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CN110760481B - Improved RPMI1640 culture medium and application thereof - Google Patents

Improved RPMI1640 culture medium and application thereof Download PDF

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CN110760481B
CN110760481B CN201911043694.6A CN201911043694A CN110760481B CN 110760481 B CN110760481 B CN 110760481B CN 201911043694 A CN201911043694 A CN 201911043694A CN 110760481 B CN110760481 B CN 110760481B
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包乐媛
李会
廖兴华
张同存
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Wuhan University of Science and Technology WHUST
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Abstract

本发明提出了一种改良RPMI1640培养基，改变了RPMI1640培养基中的氯化钠和氯化钾浓度。与RPMI1640培养基相比，本发明培养基在培养CAR‑T细胞时，能保持CAR‑T细胞的正常增殖，同时能降低CAR‑T细胞的耗竭，增强其抗肿瘤活性，具有良好的应用前景。

Figure 201911043694

The invention provides an improved RPMI1640 medium, which changes the sodium chloride and potassium chloride concentrations in the RPMI1640 medium. Compared with the RPMI1640 medium, the medium of the present invention can maintain the normal proliferation of CAR-T cells when culturing CAR-T cells, while reducing the exhaustion of CAR-T cells and enhancing its anti-tumor activity, and has good application prospects. .

