CN108949696A - Immune cell media is applied with it - Google Patents

Immune cell media is applied with it Download PDF

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Publication number
CN108949696A
CN108949696A CN201810955224.6A CN201810955224A CN108949696A CN 108949696 A CN108949696 A CN 108949696A CN 201810955224 A CN201810955224 A CN 201810955224A CN 108949696 A CN108949696 A CN 108949696A
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cell
car
culture
hydrochloride
immune cell
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吴建勇
易小萍
滕小锘
张大鹤
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Suzhou Misu Biotechnology Co Ltd
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Suzhou Misu Biotechnology Co Ltd
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Abstract

The invention discloses a kind of immune cell medias, and it includes the components such as a variety of amino acid such as the plurality of inorganic salt such as anhydrous calcium chloride, L-Leu, interleukin-22, IL-4.Immune cell media of the invention can significantly improve the proliferation times of cell when cultivating CAR-T cell, substantially extend cell culture number of days, and can effectively keep expression surface marker, while it also has many advantages, such as long shelf-life, easily prepared.

Description

Immune cell media is applied with it
Technical field
The present invention relates to a kind of immune cell medias and its application in culture CAR-T cell, belong to biologic medical Technical field.
Background technique
T cell with Chimeric antigen receptor (chimericantigenreceptor, CAR) modification is the tumor target of representative To immunization therapy in vitro with good targeting, lethal and persistence are shown in clinical test, illustrate huge answer With potentiality and development prospect.The principle of CAR-T cell therapy is the T cell modified through Chimeric antigen receptor, can specificity Ground identifies tumor associated antigen, the immunocyte for applying targeting, killing activity and the persistence of effector T cell more routinely Height, and tumor by local immunosupress microenvironment can be overcome and break host immune tolerance status, i.e., the T cell of expression CAR is with anti- The mode combination tumour antigen that original relies on, non-MHC is limited, starts and activation specific kills tumor response.
One important link of CAR-T cell therapy is to carry out the amplification of CAR-T cell, and one in this link is very Crucial factor is the effective culture medium of selection.
So far, numerous producers develop panimmunity cell culture medium, for example, such as X-VIVO (Lonza product), KBM- 551K, 15 culture medium of KBM-581, LONZA X-VIVO (healthy and free from worry product) etc..These culture base products are extensive at present It uses.But these culture mediums all contain animal source component, such as fetal calf serum, these animal source component systems are from animal blood serum Or isolated and purified out in tissue, animal pathogen may be contained, therefore have the danger for causing disease.
Although the GT-T551H3 of Stemline, Takara company of AIM-V, Sigma company of such as Gibco company, The products such as the TexMACS of Miltenyi company claim to be serum free medium, but it is when in use, if not addition serum at Point, then the proliferation of lymphocyte is more slow, it is difficult to meet cell quantity required for clinical cytology is treated.
In addition, the defect that existing immune cell media is shorter there is also the shelf-life, in the period of culture CAR-T cell It is interior, it can be the problem of the later period cell Proliferation efficiency being caused sharply to glide.
Summary of the invention
The main purpose of the present invention is to provide a kind of immune cell medias to apply with it, in the prior art to overcome It is insufficient.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
