 10 ⁷ peripheral blood mononuclear cells. Lymphocytes mainly composed of killer T cells, which are efficiently amplified from about 100 to about 1000 times, avoiding the need for traditional immune cell therapy to use a single mining machine to take a large amount of starting peripheral blood mononuclear cells to the patient's overall immune cells. The destructive effect of the system.

3. Cell products have high activity and good stability

After conventional non-specific cell culture, the cell activity is generally maintained at about 2 hours. The method of the present invention produces cell activity and stability, and the cells can be stored for 12 hours without affecting the therapeutic effect.

4. Can achieve standardized, systematic production

The method of the invention has simple operation, uniformity of steps, low risk of error, and is particularly suitable for large-scale System production.

5. Cell products can meet uniform quality standards

After amplification by the method of the present invention, lymphocytes having relatively consistent quantity and quality can be obtained, which conform to uniform quality standards and are advantageous for commercial applications. Example

The invention is further illustrated by the following examples, which are not intended to limit the invention. Example 1 : Activated lymphocyte expansion in 143 patients

(1) Peripheral blood was collected from a total of 143 patients, with different genders, ages, and tumor types. The basic information of patients is shown in Tables 1, 2 and 3.

Table 1: Patient Sex

Table 2: Patient age

Table 3: Patient tumor types

Tumor type

Blood tumor 35

Gastrointestinal tumors 39

Sarcoma 3

Hepatobiliary tumor 21

Respiratory tumors 22

Gynecologic Oncology 12

Breast cancer 4

Urinary system tumor 2

Other 5

Total 143 (2) Isolation of peripheral blood mononuclear cells (PBMC) and inoculation:

Place the collected peripheral blood (18-100ml, depending on the patient:) into a new 250mL centrifuge tube, add an equal volume of saline for injection to the blood sample, mix well and slowly add to the 15ml of each tube that has been dispensed. (US GE, item 17-5442-03) in a 50 mL centrifuge tube, centrifuged at 931 X g for 20 min. Pipette the cell layer between the interfaces into a new 50 mL centrifuge tube, add 1:1 physiological saline solution for injection, mix well, and centrifuge at 600 X g for 10 min. The supernatant was decanted, resuspended in 50 mL of saline, and centrifuged at 335 X g for 10 min. The supernatant was decanted, and 5 mL of the injection was resuspended in physiological saline, and the cells were counted by mixing the ΙΟμΙ^ cell suspension. Then, the physiological saline for injection was supplemented to 50 mL, and centrifuged at 223 X g for 10 minutes. The supernatant was decanted, resuspended in 50 mL of serum-free cell medium IMSFIOO, and inoculated into a T225 flask coated with anti-human CD3 antibody, and placed in a cell culture incubator for initiation.

(3) Add serum-free cell culture medium for the first time:

After inoculation for 3 days, the cell culture flask was taken out from the incubator, and the serum-free cell culture medium IMSFIOO (containing IL-2 1000 U/ml, IL-7 20 U/ml, INF- y lOOOU/ml) was poured into 50 mL. The cell culture flask is returned to the cell culture incubator for cultivation. The first addition of the step can be carried out for a period of not more than three days, for example, at the latest on the sixth day after the inoculation.

(4) Add serum-free cell culture medium for the second time:

After the first addition, culture for another day, remove the cell culture flask from the incubator, and measure the serum-free cell culture medium IMSFIOO (containing IL-2 1000 U/ml, IL-7 20 U/ml, INF- y lOOOU/ml). 140 mL was poured into a cell culture flask and returned to the cell culture incubator for cultivation. This second addition step can be postponed no more than two days, for example at the latest on the third day after the first addition.

(5) Add the serum-free cell culture medium for the third time and transfer to the cell culture bag:

After the second addition, the cells were cultured for another day, and the cell culture flask was taken out from the incubator, and the wall was tapped to completely detach the cells from the bottom of the bottle, and the cells were counted. For samples with a cell density of not less than IX 10 ⁶ / mL (inclusive), do the following: Cell suspension in culture flask and 750 mL serum-free cell culture medium IMSFIOO (with IL-2 1000 U/ml) through a 60 mL syringe sleeve , IL-7 20U/ml, INF- y lOOOU/ml) All were poured into a new cell culture bag, the tube was closed, and samples were taken for sterility inspection. The cell culture bag is returned to the incubator for cultivation. The third addition step can be postponed no more than two days, for example at the latest on the third day after the second addition.

(6) The fourth addition of serum-free cell culture medium and divided into two bags: After the third addition, culture for another 4 days, take a new cell culture bag to the biosafety cabinet, and pour 1000 mL or 1250 mL serum-free cell culture medium IMSF100 (containing IL-2 1000 U/ml, IL- through a 60 mL syringe sleeve). 7 20U/ml, INF- y lOOOU/ml) (Bag A), closed nozzle. The cell culture bag (bag B) containing the cells in the culture is taken out from the incubator, and the two bags are connected by a sterile bonding machine. First, the liquid in the bag A is completely flowed into the bag B, and then mixed, and then returned to the bag A, 1000 mL. The amount of liquid in both bags was 1000 mL. The tube is sealed and samples are taken for sterility inspection. Two bags of cells were returned to the incubator for culture. The fourth addition step can be postponed no more than two days, for example at the latest on the sixth day after the third addition.

