High-purity peripheral blood source tool B lymphocyte culture system and method

Abstract

The invention discloses a high-purity peripheral blood source tool B lymphocyte culture system and method. The culture system disclosed by the invention comprises an hBAFF-hCD40L 3T3 culture system and a B cell culture medium, the preparation method of the hBAFF-hCD40L 3T3 culture system comprises the following steps: transfecting a 3T3 cell by using an hBAFF-hCD40L-eGFP plasmid, and carrying out screening culture by using puromycin, so as to obtain the hBAFF-hCD40L 3T3 culture system. The B cell culture medium is prepared from the following raw materials: an IMDM culture medium, a human serum substitute, hIL-2, hIL-4, hIL-21 and the like. The culture system has the advantages of being rapid in amplification, good in activity, stable in proliferation, free of differentiation and the like, on the premise that the directional differentiation potential of the B cells is well reserved, the preparation stability of the engineered B cells can be improved, and therefore yield preparation of the tool B lymphocytes can be achieved, and therefore the culture system and the method have good application prospects.

Description

Technical Field

[0001] The invention relates to a high-purity peripheral blood-derived tool B lymphocyte culture system and method, belonging to the field of biotechnology. Background Art

[0002] Human B lymphocytes, or B cells for short, are multipotent stem cells derived from the bone marrow. The progenitor cells of B lymphocytes are present in the island of hematopoietic cells in the fetal liver (14 days for embryonic mice or 8-9 weeks for normal birth), and the bone marrow gradually replaces the production and differentiation sites of B lymphocytes. Mature B cells mainly reside in the lymph nodules in the superficial layer of the lymph node cortex and the lymph nodules in the red and white pulp of the spleen. B cells can differentiate into plasma cells under antigen stimulation, and plasma cells can synthesize and secrete antibodies (immunoglobulins), mainly performing the body's humoral immunity. In addition, the role of B cells as antigen presenting cells has gradually been recognized by people: ① When the antigen concentration is very low, B cells are the most effective antigen presenting cells; ② In the secondary immune response, B cells are the most important antigen presenting cells.

[0003] With the continuous improvement of the understanding of B cells, the demand for engineered B cells in scientific research or medical research and development is becoming increasingly strong. At present, the main sources of engineered B cells are bone marrow and peripheral blood. Bone marrow blood collection is extremely difficult, so peripheral blood mononuclear cells (PBMC) extracted from peripheral blood have become the main source of B cells. However, compared with T cells, B cells are more difficult to culture in vitro, and the literature records and mature method experience related to B cell in vitro culture are also relatively scarce. The existing in vitro culture of B cells mainly relies on the CD40L system; CD40L is an indispensable factor for maintaining the survival, activation, proliferation and differentiation of mature B lymphocytes. However, its limitations are also extremely fatal, because the rapid expansion of B cells is often accompanied by their rapid and large-scale entry into apoptosis, resulting in nonspecific immune activation of immune cells, which greatly affects the research and development and application of engineered B cells.

[0004] In addition, in previous studies, 293T cells were used as feeder cells to culture B cells, but the system had huge defects. 293T cells grow too fast as feeder cells, and usually need to be irradiated or treated with mitomycin C (MMC) in advance to inhibit their growth, thus prolonging the experimental period; the culture system often requires the addition of exogenous stimulating factors, which is cumbersome and uneconomical, and cannot systematically analyze the development, differentiation and function of B cells.

[0005] As people's research on B cells continues to deepen, researchers have found that B-cell-activating factor of the tumournecrosis-factor family (BAFF) is an important factor in maintaining the survival and expansion of B cells. In the past, the method of adding soluble BAFF to the culture supernatant has been used. This method is cumbersome and troublesome, and its concentration fluctuates, which is not conducive to the survival and expansion of B cells.

[0006] More importantly, the existing culture systems are unclear about the supplementary factors for B cell culture. Most existing research systems only add CD40L and BAFF during culture and lack other exogenous cytokine supplements, which leads to B cell differentiation.

