CN108865995A - Promote Fiber differentiation composition and its application of the CD8 positive T cell proliferation in spleen source - Google Patents
Promote Fiber differentiation composition and its application of the CD8 positive T cell proliferation in spleen source Download PDFInfo
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Abstract
The present invention relates to a kind of Fiber differentiation composition of CD8 positive T cell proliferation for promoting spleen source and its applications.The Fiber differentiation composition includes cell culture medium and the inducible factor for making an addition to cell culture medium, inducible factor includes phytohemagglutin phytolectin, CD3 antibody and CD28 antibody, the final concentration of 1 μ μ of g/mL~10 g/mL of phytohemagglutin phytolectin, the final concentration of 1 μ μ of g/mL~10 g/mL of the final concentration of 1 μ μ of g/mL~10 g/mL, the CD28 antibody of CD3 antibody.Above-mentioned Fiber differentiation composition can promote the CD8 positive T cell in spleen source to be largely proliferated.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of CD8 positive T cell proliferation for promoting spleen source
Fiber differentiation composition and its application.
Background technique
T lymphocyte derives from the multipotential stem cell of marrow.In embryonic period, embryonic phase and nascent phase, a part of multipotency in marrow is dry
Cell or pre-T cell move in thymus gland, the differentiation and maturation under the induction of thymin, become with immunocompetent T lymph
Cell.T lymphocyte is different according to function in immune response, can be divided into T helper cell (Th), suppressor T lymphocyte (TS), effect
T cell (Te) and cytotoxic T cell (Tc).Wherein, cytotoxic T cell is also known as CD8 positive T cell, surface expression CD8
Molecule is a kind of immunocyte with killing target cell.CD8 positive T cell is in fighting external antigen or tumour cell
Play the role of very important, referred to as " killer " T cell.Therefore, the ratio of CD8 positive T cell how is improved to guarantee T leaching
The immunocompetence of bar cell is most important.
The immunocyte of peripheral blood is extracted and to carry out in-vitro multiplication to it thin to obtain CD8 positive T currently, mainly passing through
Born of the same parents.However periphery blood lymphocyte accounting is less, is unfavorable for largely obtaining CD8 positive T cell.Some researchs pass through immune
The stimulating factors such as interleukin 2 are added during cell Proliferation to promote CD8 positive T cell to expand, but what it was obtained
The amount of CD8 positive T cell is still lower, is not able to satisfy actual demand.
Summary of the invention
Based on this, it is necessary to provide a kind of Fiber differentiation combination that the CD8 positive T cell for promoting spleen source is largely proliferated
Object and its application.
A kind of Fiber differentiation composition for the CD8 positive T cell proliferation promoting spleen source, including cell culture medium and add
It is added on the inducible factor of the cell culture medium, the inducible factor includes phytohemagglutin phytolectin, CD3 antibody and CD28 antibody, institute
State the final concentration of 1 μ μ of g/mL~10 g/mL of phytohemagglutin phytolectin, the final concentration of 1 μ g/mL~10 μ g/mL of the CD3 antibody, institute
State the final concentration of 1 μ μ of g/mL~10 g/mL of CD28 antibody.
This research by being added suitable phytohemagglutin phytolectin, CD3 antibody and CD28 antibody, Neng Gouyou in cell culture medium
Effect ground promotes the CD8 positive T cell in spleen source to be largely proliferated, to obtain a large amount of CD8 positive T cell.Experiment proves that
Cell number of the CD8 positive T cell in spleen source after above-mentioned Fiber differentiation composition culture is through being not added with inducible factor
2 times~5 times after cell culture medium culture.
The final concentration of 4 μ μ of g/mL~6 g/mL of the phytohemagglutin phytolectin in one of the embodiments, the CD3 antibody
The final concentration of 4 μ μ of g/mL~6 g/mL, the final concentration of 4 μ μ of g/mL~6 g/mL of the CD28 antibody.
The cell culture medium includes basal medium, makes an addition to the basal medium in one of the embodiments,
In fetal calf serum and make an addition to Sodium Pyruvate in the basal medium, the volumn concentration of the fetal calf serum is
10%~15%, final concentration of 0.8mM~1.2mM of the Sodium Pyruvate.
The basal medium includes final concentration of 1800mg/L~2200mg/L containing sugar in one of the embodiments,
The mineral member of component, the amino acid composition of final concentration of 908mg/L~1247mg/L, final concentration of 6955mg/L~7505mg/L
The vitamin component of plain component and final concentration of 33.6mg/L~45.7mg/L.
The saccharic composition that contains includes glucose in one of the embodiments,.
In one of the embodiments, in terms of mass fraction, the amino acid composition includes 270 parts~310 parts of L- essence
Propylhomoserin, 40 parts~60 parts of L- asparagine, 15 parts~25 parts of L-ASPARTIC ACID, 55 parts~75 parts of two hydrochloric acid of l-cysteine
Salt, 15 parts~25 parts of Pidolidone, 7 parts~13 parts of glycine, 10 parts~20 parts of L-Histidine, 15 parts~25 parts of L-
Hydroxyproline, 40 parts~60 parts of l-Isoleucine, 40 parts~60 parts of L-Leu, 35 parts~45 parts of L-lysine hydrochloric acid
Salt, 10 parts~20 parts of l-methionine, 10 parts~20 parts of L-phenylalanine, 15 parts~25 parts of L-PROLINE, 20 parts~
40 parts of Serine, 15 parts~25 parts of L-threonine, 4 parts~6 parts of L-Trp, 17 parts~28 parts of l-tyrosine,
260 parts~340 parts of L-Glutamine and 15 parts~25 parts of Valine.
In one of the embodiments, in terms of mass fraction, the mineral element component includes 8 parts~12 parts of nitric acid
Calcium, 4.5 parts~5.5 parts of magnesium sulfate, 65 parts~70 parts of sodium dihydrogen phosphate, 38 parts~43 parts of potassium chloride and 580 parts~620
The sodium chloride of part.