Figure 201911043694

Description

Improved RPMI1640 culture medium and application thereof
Technical Field
The invention relates to the field of biomedical cell therapy, in particular to an improved RPMI1640 culture medium and application thereof.
Background
According to the statistics of global cancer reports, blood tumors mainly including leukemia, lymphoma, multiple myeloma and the like seriously threaten the life and health of human beings, are the eighth most common malignant tumors, and have the increasing incidence rate year by year. Immunotherapy has brought new hopes for the treatment of human blood tumor in recent years, wherein the immunotherapy using genetic engineering technology to modify T cells to express antibodies capable of recognizing tumor antigens, target tumor cells, activate T cells and kill tumor cells, i.e., chimeric Antigen Receptor T cells (CAR-T) is the most promising tumor immunotherapy and research hotspot in recent years. Currently, CAR-T cell immunotherapy has achieved exciting results in clinical hematological disorders, and there are two CAR-T cell products against the CD19 antigen from B cell leukemia that have been approved by the FDA for clinical treatment of leukemia.
The main processes of CAR-T cell therapy are: isolating immune T cells from peripheral blood of a cancer patient; preparing CAR-T cells through in vitro gene modification; further expanding the CAR-T cells in vitro cell culture medium; the expanded CAR-T cells are reinfused back into the patient.
The conventional medium used for the in vitro culture of CAR-T cells is RPMI1640 medium. However, during the expansion of CAR-T cells in vitro, T cells are depleted, affecting the killing ability of T cells.
Disclosure of Invention
In view of the above, the invention provides an improved RPMI1640 culture medium, which changes the concentration of potassium ions and sodium ions in the RPMI1640 culture medium, so as to overcome the defect that CAR-T cells are easy to be exhausted in vitro culture and improve the tumor killing capacity of the CAR-T cells.
The technical scheme of the invention is realized as follows:
in a first aspect, the invention provides a modified RPMI1640 medium, wherein the concentration of sodium chloride in the medium is 53.4mM, and the concentration of potassium chloride in the medium is 55.3 mM; alternatively, the concentration of sodium chloride in the medium is 63.4mM and the concentration of potassium chloride in the medium is 45.3 mM.
On the basis of the technical scheme, preferably, the modified RPMI1640 culture medium comprises the following components: glycine 0.133mM, L-arginine 1.15mM, L-asparagine 0.379mM, L-aspartic acid 0.15mM, L-cysteine 0.208mM, L-glutamic acid 0.136mM, L-alanylglutamine 2.05mM, L-histidine 0.0968mM, L-hydroxyproline 0.153mM, L-isoleucine 0.382mM, L-leucine 0.382mM, L-lysine hydrochloride 0.219mM, L-methionine 0.101mM, L-phenylalanine 0.0909mM, L-proline 0.174mM, L-serine 0.286mM, L-threonine 0.168mM, L-tryptophan 0.0245mM, L-tyrosine 0.111mM, L-valine 0.171mM, biotin 0.00082mM, choline chloride 0.0214mM, D-calcium pantothenate 0.000524mM, folic acid 57 mM, nicotinamide 0.0082mM, p-aminobenzoic acid 0.0073mM, pyridoxine 0.82 mM, pyridoxine hydrochloride 82mM, 0.000532mM of riboflavin, 0.00297mM of thiamine hydrochloride, 0.0000037mM of vitamin B120.0000037mM of i-inositol, 0.194mM of calcium nitrate tetrahydrate, 0.407mM of magnesium sulfate heptahydrate, 23.81mM of sodium bicarbonate, 5.63mM of anhydrous disodium hydrogen phosphate, 53.4-63.4mM of sodium chloride, 45.3-55.3mM of potassium chloride, 11.11mM of D-glucose, 0.00326mM of reduced glutathione and 0.0133mM of phenol red.
In a second aspect, the invention provides the use of a modified RPMI1640 medium according to the first aspect in CAR-T cell culture.
In a third aspect, the invention also provides a method of CAR-T cell culture comprising: CAR-T cells were cultured using the modified RPMI1640 medium of the first aspect.
Compared with the prior art, the improved RPMI1640 culture medium has the following beneficial effects:
(1) the improved culture medium changes the metabolic environment of the CAR-T cells by changing the potassium ion concentration of the RPMI1640 culture medium, so that the CAR-T cells are in a stem cell state in the amplification process, excessive activation of the CAR-T cells is avoided, and apoptosis of the CAR-T cells is reduced, so that the killing function of the CAR-T cells is improved;
(2) the improved culture medium only changes the concentration of potassium ions and sodium ions of the RPMI1640 culture medium, fetal bovine serum is only added on the basis when the CAR-T cells are cultured, and growth factors such as various amino acids and vitamins are not required to be additionally added, so that the CAR-T cells can keep a good proliferation state, the apoptosis of the CAR-T cells is reduced, the tumor killing capability of the CAR-T cells is improved, the operation is simple and convenient, and the effect is obvious.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the proliferation of CD19-CAR-T cells in different media according to the invention;
FIG. 2 shows the expression of Fas (CD95) in CD19-CAR-T cultured in different media according to the present invention;
FIG. 3 shows the tumoricidal effect of CD19-CAR-T cultured in different media according to the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Interpretation of terms:
chimeric Antigen Receptor (CAR): is a core component of CAR-T, conferring on T cells the ability of Human Lymphocyte Antigen (HLA) to recognize tumor antigens in an independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell antigen receptors (TCRs). The basic design of a CAR includes a tumor-associated antigen (TAA) binding region (usually the scFV fragment from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.
