CN105420190B - Application of the B27 additives and the like in using serum free medium culture lymphocyte - Google Patents
Application of the B27 additives and the like in using serum free medium culture lymphocyte Download PDFInfo
- Publication number
- CN105420190B CN105420190B CN201610011671.7A CN201610011671A CN105420190B CN 105420190 B CN105420190 B CN 105420190B CN 201610011671 A CN201610011671 A CN 201610011671A CN 105420190 B CN105420190 B CN 105420190B
- Authority
- CN
- China
- Prior art keywords
- lymphocyte
- additives
- application
- culture
- pbmc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/71—Oxidoreductases (EC 1.)
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides application of the B27 additives and the like in using serum free medium culture lymphocyte, existing lymphocyte serum is solved in the case of no any blood serum substituting additive, the amplification in vitro of lymphocyte slowly, be difficult to meet the problem of clinical cytology treats required cell quantity.
Description
Technical field
The present invention relates to field of cell culture, relate more specifically to B27 additives and the like and are trained using serum-free
Support the application in base culture lymphocyte.
Background technology
In recent years, with the rapid development of immunotherapy techniques, the various types of cells immunization therapy based on lymphocyte
Have become the focus of people's growing interest.All kinds of lymphs with lymphocyte technology and by technique for gene engineering modification are thin
Born of the same parents are that the immunotherapy techniques of carrier have become antineoplaston effective means by force, for Refractory and relapsed acute leukemia patient
Clinical Treatment Test in show prominent curative effect.However the performance of Lymphocyte immunotherapy effect depends on sufficient amount
Lymphocyte, this make lymphocyte it is external it is effective amplification as all cellular immunotherapies key problem in technology.
It is needed to make it activate and in specific cell factor make by certain mode by the lymphocyte extracted in vivo
The lymphocyte of active proliferation and specific killing ability is converted under.Lymphocyte is all in addition people source at present
Or in vitro culture and proliferation are carried out under conditions of animal sources serum.Serum as traditional Lymphocyte expansion main additive it
One, it is most important to the quality of Lymphocyte expansion.However, no matter people source serum or animal sources serum all have certain safety
Doubt, for example, pathogeny, repulsion or allergy, to be not suitable for clinical application.Which increases the self lymph of amplification in vitro is thin simultaneously
The quality testing of born of the same parents and the difficulty of monitoring.Just because of disadvantages mentioned above, serum-free or chemically defined culture medium have been increasingly becoming
Cell produces and the necessary culture medium of clinical application cell injuring model.The culture medium of specific chemical components is by a series of high-quality
The culture medium that amount, the organic compound of high-purity and inorganic compound collectively constitute.Serum free medium and specific chemical components
Culture medium application technique that cells in vitro is produced and end point cell control of product quality it is most important.
Multiple commercial vendors all have developed the serum free medium suitable for culture in vitro of lymphocytes in succession currently on the market,
Such as GT-T551H3, Miltenyi company of Stemline, Takara company of AIM-V, Sigma company of Gibco companies
TexMACS etc..Although these lymphocytes culture mediums are labeled as the serum-free training of serum free medium or specific chemical components
Base is supported, but in the case of no any blood serum substituting additive, the proliferation of lymphocyte is still slow, it is difficult to meet clinical
The required cell quantity of cell therapy.The X-VIVO 15 of Lonza companies is to develop to expand for lymphocytes in vitro
Serum free medium.In the case where not adding other serum and substituting the condition of culture of additive, lymphocytes in vitro increases the culture medium
The effect grown is better than the serum free medium of other brands.But compared with the condition of culture of addition serum, cultivation effect is still
Gap is larger.
Commercially available B-27 additives (also abbreviation B27) are used as serum-free additive, be currently known for hippocampal neuron and
It the growth of other central nervous system (CNS) neurons and keeps its short-term or long period of activity, a concentration of 2% is used, low
The ineffective of cell is cultivated when 2% concentration.Currently, whether being suitable for other cell lines for B-27 additives, whether may be used
To use lower concentration culture cell to be not reported.