In one of the embodiments, in terms of mass fraction, the vitamin component includes 0.1 part~0.3 part of biology
Element, 0.2 part~0.3 part of D-VB5 calcium, 0.8 part~1.2 parts of folic acid, 30 parts~40 parts of inositol, 0.8 part~1.2 parts
Niacinamide, 0.8 part~1.2 parts of puridoxine hydrochloride, 0.1 part~0.3 part of riboflavin and 0.8 part~1.2 parts of thiamine hydrochloride
Element.
A method of the CD8 positive T cell proliferation promoting spleen source includes the following steps：
The CD8 positive T cell in spleen source is inoculated in the described in any item Fiber differentiation compositions of above-described embodiment
Culture.
The inoculum concentration of the CD8 positive T cell is 0.9x10 in one of the embodiments,5A/mL~1.5 × 105A/
mL。
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.It is shown below of the invention
Preferred embodiment.But the invention can be realized in many different forms, however it is not limited to implementation described herein
Example.It is made the disclosure of the present invention more thorough and comprehensive on the contrary, purpose of providing these embodiments is.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
This research provides a kind of Fiber differentiation composition of CD8 positive T cell proliferation for promoting spleen source comprising thin
Born of the same parents' culture medium and the inducible factor for making an addition to cell culture medium, inducible factor include phytohemagglutin phytolectin, CD3 antibody and CD28 anti-
Body, the final concentration of 1 μ μ of g/mL~10 g/mL, CD28 of the final concentration of 1 μ μ of g/mL~10 g/mL, the CD3 antibody of phytohemagglutin phytolectin
The final concentration of 1 μ μ of g/mL~10 g/mL of antibody.
It should be noted that final concentration refers to the final concentration in Fiber differentiation composition.Such as：The end of phytohemagglutin phytolectin
Concentration is that 1 μ g/mL refers to final concentration of 1 μ g/mL of the phytohemagglutin phytolectin in Fiber differentiation composition.
Phytohemagglutin phytolectin (PHA) is the compound of oligosaccharide and protein, belongs to macromolecule glycoprotein, has to red blood cell
Certain agglutination.
The final concentration of 4 μ μ of g/mL~6 g/mL of phytohemagglutin phytolectin in one of the embodiments,.Some implementations wherein
In example, final concentration of 1 μ g/mL, 2 μ g/mL, 4 μ g/mL, 5 μ g/mL, 6 μ g/mL, 8 μ g/mL or the 10 μ g/mL of phytohemagglutin phytolectin.
CD3 (differentiation cluster 3) is a kind of important leukocyte differentiation antigen, is present in T cell surface.CD3 passes through salt bridge and T
Cell antigen receptor (TCR) is connected, and participates in the signal transduction of T cell.CD3 antibody is the antibody for being directed to CD3.
The final concentration of 4 μ μ of g/mL~6 g/mL of CD3 antibody in one of the embodiments,.Some embodiments wherein
In, final concentration of 1 μ g/mL, 2 μ g/mL, 4 μ g/mL, 5 μ g/mL, 6 μ g/mL, 8 μ g/mL or the 10 μ g/mL of CD3 antibody.
CD28 (differentiation cluster 28) is used as a kind of T cell surface molecular, and a kind of important leukocyte differentiation antigen.CD28
Antibody is the antibody for being directed to CD28.
In wherein some embodiments, the final concentration of 4 μ μ of g/mL~6 g/mL of CD28 antibody.Some embodiments wherein
In, final concentration of 1 μ g/mL, 2 μ g/mL, 4 μ g/mL, 5 μ g/mL, 6 μ g/mL, 8 μ g/mL or the 10 μ g/mL of CD28 antibody.
The ratio between final concentration of phytohemagglutin phytolectin and CD3 antibody is 1: 2~1: 0.5 in one of the embodiments,.Using this
Concentration ratio is more advantageous to a large amount of proliferation of the CD8 positive T cell in spleen source.Preferably, phytohemagglutin phytolectin and CD3 antibody
The ratio between final concentration is 1: 1.
Cell culture medium includes basal medium, makes an addition to tire ox in basal medium in one of the embodiments,
Serum and make an addition to Sodium Pyruvate in basal medium.
Basal medium includes final concentration of 1800mg/L~2200mg/L containing sugared group in one of the embodiments,
Divide, the mineral element of the amino acid composition of final concentration of 908mg/L~1247mg/L, final concentration of 6955mg/L~7505mg/L
The vitamin component of component and final concentration of 33.6mg/L~45.7mg/L.It is trained it should be noted that final concentration refers in cell
Support the final concentration in base.Such as：Final concentration of 1800mg/L containing saccharic composition refers to the end containing saccharic composition in cell culture medium
Concentration is 1800mg/L.
Further, basal medium includes final concentration of 1900mg/L~2100mg/L containing saccharic composition, final concentration of
The amino acid composition of 1000mg/L~1100mg/L, the mineral element component of final concentration of 7000mg/L~7300mg/L and end are dense
Degree is the vitamin component of 38mg/L~42mg/L.Further, basal medium includes containing for final concentration of 2000mg/L
Saccharic composition, the amino acid composition of final concentration of 1080mg/L, the mineral element component of final concentration of 7225mg/L and final concentration of
The vitamin component of 40mg/L.
It include in one of the embodiments, glucose containing saccharic composition.It should be noted that being not limited to Portugal containing saccharic composition
Grape sugar can also include other containing saccharic composition, such as can also include fructose.
In one of the embodiments, in terms of mass fraction, amino acid composition include 270 parts~310 parts L-arginine,
40 parts~60 parts of L- asparagine, 15 parts~25 parts of L-ASPARTIC ACID, 55 parts~75 parts of l-cysteine dihydrochloride, 15
Part~25 parts of Pidolidone, 7 parts~13 parts of glycine, 10 parts~20 parts of L-Histidine, 15 parts~25 parts of L- hydroxyl dried meat
Propylhomoserin, 40 parts~60 parts of l-Isoleucine, 40 parts~60 parts of L-Leu, 35 parts~45 parts of L lysine HCL,
10 parts~20 parts of l-methionine, 10 parts~20 parts of L-phenylalanine, 15 parts~25 parts of L-PROLINE, 20 parts~40 parts
Serine, 15 parts~25 parts of L-threonine, 4 parts~6 parts of L-Trp, 17 parts~28 parts of l-tyrosine, 260 parts
~340 parts of L-Glutamine and 15 parts~25 parts of Valine.
