CN110438074A - A kind of immunocyte recovery culture solution and preparation method thereof - Google Patents

A kind of immunocyte recovery culture solution and preparation method thereof Download PDF

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Publication number
CN110438074A
CN110438074A CN201910641433.8A CN201910641433A CN110438074A CN 110438074 A CN110438074 A CN 110438074A CN 201910641433 A CN201910641433 A CN 201910641433A CN 110438074 A CN110438074 A CN 110438074A
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culture solution
acid
immunocyte
recovery culture
immunocyte recovery
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徐智峰
张新
黄智斌
洪加津
陈海明
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Guangzhou Shaai Biological Technology Co ltd
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Guangzhou Shaai Biological Technology Co ltd
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Abstract

The present invention provides a kind of immunocyte recovery culture solution, including amino acid, phosphonic acids compounds, vitamin, salt, albumen or polypeptide, enzyme or coenzyme, cell factor, plant extracts.Immunocyte recovery culture solution of the present invention can effectively keep physiological function and biological characteristics after immunocyte recovery, can be applied to clinical treatment；A possibility that being free of animal blood serum in immunocyte recovery culture solution of the invention, therefore will not introduce foreign protein, reducing animal pathogenic pollution, will not have an impact human body adoptive immunotherapy；It is at low cost compared with import immunocyte recovery culture solution, inexpensively；Compared with traditional immunocyte recovery culture solution, the anabiosis rate of immunocyte is high, and immune cell propagation activity is high, can save for a long time.

Description

A kind of immunocyte recovery culture solution and preparation method thereof
Technical field
The present invention relates to technical field of cell culture more particularly to a kind of immunocyte recovery culture solution and its preparation sides Method.
Background technique
Immune cell therapy has become refractory disease (such as malignant tumour) is treated in whole world medical field New important medical procedure, such as tumor vaccine and adoptive immunotherapy, control B cell lymphoma using CAR-T cell It treats, is mainly then fed back by self immunocyte through stimulated in vitro, culture, amplification to improve the immunity of patient, this For scientific circles and medical profession, the acquisition immunocyte that quality is reliable and stable, function is excellent is good to acquisition to be faced a little methods Bed therapeutic effect is very crucial.With the rise of immunocyte personalization technology, it is now recognized that external rapid amplifying native form It is fed back in lactation organism again after the immunocyte of immunocyte or genetic modification, is conducive to the immune defense energy for improving body Power shows the antitumor equal disease treatments ability of body that can be improved, makes histoorgan rejuvenation and extend organism life-span Ability.
Such cell mainly has Dendritic Cells (DC) at present, cytokine induced kill cell (CIK), natural kill Cell (NK), the killing cell (DC-CIK) of Dendritic Cells Induced, Chimeric antigen receptor T cell (CAR-T) etc..These are through luring The immunocyte with amplification cultivation is led, its initial cell source is all the mononuclearcell of peripheral blood or bleeding of the umbilicus, and is straight It connects and is separately cultured.
It is the key that immunocyte culture that how immunocyte culture, which realizes rapid amplifying and do not influence its function, however existing There is its growth rate when cultivating immunocyte in technology slow.
Summary of the invention
A kind of immunocyte recovery culture is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place Liquid and preparation method thereof.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of immunocyte recovery culture solution, including amino acid, phosphonic acids compounds, vitamin, salt, albumen or more Peptide, enzyme or coenzyme, cell factor, plant extracts.
Preferably, the amino acid include the sweet methionine of S- gland, arginine, asparagine, aspartic acid, cysteine, Hippuroyl-histidyl--leucine, homocysteinic acid, D- hydroxyproline, DL- homocystine, DL-3- aminobutyric acid, poly- essence One of propylhomoserin is a variety of.
Preferably, the phosphonic acids compounds include zoledronic acid, Etidronic Acid, ibandronic acid, pamidronic acid, A Lun phosphine One of acid, Risedronic Acid, rice sieve phosphoric acid are a variety of.
The vitamin includes vitamin E, vitamin B12, hydrochloric tiamide, riboflavin, retinoic acid, puridoxine hydrochloride, D- Calcium pantothenate, niacin, menadione, n-hydroxysuccinimide biotin, folic acid, D-Biotin, sodium ascorbate, in ascorbic acid It is one or more.