The present invention adds not using commercially available fetal calf serum as reference substance in various commercially available lymphocyte serums
27 additive of cell culture additive B in proportion carries out the comparative study of in-vitro multiplication, and investigates different active modes pair
The influence of cell Proliferation being capable of commercially available cell culture addition of the alternative serum for external effectively amplifying lymphocyte to filter out
Agent, and find its it is lower using concentration to reduce cost, for lymphocyte in-vitro multiplication production technology exploitation, with promote
Into the industrialization of cellular immunotherapy.
Invention content
In view of existing lymphocyte serum in the case of no any blood serum substituting additive, lymphocyte
Amplification in vitro it is slow, it is difficult to meet clinical cytology and treat required cell quantity, the purpose of the present invention is to provide B27 to add
Add new opplication of the agent and the like in using serum free medium culture lymphocyte.
B27 additives and the like of the present invention using the application in serum free medium culture lymphocyte to carry
While the amplification in vitro efficiency of high existing lymphocyte serum, lymphocyte activity is kept, and thus provide
The lymphocytes in vitro amplification culture medium of serum-free suitable for human cell's treatment use.
One aspect of the present invention provides B27 additives and the like thin using serum free medium culture lymph
Application in born of the same parents.
Wherein, the volumetric concentration of the B27 additives is 0.20-4%, it is therefore preferable to 0.25-1.75%, more preferably
0.25-1.5%.
In one embodiment of the invention, described B27 additives and the like include biotin, vitamin A (vinegar
Acid), catalase, superoxide dismutase, cortisone, ethanol amine hydrochloric acid, glutathione (reduction), l-cn hydrochloric acid,
Progesterone, putrescine dihydrochloride and trilute or combinations thereof.In another embodiment of the present invention, the B27
Additive and the like still further comprises DL- α-D-α-tocopherol acetate, DL- alpha-tocopherols, bovine serum albumin, recombined human pancreas islet
Element, human transferrin, D- galactolipins, sodium selenite, Phosphatidylcholin, cholesterol, lecithin, linoleic acid, leukotrienes or its group
It closes.
In the present invention, the lymphocyte is human lymphocyte, it is therefore preferable to the lymph in human peripheral blood single nucleus cell
Cell.
In the present invention, the B27 additives are added in lymphocytes culture medium, and the lymphocytes culture medium includes
X-VIVO 15 (be purchased from Lonza companies, article No. 04-418Q), AIM-V (being purchased from Gibco companies, article No. 12055-091) and
1640 (are purchased from Gibco companies, article No. 22400-089), Stemline (being purchased from Sigma companies, article No. S1694-1L),
GT-T551H3 (be purchased from Takara companies, article No. WK593S) and TexMACS (be purchased from Miltenyi companies, article No. 170-
076-309)。
Another aspect of the present invention additionally provides B27 additives and the like in using medium culture lymphocyte
Application.
Beneficial effects of the present invention
The present invention provides B27 additives to substitute people source or animal sources serum proliferation in lymphocyte serum-free medium
The new application of lymphocyte, this is conducive to the stably and controllable and lymphocyte treatment product of all kinds of Lymphocyte Proliferation in Vitro techniques
Scale and industrialization, be also beneficial to improve the cell therapy product through amplification in vitro during clinical patient adoptive therapy
Immunological safety.
Specifically, by the culture of B27 additive applications to lymphocyte, have the advantages that：
(1) after lymphocyte activator, B27 additives are added in the tested commercially available culture medium of institute, lymphocyte
Proliferation times are more than 200 times in 7 days, and more than 400 times in 14 days, this cultivation effect is more than or is at least equal to fetal calf serum
Effect.
(2) after lymphocyte activator, B27 additives are added in the tested commercially available culture medium of institute and are proliferated, is proliferated
Terminal CD3 positive lymphocyte ratios are more than 90%.
(3) in breeding and proliferation end-points, lymphocyte subgroup distribution with add fetal calf serum and be not added with any
The lymphocyte subgroup distribution of additive proliferation is almost the same.
(4) when being transformed to cell using technique for gene engineering, transduction efficiency fetal calf serum and is not added with addition
Any additive proliferation conditions effect is consistent.
(5) lymphocyte after technique for gene engineering modification is used, in the commercially available culture medium for being added to B27 additives,
Proliferation times and unmodified lymphocyte are almost the same.
