WO2021223274A1 - 免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立 - Google Patents

免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立 Download PDF

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WO2021223274A1
WO2021223274A1 PCT/CN2020/092287 CN2020092287W WO2021223274A1 WO 2021223274 A1 WO2021223274 A1 WO 2021223274A1 CN 2020092287 W CN2020092287 W CN 2020092287W WO 2021223274 A1 WO2021223274 A1 WO 2021223274A1
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immune cells
cells
medium
special
immune
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PCT/CN2020/092287
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French (fr)
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张炳强
陈梦梦
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青岛瑞思德生物科技有限公司
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Priority to EP20934683.2A priority Critical patent/EP4148123A4/en
Priority to KR1020227004434A priority patent/KR20230008691A/ko
Priority to US17/634,556 priority patent/US20230051425A1/en
Priority to JP2022507332A priority patent/JP7332787B2/ja
Publication of WO2021223274A1 publication Critical patent/WO2021223274A1/zh

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Definitions

  • the present invention relates to the field of biotechnology, in particular to methods for culturing, inducing, activating, and freezing immune cells in vitro and methods for establishing cell banks.
  • Immune cells currently used clinically include DC-CIK cells, TIL cells, LAK cells and NK cells. Among them, CIK cells, LAK cells and NK cells are all anti-cancer systems with natural killer cells as the main body.
  • NK cells also known as natural killer cells, patrol the body while searching for and attacking cancer cells and virus-infected cells. They play an important role in the body's natural immunity. NK cells account for 10-30% of lymphocytes in the blood and contain cytotoxic factors such as perforin and granzyme. NK cells can release perforin and granzyme. Perforin perforates on the surface of target cells, allowing granzyme b to enter target cells to induce target cell apoptosis.
  • NK cells secrete a large number of cytokines at the same time, such as ifn-v, tnf-x, gm-csf, il-3, m-csf, etc., which directly act on target cells or attack target cells by further activating other types of immune cells.
  • NK cells play an important role in tumor immunity and elimination of non-self cells: it is the main component of natural immune defense and is the first line of defense in the body's defense system.
  • the killing activity of NK cells is not restricted by MHC, does not rely on antibodies, and does not require antigens.
  • Pre-sensitization can identify and kill tumors and virus-infected cells, and directly exert cytotoxic effects on tumor cells through the perforin-granzyme pathway and Fas-FasL pathway to kill tumor cells; at the same time, it can secrete many kinds of cells in the early stage of onset Cytokines and chemokines such as TNF- ⁇ , IFN- ⁇ and IL-1, etc., these cytokines participate in anti-cancer and modulate the adaptive immune response, so NK cells are also a bridge connecting natural immunity and acquired immunity.
  • NK cells have high activity, which can kill any cancer cells. So far, there are almost no cancer cells that can be killed by highly active NK cells.
  • NK cells are innate cells that can be destroyed Any cancer cell is the mainstay of immune cell therapy.
  • NK cells Although the safety and efficacy of the anti-cancer effect of NK cells have been affirmed, because it only accounts for 10%-30% of peripheral blood lymphocytes, how to obtain high-purity and high-quality NK cell products is the key to NK treatment.
  • cytokines such as IL-2, IL-15, IL-18 and IL-7 play an important role in the in vitro expansion of NK cells.
  • the effect, the multiples of their expansion of NK cells in vitro range from several to several tens of times.
  • IL-2 is an important cytokine that induces the proliferation of NK cells.
  • IL-15 and IL-7 can activate NK cells, promote NK cell proliferation and cytokine production.
  • the role of IL-15 and IL-7 is similar to that of IL-2.
  • they can also promote the directional differentiation of hematopoietic stem cells into NK cells by binding to the complex receptor ⁇ chain expressed on the surface of NK cells, and the development and differentiation of NK cells. And maintain long-term in vitro survival and other aspects to play an important role.
  • the synergistic effect of IL-15 and IL-2 also enables the two to work together for the in vitro expansion of NK cells, which is currently the most traditional combination of cytokines for the in vitro expansion of NK cells.
  • IL-18 can not only induce the secretion of activated THi cells to produce a large amount of IFN- ⁇ , but also enhance the cytotoxicity of NK cells by promoting the opening of the Fas-FasL pathway in a dose-dependent manner.
  • NK cell therapy products there are NK cell therapy products on the market, but the culture system used is complicated, the amplification factor and the tumor killing effect are not good, the recovery effect after cryopreservation is poor, and some culture systems have problems such as safety risks. Therefore, the methods of culturing, inducing, activating, and cryopreserving natural killer cells need to be improved.
  • the present invention aims to solve the technical problems existing in the prior art.
  • the present invention proposes a method for culturing, inducing, activating, and freezing immune cells in vitro, and a method for establishing a cell bank.
  • the method has high efficiency, high speed, high safety and low cost for culturing, inducing and activating immune cells, and A large number of functionally activated immune cells obtained by induction and expansion can establish a cell bank for long-term preservation, and maintain good cell viability after resuscitation.
  • the invention provides methods for culturing, inducing, activating and freezing immune cells in vitro and methods for establishing cell banks. According to an embodiment of the present invention, the method includes:
  • the special expansion medium for immune cells is supplemented with 500-2000IU/ml IL-2, 500-2000IU/ml IL-10, 0.5-1ng/ml IL-1 ⁇ and 1-4ng/ml LIF, 0.5-2.5ng /ml EPO, 1-4ng/ml KGF, 2-5ng/ml testosterone, 1-4 ⁇ g/ml parathyroid hormone, 1-4 ⁇ g/ml laminin serum-free lymphocyte medium.
  • the special induction medium for immune cells is supplemented with 500-2000IU/ml IL-2, 500-2000IU/ml IL-10, 1-4ng/ml bFGF, 1-4ng/ml BMP-4, 0.2-0.8 ⁇ g/ ml rapamycin, 0.2-0.8 ⁇ g/ml icariin, 20-80ng/ml trametinib, 1-4ng/ml hydrocortisone, 1-4 ⁇ g/ml laminin serum-free lymphocytes Medium.
  • the special activation medium for immune cells is supplemented with 500-2000IU/ml IL-2, 500-2000IU/ml IL-10, 1-4ng/ml TGF- ⁇ , 1-2ng/ml Forskolin, 20-80ng/ml Serum-free lymphocyte culture medium of resveratrol, 1-4 ⁇ g/ml acetaminophen, 1-4 ⁇ g/ml laminin.
  • the cryopreservation solution for immune cells is a serum-free lymphocyte culture medium containing 5% by volume of DMSO, 1-2% by volume of albumin, 1-2% by volume of aminoethanol, and 91-93% by volume.