The embodiment of the invention provides a kind of immune cell medias comprising: anhydrous calcium chloride 112.5mg/L~ 115.8mg/L, L-Leu 57.85mg/L~58.95mg/L, cupric sulfate pentahydrate 0.0011mg/L~0.0012mg/L, L- rely Propylhomoserin hydrochloride 92.20mg/L~93.25mg/L, lipoic acid 0.101mg/L~0.108mg/L, nine water ferric nitrate 0.04mg/L ~0.05mg/L, L-Methionine 16.98mg/L~17.28mg/L, phenol red 7.8mg/L~8.3mg/L, ferrous sulfate heptahydrate 0.410mg/L~0.418mg/L, L-phenylalanine 35.42mg/L~36.55mg/L, 1,4- butanediamine dihydrochloride 0.078mg/L~0.080mg/L, potassium chloride 310.5mg/L~311.3mg/L, Serine 26.55mg/L~27.15mg/L, Sodium Pyruvate 52mg/L~58mg/L, magnesium chloride 28.61mg/L~28.38mg/L, L-threonine 53.41mg/L~53.49mg/ L, biotin 0.0025mg/L~0.0045mg/L, anhydrous magnesium sulfate 48.55mg/L~49.54mg/L, l-Alanine 4.35mg/L~4.55mg/L, ethanol amine 500.0mg/L~2000.0mg/L, D-VB5 calcium 2.21mg/L~2.26mg/L, chlorine Change sodium 6950.5mg/L~7120.5mg/L, L- asparagine 7.4mg/L~8.5mg/L, choline chloride 8.88mg/L~ 9.18mg/L, 40~180mg/L of transferrins, anhydrous sodium dihydrogen phosphate 54.15mg/L~54.55mg/L, Ritonavir 200.0nmol/L~1000.0nmol/L, ASPARTIC ACID 6.25mg/L~6.75mg/L, folic acid 2.45mg/L~ 2.65mg/L, oleic acid 100.0mg/L~1500.0mg/L, disodium hydrogen phosphate 70.52mg/L~73.22mg/L, L-cysteine Hydrochloride 16.98mg/L~17.65mg/L, IL-4 10mg/L~80mg/L, i- inositol 12.2mg/L~12.7mg/L, people Seralbumin 500.0mg/L~3000.0mg/L, gentamicin 1 × 103~5 × 106U/L, white vitriol 56.431mg/L ~210.433mg/L, rh-insulin 0.5mg/L~5mg/L, Pidolidone 7.25mg/L~7.55mg/L, ascorbic acid 25.0mg/L~150.0mg/L, niacinamide 1.96mg/L~2.0mg/L, L-arginine hydrochloride 145.5mg/L~148.5mg/ L, L-PROLINE 17.15mg/L~17.25mg/L, IL-77 μ g/L~9 μ g/L, μ g/L of IL-153 μ g/L~5, linoleic acid 100.0mg/L~1200.0mg/L, pyridoxal hydrochloride 1mg/L~2mg/L, progesterone 8mg/L~60mg/L, l-cysteine hydrochloride 30.19mg/L~32.29mg/L, L-Trp 8.72mg/L~9.22mg/L, hCAT 200.0mg/L~ 2200.0mg/L, puridoxine hydrochloride 0.028mg/L~0.035mg/L, methionine enkephalin 385mg/L~425mg/L, stem cell Growth factors 5 mg/L~40mg/L, l-tyrosine 38.2mg/L~39.4mg/L, riboflavin 0.213mg/L~0.219mg/L, Glycine 18.35mg/L~19.25mg/L, Valine 52.45mg/L~53.65mg/L, 17~70mg/L of interleukin-22, salt Allithiamine 2.12mg/L~2.18mg/L, 0.5~5mg/L of vitamin C, L-Histidine hydrochloride 31.23mg/L~32.48mg/ L, D-Glucose 3110mg/L~3165mg/L, thymidine 0.312mg/L~0.386mg/L, l-Isoleucine 54.21mg/L~ 54.83mg/L, hypoxanthine 1mg/L~3mg/L, vitamin B12 0.61mg/L~0.72mg/L, NaHCO33.0mmol/L~ 29mmol/L。
The embodiment of the invention also provides application of the immune cell media in CAR-T cell culture.
Further, the CAR-T cell is selected from CD19-CAR-T, CD20-CAR-T, K light chain-CAR-T, CD22-CAR- T, CD23-CAR-T, CD30-CAR-T or CD70-CAR-T.
The embodiment of the invention also provides a kind of CAR-T cell culture processes comprising: it is trained using the immunocyte Support base culture CAR-T cell.
Further, the cultural method includes: that described be immunized is added in CAR-T cell and CD3 monoclonal antibody In cell culture medium, cell density is adjusted, carries out cell culture, adds the culture medium and the CD3 monoclonal after cultivating 72h Antibody continues culture 4 days or more later, supplemented the immune cell media every 2~3 days.
Further, the cell density is (1~10) × 106cell/mL。
Further, the condition of the cell culture is 5%CO2,37 DEG C.
Further, the concentration of the CD3 monoclonal antibody is 20~50ng/mL.
Compared with the prior art, immune cell media of the invention can significantly improve cell when cultivating CAR-T cell Proliferation times, substantially extend cell culture number of days, and can effectively keep expression surface marker, while it also has guarantor The advantages that matter phase is long, easily prepared.
Detailed description of the invention
Fig. 1 be 1- of embodiment of the present invention embodiment 5 is respectively adopted, the culture medium of reference examples 1- reference examples 2 carry out it is immune thin The cell Proliferation curve graph of born of the same parents' culture.
Specific embodiment
In view of many defects of the existing technology, inventor is studied for a long period of time and is largely practiced, and is able to propose this The technical solution of invention relates generally to a kind of improved immune cell media and its application in culture CAR-T cell.
If not the paraphrase of specified otherwise, technical term involved in this specification is identical as the conventional paraphrase of this field, Such as:
Chimeric antigen receptor (CAR): being the core component of CAR-T, assigns the non-dependent mode of T cell HLA and identifies tumour The ability of antigen, this, which enables, identifies widely by the T cell of CAR transformation compared to nave T cell surface receptor TCR Target.It include tumor associated antigen (tumor-associated antigen, a TAA) combined area in the basic engineering of CAR (the scFV section for being typically derived from monoclonal antibody antigen bond area), an extracellular hinge area, a transmembrane region and a born of the same parents Interior signaling zone.The selection of target antigen carrys out the safety of the specificity of CAR, validity and genetic modification T cell itself Say it is all crucial determinant.