(6) use technique for gene engineering modification after lymphopoiesis during and end point cell subgroup distribution and not
The lymphocyte of modification is consistent.
(7) present invention has similar amplification to imitate in the lymphocyte for all lymphocyte activator methods activation tested
Fruit.
(8) dosage when B27 cultures lymphocyte is less, is more suitable for external large-scale culture, and technological process is simple
Easily-controllable, expanding effect is more preferably.
Description of the drawings
Fig. 1 is to show that B27 shows similar or more preferably effect figure in PBMC proliferation.
Fig. 2 is to show that B27 shows the figure of similar phenotypic analysis figure on culture PBMC.
Fig. 3 is to show that B27 is in the figure of dose-dependence in the proliferation for maintaining PBMC.
Fig. 4 is to show B27 not to influence the figure of PBMC transductions.
Fig. 5 is the figure of the cellular cytoxicity activity for the PBMC for showing B27 not and influencing transduction.
Specific implementation mode
Below by specific implementation mode and experimental data, the present invention is further illustrated.Although for clear mesh
, proprietary term is used below, but these terms are not meant to define or limit the scope of the invention.
In the present invention, term " culture medium " i.e. cell culture medium, refers to the aqueous solution of nutrients, can be used for supporting thin
Intracellular growth and amplification.In general, cell culture medium includes following component：Energy source usually will be saccharide compound, preferably Portugal
Grape sugar；Amino acid, the preferably amino acid of alkalinity group, including all required and nonessential amino acid；Vitamin and/or with low concentration
Other organic compounds needed；Free fatty；Inorganic compound, including trace element, inorganic salts；Buffer compounds；And
Nucleosides and base.
In the present invention, " additive " refers to any substance that can be added in culture medium.
In the present invention, term " B27 additives " refers to B27 additives ex hoc genus anne product, further includes B27 additives
Follow-up improvement or substitute products.Described B27 additives etc. can be by matching Mo Feishier companies (Thermo Fisher) or English lattice
The product that grace biotech firm (Engreen Biosystem Co., Ltd.s) sells can also be the similar production that other companies sell
Product, this kind of product are chiefly used in the culture of nerve cell, hippocampal neuron or CNS neurons.In the embodiment of the present invention, use
It is the B27 additives sold by matching Mo Feishier companies.
In the present invention, term " analog " refer to replace B27 additives in one or more ingredients and have and its
The additive of similar cell culture effect.
In the present invention, lymphocyte may be from peripheral blood lymphocytes.Lymphocyte to be amplified can also be biology
Imitate lymphocyte present in product, such as in upper theliolymphocyte, infiltrating lymphocyte, cancer ascites or hydrothorax
Lymphocyte infiltration etc..After the separation of lymphocytes in peripheral blood is easy, detaches due to lymphocyte survival rate height etc., because
The lymphocyte for being isolated from fresh peripheral blood is preferably used in this.It is highly preferred that lymphocyte refer to separation peripheral blood it is single
Lymphocyte in nucleus (PBMC).
The culture and activation of embodiment 1PBMC
In the present invention unless otherwise specified, PBMC that peripheral blood extracts and that use technique for gene engineering is modified, uses
After phytolectin (PHA) or AntiCD3 McAb/CD28 antibody (or antibody-coupled magnetic beads) activation, in addition various concentration ratio
In B27 (being bought from Thermo Fisher, article No. 17504044) or the commercially available culture medium of fetal calf serum, addition leucocyte is situated between
- 2 (IL-2) (100U/mL) of element are in 37 DEG C and 5%CO2Under conditions of cultivated, carried out changing liquid at interval of 2 days, and add white
Cytokine -2, amplification in vitro 10-20 days.
Embodiment 2B27 shows similar or more preferably effect in PBMC proliferation
In 4 kinds of different commercially available culture medium A IM-V (article No. 12055-091, culture mediums without containing any additive
A), X-VIVO (article No. 04-418Q, culture medium B), TexMACS (article No. 170-076-309, culture medium C) and GT-
In T551H3 (article No. WK593S, culture medium D), it is separately added into 10% fetal calf serum or 4%B27, by as described in Example 1
The primary PBMC of method culture 15 days.Accumulative cells expanded is measured by counting total cell.