  • the serum-free lymphocyte medium is X-VOVO15 serum-free medium or Other types of serum-free media are commercially available.
  • the immune cells are natural killer cells.
  • a large number of functionally activated immune cells can be obtained by using the invention to induce and expand mononuclear cells.
  • the invention has the advantages of high induction efficiency, fast amplification speed, high safety and low cost.
  • the present invention establishes a corresponding cell bank, categorizes large-scale immune cell storage, and has a long effective storage time. After resuscitation, the cells still maintain good cell viability and the cell recovery rate is high, thereby meeting the demand for a large number of immune cells in clinical treatment.
  • Figure 1 is a flow chart of the method of the present invention.
  • Figure 2 shows the expansion efficiency and cell viability of peripheral blood mononuclear cells within 14 days of the method of the present invention.
  • Figure 3 shows the amplification efficiency of target cells within 14 days of the method of the present invention.
  • Figure 4 shows the expansion efficiency and cell viability of peripheral blood mononuclear cells within 12 days of the method of the present invention.
  • Figure 5 shows the expansion efficiency of the method of the present invention on target cells within 12 days.
  • the present invention provides a method for in vitro culture, induction, activation, cryopreservation of immune cells and a method for establishing a cell bank, which specifically includes: using a special expansion medium for immune cells to carry out the first-stage expansion culture of mononuclear cells to obtain Preliminarily expanded immune cells; use the special induction medium for immune cells to conduct the second stage induction and expansion culture of the initially expanded immune cells to obtain induced immune cells; use the special activation medium for immune cells to carry out the induced immune cells
  • the third stage is to activate and expand the culture to obtain a large number of activated immune cells; use the special freezing liquid for immune cells to freeze the immune cells to obtain the frozen immune cells; save according to ABO/RH classification and HLA classification, Establish a searchable immune cell information archive and construct an immune cell bank.
  • the special expansion medium for immune cells is supplemented with 500-2000IU/ml IL-2, 500-2000IU/ml IL-10, 0.5-1ng/ml IL-1 ⁇ and 1-4ng/ml LIF , 0.5-2.5ng/ml EPO, 1-4ng/ml KGF, 2-5ng/ml testosterone, 1-4 ⁇ g/ml parathyroid hormone, 1-4 ⁇ g/ml laminin serum-free lymphocyte culture medium.
  • the special induction medium for immune cells is supplemented with 500-2000IU/ml IL-2, 500-2000IU/ml IL-10, 1-4ng/ml bFGF, 1-4ng/ml BMP-4, 0.2-0.8 ⁇ g/ml rapamycin, 0.2-0.8 ⁇ g/ml icariin, 20-80ng/ml trametinib, 1-4ng/ml hydrocortisone, 1-4 ⁇ g/ml laminin Serum-free lymphocyte medium.
  • the special activation medium for immune cells is supplemented with 500-2000IU/ml IL-2, 500-2000IU/ml IL-10, 1-4ng/ml TGF- ⁇ , 1-2ng/ml Forskolin, Serum-free lymphocyte culture medium of 20-80ng/ml resveratrol, 1-4 ⁇ g/ml acetaminophen, 1-4 ⁇ g/ml laminin.
  • the immune cells are natural killer cells.
  • the first stage of the immune cell expansion medium process passage once every 2-3 days and pass 2 times; in the second induction and expansion culture process, passage once every 2-3 days, passaging 2 times; in the third stage of large-scale activation and expansion culture process, passage once every 2 days, passage at least 2 times (multiple passages are possible). Therefore, immune cells can achieve high-purity large-scale expansion in a short time, and sufficient functionally activated immune cells can be obtained for possible clinical immunotherapy.
  • the special freezing solution for immune cells is a serum-free lymphocyte culture medium containing 5% by volume of DMSO, 1-2% by volume of albumin, 1-2% by volume of aminoethanol, and 91-93% by volume. .
  • DMSO dimethyl methoxysulfoxide
  • albumin 1-2% by volume of albumin
  • aminoethanol aminoethanol
  • 91-93% by volume 91-93% by volume.
  • the concentration of cryopreserved cells is high, which is suitable for cryopreservation of large-scale immune cells, and the cost of cryopreservation is low, and the effect of cell freezing is good, and the cell viability and cell yield after resuscitation are high.
  • the activated and expanded immune cells are stored according to ABO/RH classification and HLA classification, and immune cell information archives for retrieval are established to construct an immune cell bank.
  • the isolated mononuclear cells are induced, activated and expanded into target immune cells (NK cells), and the activity of the target immune cells is detected.
  • NK cells target immune cells
  • the hospital is required to sign an informed consent form with the donor, in triplicate.
  • One copy each for the donor and the medical institution, and the other copy is sent to the laboratory along with the specimen.
  • the hospital inquires about the donor's personal information, past treatment history, family genetic history, and whether there is a history of infectious diseases and abnormalities of the hematopoietic or immune system by means of inquiries and forms.
  • the hospital is required to obtain the consent of the donor or its authorized personnel, to consult its physical examination data, and obtain the physical examination information.
  • the donor’s physical examination information should include the following items: HIV-1/2 antibody (AIDS antibody), HBsAg (hepatitis B surface antigen), anti-HCV (hepatitis C antibody), CMV-IgM antibody (cytomegalovirus antibody), ALT ( Transaminase), Treponema pallidum antibody.
  • the testing methods are implemented respectively according to the current testing standards of the Ministry of Health.
  • the donor's physical examination should at least meet the "Blood Donor Health Examination Requirements.”
  • the donor s informed consent form, personal information collection form, inspection information, etc. shall be numbered and sealed. Any person who has access to the data shall not disclose the privacy of the donor without the consent of the donor or its authorized personnel.
  • a sterile blood collection bag with anticoagulant to collect about 100ml of human peripheral blood. Reserve 1ml of peripheral blood for quick examination and blood group identification.
  • the blood collection bag is immediately aseptically packaged, stored and transported aseptically at 4°C, and collected information is accurately recorded.
  • the peripheral blood will be sent to the GMP laboratory after the quick screening is qualified. If the quick screening is unqualified, the blood sample will be discarded.
  • step 2 Resuspend the blood cell pellet in step 2 with an equal volume of normal saline of plasma and transfer it to a 250ml sterile glass bottle, add 1/3 of the total volume of blood hydroxyethyl starch, gently shake the saline bottle to make the mixture uniform. Set aside to allow the red blood cells to settle.
  • the taken out 10ml cell suspension was centrifuged at 1800 rpm to recover the cells and perform flow cytometric antibody labeling.
  • Nude mice tumorigenicity test SPF female BALB/c nude mice, 4-6 weeks old, weighing 18-20g, raised in a rat cage with a lid in an air laminar flow rack, drinking water, standard feed and other contact with animals All products are sterilized.