Further, in terms of mass fraction, amino acid composition include 280 parts~300 parts L-arginine, 45 parts~55 parts
L- asparagine, 17 parts~22 parts of L-ASPARTIC ACID, 60 parts~70 parts of l-cysteine dihydrochloride, 17 parts~23 parts
Pidolidone, 8 parts~12 parts of glycine, 13 parts~18 parts of L-Histidine, 17 parts~23 parts of L- hydroxyproline, 45 parts~
55 parts of l-Isoleucine, 45 parts~55 parts of L-Leu, 37 parts~42 parts of L lysine HCL, 13 parts~17 parts
L-methionine, 13 parts~18 parts of L-phenylalanine, 18 parts~23 parts of L-PROLINE, 25 parts~35 parts of Serine,
17 parts~23 parts of L-threonine, 4.5 parts~5.5 parts of L-Trp, 19 parts~26 parts of l-tyrosine, 280 parts~330 parts
L-Glutamine and 17 parts~22 parts of Valine.
Further, in terms of mass fraction, amino acid composition includes 290 parts of L-arginine, 50 parts of L- door winter acyl
Amine, 20 parts of L-ASPARTIC ACID, 65 parts of l-cysteine dihydrochloride, 20 parts of Pidolidone, 10 parts of glycine, 15 parts
L-Histidine, 20 parts of L- hydroxyproline, 50 parts of l-Isoleucine, 50 parts of L-Leu, 40 parts of L-lysine hydrochloric acid
Salt, 15 parts of l-methionine, 15 parts of L-phenylalanine, 20 parts of L-PROLINE, 30 parts of Serine, 20 parts of L- Soviet Union
Propylhomoserin, 5 parts of L-Trp, 23 parts of l-tyrosine, 300 parts of L-Glutamine and 20 parts of Valine.
In one of the embodiments, in terms of mass fraction, mineral element component include 8 parts~12 parts calcium nitrate, 4.5
Part~5.5 parts of magnesium sulfate, 65 parts~70 parts of sodium dihydrogen phosphate, 38 parts~43 parts of potassium chloride and 580 parts~620 parts of chlorine
Change sodium.Further, in terms of mass fraction, mineral element component include 9 parts~11 parts calcium nitrate, 4.75 parts~5.25 parts
Magnesium sulfate, 67 parts~72 parts of sodium dihydrogen phosphate, 39 parts~41 parts of potassium chloride and 590 parts~610 parts of sodium chloride.More into one
Step ground, in terms of mass fraction, mineral element component include 10 parts of calcium nitrate, 5 parts of magnesium sulfate, 60 parts of sodium dihydrogen phosphate,
40 parts of potassium chloride and 600 parts of sodium chloride.
In one of the embodiments, in terms of mass fraction, vitamin component include 0.1 part~0.3 part biotin,
0.2 part~0.3 part of D-VB5 calcium, 0.8 part~1.2 parts of folic acid, 30 parts~40 parts of inositol, 0.8 part~1.2 parts of nicotinoyl
Amine, 0.8 part~1.2 parts of puridoxine hydrochloride, 0.1 part~0.3 part of riboflavin and 0.8 part~1.2 parts of thiamine hydrochloride.Into
One step, in terms of mass fraction, vitamin component includes 0.2 part of biotin, 0.25 part of D-VB5 calcium, 1 part of folic acid, 35
The inositol, 1 part of niacinamide, 1 part of puridoxine hydrochloride, 0.2 part of riboflavin and 1 part of thiamine hydrochloride of part.
In one of the embodiments, in terms of mass fraction, vitamin component further includes that 0.003 part~0.07 part of dimension is raw
Plain B12.Further, in terms of mass fraction, vitamin component further includes 0.005 part of vitamin B12.
Basal medium further includes the assisted group of final concentration of 3.3mg/L~4.7mg/L in one of the embodiments,
Point.Helper component is selected from least one of glutathione and choline chloride.It is trained it should be noted that final concentration refers in cell
Support the final concentration in base.Such as：The final concentration of 3.3mg/L of helper component refers to that the end of helper component in cell culture medium is dense
Degree is 3.3mg/L.
In one of the embodiments, helper component in terms of mass fraction including 0.7 part~1.2 parts glutathione and
2.5 parts~3.5 parts of choline chloride.Wherein, glutathione is reduced glutathione.Further, helper component is with mass parts
Number meter includes 1 part of glutathione and 3 parts of choline chloride.
The volumn concentration of fetal calf serum is 10%~15% in one of the embodiments,.It should be noted that this
The volumn concentration at place refers to volumn concentration in cell culture medium.Such as：The volumn concentration of fetal calf serum
Refer to that the volumn concentration of fetal calf serum in cell culture medium is 10% for 10%.Further, the volume of fetal calf serum
Percentage composition is 12%.
Final concentration of 0.8mM~1.2mM of Sodium Pyruvate in one of the embodiments,.By in cell culture medium
Sodium Pyruvate is added, is conducive to CD8 positive T cell and absorbs each nutriment, promote the growth and breeding of CD8 positive T cell.It needs
It is noted that final concentration refers to final concentration in cell culture medium.Such as：The final concentration of 1mM of Sodium Pyruvate refers to third
The final concentration of 1mm of ketone acid sodium in cell culture medium.Further, the final concentration of 1mM of Sodium Pyruvate.
The ratio between final concentration of Sodium Pyruvate and phytohemagglutin phytolectin is 8.8~132 in one of the embodiments,.This ratio
Under, CD8 positive T cell can be quickly proliferated, to obtain a large amount of CD8 positive T cell.Preferably, Sodium Pyruvate and plant
The ratio between final concentration of hemagglutinin is 22.