CAR-T cell: Chimeric antigen receptor T cell.
CD19-CAR-T: the T cell of the mosaic antigen acceptor gene modification of targeting CD19 molecule.
CD3: in immunology, the co-receptor of CD3 (differentiation cluster 3) T cell is a kind of protein complex.In mammal In, which contains a CD3 γ chain, CD3 δ chain and 2CD3 ε chain.These chains have be referred to as a molecule pair T cell by Body (TCR) and ζ-chain are to generate the T lymphocyte of activation signal.The T cell receptor that the TCR, ζ chain and CD3 molecule are constituted together Compound.In immune cell media of the invention each raw material should all be meet United States Pharmacopeia or " national standard chemistry reagent " or Pharmacopoeia of the People's Republic of China version second ultrapure or analytical reagents in 2010, is reasonably wanted with meeting clinical safety It asks.
Immune cell media of the invention is free of any animal sources composition, and biological source ingredient therein is pharmaceutical grade Or highly purified source of people substance, therefore safety is guaranteed.
Cell, original used in each component and cell culture processes in the immune cell media addressed in this specification Material is available on the market.
More specific detail is made to technical solution of the present invention below in conjunction with several embodiments.
The component of the immune cell media of embodiment 1- embodiment 5 is detailed in following table:
The preparation method of each immune cell media includes: by above-mentioned culture medium each group in previous embodiment 1- embodiment 5 Divide and mixed in proportion with high-purity aqua sterilisa, 4 DEG C of concussions mix 1 hour, then stand-by by 0.22 μm of membrane filtration degerming.
Cell culture is carried out using the immune cell media of embodiment 1- embodiment 5, process is as follows: by commercially available expression The culture medium of the T cell of Chimeric antigen receptor CD19CAR embodiment 1- embodiment 5 is resuspended, while the CD3 of 30ng/mL is added Monoclonal antibody adjusts 1 × 106cell/mL of inoculum density, is put into 5%CO2, cultivate in 37 DEG C of incubators.According to 5 after culture 72 hours The density of × 105cell/mL adds the culture medium of embodiment 1- embodiment 5, while adding the CD3 monoclonal antibody of 30ng/mL.It is every later The supplement culture medium is primary within 2-3 days, keeps cell density in 5 × 105cell/mL；Later, the growth curve of cell is calculated, and Culture to 7 days and 21 days cells are taken to carry out flow cytometer detection.
Cell detection specifically: the cell being collected into is cleaned 2 times with PBS, its surface marker is detected by streaming instrument The expression rate of CD4+, CD8+ and CD19CAR.
In addition, the culture medium of X-VIVO, KBM-581 culture medium alternate embodiment 1- embodiment 5 is respectively adopted, according to phase Tongfang Formula carries out cell culture, and is tested using same way, and as a comparison case 1,2.
Test result:
1, the cell Proliferation curve measured is detailed in lower Fig. 1.Wherein the corresponding curve of embodiment 1-5 is to be named as " embodiment " Curve (data used be average test data), comparative example 1,2 response curves are named as " comparative example 1 ", " comparative example 2 ".
2, flow cytometer detection result:
Remarks: " embodiment " related data is the average value of 5 the data obtained of embodiment 1- embodiment in upper table.
Obviously, the immune cell media of the embodiment of the present invention, which at least has the advantages that, can greatly improve a variety of exempt from The proliferation times of epidemic disease cell, and cell culture number of days can be extended, cultivated days can reach 21 days or more, and can extraordinary guarantor Hold the surface markers such as expression CD4+, CD8+, CD19CAR.
It should be appreciated that the above description is only an embodiment of the present invention, it is not intended to limit the scope of the invention, it is all Using equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, it is applied directly or indirectly in other Relevant technical field, is included within the scope of the present invention.