  • 1.1 Cells can be expanded 220 times after 14 days of culture expansion
  • peripheral blood mononuclear cells separated from the peripheral blood lymphocyte separation solution were inoculated into a 20 ml culture system, and the cells quickly entered the logarithmic growth stage. After 14 days of culture and expansion, the culture system was expanded to 4L, the cell number was expanded to 1.3 ⁇ 10 10 , the expansion factor was up to 220 times, and the number of viable cells was more than 95%. The results of cell viability are shown in Figure 2.
  • NK cells in lymphocytes (CD3 - CD56 + ) increased from 18.15% to 97.64%, while the proportion of T lymphocytes (CD3 + ) dropped from 71.47% to 3.22%, both helper T cells (Th, CD3 + CD4 + ) and cytotoxic T cells (Tc, CD3 + CD8a + ) were reduced, B lymphocytes (CD3 - CD19 + ) basically disappeared, and cell homogeneity was significantly improved;
  • NK cells expanded by 1165 times after 14 days of culture and the ratio of lymphocytes in peripheral blood mononuclear cells before and after 14 days of culture is shown in Figure 3.
  • NK cells CD3 - CD56 + ; T cells: CD3 + ; helper T cells (Th): CD3 + CD4 + ; cytotoxic T cells (Tc cells): CD3 + CD8a + ; B cells: CD3 - CD9 + .
  • peripheral blood mononuclear cells separated from the peripheral blood lymphocyte separation solution were inoculated into a 20 ml culture system, and the cells quickly entered the logarithmic growth stage. After culturing for 12 days, the number of cells expanded to 9.7 ⁇ 10 9 , the expansion fold reached 162 times, and the number of viable cells was more than 95%.
  • the experimental results are shown in Figure 4 for details.
  • lymphocytes After 12 days of expansion of peripheral blood mononuclear cells, the proportion of NK cells in lymphocytes (CD3 - CD56 + ) increased from 21.47% on day 6 to 93.22% on day 12. At the same time, T lymphocytes (CD3 + ) Dropped to 3.86%, helper T cells (Th, CD3 + CD4 + ) and cytotoxic T cells (Tc, CD3 + CD8a + ) were reduced, B lymphocytes (CD3 - CD19 + ) basically disappeared, and the cells were uniform The performance is significantly improved. The ratio of lymphocytes in peripheral blood mononuclear cells before and after 12 days of culture is shown in Figure 5.
  • the cultured cells all secreted IFN- ⁇ , TNF- ⁇ , and Perforin. The results are shown in Table 1.
  • Nude mice tumorigenicity experiment results A group of physiological saline control group (the number of tumor-forming mice/the number of mice in the group, 0/5), the group B Raji cell control group (3/5), the C group K562 cell control group ( 4/5), the NK cell group in group D (0/5), that is, the mice in the 0.2ml saline group and 3 ⁇ 10 7 cells/0.2ml cultured for 28 days in the NK cell group during the 2 months observation period were both There was no tumor formation. 4/5 and 5/5 mice in group B of mice injected with the same amount of Raji cells and K562 cells formed visible tumors, respectively. This result shows that even if it is cultured for 28 days, NK cells are still safe and effective and will not cause tumor formation.
  • Example 1 The NK cells induced and expanded in vitro in Example 1 were cryopreserved, and the effects of cryopreservation were compared.
  • the NK cells obtained on the 14th day of in vitro induction and expansion in Example 1 were cryopreserved in the following four types of cryopreservation solutions, and the components of the four types of cryopreservation solutions were as follows:
  • Cryopreservation solution 1 5% by volume of DMSO, 1% by volume of albumin, 1% by volume of aminoethanol, 93% by volume of X-VOVO15 serum-free medium;
  • Cryopreservation solution 2 5% by volume of DMSO, 2% by volume of albumin, 2% by volume of aminoethanol, 91% by volume of X-VOVO15 serum-free medium;
  • Cryopreservation solution 3 5 vol% DMSO, 2 vol% albumin, 93 vol% X-VOVO15 serum-free medium;
  • Cryopreservation solution 4 5% by volume of DMSO, 2% by volume of aminoethanol, 93% by volume of X-VOVO15 serum-free medium;
  • Cryopreservation Solution 5 5% by volume of DMSO, 95% by volume of X-VOVO15 serum-free medium;
  • Cryopreservation solution 6 5% by volume of DMSO, 10% by volume of autologous plasma, 85% by volume of X-VOVO15 serum-free medium;
  • Cryopreservation solution 7 10% by volume of DMSO, 2% by volume of albumin, 2% by volume of aminoethanol, 86% by volume of X-VOVO15 serum-free medium.
  • Freeze NK cells according to the following steps: After mixing the cryopreservation solution and the cells, quickly transfer them into the cryopreservation tube, and put them in the cryopreservation box, cool down overnight by program at -70°C, and transfer to liquid nitrogen the next day. Wherein each 107 NK cells using 1ml freezing medium. NK cells were cryopreserved for 60 days and then resuscitated. Detect the survival rate of cells before and after cryopreservation, and the recovery rate of cells after resuscitation. Specifically, the cell survival rate calculation method before cryopreservation and after cryopreservation and resuscitation is: [number of living cells/(number of living cells+number of dead cells)] ⁇ 100%. The calculation method of the cell recovery rate after resuscitation is: (number of viable cells after resuscitation/number of viable cells during cryopreservation) ⁇ 100%.
  • cryopreservation solution 1 As shown in Table 2, the survival rate of cells after resuscitation was lower than before cryopreservation. Specifically, the cell viability and cell yield of cryopreservation solutions 1 and 2 were significantly better than those of cryopreservation solutions 3 and 4. There was no difference between freezing solutions 1 and 2, and there was no difference between freezing solutions 3 and 4. The difference indicates that adding 1-2% by volume albumin and 1-2% by volume aminoethanol has a better protective effect on NK cells. The cell viability and cell yield of cryopreservation solution 1 is significantly better than that of cryopreservation solution 7, indicating that 5% DMSO concentration is the optimal concentration. Increasing the DMSO concentration will increase the cytotoxic effect of DMSO, but will not increase the NK cell viability and Cell yield.
  • cryopreservation fluid 5 and cryopreservation fluid 6 are not much different, and their viability is significantly lower than cryopreservation fluid 1 and cryopreservation fluid 2, indicating that the addition of autologous plasma can not increase cell yield and cell viability .
  • the cell viability and cell yield of the immune cell-specific cryopreservation fluid (ie cryopreservation fluid 1 and cryopreservation fluid 2) of the present invention is significantly higher than that of other types of cryopreservation fluids.