Cell culture medium further includes the antibiosis that volumn concentration is 0.8%~1.2% in one of the embodiments,
Element, so as to inhibit the growth of contaminating microorganisms.Antibiotic is selected from least one of penicillin and streptomysin.Further, carefully
Born of the same parents' culture medium further includes the penicillin that volumn concentration is 0.4%~0.6% and volumn concentration is 0.4%~0.6%
Streptomysin.Further, cell culture medium further includes the penicillin and volumn concentration that volumn concentration is 0.5%
For 0.5% streptomysin.
Cell culture medium further includes pH adjusting agent in one of the embodiments, so that the pH of cell culture medium is maintained at
7.2~7.4.Further, pH adjusting agent is sodium bicarbonate.Sodium bicarbonate is final concentration of in one of the embodiments,
1.5g/L~2.5g/L.Further, the final concentration of 2g/L of sodium bicarbonate.
It should be noted that cell culture medium is not limited to the above-mentioned cell culture medium pointed out, cell culture medium may be
RPMI-1640 culture medium or DMEM culture medium.
The CD8 positive T cell in spleen source is the CD8 positive T cell or reality of the spleen of people in one of the embodiments,
Test the CD8 positive T cell of the spleen of animal.Since spleen is the maximum immune organ of body, total lymphoid tissue total amount is accounted for
25%, contain a large amount of lymphocyte and macrophage.Using spleen as the source of CD8 positive T cell, be conducive to largely obtain
CD8 positive T cell.
The CD8 positive T cell in spleen source is the CL- for being purchased from Wuhan Pu Nuosai company in one of the embodiments,
The CTL spleen cell of 0331 lot number.It should be noted that the CD8 positive T cell in spleen source be not limited to it is above-mentioned commercially available
Cell is also possible to the CD8 positive T cell isolated by spleen.
This research by being added suitable phytohemagglutin phytolectin, CD3 antibody and CD28 antibody, Neng Gouyou in cell culture medium
Effect ground promotes the CD8 positive T cell in spleen source to be largely proliferated, to obtain a large amount of CD8 positive T cell.Meanwhile this research
Stimulating factor used is easy to obtain and can be obviously improved the number of CD8 positive T cell, is conducive to large-scale industry metaplasia
CD8 positive T cell is produced, to be applied in the drug of preparation treatment entity tumor or liquid tumors.Experiment proves that spleen source
Cell number of the CD8 positive T cell after above-mentioned Fiber differentiation composition culture be through being not added with the cell culture of inducible factor
2 times~5 times after base culture.
This research also provides a kind of method of CD8 positive T cell proliferation for promoting spleen source comprising following operation：
The CD8 positive T cell in spleen source is inoculated in above-mentioned Fiber differentiation composition and is cultivated.
The inoculum concentration of CD8 positive T cell is 0.9 × 10 in one of the embodiments,5A/mL~1.5 × 105A/mL.
Incubation time is 3 days~7 days in one of the embodiments, adds Fiber differentiation composition every 2 days~3 days.
Further, the Fiber differentiation composition and this time added every time have been used for the Fiber differentiation of culture CD8 positive T cell before adding
The volume ratio of composition is 1: 3~1: 1.
The CD8 positive T cell in spleen source is the CD8 positive T cell or reality of the spleen of people in one of the embodiments,
Test the CD8 positive T cell of the spleen of animal.Since spleen is the maximum immune organ of body, total lymphoid tissue total amount is accounted for
25%, contain a large amount of lymphocyte and macrophage.Using spleen as the source of CD8 positive T cell, be conducive to largely obtain
CD8 positive T cell.
The CD8 positive T cell in spleen source is Wuhan Pu Nuosai company CL-0331 lot number in one of the embodiments,
CTL spleen cell.It should be noted that the CD8 positive T cell in spleen source is not limited to above-mentioned commercially available cell,
It can be the CD8 positive T cell isolated by spleen.
Separating CD8 positive T cell from spleen in one of the embodiments, includes following operation S111~S112：
S111, mononuclearcell is separated from spleen.
Mononuclearcell is the cell of only one nucleus.The mononuclearcell separated in spleen includes that lymph is thin
Born of the same parents and monocyte.
The operation of S111 in one of the embodiments, is specially：Spleen is broken, sieving obtains cell liquid, by cell
Liquid density gradient centrifugation, obtains mononuclearcell.
In one of the embodiments, that spleen is broken, sieving obtains in the operation of cell liquid, and the mode of broken spleen is selected
From at least one of shredding and grind.
In one of the embodiments, that spleen is broken, sieving obtains in the operation of cell liquid, by broken spleen mistake
200 mesh~400 mesh obtain cell liquid.
It is specially by the operation of cell liquid density gradient centrifugation in one of the embodiments,：Cell liquid is added to leaching
In bar cell density gradient centrifugation separating liquid, it is centrifuged 10 minutes~20 minutes, collects in 2 DEG C~8 DEG C, 1000rpm~2000rpm
Tunica albuginea confluent monolayer cells, as mononuclearcell.
It should be noted that during cell liquid is added to lymphocyte density gradient centrifugation separating liquid, cell liquid
It should be flowed into as far as possible along chamber wall, so as to form clearly boundary between lymphocyte density gradient centrifugation separating liquid and cell liquid
Limit, to be conducive to efficiently separating for mononuclearcell.
It further include diluting cell liquid in one of the embodiments, by before the operation of cell liquid density gradient centrifugation
It is 10 to total cell number6A/mL~107The operation of a/mL.It should be noted that if the cell number of cell liquid is 106A/
ML~107A/mL omits the diluted operation of cell liquid.
The volume of cell liquid and the volume ratio of lymphocyte density gradient centrifugation separating liquid are in one of the embodiments,
1~3, lymphocyte density gradient centrifugation separating liquid is ficoll lymphocyte separation medium.Further, ficoll lymphocyte
Separating liquid is up to the ficoll lymphocyte separation medium that section is producer.It should be noted that lymphocyte density gradient centrifugation point
Chaotropic is not limited to the above-mentioned separating liquid pointed out, or other separating liquids, such as can be the ficoll lymph of Cedarlane
Cell separating liquid, the ficoll lymphocyte separation medium of GE or ficoll lymphocyte separation medium of Sigam etc..
Spleen is commercially available spleen, such as the spleen of pig etc. in one of the embodiments,.