The salt includes calcium chloride, potassium chloride, bitter salt, sodium selenite, sodium chloride, sodium dihydrogen phosphate, carbon One of sour hydrogen sodium is a variety of.
Preferably, the albumen or polypeptide include collagen, histone, poly-D-lysine, fibronectin, layer adhesion One of albumen, actrapid monotard, γ-human immunoglobulin are a variety of.
Preferably, the enzyme or coenzyme include galactose oxidase, glutamine transaminage, Co-Q10, L-Asparaginasum One of or it is a variety of.
Preferably, the cell factor includes epidermal growth factor, platelet derived growth factor, insulin-like growth factor Son, vascular endothelial growth factor, human serum albumins, colony stimulating factor, tumor necrosis factor, macrophage colony thorn Swash one of the factor, interleukin, interferon or a variety of.
Preferably, the plant extracts include dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract one Kind is a variety of.
Preferably, the mass ratio of the dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract are as follows: dragon's blood element: Rhodioside: Herba Dendrobii extract: Moringa seed extract=1~3:1~3:2~4:1~4.
Preferably, the mass ratio of the dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract are as follows: dragon's blood element: Rhodioside: Herba Dendrobii extract: Moringa seed extract=3:1:3:4.
Preferably, the dragon's blood element is 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one or/and lourerin B.
Dragon's blood element is the main component of Sanguis Draxonis flavone, the big internal organs of blood flow is distributed mainly on, such as liver, kidney.Tool Play the role of significantly preventing and treating liver fibrosis.Resina Draconis can effectively reduce the degree of fibrosis of lung fibrosis in rats lung tissue, The rat liver fibrosis that lourerin B can also induce thioacetamide has certain preventive and therapeutic action.3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one is to focal Property cerebral ischemia re-pouring caused by damage have certain protective effect.
Rhodioside is the one kind extracted from the drying root and rhizome or dry herb of crassulaceae plants rhodiola kirilowii Regel Compound has pre- preventing tumor, enhances immune function, delays senescence, antifatigue, anti anoxia, radiation protection, bidirectional modulation maincenter mind The effects of through, restoration and protection body.It is clinically used for treatment neurasthenia and neurosis；As excitor nerve agent, for mentioning High intelligent Force, improvement vegetative nerve-antiotasis obstacle have myasthenia etc.；For the illness that free radical increases, such as tumour, radiation Damage, pulmonary emphysema, senile cataract etc..Rhodioside preparation is also used to sports medical science and aerospace medicine, for various special The health protection of staff under environmental condition.
Root of kirilow rhodiola has adaptive significances and dual regulation.Monoamine neurotransmitter, spleen in mouse brain through microwave radiation And there is inhibition variation in cyclic adenosine monophosphate, lymphocyte transformation rate, serum hemolysin etc. in thymus gland, root of kirilow rhodiola can be allowed to restore Normally.After injecting rhodioside, the thyroid function and adrenal function of rabbit can be enhanced, and can excited mouse eggs endocrine function Energy.Improve attention and memory.β-indoxyl level in blood plasma is improved, the variation of stress hormone is prevented.
Herba Dendrobii extract, which has, increases the strong immunity of body, and antitumor action, especially dendrobium nobile water-soluble polysaccharide is to external people Tumor cell of liver and Human neuroblastoma cell also have obvious inhibiting effect.Dendrobium polysaccharide has very strong oxidation resistance, has Compared with the effect of high inhibition DNA damage.Studies on chemical constitutents from plants of Dendrobium Sw is rich and varied, and pharmacological action is also extensive.
Moringa seed extract is a kind of green or brown powder.With strengthen immunity, toxin expelling, body shaping, anti-aging, anti- Cancer simultaneously has greatly improvement effect to a variety of chronic and major disease.Moringa seeds are natural green foods, contain human body institute The complete nutrients matter needed, it may replace multi-vitamins, calcium tablet, cod-liver oil etc..Very to control blood glucose, lowering blood pressure and blood fat There is strong effect.