Substantially it is difficult the increasing for maintaining PBMC as shown in Figure 1, when with commercially available medium culture PBMC without any addition
It grows.When cultivating PBMC in culture medium A or B, B27 shows similar effect with fetal calf serum in PBMC proliferation, such as
Lymphopoiesis multiple in 7 days more than 200 times, more than 400 times in 14 days；And works as and cultivate PBMC in culture medium C and D
When, fetal calf serum cannot maintain the proliferation of PBMC, B27 to have the effect of far better than fetal calf serum.Therefore, B27 increases in PBMC
It grows and shows similar or more preferably effect.
Embodiment 3B27 shows similar phenotypic analysis figure on culture PBMC
In 4 kinds of different commercially available culture medium A IM-V (article No. 12055-091, culture mediums without containing any additive
A), X-VIVO 15 (article No. 04-418Q, culture medium B), TexMACS (article No. 170-076-309, culture medium C) and GT-
In T551H3 (article No. WK593S, culture medium D), it is separately added into 10% fetal calf serum or 4%B27 (is purchased from ThermoFisher
, article No. 17504044), by the primary PBMC of method culture as described in Example 1 15 days.The culture of different time points is thin
PBS (Hyclone are used after born of the same parents' sample:SH30028.02B) centrifuge washing sucks supernatant, 300 μ L PBS weights of cell precipitation
Mixing is hanged, the cell suspension after mixing is divided into three pipes (number (1), (2) and (3)) often 100 μ L cell suspensions of pipe：
(1) it is used as negative control, is not added with any detection antibody.
(2) each 2 μ L of detection antibody of fluorescent marker are added in CD3/4/8 detection pipes into the pipe successively：
Anti Human CD3 FITC(Tonbo Biosciences:35-0037-T100)
Anti Human CD4 PE(Tonbo Biosciences:50-0048-T100)
Anti Human CD8 PreCP(Tonbo Biosciences:65-0088-T100)
(3) each 2 μ L of detection antibody of fluorescent marker are added in CD19/56 detection pipes into the pipe successively：
PE-Mouse anti Human CD56(BD Biosciences:561903)
PreCP-anti Human CD19(Tonbo Biosciences:65-0199-T100)
After various kinds quality control vibrates mixing, 4 DEG C are protected from light incubation 30 minutes.Sample cell is taken out, often pipe is added 1mL PBS centrifugations and washes
It washs, removes supernatant, mixing is resuspended with 200 μ L PBS respectively in cell precipitation.Above-mentioned three solencytes sample is one group, is transferred to respectively
96 hole round bottom plates, and a hole PBS (200 μ L) is added between CD3/4/8 detection holes and CD19/56 detection holes.Multiple cell samples
Product carry out dyeing and preparation of samples according to aforesaid operations.Cell sample is carried out in verifying the Guava Millipore passed through in advance
Flow cytometer detection.
Analyze T lymphocytes (CD3+), CD4+T lymphocytes (CD3+CD4+), CD8+T lymphocytes (CD3+CD8+), B
The percentage of cell (CD19+) and NK cells (CD56+).
As shown in Fig. 2, in the culture medium of addition B27, proliferation end-points CD3 positive lymphocyte ratios are more than 90%, proliferation
Terminal CD4 or CD8 positive lymphocyte ratio is also very close with fetal calf serum.That is, PBMC is cultivated with fetal calf serum and B27,
Similar figure is shown in phenotypic analysis.Therefore, in breeding and proliferation end-points, lymphocyte subgroup is distributed and adds
The lymphocyte subgroup distribution for adding fetal calf serum and being not added with any additive proliferation is almost the same.
Embodiment 4B27 is in dose-dependence in the proliferation for maintaining PBMC
By PBMC respectively in X-VIVO 15 (article No. 04-418Q, culture medium B) and TexMACS (article No. 170-076-
309, culture medium C) in, by method described in embodiment 1, PBMC is cultivated 12 days with the B27 of various concentration, was measured every 3 days tired
Count cells expanded.Accumulative cells expanded is measured by counting total cell.