Claims (8)

1. a kind of immune cell media, characterized by comprising: the bright ammonia of anhydrous calcium chloride 112.5mg/L~115.8mg/L, L- Sour 57.85mg/L~58.95mg/L, cupric sulfate pentahydrate 0.0011mg/L~0.0012mg/L, L lysine HCL 92.20mg/L~93.25mg/L, lipoic acid 0.101mg/L~0.108mg/L, nine water ferric nitrate 0.04mg/L~0.05mg/L, L-Methionine 16.98mg/L~17.28mg/L, phenol red 7.8mg/L~8.3mg/L, ferrous sulfate heptahydrate 0.410mg/L~ 0.418mg/L, L-phenylalanine 35.42mg/L~36.55mg/L, 1,4- butanediamine dihydrochloride 0.078mg/L~ 0.080mg/L, potassium chloride 310.5mg/L~311.3mg/L, Serine 26.55mg/L~27.15mg/L, Sodium Pyruvate 52mg/L~58mg/L, magnesium chloride 28.61mg/L~28.38mg/L, L-threonine 53.41mg/L~53.49mg/L, vitamin H 0.0025mg/L~0.0045mg/L, anhydrous magnesium sulfate 48.55mg/L~49.54mg/L, l-Alanine 4.35mg/L~ 4.55mg/L, ethanol amine 500.0mg/L~2000.0mg/L, D-VB5 calcium 2.21mg/L~2.26mg/L, sodium chloride 6950.5mg/L~7120.5mg/L, L- asparagine 7.4mg/L~8.5mg/L, choline chloride 8.88mg/L~9.18mg/ L, 40~180mg/L of transferrins, anhydrous sodium dihydrogen phosphate 54.15mg/L~54.55mg/L, Ritonavir 200.0nmol/L ~1000.0nmol/L, ASPARTIC ACID 6.25mg/L~6.75mg/L, folic acid 2.45mg/L~2.65mg/L, oleic acid 100.0mg/L~1500.0mg/L, disodium hydrogen phosphate 70.52mg/L~73.22mg/L, L-cysteine hydrochloride 16.98mg/ L~17.65mg/L, IL-4 10mg/L~80mg/L, i- inositol 12.2mg/L~12.7mg/L, human serum albumins 500.0mg/L~3000.0mg/L, gentamicin 1 × 103~5 × 106U/L, white vitriol 56.431mg/L~ 210.433mg/L, rh-insulin 0.5mg/L~5mg/L, Pidolidone 7.25mg/L~7.55mg/L, ascorbic acid 25.0mg/L~150.0mg/L, niacinamide 1.96mg/L~2.0mg/L, L-arginine hydrochloride 145.5mg/L~148.5mg/ L, L-PROLINE 17.15mg/L~17.25mg/L, IL-77 μ g/L~9 μ g/L, μ g/L of IL-153 μ g/L~5, linoleic acid 100.0mg/L~1200.0mg/L, pyridoxal hydrochloride 1mg/L~2mg/L, progesterone 8mg/L~60mg/L, l-cysteine hydrochloride 30.19mg/L~32.29mg/L, L-Trp 8.72mg/L~9.22mg/L, hCAT 200.0mg/L~ 2200.0mg/L, puridoxine hydrochloride 0.028mg/L~0.035mg/L, methionine enkephalin 385mg/L~425mg/L, stem cell Growth factors 5 mg/L~40mg/L, l-tyrosine 38.2mg/L~39.4mg/L, riboflavin 0.213mg/L~0.219mg/L, Glycine 18.35mg/L~19.25mg/L, Valine 52.45mg/L~53.65mg/L, 17~70mg/L of interleukin-22, salt Allithiamine 2.12mg/L~2.18mg/L, 0.5~5mg/L of vitamin C, L-Histidine hydrochloride 31.23mg/L~32.48mg/ L, D-Glucose 3110mg/L~3165mg/L, thymidine 0.312mg/L~0.386mg/L, l-Isoleucine 54.21mg/L~ 54.83mg/L, hypoxanthine 1mg/L~3mg/L, vitamin B12 0.61mg/L~0.72mg/L, NaHCO33.0mmol/L~ 29mmol/L。
2. application of the immune cell media described in claim 1 in CAR-T cell culture.
3. application as claimed in claim 2, it is characterised in that: the CAR-T cell is selected from CD19-CAR-T, CD20-CAR- T, K light chain-CAR-T, CD22-CAR-T, CD23-CAR-T, CD30-CAR-T or CD70-CAR-T.
4. a kind of CAR-T cell culture processes, characterized by comprising: use immunocyte culture as described in claim 1 Base culture CAR-T cell.
5. cultural method according to claim 6, characterized by comprising: add CAR-T cell and CD3 monoclonal antibody Enter in the immune cell media, adjust cell density, carry out cell culture, cultivate add after 72h the culture medium and The CD3 monoclonal antibody continues culture 4 days or more later, supplemented the immune cell media every 2~3 days.
6. cultural method according to claim 5, which is characterized in that the cell density is (1~10) × 106cell/ mL。
7. cultural method according to claim 5, which is characterized in that the condition of the cell culture is 5%CO2,37 DEG C.
8. cultural method according to claim 5, which is characterized in that the concentration of the CD3 monoclonal antibody be 20~ 50ng/mL。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110184239A (en) * 2019-06-12 2019-08-30 兰莲(杭州)生物科技有限公司 A kind of preparation method for activating T lymphocyte culture solution and its activating T lymphocyte
CN110894466A (en) * 2019-12-20 2020-03-20 广州海润康华生物科技有限公司 Culture medium for stem cell culture
CN117070454A (en) * 2023-10-18 2023-11-17 北京细胞治疗集团有限公司 Preparation method of CAR-T cells