  • the isolated mononuclear cells are induced and expanded into immune cells, stored according to ABO/RH classification and HLA classification, and immune cell information for retrieval is established Archives, build an immune cell bank.
  • the hospital asks for the donor’s personal information, past treatment history, family genetic history, and whether there is a history of infectious diseases and abnormalities in the hematopoietic or immune system by means of inquiries and forms.
  • the hospital must sign an informed consent form with the donor to obtain the consent of the donor or its authorized personnel, check its physical examination data, and obtain the physical examination information.
  • the donor’s physical examination information should include the following items: HIV-1/2 antibody, HBsAg, anti-HCV, CMV-IgM antibody, ALT, Treponema pallidum antibody.
  • Personal information collection forms, informed consent forms, inspection information, etc. need to be numbered and sealed, and a searchable immunization donor archive information database can be established.
  • a sterile blood collection bag with anticoagulant to collect about 100ml of human peripheral blood, and reserve 5ml of peripheral blood for quick test (HIV-1/2 antibody, HBsAg, anti-HCV, CMV-IgM antibody, Treponema pallidum antibody) and ABO/RH typing and HLA typing identification
  • blood collection bags are immediately aseptically packaged, stored and transported aseptically at 4°C, and collected information is accurately recorded.
  • the peripheral blood will be sent to the GMP laboratory after the quick screening is qualified. If the quick screening is unqualified, the blood sample will be discarded.
  • step 2 Resuspend the blood cell pellet in step 2 with an equal volume of normal saline of plasma and transfer it to a 250ml sterile glass bottle, add hydroxyethyl starch of 1/3 of the total volume of blood, gently shake the saline bottle to mix evenly. Set aside to allow the red blood cells to settle.
  • the medium ratio is a special expansion medium for immune cells, that is, 1500IU/ml IL-2, 1500IU/ml IL-10, 0.7ng/ml ml IL-1 ⁇ and 2.5ng/ml LIF, 1.5ng/ml EPO, 2.5ng/ml KGF, 3.5ng/ml testosterone, 2.5 ⁇ g/ml parathyroid hormone, 2.5 ⁇ g/ml laminin X-VOVO15 no Serum culture medium, 37°C, 5% CO 2 aseptic culture, recorded as the 0th day. Observe the cells daily, and change the medium every 2-3 days according to the color of the medium.
  • the cell suspension is tested: mycoplasma, endotoxin, microorganism and virus.
  • Tested and immune cells immune cells per 107 configured using the above-described immune cells 1ml special cryopreservation solution, for cryopreservation, press ABO / RH typing and HLA typing be saved, for the establishment of immune cell search Information archives to build an immune cell bank.

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Abstract

一种免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立。该方法包括：利用免疫细胞专用扩增培养基将单个核细胞进行第一阶段扩增培养，得到初步扩增的免疫细胞；利用免疫细胞专用诱导培养基将初步扩增的免疫细胞进行第二阶段诱导和扩增培养，得到诱导的免疫细胞；利用免疫细胞专用激活培养基将诱导的免疫细胞进行第三阶段激活和扩增培养，获得大量的激活功能的免疫细胞；利用免疫细胞专用冻存液冻存免疫细胞，得到冻存的免疫细胞；按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，构建免疫细胞库。