CAR-T cells: chimeric antigen receptor T cells.
CD 19-CAR-T: chimeric antigen receptor genetically modified T cells targeting the CD19 molecule.
CD 3: in immunology, the co-receptor of CD3 (cluster of differentiation 3) T cells is a protein complex, which consists of four distinct chains. In mammals, the complex contains one CD3 γ chain, one CD3 δ chain, and two CD3 epsilon chains. These chains have T lymphocytes called a molecular parat cell receptor (TCR) and zeta-chains to generate activation signals. The TCR, zeta chain and CD3 molecules together form a T cell receptor complex.
Pbmc (periheral blood mononar cell): peripheral blood mononuclear cells are cells having a single nucleus in peripheral blood, and include lymphocytes, monocytes, and the like.
All raw materials in the improved RPMI1640 culture medium disclosed by the invention are ultrapure or analytically pure reagents which meet the second division of the United states Pharmacopeia, national Standard chemical reagents or Chinese pharmacopoeia 2010 edition so as to meet the requirements of clinical safety and reasonability.
RPMI1640 medium was purchased from Gibco, CD3 antibody was purchased from Milteny biotec, anti-fas antibody was purchased from Biolegend, interleukin 2(IL-2) was purchased from Peprotech, HEK 293T cells and Raji cells were purchased from ATCC cell bank, and LDH kit was purchased from Sigma. The lentiviral plasmid for CD19 CAR-T was constructed by the laboratory.
The invention is further illustrated by the following examples:
the media components of examples 1-3 and comparative examples are detailed in the following table:
Figure GDA0002851824350000051
examples 1-3 the medium was modified RPMI1640 medium, as indicated by MTM1-MTM 3; the comparative example is RPMI1640 medium.
The preparation method of each medium in the above examples 1 to 3 and comparative example includes: mixing the components of the culture medium with high-purity sterilized water in proportion, shaking and uniformly mixing for 1 hour at 4 ℃, and then filtering and sterilizing through a filter membrane of 0.22 mu m for later use.
Each of the following experiments using the culture media of examples 1-3 and comparative examples was repeated in T cells of three healthy adults.
PBMC and T cell isolation: peripheral blood from healthy adult donors was drawn, PBMC separated using Ficoll, followed by T cell separation using anti-CD3 magnetic beads.
The preparation method of the CD19-CAR-T cell comprises the following steps: transfecting HEK 293T cells with a lentiviral plasmid of CD19-CAR-T, collecting cell culture supernatant after 5 days, and preparing lentivirus; t cells were transfected with MOI 5:1 (lentivirus: T cells).
The culture medium of example 1-example 3 and comparative example was used for culturing CD19-CAR-T cells as follows: the CD19-CAR-T cells were resuspended in the culture media of examples 1-3 and comparative examples, 10% of fetal bovine serum, 30ng/mL of OKT3 antibody and 4 allogeneic PBMCs (1: 200 volume ratio of T cells to PBMCs) were added simultaneously, and the seeding density of CD19-CAR-T cells was adjusted to 1X 105cell/mL, 5% CO2And culturing in an incubator at 37 ℃. During the culture, every other timeAdding 200IU IL-2 in one day, and culturing for 12 days.
The CD19-CAR-T cells cultured according to the above steps by using the culture media of example 1-example 3 and comparative example were tested, specifically:
1) cells cultured for 6 days and 12 days were collected and counted by trypan blue staining.
2) CD19-CAR-T cells cultured for 12 days were stained with anti-Fas antibody, and the expression rate of cell surface death receptor factor Fas (CD95) was examined by flow cytometry.
3) The CD19-CAR-T cells cultured for 12 days and cultured leukemia Raji cells are co-cultured according to the volume ratio of 3:1, and the tumor killing rate of the CD19-CAR-T cells is detected and calculated by using an LDH kit.
The test results are as follows:
1) cell proliferation status
Cell proliferation after culture of CD19-CAR-T cells using the media of example 1-example 3 and comparative example is shown in figure 1, and the results show that the modified media MTM1, MTM2 and MTM3 have no effect on the proliferation of CD19 CAR-T cells compared to RPMI1640 medium.
2) Results of detection of Fas (CD95) expression Rate
The expression of Fas (CD95) in CD19-CAR-T cultured in the culture media of example 1-example 3 and comparative example is shown in FIG. 2, and the modified culture media MTM1 and MTM2 can significantly reduce the expression of Fas (CD95) on the surface of CD19-CAR-T cells.
3) Detection result of CD19-CAR-T cell tumor killing rate
The tumor killing rate of CD19-CAR-T cultured by the culture media of example 1-example 3 and comparative example is shown in FIG. 3, and after the CD19 CAR-T cultured by the modified culture media MTM1 and MTM2 is co-cultured with leukemia cells, the tumor killing capacity of CD19-CAR-T cells can be remarkably enhanced.
Compared with the RPMI1640 culture medium, the improved RPMI1640 culture medium provided by the invention has no influence on the proliferation of CD19-CAR-T cells when the CD19-CAR-T cells are cultured, but can down-regulate the expression of Fas (CD95) on the surface of the CD19-CAR-T cells, so that Fas-mediated T cell apoptosis is inhibited, and the tumor killing capacity of the CD19-CAR-T cells is remarkably enhanced.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (2)