[0007] In summary, the existing engineered B cell culture system cannot meet the demand. The soluble factor system is added in multiple times and stages, which is cumbersome to operate and increases the potential risks of culture system contamination and batch effect. The CD40L system causes cell proliferation and apoptosis at the same time, and the preparation rate of engineered B cells is low. The 293T culture system requires irradiation before B cell culture, which increases the difficulty of operation. In addition, all the above systems do not take into account the immortalization of B cells, and have obvious limitations in practical applications. Therefore, there is an urgent need to develop a simple and easy-to-use engineered B cell culture system based on human peripheral blood. Summary of the invention

[0008] The purpose of the present invention is: in view of the deficiencies of the prior art, the present invention provides a high-purity peripheral blood-derived tool B lymphocyte culture system and method; the B cell culture system provided by the present invention can improve the stability of engineered B cell preparation while well retaining the B cell directional differentiation potential, thereby facilitating the realization of its mass production.

[0009] In order to achieve the above object, the present invention provides a culture system for culturing high-purity peripheral blood-derived tool B lymphocytes, comprising a BAFF-CD40L 3T3 culture system and a B cell culture medium;

[0010] The preparation method of the hBAFF-hCD40L 3T3 culture system comprises: using hBAFF-hCD40L-eGFP plasmid to transfect 3T3 cells, and then using puromycin to perform screening and culture, thereby obtaining;

[0011] The main components of the B cell culture medium include: IMDM culture medium, human serum substitute and MEM non-essential amino acids (preferably the three are prepared in a volume ratio of 89:10:1), and are supplemented with hIL-2 (preferably added at a concentration of 50 ng / ml), hIL-4 (preferably added at a concentration of 10 ng / ml), hIL-21 (preferably added at a concentration of 10 ng / ml), 2-mercaptoethanol (preferably added at a concentration of 55 μM), penicillin (preferably added at a concentration of 100 U / ml), streptomycin (preferably added at a concentration of 100 μg / ml), HEPES (biological buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), preferably added at a concentration of 10 mM) and sodium pyruvate (preferably added at a concentration of 1 mM).

[0012] Preferably, it also includes: EBV virus or culture medium containing EBV virus.

[0013] Preferably, the EBV virus is: EBV extracted from the cell culture supernatant of B95-8.

[0014] The present invention also provides the use of the above culture system in culturing high-purity peripheral blood-derived tool B lymphocytes.

[0015] The present invention also provides a method for culturing high-purity peripheral blood-derived tool B lymphocytes, comprising: washing PBMCs newly extracted from peripheral blood or thawed and frozen, performing cell activity staining and cell counting, resuspending them in a B cell culture medium, transferring them to a hBAFF-hCD40L 3T3 culture system for culture, then passage and amplify them, and collecting cell precipitates after centrifugation to obtain B lymphocytes; the B cell culture medium is the B cell culture medium in the above-mentioned culture system, and the hBAFF-hCD40L3T3 culture system is the hBAFF-hCD40L 3T3 culture system in the above-mentioned culture system.

[0016] Preferably, the method further comprises an immortalization step: aspirating the supernatant after passage and amplification, adding EBV culture medium, centrifuging, removing the supernatant, collecting the cell precipitate, resuspending the cell precipitate in B cell culture medium, and then inoculating the cell precipitate into a new BAFF-CD40L 3T3 culture system for culture.

[0017] Preferably, the method for preparing the EBV culture medium comprises: after culturing B95-8 cells, adding tetradecanoylphorbol acetate to the culture supernatant for incubation, then removing the supernatant, replacing with a new culture medium for culturing; collecting the culture supernatant as the EBV culture medium.

[0018] Preferably, the culture medium used for culturing B95-8 cells is RPMI 1640 culture medium.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) Fast expansion: The culture system of the present invention can expand to 30 times within 15 days of culture, which exceeds other existing culture systems;

[0021] (2) Good activity: The B cell culture system provided by the present invention can better ensure the activity of B cells and avoid B cell apoptosis caused by BAFF deficiency or level fluctuation; and the 3T3 feeder cells culture system ensures that cytokines are continuously secreted and maintained at a certain level;

[0022] (3) Stable proliferation without differentiation: The culture medium of this culture system contains human interleukin 2 (hIL-2), human interleukin 4 (hIL-4), and human interleukin 21 (hIL-21); it can better maintain B cell proliferation without promoting B cell differentiation;

[0023] (4) Immortality: Engineered B cells need to ensure long-term stability, so choosing a suitable method to immortalize the cells can avoid the effects of cell aging; the immortalization step of the method of the present invention is only performed during cell centrifugation and will not contaminate the feeder cells system.