Description

免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立 技术领域
本发明涉及生物技术领域，具体地，涉及免疫细胞体外培养、诱导、激活、冻存的方法及其细胞库建立的方法。
背景技术
细胞生物治疗己经成为继手术、放化疗及内分泌治疗之后的第五大治疗模式。过继性免疫细胞治疗是细胞生物治疗方法之一，它是指向肿瘤患者输注具有抗肿瘤活性的免疫细胞，直接杀伤肿瘤细胞或激发机体免疫反应来杀伤肿瘤细胞，达到治疗肿瘤的目的。目前临床上应用的免疫细胞包括DC-CIK细胞、TIL细胞、LAK细胞及NK细胞，其中，CIK细胞、LAK细胞和NK细胞都是以自然杀伤细胞为主体的抗癌体系。
NK细胞又称自然杀伤细胞(natural killer cell)，在人体全身巡逻的同时搜寻癌细胞及病毒感染细胞等并对其进行攻击，在人体自然免疫中发挥着重要的作用。NK细胞占血液中淋巴细胞的10-30％，含穿孔素、颗粒酶等细胞毒性因子。NK细胞可释放穿孔素、颗粒酶，穿孔素在靶细胞表面穿孔，使颗粒酶b进入靶细胞诱导靶细胞凋亡。NK细胞同时分泌大量的细胞因子，如ifn-v、tnf-x、gm-csf、il-3、m-csf等，直接作用于靶细胞，或通过进一步激活其他种类免疫细胞攻击靶细胞。
NK细胞在肿瘤免疫、清除非己细胞等方面发挥重要作用：它是天然免疫防御的主要组成，位于机体防御体系的第一道防线，NK细胞的杀伤活性无MHC限制，不依赖抗体，无需抗原预先致敏即可识别并杀死肿瘤及病毒感染的细胞，通过穿孔素-颗粒酶途径和Fas-FasL通路直接对肿瘤细胞发挥细胞毒作用杀伤肿瘤细胞；同时它又能在发病早期分泌多种细胞因子和趋化因子如TNF-α、IFN-γ和IL-1等，这些细胞因子参与抗癌和调节获得性免疫应答，故NK细胞也是连接天然免疫与获得性免疫的桥梁。
NK细胞疗法的优势：(1)NK细胞活性高，可杀伤任何癌细胞，目前为止几乎没有发现高活性NK细胞杀不死的癌细胞；(2)有效刺激免疫力，激活免疫系统，没有毒副作用；(3)有效去除手术、放疗后残留的癌细胞；(4)NK细胞能够增强患者的免疫功能，同时具有抗恶性肿瘤和抗病毒的作用；(5)NK细胞是先天性细胞，可破坏任何癌细胞，是免疫细胞治疗的主力队员。
虽然NK细胞的抗癌效果的安全性和疗效得到肯定，但由于它仅占外周血淋巴细胞的10％-30％，因而如何获得高纯度、高质量地NK细胞产品是NK治疗的关键。近几年发现通过体外的刺激培养可进行相对大规模的NK细胞制备，己知IL-2、IL-15、IL-18和IL-7等细胞因子在对NK细胞的体外扩增上发挥重要作用，它们体外扩增NK细胞的倍数在几倍至数十倍不等。IL-2是重要的诱导NK细胞增殖的细胞因子，它可以激活NK细胞、促进NK细胞增殖和细胞因子的产生。IL-15和IL-7的作用与IL-2相似，同时它们还可通过与NK细胞表面表达的复合受体γ链结合，促进造血干细胞定向分化为NK细胞，且对NK细胞的发育、分化和维持长期体外存活等方面发挥重要作用。IL-15与IL-2的协同作用也使两者联合作用于NK细胞的体外扩增，是目前NK细胞体外扩增最传统的细胞因子组合。据报道IL-18不但能诱导活化的THi细胞分泌产生大量的IFN-γ，更能通过促进Fas-FasL通路的开放，增强NK细胞的细胞毒性，且呈剂量依赖性。
目前市场上已有NK细胞治疗产品，但使用的培养体系繁杂、扩增倍数及杀瘤效果不佳、冻存后复苏效果差、部分培养体系存在安全风险等问题。由此，自然杀伤细胞的培养、诱导、激活、冻存方法有待改进。
发明内容
本发明旨在解决现有技术中存在的技术问题。为此，本发明提出免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立的方法，该方法培养、诱导和激活免疫细胞的效率高、速度快、安全性高且成本低，并且诱导扩增获得的大量功能激活的免疫细胞可建立细胞库进行长期保存，复苏后依然保持良好的细胞活性。
本发明提供了免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立的方法。根据本发明的实施例，该方法包括：
利用免疫细胞专用扩增培养基将单个核细胞进行第一阶段扩增培养，得到初步扩增的免疫细胞；利用免疫细胞专用诱导培养基将初步扩增的免疫细胞进行第二阶段诱导和扩增培养，得到诱导的免疫细胞；利用免疫细胞专用激活培养基将诱导的免疫细胞进行第三阶段激活和扩增培养，获得大量的激活功能的免疫细胞；利用免疫细胞专用冻存液冻存免疫细胞，得到冻存的免疫细胞；按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，构建免疫细胞库。
所述免疫细胞专用扩增培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、0.5-1ng/ml IL-1α和1-4ng/ml LIF、0.5-2.5ng/ml EPO、1-4ng/ml KGF、2-5ng/ml睾酮、1-4μg/ml甲状旁腺激素、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
所述免疫细胞专用诱导培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、1-4ng/ml bFGF、1-4ng/ml BMP-4、0.2-0.8μg/ml雷帕霉素、0.2-0.8μg/ml淫羊藿苷、20-80ng/ml曲美替尼、1-4ng/ml氢化可的松、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
所述免疫细胞专用激活培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、1-4ng/ml TGF-β、1-2ng/ml Forskolin、20-80ng/ml白藜芦醇、1-4μg/ml对乙酰氨基酚、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
所述免疫细胞专用冻存液是包含5体积％的DMSO、1-2体积％的白蛋白、1-2体积％氨基乙醇、91-93体积％的无血清淋巴细胞培养基。
所述免疫细胞专用扩增培养基、免疫细胞专用诱导培养基、免疫细胞专用激活培养基和免疫细胞专用冻存液中，所述的无血清淋巴细胞培养基为X-VOVO15无血清培养基或市售其他类型无血清培养基。
根据本发明的实施例，所述免疫细胞为自然杀伤细胞。
利用本发明对单个核细胞进行诱导扩增培养可得到大量功能激活的免疫细胞。本发明具有诱导效率高、扩增速度快、安全性高和成本低等优点。且本发明建立相应的细胞库，分类进行大规模的免疫细胞存储，有效保存时间长，复苏后细胞依然保持良好的细胞活力，细胞回收率高，从而满足临床治疗中大量免疫细胞的需求。
附图说明
图1是本发明方法的流程图。
图2是本发明方法14天内对外周血单个核细胞的扩增效率和细胞活率。
图3是本发明方法14天内对目标细胞的扩增效率。
图4是本发明方法12天内对外周血单个核细胞的扩增效率和细胞活率。
图5是本发明方法12天内对目标细胞的扩增效率。
具体实施方式