1.一种改良RPMI1640培养基在CAR-T细胞培养中的应用，其特征在于：所述培养基中的氯化钠浓度为53.4mM，氯化钾浓度为55.3mM；或者所述培养基中的氯化钠浓度为63.4mM，氯化钾浓度为45.3mM；1. an application of an improved RPMI1640 medium in CAR-T cell culture, characterized in that: the sodium chloride concentration in the medium is 53.4mM, and the potassium chloride concentration is 55.3mM; or in the medium The sodium chloride concentration is 63.4mM and the potassium chloride concentration is 45.3mM; 包括以下组分：甘氨酸0.133mM、L-精氨酸1.15mM、L-天冬酰胺0.379mM、L-天冬氨酸0.15mM、L-半胱氨酸0.208mM、L-谷氨酸0.136mM、L-丙氨酰谷氨酰胺2.05mM、L-组氨酸0.0968mM、L-羟脯氨酸0.153mM、L-异亮氨酸0.382mM、L-亮氨酸0.382mM、L-赖氨酸盐酸盐0.219mM、L-甲硫氨酸0.101mM、L-苯丙氨酸0.0909mM、L-脯氨酸0.174mM、L-丝氨酸0.286mM、L-苏氨酸0.168mM、L-色氨酸0.0245mM、L-酪氨酸0.111mM、L-缬氨酸0.171mM、生物素0.00082mM、氯化胆碱0.0214mM、D-泛酸钙0.000524mM、叶酸0.00227mM、烟酰胺0.0082mM、对氨基苯甲酸0.0073mM、盐酸吡哆醇0.00485mM、核黄素0.000532mM、盐酸硫胺素0.00297mM、维生素B12 0.0000037mM、i-肌醇0.194mM、四水硝酸钙0.424mM、七水硫酸镁0.407mM、碳酸氢钠23.81mM、无水磷酸氢二钠5.63mM、氯化钠53.4-63.4mM、氯化钾45.3-55.3mM、D-葡萄糖11.11mM、还原型谷胱甘肽0.00326mM、酚红0.0133mM。Including the following components: Glycine 0.133mM, L-Arginine 1.15mM, L-Asparagine 0.379mM, L-Aspartate 0.15mM, L-Cysteine 0.208mM, L-Glutamate 0.136mM , L-Alanylglutamine 2.05mM, L-Histidine 0.0968mM, L-Hydroxyproline 0.153mM, L-Isoleucine 0.382mM, L-Leucine 0.382mM, L-Lysine Hydrochloride 0.219mM, L-Methionine 0.101mM, L-Phenylalanine 0.0909mM, L-Proline 0.174mM, L-Serine 0.286mM, L-Threonine 0.168mM, L-Chromo Amino Acid 0.0245mM, L-Tyrosine 0.111mM, L-Valine 0.171mM, Biotin 0.00082mM, Choline Chloride 0.0214mM, D-Calcium Pantothenate 0.000524mM, Folic Acid 0.00227mM, Nicotinamide 0.0082mM, Right Aminobenzoic acid 0.0073mM, pyridoxine hydrochloride 0.00485mM, riboflavin 0.000532mM, thiamine hydrochloride 0.00297mM, vitamin B12 0.0000037mM, i-inositol 0.194mM, calcium nitrate tetrahydrate 0.424mM, magnesium sulfate heptahydrate 0.407 mM, sodium bicarbonate 23.81mM, anhydrous disodium hydrogen phosphate 5.63mM, sodium chloride 53.4-63.4mM, potassium chloride 45.3-55.3mM, D-glucose 11.11mM, reduced glutathione 0.00326mM, phenol red 0.0133mM. 2.一种CAR-T细胞培养的方法，其特征在于：采用权利要求1所述的改良RPMI1640培养基培养CAR-T细胞。2. A method for culturing CAR-T cells, characterized in that: using the modified RPMI1640 medium according to claim 1 to cultivate CAR-T cells.
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