(7) Purification and concentration of collected cells:

After the fourth addition, culture for another 4 days, take a bag of cells from the incubator to the biosafety cabinet, divide the cell suspension into four 250 mL centrifuge tubes, and centrifuge at 931 X g for 5 minutes. Pour off the supernatant and shake off the sediment. Another bag of cells from the same batch was removed from the incubator, and the cell suspension was equally divided into four identical 250 mL centrifuge tubes and centrifuged at 931 X g for 5 minutes. Pour off the supernatant and shake off the pellet. Four tubes of cells were combined into two tubes, and the physiological saline for injection was separately replenished to 250 mL, and centrifuged at 931 Xg for 5 minutes. The supernatant was decanted and the pellet was shaken off. Then, the cells in the two tubes were washed three times with 200 mL of physiological saline for injection, combined into one tube, and mixed and sampled for cell counting. After centrifugation at 931 X g for 5 minutes, the supernatant was decanted and the cells were resuspended in saline with injection containing 1% human albumin. The cell suspension is then transferred to a disposable plastic transfer bag to form a cell preparation. The harvesting step can be postponed no more than two days, for example at the latest on the sixth day after the fourth addition.

The amplification results are listed in Table 4 below and summarized in Table 5.

IICC80/M0ZN3/X3d I6Z 0/SI0Z OAV

89CC80/M0ZN3/X3d I6Z 0/SI0Z OAV

CI

9CC80/M0ZN3/X3d I6Z 0/SI0Z OAV

:, to

Table 5: Comparison of cell differences before and after culture

As can be seen from the above results, lymphocytes of all 143 patients obtained high-efficiency amplification using the method of the present invention. Moreover, the number of cells and the cell phenotype after culture were significantly lower than those before culture, and the decrease was 62-85%, that is, the cell product with more consistent quality could be obtained by the method of the present invention. Example 2: Stability analysis of amplified activated lymphocytes

The activated lymphocyte products amplified by the three batches according to the method of the present invention are divided into two parallel groups, one group is stored at 2-8 ° C, and one group is stored at room temperature (15-25 ° C), every 4 The cell activity was measured at half hour (Trypan blue staining and viable cell count under the microscope:), and the cell activity time curve was plotted. The results are shown in Table 6 below and Figure 2.

It can be seen from the above that the activated lymphocytes expanded according to the method of the present invention are stored under two temperature conditions, and there is no significant difference in cell activity changes (p>0.05), and can be maintained at more than 85% within 12 hours, which is consistent with the return of the human body. Requirements.

Table 6

Group A samples were stored at 2-8 ° C, and Group B samples were stored at room temperature. "Al, 2, 3" represent three batches of cell products, respectively. Common cell cultures reported by (GIBCO), TexMACS (Miltenyi Biotech) and IMSF100 (LYMMU OTECH), and literature (Hoyle, C., et al., Blood, 1998. 92(9): p. 3318-27.) Base RPMI-1640 (GIBCO) performance parameters in the culture method of the present invention.

Peripheral blood from 3 normal individuals was collected and mononuclear cells were isolated. The cells were divided into five groups (RPMI-1640 containing 10% fetal bovine serum, X-VIV015, TexMACS, AIM-V and IMSF100), and the same number of cells were inoculated, and culture was carried out using the culture step of the present invention.

1. Separation of peripheral blood mononuclear cells

Peripheral blood (120 ml/person) of 3 normal humans was collected, and PBMC of peripheral blood mononuclear cells was obtained by density gradient centrifugation (Ficoll method) under sterile conditions:). Each person's cells were divided into 5 equal parts, using 50 ml of five mediums (both cytokine IL-2 1000 U/ml, IL-7 20 U/ml, INF- y 1000 U/ml, see table below) 7) After resuspending, inoculate the LC-AC T225 cell culture flask coated with CD3+ antibody, and the number of cells inoculated is 1.2 X 10 ⁷ /bottle, and the number of cells inoculated in each group is the same.

Table 7

2. Amplification of activated lymphocytes

15 cell culture flasks were placed in a cell culture incubator (37 ° C, 7.5% C0 ₂ ).

After 72 hours of culture, the flask was taken out, and the growth of the cells was observed under a microscope. Each cell culture flask was supplemented with 50 ml of the corresponding component medium.

After 96 hours of culture, the flask was taken out, and the growth of the cells was observed under a microscope. Each cell culture flask was supplemented with 140 ml of the corresponding component medium.

After 6 days of culture, the flask was taken out and the growth of the cells was observed under a microscope. If the cell density is consistent with Request (0.9 X 10 ⁶ -1.4 X 10 ⁶ /ml) for bagging culture.

After 10 days of culture, the color of the culture medium and the state of the cells were observed, and the bag culture was carried out.

After 14 days of culture, the color and cell state of the culture medium in the bag were observed. Each cell of each sample was harvested separately, and appropriate cell samples were taken for cell counting and activity detection and cell phenotyping.