本发明提供了一种免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立的方法，具体包括：利用免疫细胞专用扩增培养基将单个核细胞进行第一阶段扩增培养，得到初步扩增的免疫细胞；利用免疫细胞专用诱导培养基将初步扩增的免疫细胞进行第二阶段诱导和扩增培养，得到诱导的免疫细胞；利用免疫细胞专用激活培养基将诱导的免疫细胞进行第三阶段激活和扩增培养，获得大量的激活功能的免疫细胞；利用免疫细胞专用冻存液冻存免疫细胞，得到冻存的免疫细胞；按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，构建免疫细胞库。
根据本发明的实施例，免疫细胞专用扩增培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、0.5-1ng/ml IL-1α和1-4ng/ml LIF、0.5-2.5ng/ml EPO、1-4ng/ml KGF、2-5ng/ml睾酮、1-4μg/ml甲状旁腺激素、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
根据本发明的实施例，免疫细胞专用诱导培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、1-4ng/ml bFGF、1-4ng/ml BMP-4、0.2-0.8μg/ml雷帕霉素、0.2-0.8μg/ml淫羊藿苷、20-80ng/ml曲美替尼、1-4ng/ml氢化可的松、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
根据本发明的实施例，免疫细胞专用激活培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、1-4ng/ml TGF-β、1-2ng/ml Forskolin、20-80ng/ml白藜芦醇、1-4μg/ml对乙酰氨基酚、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
根据本发明的实施例，所述免疫细胞为自然杀伤细胞。
根据本发明的实施例，第一阶段免疫细胞扩增培养基过程中，每2-3天传代一次，传代2次；第二诱导和扩增培养过程中，每2-3天传代一次，传代2次；第三阶段大规模激活和扩增培养过程中，每2天传代一次，传代至少2次(可多次传代)。因此，免疫细胞可在短时间内实现高纯度规模化扩增，获得充足的功能激活的免疫细胞，用于可能的临床免疫治疗。
根据本发明的实施例，免疫细胞专用冻存液是包含5体积％的DMSO、1-2体积％的白蛋白、1-2体积％氨基乙醇、91-93体积％的无血清淋巴细胞培养基。每10 7-10 8个免疫细胞采用1ml上述免疫细胞专用冻存液。因此，冻存的细胞浓度高，适于大规模免疫细胞的冻存，冻存成本低，并且细胞冻存效果好，复苏后细胞活率和细胞得率高。
根据本发明的实施例，将激活扩增的免疫细胞，按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，构建免疫细胞库。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
利用本发明的免疫细胞培养、诱导、激活扩增方法，将分离的单个核细胞诱导激活扩增为目的免疫细胞(NK细胞)，并检测目的免疫细胞的活性。
一、实验方法
1.供体筛选
1.1要求医院须与供体签署知情同意书，一式3份。供体、医疗机构各一份，另一份随标本送交实验室。
1.2医院以询问和填表方式征询供体个人信息、过往治疗史、家族遗传史，以及是否有传染病史及造血或免疫系统的异常情况等信息。要求医院取得供体本人或其授权人员的同意，查阅其体检资料，获得体检信息。供体体检信息应包括如下项目：HIV-1/2抗体(艾滋病抗体)、HBsAg(乙肝表面抗原)、抗-HCV(丙型肝炎抗体)、CMV-IgM抗体(巨细胞病毒抗体)、ALT(转氨酶)、梅毒螺旋体抗体。检测方法分别执行卫生部现行检测标准。供体体检至少应符合《献血者健康检查要求》。
1.3由指定的有经验的医疗人员对收集到的信息进行综合评价，确定供体是否符合要求。供体知情同意书、个人信息采集表、检查信息等需编号密封保存。任何接触资料的人员未经供体本人或其授权人员的同意，不得泄露其隐私。
1.4按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫供体档案信息库。
2.采集外周血，分离外周血血浆和单个核细胞
2.1用加入抗凝剂的无菌采血袋采集人外周血约100ml，预留1ml外周血做快检和血型鉴定， 采血袋立即无菌封装，无菌4℃保存运输，准确记录采集信息。快检筛选合格后将外周血送入GMP实验室，若快检不合格，血样废弃处理。
2.2在GMP实验室中取出血袋，酒精消毒采血袋，观察无凝血和溶血后在超净台中打开血袋，将血液转移到至50ml无菌离心管中(≤45ml/管)，2500rpm离心10min。
2.3转移上层血浆到另一离无菌离心管中，3500rpm离心10min，收集上清血浆到新的无菌离心管中，用口膜密封离心管口，血细胞用于分离单个核细胞。
2.4将血浆放到56℃水浴锅中水浴30-50min灭活补体，3500rpm离心15min去除补体，10ml每支分装到15ml无菌离心管中，-20℃冻存备用；并留取7.5ml血浆进行慢检：病毒五项、支原体、内毒素和微生物。
2.5将步骤2中的血细胞沉淀用血浆等体积的生理盐水重悬后转移到250ml无菌玻璃瓶中，加入血液总体积1/3量的羟乙基淀粉，轻轻晃动盐水瓶使混合均匀，静置使红细胞沉降。
2.6待红细胞层沉降分层后，将上层乳白色悬液轻轻吸取到无菌离心管，1800rpm离心5min，弃去上清，沉淀用10ml生理盐水重悬。
2.7取无菌15ml离心管2支，各加入5ml常温人外周血淋巴细胞分离液，分别在上层轻轻加入各5ml细胞悬液。室温2000rpm慢升慢降离心25min。
2.8轻轻取出离心管，小心吸取界面中间云雾层白细胞至新的15ml离心管中，补加生理盐水洗涤2次。
2.9 1ml生理盐水重悬细胞，取5μl细胞加入到245μl生理盐水中稀释50倍，计数，并用台盼蓝染色计细胞存活率。
2.10预留4.5×10 6个细胞进行流式抗体标记检测NK细胞的比例；剩余的单个核细胞细胞接种到加有20ml培养基的培养瓶中，加入免疫细胞专用扩增培养基,即添加了1000IU/ml IL-2、1000IU/ml IL-10、0.8ng/ml IL-1α和2ng/ml LIF、1.5ng/ml EPO、2ng/ml KGF、3ng/ml睾酮、2μg/ml甲状旁腺激素、2μg/ml层粘连蛋白的X-VOVO15无血清培养基，置于37℃，5％CO 2培养箱中无菌培养，记为第0天。每日观察细胞，根据培养基的颜色，每2-3天换液传代一次。
2.11换液2次以后，当培养体系已经达到2L时，更换培养体系为免疫细胞专用诱导培养基， 即添加了1000IU/ml IL-2、1000IU/ml IL-10、2ng/ml bFGF、2ng/ml BMP-4、0.6μg/ml雷帕霉素、0.6μg/ml淫羊藿苷、50ng/ml曲美替尼、2ng/ml氢化可的松、2μg/ml层粘连蛋白的X-VOVO15无血清培养基。每日观察细胞，根据培养基的颜色，每2-3天换液传代一次。