The present invention also provides a kind of preparation methods of immunocyte recovery culture solution as described above, comprising the following steps:
(1) said vitamin is added in the deionized water of certain volume, is stirred evenly；
(2) above-mentioned Chinese medical extract, albumen or polypeptide, enzyme or coenzyme, salt are sequentially added into the solution in step (1), 30~35 DEG C of 30~40min of water-bath, stir evenly；
(3) above-mentioned amino acid, phosphonic acids compounds, cell factor are sequentially added into the solution in step (2), 30~35 DEG C 30~40min of water-bath, stirs evenly, and stands 30~40min to get immunocyte recovery culture solution of the invention.
Beneficial effects of the present invention: after immunocyte recovery culture solution of the present invention can effectively keep immunocyte to recover Physiological function and biological characteristics, can be applied to clinical treatment；Animal blood is free of in immunocyte recovery culture solution of the invention Clearly, a possibility that therefore foreign protein will not being introduced, reducing animal pathogenic pollution, human body adoptive immunotherapy will not be generated It influences；It is at low cost compared with import immunocyte recovery culture solution, inexpensively；Compared with traditional immunocyte recovery culture solution, The anabiosis rate of immunocyte is high, and immune cell propagation activity is high, can save for a long time.
Specific embodiment
For more concise displaying technical solution of the present invention, objects and advantages, combined with specific embodiments below The present invention is described in further detail.
Embodiment 1
The immunocyte recovery culture solution of the present embodiment, it includes following weight raw material that culture medium described in 1L, which is made, mainly:
Amino acid: the sweet methionine 5mg of S- gland, alanine 5mg, arginine 8mg, asparagine 10mg, aspartic acid 20mg, Cysteine 15mg, hippuroyl-histidyl--leucine 10mg, homocysteinic acid 10mg, D- hydroxyproline 5mg, DL- high Guang Propylhomoserin 8mg, DL-3- aminobutyric acid 15mg, poly arginine 20mg.
Phosphonic acids compounds: zoledronic acid 5mg, Etidronic Acid 2mg, ibandronic acid 5mg, pamidronic acid 3mg, alendronic acid 5mg, Risedronic Acid 1mg, rice sieve phosphoric acid 3mg.
Vitamin: 10 μ g of vitamin E, 5 μ g of vitamin B12,10 μ g of hydrochloric tiamide, 5 μ g of riboflavin, 6 μ g of retinoic acid, salt Sour 8 μ g of pyridoxol, 10 μ g of D-VB5 calcium, 15 μ g of niacin, 5 μ g of menadione, 5 μ g of n-hydroxysuccinimide biotin, 10 μ of folic acid G, 5 μ g of D-Biotin, 20 μ g of sodium ascorbate, 25 μ g of ascorbic acid.
Salt: calcium chloride 5mg, potassium chloride 5mg, bitter salt 10mg, sodium selenite 5mg, sodium chloride 15mg, phosphoric acid Sodium dihydrogen 10v, sodium bicarbonate 5mg.
Albumen or polypeptide: collagen 30mg, Histone 4 0mg, poly-D-lysine mg, fibronectin mg, layer adhesion egg White 20mg, actrapid monotard 20mg, γ-human immunoglobulin 30mg.
Enzyme or coenzyme: 10 μ g of galactose oxidase, 15 μ g of glutamine transaminage, 20 μ g of Co-Q10, L-Asparaginasum 10μg。
Cell factor: 20 μ g of epidermal growth factor, 15 μ g of platelet derived growth factor, 5 μ of type-1 insulin like growth factor G, 5 μ g of vascular endothelial growth factor, 15 μ g of human serum albumins, 5 μ g of colony stimulating factor, 5 μ g of tumor necrosis factor, huge 5 μ g of phagocyte colony stimulating factor, IL-10 μ g, 5 μ g of interferon.
Plant extracts: 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one 20mg, rhodioside 20mg, Herba Dendrobii extract 15mg, Moringa seed extract 10mg.
Embodiment 2
The method for preparing the immunocyte recovery culture solution of embodiment 1, comprising the following steps:
(1) said vitamin is added in the deionized water of certain volume, is stirred evenly；
(2) above-mentioned Chinese medical extract, albumen or polypeptide, enzyme or coenzyme, salt are sequentially added into the solution in step (1), 35 DEG C of water-bath 30min, stir evenly；
(3) above-mentioned amino acid, phosphonic acids compounds, cell factor are sequentially added into the solution in step (2), 35 DEG C of water-baths 30min is stirred evenly, and stands 30min to get immunocyte recovery culture solution of the invention.