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014165707A2 (en) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Effective generation of tumor-targeted t-cells derived from pluripotent stem cells
CN104450614A (en) * 2014-12-05 2015-03-25 上海安集协康生物技术有限公司 Animal protein-free immune cell serum-free medium and using method thereof
CN104818248A (en) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 Immunocyte culture medium, and culture method and application of immunocytes
CN105154402A (en) * 2015-10-28 2015-12-16 广州赛莱拉干细胞科技股份有限公司 Culture medium, application thereof and culture method
CN105176925A (en) * 2015-09-26 2015-12-23 友康恒业生物科技（北京）有限公司 Immune cell serum-free medium and preparation and application thereof
CN106834229A (en) * 2017-01-25 2017-06-13 华东理工大学 For the serum free medium of people's immunologic cytotoxicity cell expansion ex vivo
WO2017219528A1 (en) * 2016-06-21 2017-12-28 林志国 Preparation method for cd70-containing chimeric antigen receptor-modified t cells specifically recognizing egfrviii

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014165707A2 (en) * 2013-04-03 2014-10-09 Memorial Sloan-Kettering Cancer Center Effective generation of tumor-targeted t-cells derived from pluripotent stem cells
CN104450614A (en) * 2014-12-05 2015-03-25 上海安集协康生物技术有限公司 Animal protein-free immune cell serum-free medium and using method thereof
CN104818248A (en) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 Immunocyte culture medium, and culture method and application of immunocytes
CN105176925A (en) * 2015-09-26 2015-12-23 友康恒业生物科技（北京）有限公司 Immune cell serum-free medium and preparation and application thereof
CN105154402A (en) * 2015-10-28 2015-12-16 广州赛莱拉干细胞科技股份有限公司 Culture medium, application thereof and culture method
WO2017219528A1 (en) * 2016-06-21 2017-12-28 林志国 Preparation method for cd70-containing chimeric antigen receptor-modified t cells specifically recognizing egfrviii
CN106834229A (en) * 2017-01-25 2017-06-13 华东理工大学 For the serum free medium of people's immunologic cytotoxicity cell expansion ex vivo

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALNABHAN, R等: "Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells", 《CYTOTHERAPY》 *
CHAVEZ, AT等: "Impact of Different Culture Media Supplements on CAR T Cell Function", 《MOLECULAR THERAPY》 *
PTACKOVA, P等: "A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21", 《CYTOTHERAPY》 *
高群等: "不同培养基与血清的培养体系对细胞因子诱导杀伤细胞增殖和功能的影响", 《中国肿瘤生物治疗杂志》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110184239A (en) * 2019-06-12 2019-08-30 兰莲(杭州)生物科技有限公司 A kind of preparation method for activating T lymphocyte culture solution and its activating T lymphocyte
CN110184239B (en) * 2019-06-12 2021-04-06 蓝莲(杭州)生物科技有限公司 Activated T lymphocyte culture solution and preparation method of activated T lymphocyte
CN110894466A (en) * 2019-12-20 2020-03-20 广州海润康华生物科技有限公司 Culture medium for stem cell culture
CN117070454A (en) * 2023-10-18 2023-11-17 北京细胞治疗集团有限公司 Preparation method of CAR-T cells

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