[0024] (5) High purity: The preliminary magnetic separation and subsequent long-term culture designed in the present invention are suitable for the expansion of B lymphocytes. The CD19+ cell B lymphocyte ratio obtained by long-term culture is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the B cell collection, sorting, and culture process;

[0026] Figure 2 The pictures during the culture process are as follows: ① After blood collection, the blood sample is stored in a 10ml EDTA anticoagulant tube; ② After gradient centrifugation using lymphocyte separation solution, the light yellow upper liquid is plasma, the middle white flocculent layer is peripheral blood lymphocytes, the lower transparent layer is lymphocyte separation solution, and the bottom dark red layer is red blood cells and platelets; ③ The white precipitate at the bottom of the test tube is PBMC after washing; ④ The microscopic picture of B lymphocytes at the starting point after sorting; ⑤ The microscopic picture of B lymphocytes after 2 days of co-culture; ⑥ The microscopic picture of B lymphocytes after 6 days of co-culture (after 3 co-culture cycles and 2 separations);

[0027] Figure 3 is the hBAFF-hCD40L plasmid spectrum;

[0028] Figure 4 B cell expansion curves in different culture methods;

[0029] Figure 5The percentage of B cell apoptosis after 15 days in different culture systems;

[0030] Figure 6 CD19 after 15 days of culture using different methods + B lymphocyte ratio. DETAILED DESCRIPTION

[0031] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Example

[0034] A high-purity peripheral blood-derived tool B lymphocyte culture system and method:

[0035] (I) B cell culture and differentiation system:

[0036] (1) Preparation of culture system: The B cell hBAFF-hCD40L 3T3 culture system involved in this example is mainly divided into two parts: culture background and culture reagents.

[0037] (2) Culture background: The hBAFF-hCD40L-eGFP plasmid and NIH 3T3 cells were used for construction; 3T3 cells were transfected with the hBAFF-hCD40L-eGFP plasmid and then screened and cultured with puromycin; stably transfected 3T3 cells were plated at a certain cell ratio for backup.

[0038] (3) Culture reagents: Prepare engineered B cell culture medium, including culture materials including main components and additives: ① Main components: IMDM culture medium (gibco, IMDM 1X + GlutaMAX TM ), human serum replacement (gibco, CTSTMImmune cell SR), MEM non-essential amino acids, the volume ratio of the three is 89:10:1; ② Added ingredients: hIL-2 (PeproTech, 50ng / ml), hIL-4 (PeproTech, 10ng / ml), hIL-21 (PeproTech, 10ng / ml), 2-mercaptoethanol (Invitrogen, 55μM), double anti-penicillin / streptomycin (gibco, 100U / ml), HEPES (Invitrogen, 10mM), sodium pyruvate (Invitrogen, 1mM).

[0039] (4) Immortality method: At present, the immortalization method of B cells mainly adopts immortalization sequence virus (Bcl-6-BCL-xl-eGFP) and EBV virus infection. The present invention mainly extracts EBV (Epstein-Barr virus, EB virus) from the cell culture supernatant of B95-8 to immortalize B cells. Centrifugal infection is adopted to significantly improve the infection rate and has no effect on the BAFF-CD40L 3T3 culture system.

[0040] (II) Cultivation methods (cultivation process such as Figures 1-2 (shown)

[0041] (1) PBMCs were prepared from fresh peripheral blood, washed once, and then stained for cell viability and counted.

[0042] (2) Using the B cell-specific culture medium of the present invention, 5×10 5 Resuspend B cells at a density of 1:1 / ml and transfer to the hBAFF-hCD40L 3T3 culture system (6-well plate or 24-well plate) that suits the needs.