2.12继续再换液2次以后，更换培养体系为免疫细胞专用激活培养基，即添加了1000IU/ml IL-2、1000IU/ml IL-10、2ng/ml TGF-β、2ng/ml Forskolin、50ng/ml白藜芦醇、2μg/ml对乙酰氨基酚、2μg/ml层粘连蛋白的X-VOVO15无血清培养基。每日观察细胞，每2天换液传代一次。
2.13培养扩增14天时，进行细胞回收，留取10ml培养基上清进行Elisa分泌因子检测。
2.14将回收的细胞用200ml生理盐水重悬，并加入10ml人血清白蛋白，混匀；并从中取10ml细胞悬液待检，剩余的细胞可注入回输袋中准备回输。从回输袋中抽取10ml细胞悬液进行检测：支原体、内毒素、微生物和病毒五项。
3.流式细胞仪检测细胞中淋巴细胞谱系
将取出的10ml细胞悬液，1800rpm离心回收细胞，进行流式抗体标记。设置同型对照、单标样品和染色管，每管样品细胞数约5×10 5个，然后加入对应抗体染色。4℃，30min放置，用生理盐水洗涤，然后上机检测分析淋巴细胞群中的NK细胞比例。
4.将剩余10ml细胞悬液上清，进行分装，用以检测病毒五项、内毒素、支原体、微生物。
5.裸鼠致瘤试验SPF级雌性BALB/c裸鼠，4-6周龄，体重18-20g，于空气层流架中带盖鼠笼内饲养，饮用水、标准饲料及其它与动物接触品均经灭菌处理。取阳性对照Ragi细胞和K562细胞及待检测的体外诱导分化的第28天的NK细胞按3×10 7个/0.2ml接种裸鼠肋部皮下，用苦味酸标记，为期2个月观察成瘤情况。
6.将收细胞的培养基上清进行Elisa分泌因子检测，即IFN-γ、TNF-α和Perforin检测。
二、实验结果
1.培养扩增14天的实验结果
1.1培养扩增14天后细胞可扩增220倍
所述取外周血淋巴细胞分离液分离后的6.0×10 7个外周血单个核细胞接种到20ml培养体系中，细胞很快进入对数生长阶段。培养扩增14天后，培养体系扩大到4L，细胞数扩增到1.3×10 10， 扩增倍数在最高达220倍，活细胞数在95％以上，培养不同时间的外周血单个核细胞数和细胞活性的结果详见图2。
1.2扩增后的外周血单个核细胞中NK细胞比例明显升高
外周血单个核细胞经14天培养扩增后，淋巴细胞中的NK细胞比例(CD3 -CD56 +)由18.15％上升到97.64％，与此同时T淋巴细胞(CD3 +)比例由71.47％下降到3.22％，辅助性T细胞(Th,CD3 +CD4 +)和细胞毒性T细胞(Tc,CD3 +CD8a +)都有降低，B淋巴细胞(CD3 -CD19 +)基本消失，细胞均一性显著提高；结合图1中的细胞计数计算可知，培养14天后NK细胞扩增1165倍，培养14天前后的外周血单个核细胞中的淋巴细胞比例的结果见图3，其中，NK细胞：CD3 -CD56 +；T细胞：CD3 +；辅助性T细胞(Th)：CD3 +CD4 +；细胞毒性T细胞(Tc细胞)：CD3 +CD8a +；B细胞：CD3 -CD9 +。
1.3培养扩增得到的细胞产品检测未发现病原体感染
我们将培养后的细胞和细胞悬液委托检测平台检测了乙肝表面抗原、丙肝抗原、人类免疫缺陷病毒抗体、梅毒螺旋体特异性抗体、巨噬细胞病毒以及支原体、细菌和内毒素，检测结果均呈阴性，说明该批次产品是安全的，培养过程中没有造成污染。
2.培养12天的实验结果
2.1培养12天后细胞可扩增162倍
取外周血淋巴细胞分离液分离后的6.0×10 7个外周血单个核细胞接种到20ml培养体系中，细胞很快进入对数生长阶段。培养12天后，细胞数扩增到9.7×10 9，扩增倍数在达162倍，活细胞数在95％以上，实验结果详见图4。
2.2扩增后的外周血单个核细胞中的NK细胞比例明显升高
外周血单个核细胞经12天扩增后，淋巴细胞中的NK细胞比例(CD3 -CD56 +)从第6天的21.47％上升到第12天的93.22％，与此同时T淋巴细胞(CD3 +)比例下降到3.86％，辅助性T细胞(Th,CD3 +CD4 +)和细胞毒性T细胞(Tc,CD3 +CD8a +)都有降低，B淋巴细胞(CD3 -CD19 +)基本消失，细胞均一性显著提高，培养12天前后的外周血单个核细胞中的淋巴细胞比例详见图5。
2.3培养扩增得到的细胞产品检测未发现病原体感染
我们将培养后的细胞和细胞悬液委托检测平台检测了乙肝表面抗原、丙肝抗原、人类免疫缺陷 病毒抗体、梅毒螺旋体特异性抗体、巨噬细胞病毒以及支原体、细菌和内毒素，检测结果均呈阴性，说明该批次产品是安全的，培养过程中没有造成污染。
3.所培养的细胞均有IFN-γ、TNF-α、Perforin的分泌,结果如表1所示。
表1.培养基上清进行Elisa检测
Figure PCTCN2020092287-appb-000001
4.培养扩增后的NK细胞不会在体内形成肿瘤
裸鼠致瘤实验结果：A组生理盐水对照组(成瘤小鼠数/组内小鼠数，0/5)，B组Raji细胞对照组(3/5)，C组K562细胞对照组(4/5)，D组NK细胞组(0/5)，即在2个月观察期内皮下注射0.2ml生理盐水组和3×10 7个/0.2ml培养28天的NK细胞组小鼠均未见肿瘤形成，注射等量Raji细胞和K562细胞的小鼠B组分别有4/5和5/5只小鼠形成了可见的肿瘤。该结果说明即使培养到28天，NK细胞依然是安全有效的，不会导致肿瘤的形成。
实施例2
对实施例1体外诱导激活扩增的NK细胞进行冻存，比较冻存效果。
将实施例1体外诱导扩增第14天获得的NK细胞分别采用以下四种冻存液进行冻存，其中四种冻存液的成份如下：
冻存液1：5体积％的DMSO、1体积％的白蛋白、1体积％氨基乙醇、93体积％的X-VOVO15无血清培养基；
冻存液2：5体积％的DMSO、2体积％的白蛋白、2体积％氨基乙醇、91体积％的X-VOVO15无血清培养基；
冻存液3：5体积％的DMSO、2体积％的白蛋白、93体积％的X-VOVO15无血清培养基；
冻存液4：5体积％的DMSO、2体积％的氨基乙醇、93体积％的X-VOVO15无血清培养基；
冻存液5：5体积％的DMSO、95体积％的X-VOVO15无血清培养基；
冻存液6：5体积％的DMSO、10体积％自体血浆、85体积％X-VOVO15无血清培养基；
冻存液7：10体积％的DMSO、2体积％的白蛋白、2体积％氨基乙醇、86体积％的X-VOVO15无血清培养基。
按照以下步骤冻存NK细胞：将冷冻保存液与细胞混匀后，速移入冻存管，并放入冻存盒中，-70℃程序降温过夜，次日转入液氮内。其中，每10 7个NK细胞采用1ml冻存液。冷冻保存NK细胞60天，然后进行复苏。检测冻存前后细胞的存活率，以及复苏后细胞回收率。具体地，冻存前以及冻存并复苏后的细胞存活率计算方法为：【活细胞数/(活细胞数+死细胞数)】×100％。复苏后细胞回收率的计算方法为：(复苏后活细胞数/冻存时活细胞数)×100％。
复苏NK细胞检查结果：
表2.不同冻存液冻存复苏效果比较
Figure PCTCN2020092287-appb-000002
如表2所示，复苏后细胞存活率均低于冻存前。具体地，冻存液1和2保存的细胞活率和细胞得率明显优于冻存液3和4，冻存液1和2之间未见差别，冻存液3和4之间未见差别，表明添加1-2体积％白蛋白和1-2体积％氨基乙醇对NK细胞具有较好的保护作用。冻存液1保存的细胞活率和细胞得率明显优于冻存液7，表明5％DMSO浓度是最适浓度，增加DMSO浓度将增加DMSO的细胞毒性作用，反而不能提高NK细胞活率和细胞得率。冻存液5和冻存液6细胞活率和细胞得率差别不大，且其活率显著低于冻存液1和冻存液2，说明添加自体血浆不能提高细胞得率和细胞活率。 综上，本发明的免疫细胞专用冻存液(即冻存液1和冻存液2)细胞活率和细胞得率显著高于其他类型冻存液。