S112, CD8 positive T cell is separated from mononuclearcell.
The mode for separating CD8 positive T cell from mononuclearcell in one of the embodiments, is magnetic bead sorting.Its
In, magnetic bead used in magnetic bead sorting is the magnetic bead with the bis- positive indications of CD8 and CD3.Preferably, magnetic bead used in magnetic bead sorting
The magnetic bead with the bis- positive indications of CD8 and CD3 that lot number for Mei Tian girl company is 130-090-859.
In one of the embodiments, during magnetic bead sorting, total cell number is 10 in mononuclearcell6A/mL
~107A/mL.The volume ratio of mononuclearcell and magnetic bead is 4: 1.
The method for promoting the CD8 positive T cell proliferation in spleen source in this research is easy to operate, and stimulating factor used is equal
It is easily obtained and can be obviously improved the number of the CD8 positive T cell in spleen source, is conducive to large-scale industrial production spleen
The CD8 positive T cell in source.It is controlled in addition, the CD8 positive T cell obtained by above embodiment can also be applied to preparation
It treats in entity tumor or the drug of liquid tumors.
The following are specific embodiment parts：
In following embodiment, unless otherwise instructed, test method without specific conditions, usually according to normal condition,
For example, see Pehanorm Brooker, EF, not the written molecular cloning of Ritchie, T Manny A Disi etc. (Jin Dongyan, Li Mengfeng etc. are translated) is real
Test (the Beijing guide [M]：Science Press, 1992) method that condition or kit manufacturer described in are recommended is realized.
Reagent used in embodiment is commercially available.
In following embodiment, unless otherwise instructed, SD rat is purchased from Guangdong Province's animal center.HBSS buffer is purchased from
Sigma company.It is company that ficoll lymphocyte separation medium, which is purchased from up to section,.The pH of PBS buffer solution is 7.2~7.4, and is purchased from
Hyclone company.RPMI1640 culture medium is purchased from Gibco company.DMEM culture medium is purchased from Gibco company.Dual anti-magnetic bead is band
There is the magnetic bead of the bis- positive marks of CD8 and CD3, and is purchased from Mei Tian girl company.Phytohemagglutin phytolectin is purchased from Sigma company.The purchase of CD3 antibody
In BD company.CD28 antibody is purchased from BD company.IL-2 (interleukin 2) is purchased from BD company.Penicillin is purchased from Gibco company.
Streptomysin is purchased from Gibco company.
In following embodiment, unless otherwise instructed, " part " refers both to mass fraction.
Embodiment 1
Extract the total cell of spleen：
(1) SD rat is taken, after giving anesthesia with anesthetic, superclean bench is transferred to, cuts spleen group after opening abdominal cavity
It knits, is put into the pre- HBSS buffer for being cooled to 4 DEG C.
(2) during the spleen tissue being placed in HBSS buffer is transferred between the cell manipulation after disinfection.It will be clean
The Tissue Culture Dish of 60mm is placed on ice, and the HBSS caching liquid after 3mL, pre-cooling is added in Tissue Culture Dish, by spleen group
It knits and is transferred in the Tissue Culture Dish equipped with HBSS buffer.
(3) spleen tissue is shredded with scissors, the spleen tissue after shredding is ground, by the spleen tissue after grinding
200 mesh screens are placed in, and rinse the sieve with the HBSS buffer of 1mL, collect filtrate, the as cell containing spleen total cell
Liquid.
Embodiment 2
Extract mononuclearcell：
(1) centrifuge tube of clean 15mL is taken, ficoll lymphocyte separation medium is packed into, is added along the tube wall of centrifuge tube real
The cell liquid that example 1 obtains is applied, so as to form clearly boundary between ficoll lymphocyte separation medium and cell liquid.Wherein, carefully
Total cell number is 5 × 10 in cytosol6The volume ratio of a/mL, ficoll lymphocyte separation medium and cell liquid is 1: 2.
(2) centrifuge tube is carefully placed into horizontal centrifuge (Hunan instrument company's T D5A-WS), is centrifuged 15 points in 25 DEG C, 1500rpm
Clock.After centrifugation, centrifuge tube is carefully taken out, forms tunica albuginea layer between ficoll lymphocyte separation medium layer and cell liquid layer,
As mononuclearcell layer.
(3) tunica albuginea confluent monolayer cells are taken out with pipette, is transferred in the centrifuge tube of another clean 15mL.3mL is added
PBS buffer solution wash tunica albuginea confluent monolayer cells, in 25 DEG C, 1000rpm be centrifuged 10 minutes, abandon supernatant；Add PBS buffer solution repetition
It washed once, collect precipitating, as mononuclearcell.
Embodiment 3
CD8 positive T cell is separated from mononuclearcell：
(1) precipitating for obtaining embodiment 2 is resuspended with RPMI1640 culture medium, obtains re-suspension liquid, the cell in re-suspension liquid
Number is 5 × 106A/mL.
(2) re-suspension liquid is transferred in the culture bottle equipped with RPMI1640 culture medium, is placed in 37 DEG C, 5%CO2In incubator
Culture 1 day, obtains culture solution, and the volume ratio of re-suspension liquid and RPMI1640 culture medium is 10.By culture solution in 4 DEG C, 1600rpm from
The heart 10 minutes, obtain sediment.
(3) sediment is resuspended with PBS buffer solution, dual anti-magnetic bead is added and is protected from light incubation 10min in 4 DEG C.Wherein, PBS is slow
Fliud flushing additive amount is：4×107The PBS buffer solution of 160 μ L is added in a mononuclearcell.The additive amount of dual anti-magnetic bead is：4×107
The dual anti-magnetic bead of 40 μ L is added in a mononuclearcell.
(4) after being incubated for, PBS buffer solution is added again and dual anti-magnetic bead is protected from light in 4 DEG C and is incubated for 15min.Wherein, PBS
Buffer additive amount is：4×107The PBS buffer solution of 320 μ L is added in a mononuclearcell.The additive amount of dual anti-magnetic bead is：4×
107The dual anti-magnetic bead of 80 μ L is added in a mononuclearcell.