As shown in figure 3, the B27 of higher concentration causes the PBMC more than 1600 times (such as 2% and 4%) in culture medium B
Proliferation, and the B27 (such as 0.25%, 0.5%, 1%, 1.25%, 1.5% and 1.75%) of low concentration is also enough that PBMC is maintained to increase
Grow (>800 times of amplifications).Similarly, the B27 (such as 2% and 4%) of higher concentration causes the PBMC more than 650 times in culture medium C
Proliferation, and the B27 (such as 0.25%, 0.5%, 1%, 1.25%, 1.5% and 1.75%) of low concentration is also enough to maintain PBMC
Proliferation (>350 times of amplifications).Therefore, B27 maintain PBMC proliferation on be in dose dependent, and with the routine of nerve cell
It is compared using concentration 2%, only needs 0.25%B27 that can maintain PBMC normal proliferatives in lymphocyte, added to minimize
Add possible adverse effect of the agent to cell.
In addition, in culture medium B, 10% fetal calf serum only has the proliferation of 900 times or so of PBMC, and in culture medium C
The proliferation of only 100 times or so of PBMC.That is, low concentration B27 (such as 0.25%, 0.5%, 1%, 1.25%,
1.5% and cultivation effect 1.75%) be better than 10% fetal calf serum cultivation effect.
Embodiment 5B27 does not influence PBMC transductions
In the culture medium B without containing any additive, 10% fetal calf serum or 4%B27 is added, as described in Example 1
Method culture activation primary PBMC, the slow virus carrier containing Chimeric antigen receptor (CAR) and GFP reporter genes turn be used in combination
It leads.Quantify transduction efficiency by the percentage of GFP positive cells when 3 days after the transduction, 6 days and 10 days.In addition, accumulative cell
Amplification times are measured by counting total cell.
As shown in fig. 4 a, the transduction efficiency of PBMC is similar in the culture medium containing fetal calf serum with B27.In addition,
As shown in Figure 4 b, for the PBMC after transduction compared with the PBMC of fetal calf serum culture, amplification rate is also similar.Therefore, B27
The transduction efficiency and the amplification rate after transduction for not influencing PBMC.
Embodiment 6B27 does not influence the cellular cytoxicity activity of the PBMC of transduction
With the PBMC containing Chimeric antigen receptor (CAR) and the slow virus carrier transduction activation of GFP reporter genes.Transduction 6
After it, PBMC is co-cultured with human tumor cell line, carries out cytotoxicity assay.
In Figure 5, UT indicates that the PBMC that do not transduce, TT indicate the PBMC of CAR transductions.As shown in Figure 5 a, the PBMC of CAR transductions
Show the specific cytotoxic activity to tumour cell.As illustrated in figures 5 b and 5 c, with the increase of the concentration of hIFNg and hIL2
Show stimulation and the proliferation degree of T cell.It can be seen that B27 does not influence the cellular cytoxicity activity of the PBMC of transduction.
More than, it is illustrated based on embodiments of the present invention, but the present invention is not limited thereto, those skilled in the art
Member it should be understood that can be implemented in a manner of carrying out modifications and changes in the range of the purport of the present invention, such deformation and
The mode of change ought to belong to the scope of protection of the present invention.
Claims (5)
- Application of the 1.B27 additives in using serum free medium culture lymphocyte, the B27 additives include biology It is element, axerophtholum aceticum, catalase, superoxide dismutase, cortisone, ethanol amine hydrochloric acid, reduced glutathione, left-handed The volumetric concentration of carnitine hydrochloric acid, progesterone, putrescine dihydrochloride and trilute, the B27 additives is 0.20- 4%, the lymphocyte is the lymphocyte in human peripheral blood single nucleus cell.
- 2. application as described in claim 1, wherein a concentration of 0.25-1.75% of the B27 additives.
- 3. application as described in claim 1, wherein the B27 additives further include DL- α D-α-tocopherol acetates, DL- α-fertility Phenol, bovine serum albumin, rh-insulin, human transferrin, D- galactolipins, sodium selenite, Phosphatidylcholin, cholesterol, ovum Phosphatide, linoleic acid, leukotrienes or combinations thereof.
- 4. application as described in any one of claims 1-3, wherein the B27 additives are added in lymphocytes culture medium, The lymphocytes culture medium includes X-VIVO 15, AIM-V, 1640, Stemline, GT-T551H3, TexMACS or its group It closes.