实施例3
利用本发明实施例的免疫细胞的培养、诱导、激活方法，将分离的单个核细胞诱导扩增为免疫细胞，按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，构建免疫细胞库。
1.供体筛选
医院以询问和填表方式征询供体个人信息、过往治疗史、家族遗传史，以及是否有传染病史及造血或免疫系统的异常情况等信息。医院须与供体签署知情同意书取得供体本人或其授权人员的同意，查阅其体检资料，获得体检信息。供体体检信息应包括如下项目：HIV-1/2抗体、HBsAg、抗-HCV、CMV-IgM抗体、ALT、梅毒螺旋体抗体。个人信息采集表、知情同意书、检查信息等需编号密封保存，建立可供检索的免疫供体档案信息库。
2.采集外周血，分离外周血血浆和单个核细胞
2.1用加入抗凝剂的无菌采血袋采集人外周血约100ml，预留5ml外周血做快检(HIV-1/2抗体、HBsAg、抗-HCV、CMV-IgM抗体、梅毒螺旋体抗体)和ABO/RH分型、HLA分型鉴定，采血袋立即无菌封装，无菌4℃保存运输，准确记录采集信息。快检筛选合格后将外周血送入GMP实验室，若快检不合格，血样废弃处理。
2.2在GMP实验室中取出血袋，酒精消毒采血袋，观察无凝血和溶血后在超净台中打开血袋，将血液转移到至50ml无菌离心管中(≤45ml/管)，2500rpm离心10min。
2.3转移上层血浆到另一离无菌离心管中，3500rpm离心10min，收集上清血浆到新的无菌离心管中，用口膜密封离心管口，血细胞用于分离单个核细胞。
2.4将步骤2中的血细胞沉淀用血浆等体积的生理盐水重悬后转移到250ml无菌玻璃瓶中，加入血液总体积1/3量的羟乙基淀粉，轻轻晃动盐水瓶使混合均匀，静置使红细胞沉降。
2.5待红细胞层沉降分层后，将上层乳白色悬液轻轻吸取到无菌离心管，1800rpm离心5min，弃去上清，沉淀用10ml生理盐水重悬。
2.6取无菌15ml离心管2支，各加入5ml常温人外周血淋巴细胞分离液，分别在上层轻轻加 入各5ml细胞悬液。室温2000rpm慢升慢降离心25min。
2.7轻轻取出离心管，小心吸取界面中间云雾层白细胞至新的15ml离心管中，补加生理盐水洗涤2次。
2.8 1ml生理盐水重悬细胞，取5μl细胞加入到245μl生理盐水中稀释50倍，计数，并用台盼蓝染色计细胞存活率。
2.9单个核细胞细胞接种到加有20ml培养基的培养瓶中，培养基配比为免疫细胞专用扩增培养基,即添加了1500IU/ml IL-2、1500IU/ml IL-10、0.7ng/ml IL-1α和2.5ng/ml LIF、1.5ng/ml EPO、2.5ng/ml KGF、3.5ng/ml睾酮、2.5μg/ml甲状旁腺激素、2.5μg/ml层粘连蛋白的X-VOVO15无血清培养基，37℃，5％CO 2无菌培养，记为第0天。每日观察细胞，根据培养基的颜色，每2-3天换液传代一次。
2.10换液2次以后，当培养体系已经达到2L时，更换培养体系为免疫细胞专用诱导培养基，即添加了1500IU/ml IL-2、1500IU/ml IL-10、2.5ng/ml bFGF、2.5ng/ml BMP-4、0.5μg/ml雷帕霉素、0.5μg/ml淫羊藿苷、50ng/ml曲美替尼、2.5ng/ml氢化可的松、2.5μg/ml层粘连蛋白的X-VOVO15无血清培养基。每日观察细胞，根据培养基的颜色，每2-3天换液传代一次。
2.11继续再换液2次以后，更换培养体系为免疫细胞专用激活培养基，即添加了1500IU/ml IL-2、1500IU/ml IL-10、2.5ng/ml TGF-β、1.5ng/ml Forskolin、50ng/ml白藜芦醇、2.5μg/ml对乙酰氨基酚、2.5μg/ml层粘连蛋白的X-VOVO15无血清培养基。每日观察细胞，每2天换液传代一次。
2.12按5体积％的DMSO、2体积％的白蛋白、2体积％氨基乙醇、91体积％的X-VOVO15无血清培养基的比例配置免疫细胞专用冻存液。
2.13收集上述激活扩增的免疫细胞。将细胞悬液进行检测：支原体、内毒素、微生物和病毒五项。检测合格的免疫细胞，每10 7个免疫细胞采用1ml上述配置好的免疫细胞专用冻存液，进行冻存操作，按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，构建免疫细胞库。

Claims (7)

  1. 一种免疫细胞体外培养、诱导、激活、冻存方法及其细胞库建立，其特征在于，包括：利用免疫细胞专用扩增培养基将单个核细胞进行第一阶段扩增培养，得到初步扩增的免疫细胞；利用免疫细胞专用诱导培养基将初步扩增的免疫细胞进行第二阶段诱导和扩增培养，得到诱导的免疫细胞；利用免疫细胞专用激活培养基将诱导的免疫细胞进行第三阶段激活和扩增培养，获得大量的激活功能的免疫细胞；利用免疫细胞专用冻存液冻存免疫细胞，得到冻存的免疫细胞；按ABO/RH分型和HLA分型进行保存，建立可供检索的免疫细胞信息档案，建免疫细胞库。
  2. 根据权利要求1所述的方法，其特征在于，所述免疫细胞专用扩增培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、0.5-1ng/ml IL-1α和1-4ng/ml LIF、0.5-2.5ng/ml EPO、1-4ng/ml KGF、2-5ng/ml睾酮、1-4μg/ml甲状旁腺激素、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
  3. 根据权利要求1所述的方法，其特征在于，所述免疫细胞专用诱导培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、1-4ng/ml bFGF、1-4ng/ml BMP-4、0.2-0.8μg/ml雷帕霉素、0.2-0.8μg/ml淫羊藿苷、20-80ng/ml曲美替尼、1-4ng/ml氢化可的松、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
  4. 根据权利要求1所述的方法，其特征在于，所述免疫细胞专用激活培养基是添加了500-2000IU/ml IL-2、500-2000IU/ml IL-10、1-4ng/ml TGF-β、1-2ng/ml Forskolin、20-80ng/ml白藜芦醇、1-4μg/ml对乙酰氨基酚、1-4μg/ml层粘连蛋白的无血清淋巴细胞培养基。
  5. 根据权利要求1所述的方法，其特征在于，所述免疫细胞专用冻存液是包含5体积％的DMSO、1-2体积％的白蛋白、1-2体积％氨基乙醇、91-93体积％的无血清淋巴细胞培养基。
  6. 根据权利要求2、3、4、5所述的方法，其特征在于，所述免疫细胞专用扩增培养基、免疫细胞专用诱导培养基、免疫细胞专用激活培养基和免疫细胞专用冻存液中，所述的无血清淋巴细胞培养基为X-VOVO15无血清培养基或市售其他类型无血清培养基。
  7. 根据权利要求1所述的方法，其特征在于，所述免疫细胞为自然杀伤细胞。
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