(5) slow with PBS by the excessively magnetic microtrabeculae (the lot number 130-090-859 of Mei Tian girl company) of Incubating Solution after being incubated for
Fliud flushing rinses lower CD8 negative cells.It after removing magnetic field, is rinsed with PBS buffer solution, collects flushing liquor, as contain CD8 positive T
The flushing liquor of cell.
(6) flushing liquor is centrifuged 10 minutes in 25 DEG C, 1200rpm, is discarded supernatant.The PBS washing for adding 5mL, in 25
DEG C, 1200rpm be centrifuged 10 minutes, discard supernatant, obtain CD8 positive T cell.
Embodiment 4
The present embodiment is as follows during expanding to CD8 positive T cell：
Amplification cultivation is carried out to CD8 positive T cell using culture plate, the Fiber differentiation composition of 4mL is added in each hole,
And 4.5 × 10 are accessed in every hole5A CD8 positive T cell, is placed in 37 DEG C, 5%CO2Incubator culture, incubation time are 3 days.
Wherein, the hole cultivated is 3 holes.CD8 positive T cell used is the CD8 positive T cell that embodiment 3 obtains.Induction training
Support includes cell culture medium, PHA, CD3 antibody and CD28 antibody in composition.Final concentration of 1 μ g/mL, the CD3 antibody of PHA
The final concentration of 1 μ g/mL of final concentration of 1 μ g/mL, CD28 antibody.Cell culture medium includes basal medium, makes an addition to basic training
The fetal calf serum in base is supported, Sodium Pyruvate in basal medium is made an addition to, makes an addition to penicillin in basal medium, addition
Streptomysin in basal medium and sodium bicarbonate in basal medium is made an addition to, the volumn concentration of fetal calf serum is
10%, the final concentration of 0.8mM of Sodium Pyruvate, the penicillin that volumn concentration is 0.4%, the volumn concentration of streptomysin
It is 0.4%, the final concentration of 1.5g/L of sodium bicarbonate.
Wherein, basal medium includes the amino containing saccharic composition, final concentration of 1000mg/L of final concentration of 1900mg/L
The vitamin component of acid constituents, the mineral element component of final concentration of 7000mg/L and final concentration of 38mg/L.It is containing saccharic composition
Glucose.Amino acid composition includes 270 parts of L-arginine, 40 parts of L- asparagine, 15 parts of L-ASPARTIC ACID, 55 parts
L-cysteine dihydrochloride, 15 parts of Pidolidone, 7 parts of glycine, 10 parts of L-Histidine, 15 parts of L- hydroxyproline,
40 parts of l-Isoleucine, 40 parts of L-Leu, 35 parts of L lysine HCL, 10 parts of l-methionine, 10 parts
L-phenylalanine, 15 parts of L-PROLINE, 20 parts of Serine, 15 parts of L-threonine, 4 parts of L-Trp, 17 parts
L-tyrosine, 260 parts of L-Glutamine and 15 parts of Valine.Mineral element component include 8 parts calcium nitrate, 4.5 parts
Magnesium sulfate, 65 parts of sodium dihydrogen phosphate, 38 parts of potassium chloride and 580 parts of sodium chloride.Vitamin component includes 0.1 part of life
Object element, 0.2 part of D-VB5 calcium, 0.8 part of folic acid, 30 parts of inositol, 0.8 part of niacinamide, 0.8 part of puridoxine hydrochloride,
0.1 part of riboflavin and 0.8 part of thiamine hydrochloride.
Embodiment 5
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 4, the difference is that：
The Fiber differentiation composition of 3mL is added in each hole, final concentration of 10 μ g/mL, the CD3 antibody of PHA in Fiber differentiation composition
Final concentration of 10 μ g/mL, CD28 antibody final concentration of 10 μ g/mL.Cell culture medium includes basal medium, makes an addition to base
Fetal calf serum in basal culture medium, make an addition to Sodium Pyruvate in basal medium, make an addition to penicillin in basal medium,
The streptomysin that makes an addition in basal medium and sodium bicarbonate in basal medium is made an addition to, the volume basis of fetal calf serum contains
Amount is 15%, the final concentration of 1.2mM of Sodium Pyruvate, the penicillin that volumn concentration is 0.6%, the volume basis of streptomysin
Content is 0.6%, the final concentration of 2.5g/L of sodium bicarbonate.
Wherein, basal medium includes the amino containing saccharic composition, final concentration of 1247mg/L of final concentration of 2200mg/L
The vitamin component of acid constituents, the mineral element component of final concentration of 7505mg/L and final concentration of 45.7mg/L.Containing saccharic composition
For glucose.Amino acid composition includes 310 parts of L-arginine, 60 parts of L- asparagine, 25 parts of L-ASPARTIC ACID, 75 parts
L-cysteine dihydrochloride, 25 parts of Pidolidone, 13 parts of glycine, 20 parts of L-Histidine, 25 parts of L- hydroxyl dried meat ammonia
Acid, 60 parts of l-Isoleucine, 60 parts of L-Leu, 45 parts of L lysine HCL, 20 parts of l-methionine, 20 parts
L-phenylalanine, 25 parts of L-PROLINE, 40 parts of Serine, 25 parts of L-threonine, 6 parts of L-Trp, 28 parts
L-tyrosine, 340 parts of L-Glutamine and 25 parts of Valine.
Embodiment 6
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 4, the difference is that：
The Fiber differentiation composition of 4mL is added in each hole, final concentration of 5 μ g/mL, the CD3 antibody of PHA in Fiber differentiation composition
The final concentration of 5 μ g/mL of final concentration of 5 μ g/mL, CD28 antibody.Cell culture medium includes basal medium, makes an addition to basic training
The fetal calf serum in base is supported, Sodium Pyruvate in basal medium is made an addition to, makes an addition to penicillin in basal medium, addition
Streptomysin in basal medium and sodium bicarbonate in basal medium is made an addition to, the volumn concentration of fetal calf serum is
12%, the final concentration of 1mM of Sodium Pyruvate, the penicillin that volumn concentration is 0.5%, the volumn concentration of streptomysin are
0.5%, the final concentration of 2g/L of sodium bicarbonate.