- Application of the 5.B27 additives in using medium culture lymphocyte, the B27 additives include biotin, acetic acid Vitamin A, catalase, superoxide dismutase, cortisone, ethanol amine hydrochloric acid, reduced glutathione, l-cn salt The volumetric concentration of acid, progesterone, putrescine dihydrochloride and trilute, the B27 additives is 0.20-4%, described Lymphocyte is the lymphocyte in human peripheral blood single nucleus cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610011671.7A CN105420190B (en) | 2016-01-08 | 2016-01-08 | Application of the B27 additives and the like in using serum free medium culture lymphocyte |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610011671.7A CN105420190B (en) | 2016-01-08 | 2016-01-08 | Application of the B27 additives and the like in using serum free medium culture lymphocyte |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105420190A CN105420190A (en) | 2016-03-23 |
| CN105420190B true CN105420190B (en) | 2018-07-31 |
Family
ID=55498733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610011671.7A Active CN105420190B (en) | 2016-01-08 | 2016-01-08 | Application of the B27 additives and the like in using serum free medium culture lymphocyte |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105420190B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754697A (en) * | 2016-12-23 | 2017-05-31 | 江西宜信堂医疗科技有限公司 | A kind of serum-free lymphocytes culture medium |
| CN109652371A (en) * | 2019-01-14 | 2019-04-19 | 温州医科大学附属第医院 | A kind of configuration method obtaining memory T-lymphocyte subgroup culture medium |
| CN112481193A (en) * | 2020-11-26 | 2021-03-12 | 海西纺织新材料工业技术晋江研究院 | Standardized culture medium for three-dimensional culture of intestinal and intestinal cancer tissue organoid and culture method |
| WO2024114809A1 (en) * | 2022-12-02 | 2024-06-06 | 上海药明巨诺生物医药研发有限公司 | Composition for cell culture |
| CN117965445B (en) * | 2024-04-02 | 2024-06-25 | 上海药明巨诺生物医药研发有限公司 | Composition for cell culture |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352343A (en) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| CN102703385A (en) * | 2012-06-26 | 2012-10-03 | 亚太干细胞科研中心有限公司 | A kind of mesenchymal stem cell culture medium |
| WO2013131045A1 (en) * | 2012-03-02 | 2013-09-06 | The Regents Of The University Of California | Expansion of alloantigen-reactive regulatory t cells |
| CN104371972A (en) * | 2013-08-12 | 2015-02-25 | 英普乐孚生物技术(上海)有限公司 | T lymphocyte culture medium without any animal original or human original component and preparation method thereof |
| CN104830765A (en) * | 2015-04-30 | 2015-08-12 | 上海泰坦科技股份有限公司 | High efficiency fetal calf serum substitute |
| CN104946587A (en) * | 2015-07-27 | 2015-09-30 | 广州市达晖生物技术有限公司 | Lymphocyte serum-free medium, as well as preparation method and application thereof |
| CN105039251A (en) * | 2015-07-27 | 2015-11-11 | 广州市达晖生物技术有限公司 | Lymphocyte serum-free medium with stable pH value as well as preparation method and application of lymphocyte serum-free medium |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8859278B2 (en) * | 2005-08-26 | 2014-10-14 | The Trustees Of Columbia University In The City Of New York | Fully human hybridoma fusion partner cell lines |
-
2016
- 2016-01-08 CN CN201610011671.7A patent/CN105420190B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352343A (en) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| WO2013131045A1 (en) * | 2012-03-02 | 2013-09-06 | The Regents Of The University Of California | Expansion of alloantigen-reactive regulatory t cells |
| CN102703385A (en) * | 2012-06-26 | 2012-10-03 | 亚太干细胞科研中心有限公司 | A kind of mesenchymal stem cell culture medium |
| CN104371972A (en) * | 2013-08-12 | 2015-02-25 | 英普乐孚生物技术(上海)有限公司 | T lymphocyte culture medium without any animal original or human original component and preparation method thereof |
| CN104830765A (en) * | 2015-04-30 | 2015-08-12 | 上海泰坦科技股份有限公司 | High efficiency fetal calf serum substitute |
| CN104946587A (en) * | 2015-07-27 | 2015-09-30 | 广州市达晖生物技术有限公司 | Lymphocyte serum-free medium, as well as preparation method and application thereof |