[0043] (3) Culture at 37°C and 5% CO2 for 48 hours and then subculture and expand (can be extended to 64 hours as needed, but not more than 72 hours at most).

[0044] (4) EBV-containing culture medium: Prepared using B95-8 cells; after culturing for a certain period of time at 37°C and 5% CO2, 20 ng / ml tetradecanoyl phorbol acetate (TPA) was added to the culture supernatant; after incubation for 1 hour, the supernatant was removed and replaced with new RPMI 1640 medium for 48 hours; the culture supernatant was collected as the subsequent immortalized EBV medium.

[0045] (5) Gently shake the culture plate and aspirate the supernatant, add it to a 10 ml centrifuge tube. During centrifugation, add an equal volume of EBV supernatant (B95-8 culture supernatant) to the original culture medium, centrifuge at 800 g for 10 minutes, then remove the supernatant, resuspend in B cell-specific culture medium, and then inoculate into a new BAFF-CD40L 3T3 culture system (CD19 magnetic bead sorting can be performed before culture).

[0046] (5) After B cells are expanded to the required number, they can be used or cryopreserved at -80°C or liquid nitrogen.

[0047] Figure 4 B cell expansion curves in different culture methods; Figure 5 is the apoptotic proportion of B cells in different culture systems after 15 days; Figures 4-5 It can be seen that the culture system of the present invention has fast expansion, good activity, and a low apoptosis rate of B cells. Figure 6 CD19 after 15 days of culture using different methods + The proportion of B lymphocytes is determined by Figure 6 It can be seen that the magnetic separation in the early stage and the long-term culture in the later stage are suitable for the expansion of B lymphocytes, and CD19 + The proportion of B lymphocytes is high.

[0048] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A culture system for culturing high-purity peripheral blood-derived B lymphocytes, characterized in that: Includes hBAFF-hCD40L 3T3 culture system and B cell culture medium; The preparation method of the hBAFF-hCD40L 3T3 culture system comprises: using hBAFF-hCD40L-eGFP plasmid to transfect 3T3 cells, and then using puromycin to perform screening and culture, thereby obtaining; The main components of the B cell culture medium include: IMDM culture medium, human serum replacement and MEM non-essential amino acids, and are supplemented with hIL-2, hIL-4, hIL-21, 2-mercaptoethanol, penicillin, streptomycin, HEPES and sodium pyruvate.

2. The culture system according to claim 1, characterized in that Also includes: EBV virus or culture medium containing EBV virus.

3. The culture system according to claim 2, characterized in that The EBV virus is: EBV extracted from the cell culture supernatant of B95-8.

4. Use of the culture system according to any one of claims 1 to 3 in culturing high-purity peripheral blood-derived B lymphocytes.

5. A method for culturing high-purity peripheral blood-derived B lymphocytes, characterized in that: include: PBMCs newly extracted from peripheral blood or resuscitated and frozen are washed, stained for cell viability and counted, resuspended in B cell culture medium, transferred to hBAFF-hCD40L 3T3 culture system for culture, then passaged and amplified, and cell precipitates are collected after centrifugation to obtain B lymphocytes; the B cell culture medium is the B cell culture medium in the culture system described in any one of claims 1 to 3, and the hBAFF-hCD40L 3T3 culture system is the hBAFF-hCD40L 3T3 culture system in the culture system described in any one of claims 1 to 3.

6. The culture method according to claim 5, characterized in that The method also includes an immortalization step: after passage and expansion, the supernatant is aspirated, EBV culture medium is added, centrifugation is performed, the supernatant is removed, the cell pellet is collected, the cell pellet is resuspended in B cell culture medium, and then the cell pellet is inoculated into a new hBAFF-hCD40L 3T3 culture system for culture.

7. The culture method according to claim 6, characterized in that The preparation method of the EBV culture fluid comprises: after culturing B95-8 cells, adding tetradecanoylphorbol acetate to the culture supernatant for incubation, then removing the supernatant, replacing with a new culture medium for culturing; collecting the culture supernatant is the EBV culture fluid.

8. The culture method according to claim 7, characterized in that The culture medium used for culturing B95-8 cells is RPMI 1640 culture medium.

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