Wherein, basal medium includes the amino containing saccharic composition, final concentration of 1080mg/L of final concentration of 2000mg/L
The vitamin component of acid constituents, the mineral element component of final concentration of 7225mg/L and final concentration of 40mg/L.It is containing saccharic composition
Glucose.Amino acid composition includes 290 parts of L-arginine, 50 parts of L- asparagine, 20 parts of L-ASPARTIC ACID, 65 parts
L-cysteine dihydrochloride, 20 parts of Pidolidone, 10 parts of glycine, 15 parts of L-Histidine, 20 parts of L- hydroxyproline,
50 parts of l-Isoleucine, 50 parts of L-Leu, 40 parts of L lysine HCL, 15 parts of l-methionine, 15 parts
L-phenylalanine, 20 parts of L-PROLINE, 30 parts of Serine, 20 parts of L-threonine, 5 parts of L-Trp, 23 parts
L-tyrosine, 300 parts of L-Glutamine and 20 parts of Valine.Mineral element component include 10 parts calcium nitrate, 5 parts
Magnesium sulfate, 60 parts of sodium dihydrogen phosphate, 40 parts of potassium chloride and 600 parts of sodium chloride.Vitamin component includes 0.2 part of biology
Element, 0.25 part of D-VB5 calcium, 1 part of folic acid, 35 parts of inositol, 1 part of niacinamide, 1 part of puridoxine hydrochloride, 0.2 part
Riboflavin and 1 part of thiamine hydrochloride.
Embodiment 7
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
Basal medium includes the helper component of final concentration of 3.3mg/L.Helper component includes 1 part of glutathione and 3 parts of chlorination
Choline.Vitamin component further includes 0.005 part of vitamin B12.
Embodiment 8
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
Incubation time is 7 days, adds a Fiber differentiation composition within each three days, every time to the Fiber differentiation composition added in every hole
Volume and the hole in had been used for before secondary add culture CD8 positive T cell Fiber differentiation composition volume ratio be 1: 2.
Embodiment 9
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
Based on mass fraction, Fiber differentiation composition includes the cell culture medium of 99..85%, 0.05% PHA, 0.05% CD3 anti-
Body and 0.05% CD28 antibody.
Embodiment 10
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
Cell culture medium is DMEM culture medium.
Embodiment 11
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
Cell culture medium is RPMI1640 culture medium.
Embodiment 12
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
The final concentration of 5 μ g/mL of PHA, does not add CD3 antibody and CD28 antibody.
Embodiment 13
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
The final concentration of 5 μ g/mL of CD3 antibody, does not add PHA and CD28 antibody.
Embodiment 14
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
The final concentration of 5 μ g/mL of CD28 antibody, does not add PHA and CD3 antibody.
Embodiment 15
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
It is not added with PHA, CD3 antibody and CD28 antibody.
Embodiment 16
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
PHA, and the final concentration of 5 μ g/mL of IL-2 are substituted with IL-2.
Embodiment 17
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
CD3 antibody, and the final concentration of 5 μ g/mL of IL-2 are substituted with IL-2.
Embodiment 18
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
CD28 antibody, and the final concentration of 5 μ g/mL of IL-2 are substituted with IL-2.
Embodiment 19
The process that the CD8 positive T cell of the present embodiment is expanded is roughly the same with embodiment 6, the difference is that：
CD8 positive T cell used is the CD8 positive T cell isolated from peripheral blood.
Embodiment 20
The process that the CD8 positive T cell of the present embodiment is expanded is as follows：
The cell culture medium added with PHA of 4mL is added in each hole, is placed in 37 DEG C, 5%CO21 is cultivated in incubator
It, the final concentration of 5 μ g/mL of PHA, cell culture medium is identical as embodiment 6.Liquid is discarded supernatant after culture, then at every
The cell culture medium added with CD3 antibody and CD28 antibody of 4mL is added in hole, is placed in 37 DEG C, 5%CO22 are cultivated in incubator
It, the final concentration of 5 μ g/mL of final concentration of 5 μ g/mL, the CD28 antibody of CD3 antibody, the phase of cell culture medium and embodiment 6
Together.
Test：CD8 positive T cell after to cultivating in embodiment 4~20 carries out cell count, and measurement result is detailed in
Table 1.Wherein, the average value that cell number counts in table 1 for three holes in each embodiment.
The cell number of CD8 positive T cell after the culture of 1 embodiment 4~20 of table
| Cell number (a/mL) | |
| Embodiment 4 | 7×105 |
| Embodiment 5 | 4×105 |
| Embodiment 6 | 8×105 |
| Embodiment 7 | 9×105 |
| Embodiment 8 | 1.5×106 |
| Embodiment 9 | 8.5×105 |
| Embodiment 10 | 6.5×105 |
| Embodiment 11 | 7.5×105 |
| Embodiment 12 | 1.8×105 |
| Embodiment 13 | 0.8×105 |
| Embodiment 14 | 1.5×105 |
| Embodiment 15 | 1.7×105 |
| Embodiment 16 | 4×105 |
| Embodiment 17 | 2.1×105 |
| Embodiment 18 | 1.6×105 |
| Embodiment 19 | 1.5×105 |
| Embodiment 20 | 1.2×105 |
As it can be seen from table 1 the cell number of the CD8 positive T cell after the culture of embodiment 1~11 is at least 4 × 105
A/mL, hence it is evident that the cell number (1.7 × 10 of the CD8 positive T cell after being cultivated better than embodiment 155A/mL), in explanation
The Fiber differentiation composition for stating embodiment can effectively promote the CD8 positive T cell in spleen source and largely be proliferated, to obtain
Obtain a large amount of CD8 positive T cell.