| CN105039251A (en) * | 2015-07-27 | 2015-11-11 | 广州市达晖生物技术有限公司 | Lymphocyte serum-free medium with stable pH value as well as preparation method and application of lymphocyte serum-free medium |
Non-Patent Citations (4)
| Title |
|---|
| A serum-free medium for human primary T lymphocyte culture;Alan L. Causey等;《Journal of Immunological Methods》;19941231;第175卷;第115-121页 * |
| Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasal, a New Serum-free Medium Combination;G.J. Brewer等;《Journal of Neuroscience Research》;19931231;第35卷;第567-576页 * |
| 无血清培养基的成分改进及应用现状;杨颜慈等;《生物学杂志》;20130228;第30卷(第1期);第82-85页 * |
| 硒和B-27添加剂在神经干细胞存活与分化中的作用;田东萍等;《中华预防医学杂志》;20050930;第39卷(第5期);第324-328页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105420190A (en) | 2016-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7319638B2 (en) | Medium composition and method for culturing cells or tissues using the composition | |
| CN105420190B (en) | Application of the B27 additives and the like in using serum free medium culture lymphocyte | |
| CN107922925B (en) | Method for natural killer cell expansion | |
| US20240279597A1 (en) | Culture medium composition and method of culturing cell or tissue using thereof | |
| JP6799895B2 (en) | Production method of TCRγδ + T cells | |
| EP3134512B1 (en) | Method for automated generation of genetically modified t cells | |
| Oyer et al. | Generation of highly cytotoxic natural killer cells for treatment of acute myelogenous leukemia using a feeder-free, particle-based approach | |
| JP2022058480A (en) | Method and composition for inducing hematopoietic cell differentiation | |
| CN106434556B (en) | A method for inducing and expanding type I NKT cells in vitro | |
| AU2010266016B2 (en) | Differentiated pluripotent stem cell progeny depleted of extraneous phenotypes | |
| TWI612137B (en) | Method for producing composition containing immune cells and composition for treating cancer | |
| CN102471753A (en) | Stem cell immunomodulation application method and apparatus | |
| CN107002029A (en) | The inducing culture and method cultivated and treated for stem cell | |
| EP3831936A1 (en) | Method for producing regenerated t cell population via ips cells | |
| Hook et al. | Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays | |
| EP3114215A1 (en) | Pooled nk cells from ombilical cord blood and their uses for the treatment of cancer and chronic infectious disease | |
| CN108060129A (en) | Regulatory T cells amplification in vitro method | |
| CN109370985A (en) | Serum-free medium for large-scale culture of human umbilical cord mesenchymal stem cells | |
| CN107151654A (en) | A kind of culture medium of people source T lymphocytes and its preparation method and application | |
| KR20030032905A (en) | Method of proliferating natural killer cells | |
| CN112805015A (en) | Hematopoietic stem cell and hematopoietic progenitor cell expansion system | |
| CN107109366B (en) | Tolerogenic dendritic cells, its generation method and application thereof | |
| Torrez et al. | Derivation of neural progenitors and retinal pigment epithelium from common marmoset and human pluripotent stem cells | |
| CN109983118A (en) | Pass through stem cell induction liver cell with RNA | |
| TWI860972B (en) | Markers for hematopoietic progenitor cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180212 Address after: 214109 Huishan Road No. 1719-6, Huishan Economic Development Zone, Wuxi City, Jiangsu Province, A area Applicant after: Wuxi pharmaceutical Ming Shengji medical science and Technology Co., Ltd. Address before: 200131 Shanghai City, Pudong New Area Chinese Foot Road (Shanghai) Free Trade Zone No. 288 Building No. 1 Applicant before: Shanghai Yaoming Kangde New Medicine Development Co., Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 214109 A area 1719-6, Huishan Road, Huishan Economic Development Zone, Wuxi, Jiangsu. Patentee after: Wuxi Shengji Medicine Technology Co., Ltd. Address before: 214109 A area 1719-6, Huishan Road, Huishan Economic Development Zone, Wuxi, Jiangsu. Patentee before: Wuxi pharmaceutical Ming Shengji medical science and Technology Co., Ltd. |