3. Biological behavior of activated activated lymphocytes

The cells were counted using a cell counting plate to determine the amplification ratio. Amplified cell viability was obtained by trypan blue staining and cell counting. Phenotypic assays of the expanded cells were determined by flow cytometry using FITC-labeled CD3 antibody, PerCP-Cy5.5-labeled CD4 antibody, and APC-labeled CD8 antibody.

4. Statistical methods

Data comparison between the five groups was performed by t test, and data comparison between the two groups was analyzed by two way ANOVA. Result:

The cell count test results are shown in Table 8 below. Among them, the serum-free medium AIM-V group gradually died after 5-6 days of culture. Thus, it can be seen that AIM-V medium cannot be used in the cell culture system of the present invention. Table 8: Cell count test results

The results of cell expansion analysis are shown in Figure 3. As can be seen from Figure 3, after 14 days of culture, no The cell expansion ability of the serum cell culture medium IMSF100 group was significantly better than the other groups, ρθ.001 or p<0.05, both of which were statistically significant.

Table 9 below shows the results of cell viability assay. After 14 days of culture, there was no significant difference in cell viability. Table 9: Cell viability test results

The comparison results of cell phenotypes before and after culture are shown in Table 10. Prior to culture, the same cells were used in different media, with the same percentage of phenotype. The percentage of cell phenotypes of different media after culture is shown in Figure 4, where the table shows the P value for significant differences between different media. The percentage of CD3+ cells in IMSF100 cultured cells was significantly different from that of RPMI1640 and TexMACS. Table 10: Comparison of cell phenotypes before and after culture

Before cultivation, after cultivation

Numbering

CD3+ CD3+ CD8+ CD3+ CD4+ CD3+ CD3+ CD8+ CD3+ CD4+

A1 65.7 29.3 31.09 94.91 74.40 14.03

A2 41.39 10.4 28.35 95.08 77.82 15.38

A3 53.29 32.88 18.81 95.83 79.23 8.45

B1 65.7 29.3 31.09 98.59 92.26 2.95

B2 41.39 10.4 28.35 98.58 90.63 2.12

B3 53.29 32.88 18.81 98.12 93.23 3.86

D1 65.7 29.3 31.09 96.82 89.40 0.98

D2 41.39 10.4 28.35 98.44 92.99 1.95

D3 53.29 32.88 18.81 97.86 83.78 9.16

E1 65.7 29.3 31.09 98.41 93.48 2.34

E2 41.39 10.4 28.35 99.76 92.08 6.09

E3 53.29 32.88 18.81 98.58 90.63 2.12 Conclusion. - As can be seen from the above test results, serum-free cell culture medium in four serum-free medium

AIM-V cannot be used to amplify activated lymphocyte culture, and IMSF100 has the best effect on amplifying activated lymphocyte culture.

Claims

Claim

A method for amplifying activated lymphocytes by serum-free culture, the method comprising: a) a lymphocyte activation step, wherein a biological sample containing lymphocytes and a lymphocyte activator coated on a culture vessel are used Contact and culture in serum-free medium for 3-6 days;

b) a first amplification step, wherein the culture obtained in the step (a) is added to a serum-free medium having a volume of 0.8-1.2 times, and further cultured for 1-3 days;

c) a second amplification step, wherein the culture obtained in the step (b) is added to a serum-free medium having a volume of 1-1.5 times, and further cultured for 1-3 days;

d) a third amplification step, wherein the culture obtained in the step c) is added with a serum-free medium having a volume of 2-4 times, and cultured for 4-6 days;

e) a fourth amplification step, wherein the culture obtained in the step d) is added with a serum-free medium having a volume of 0.5 to 1.5 times, and further cultured for 4-6 days;

f) Harvesting the amplified activated lymphocytes obtained in step (e).

2. The method of claim 1, wherein the lymphocyte activator is an anti-CD3 antibody.

3. The method of claim 1 or 2, wherein the same serum-free medium is used in steps (a)-(e).

4. The method of any of claims 1-3, wherein the serum-free medium further comprises a cytokine.

5. The method of claim 4, wherein the cytokine comprises IL-2, IL-7 and INF-y.

6. The method of claim 5, wherein the concentration of IL-2 in said serum-free medium is 750-1500 U/ml, preferably 1000 U/ml.

7. The method of claim 5, wherein the concentration of IL-7 in said serum-free medium is 10-30 U/ml, preferably 20 U/ml.

8. The method of claim 5, wherein the concentration of INF-Y in the serum-free medium is 750-1500 U/ml, preferably 1000 U/ml.

9. The method of any of claims 1-8, wherein the serum-free medium is selected from the group consisting of X-VIV015, TexMACS and IMSF100 medium.

10. The method of any of claims 1-9, wherein the lymphocytes are amplified 100-1000 fold.

11. The method of any of claims 1-9, wherein the amplified lymphocytes are predominantly CD8+ T cells.

12. The method of any of claims 1-9, wherein the activity of the amplified lymphocytes obtained is maintained for at least 12 hours.