Wherein, the cell number of the CD8 positive T cell after embodiment 6 is cultivated illustrates 5 μ g/ better than embodiment 4~5
The CD28 antibody of the PHA of mL, the CD3 antibody of 5 μ g/mL and 5 μ g/mL are more advantageous to the proliferation of CD8 positive T cell.Embodiment 6 is trained
The cell number of CD8 positive T cell after supporting is better than embodiment 10~11, illustrates the cell culture medium of above embodiment more
Be conducive to the proliferation of CD8 positive T cell.The cell number of CD8 positive T cell after the culture of embodiment 6 is better than embodiment 19,
Illustrate that the Fiber differentiation composition of above embodiment is more advantageous to the proliferation of the CD8 positive T cell in spleen source.Embodiment 6
The cell number of CD8 positive T cell after culture is better than embodiment 16~18 and embodiment 20, illustrates above embodiment
The synergistic effect of PHA, CD3 antibody and CD28 antibody in Fiber differentiation composition, the CD8 positive T for being more advantageous to spleen source are thin
The proliferation of born of the same parents.The cell number of CD8 positive T cell after the culture of embodiment 7 illustrates confactor better than embodiment 6
The proliferation for being more advantageous to CD8 positive T cell is added.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of Fiber differentiation composition for the CD8 positive T cell proliferation for promoting spleen source, which is characterized in that including cell
Culture medium and the inducible factor for making an addition to the cell culture medium, the inducible factor include phytohemagglutin phytolectin, CD3 antibody and
CD28 antibody, the final concentration of 1 μ μ of g/mL~10 g/mL of the phytohemagglutin phytolectin, the final concentration of 1 μ g/mL of the CD3 antibody
~10 μ g/mL, the final concentration of 1 μ μ of g/mL~10 g/mL of the CD28 antibody.
2. Fiber differentiation composition according to claim 1, which is characterized in that final concentration of 4 μ of the phytohemagglutin phytolectin
The μ of g/mL~6 g/mL, the final concentration of 4 μ μ of g/mL~6 g/mL of the CD3 antibody, the final concentration of 4 μ g/mL of the CD28 antibody
~6 μ g/mL.
3. Fiber differentiation composition according to claim 1, which is characterized in that the cell culture medium includes basis culture
Base makes an addition to the fetal calf serum in the basal medium and makes an addition to the Sodium Pyruvate in the basal medium, the tire
The volumn concentration of cow's serum is 10%~15%, final concentration of 0.8mM~1.2mM of the Sodium Pyruvate.
4. Fiber differentiation composition according to claim 3, which is characterized in that the basal medium includes final concentration of
The amino acid composition, final concentration of containing saccharic composition, final concentration of 908mg/L~1247mg/L of 1800mg/L~2200mg/L
The mineral element component of 6955mg/L~7505mg/L and the vitamin component of final concentration of 33.6mg/L~45.7mg/L.
5. Fiber differentiation composition according to claim 4, which is characterized in that the saccharic composition that contains includes glucose.
6. Fiber differentiation composition according to claim 4, which is characterized in that in terms of mass fraction, the amino acid group
Divide includes 270 parts~310 parts of L-arginine, 40 parts~60 parts of L- asparagine, 15 parts~25 parts of L-ASPARTIC ACID, 55
Part~75 parts of l-cysteine dihydrochloride, 15 parts~25 parts of Pidolidone, 7 parts~13 parts of glycine, 10 parts~20 parts
L-Histidine, 15 parts~25 parts of L- hydroxyproline, 40 parts~60 parts of l-Isoleucine, 40 parts~60 parts of the bright ammonia of L-
Acid, 35 parts~45 parts of L lysine HCL, 10 parts~20 parts of l-methionine, 10 parts~20 parts of L-phenylalanine,
15 parts~25 parts of L-PROLINE, 20 parts~40 parts of Serine, 15 parts~25 parts of L-threonine, 4 parts~6 parts of L- color
Propylhomoserin, 17 parts~28 parts of l-tyrosine, 260 parts~340 parts of L-Glutamine and 15 parts~25 parts of Valine.
7. Fiber differentiation composition according to claim 4, which is characterized in that in terms of mass fraction, the mineral element
Component include 8 parts~12 parts of calcium nitrate, 4.5 parts~5.5 parts of magnesium sulfate, 65 parts~70 parts of sodium dihydrogen phosphate, 38 parts~
43 parts of potassium chloride and 580 parts~620 parts of sodium chloride.
8. Fiber differentiation composition according to claim 4, which is characterized in that in terms of mass fraction, the vitamin group
Divide includes 0.1 part~0.3 part of biotin, 0.2 part~0.3 part of D-VB5 calcium, 0.8 part~1.2 parts of folic acid, 30 parts~40
Part inositol, 0.8 part~1.2 parts of niacinamide, 0.8 part~1.2 parts of puridoxine hydrochloride, 0.1 part~0.3 part of riboflavin and
0.8 part~1.2 parts of thiamine hydrochloride.
9. a kind of method for the CD8 positive T cell proliferation for promoting spleen source, which is characterized in that include the following steps：
The CD8 positive T cell in spleen source is inoculated in Fiber differentiation composition according to any one of claims 1 to 8 and is trained
It supports.
10. the method for the CD8 positive T cell proliferation according to claim 1 for promoting spleen source, which is characterized in that institute
The inoculum concentration for stating CD8 positive T cell is 0.9 × 105A/mL~1.5 × 105A/mL.
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| WO2021023263A1 (en) * | 2019-08-06 | 2021-02-11 | 连云港金康和信药业有限公司 | Pharmaceutical composition producing safe amount of nitric oxide in body and use thereof |
| WO2021023264A1 (en) * | 2019-08-06 | 2021-02-11 | 连云港金康和信药业有限公司 | Pharmaceutical composition for treating african swine fever and use thereof |
| CN114340609A (en) * | 2019-08-06 | 2022-04-12 | 连云港金康和信药业有限公司 | Pharmaceutical composition for producing safe amount of nitric oxide and use thereof |
| CN112007163B (en) * | 2019-08-06 | 2023-07-28 | 连云港金康和信药业有限公司 | Pharmaceutical composition for treating African swine fever and application thereof |
| CN114149978A (en) * | 2022-02-09 | 2022-03-08 | 深圳博雅感知药业有限公司 | Method for producing CAR-T cells |
| CN114149978B (en) * | 2022-02-09 | 2022-04-26 | 深圳博雅感知药业有限公司 | Method for producing CAR-T cells |
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