Effect of the 3 immunocyte recovery culture solution of embodiment to the amplification rate of DC cell culture
The DC cell of bone marrow derived is selected to do cell culture experiments in vitro, cell presses 1 × 104It is separately added into after inoculation The culture of the immunocyte recovery culture solution (experimental group) of conventional DMEM culture medium (control group) and embodiment 1,37 DEG C, 5%CO2, Cell density is surveyed by mtt assay after 72 hours, the results are shown in Table 1:
Table 1: experimental group and cellular control unit density
Detect number Experimental group Control group
1 1.5×107 1.2×106
2 1.6×107 1.6×106
3 1.5×107 1.4×106
Average value 1.5×107 1.4×106
As can be seen from Table 1, using immunocyte recovery culture solution of the present invention to the amplification rate and DMEM culture medium of DC cell It compares, cell proliferation rate improves 10.7 times under the same conditions.
Influence of the 4 immunocyte recovery culture solution of embodiment to DC cell phenotype positive ratio
By the DC cell of bone marrow derived, adjusting concentration is 1 × 106The culture of embodiment 1 or control group is added in/mL Base (DMEM culture medium), in 37 DEG C, saturated humidity, 5%CO2Incubator culture, every 3d half, which is measured, changes liquid.After cultivating 7d, it is added glimmering Signal rat anti-mouse CD40 and CD86, room temperature, which is protected from light, is incubated for 30min, after PBS washing cell, is carried out carefully with flow cytometer Born of the same parents count, and detection cell phenotype positive ratio is percentage shared by Phenotype positive cell, the results are shown in Table 2.
Table 2: influence of the culture medium to DC cell phenotype positive ratio
Culture medium CD40 Phenotype positive ratio (%) CD68 Phenotype positive ratio (%)
Embodiment 1 21.3 32.5
DMEM culture medium 11.5 18.6
From table 2 it can be seen that D40 the and CD86 cell phenotype positive ratio of embodiment 1 is significantly higher than control group (P > 0.05), illustrate that the culture solution of embodiment 1 compared with control group (conventional DMEM), has positive effect.
Embodiment 5
Uniquely difference is the present embodiment with embodiment 1, dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract Mass ratio are as follows: dragon's blood element: rhodioside: Herba Dendrobii extract: Moringa seed extract=1:1:2:1.
Specially lourerin B 10mg, rhodioside 10mg, Herba Dendrobii extract 20mg, Moringa seed extract 10mg.
Embodiment 6
Uniquely difference is the present embodiment with embodiment 1, dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract Mass ratio are as follows: dragon's blood element: rhodioside: Herba Dendrobii extract: Moringa seed extract=2:2:3:2.
Specially lourerin B 20mg, rhodioside 20mg, Herba Dendrobii extract 30mg, Moringa seed extract 20mg.
Embodiment 7
Uniquely difference is the present embodiment with embodiment 1, dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract Mass ratio are as follows: dragon's blood element: rhodioside: Herba Dendrobii extract: Moringa seed extract=3:3:4:3.
Specially lourerin B 30mg, rhodioside 30mg, Herba Dendrobii extract 40mg, Moringa seed extract 30mg.
Embodiment 8
Uniquely difference is the present embodiment with embodiment 1, dragon's blood element, rhodioside, Herba Dendrobii extract, Moringa seed extract Mass ratio are as follows: dragon's blood element: rhodioside: Herba Dendrobii extract: Moringa seed extract=3:1:3:4.
Specially lourerin B 30mg, rhodioside 10mg, Herba Dendrobii extract 30mg, Moringa seed extract 40mg.
The effect of the plant extracts embodiment culture solution of optimization is compared, detection method is using embodiment 3 and in fact Example 4 is applied, as a result as shown in Table 3, 4:
Table 3: effect of 5~8 immunocyte recovery culture solution of embodiment to the amplification rate of DC cell culture
Grouping Cell density
Embodiment 5 1.5×107
Embodiment 6 1.7×107
Embodiment 7 1.8×107
Embodiment 8 2.1×107
Table 4: influence of 5~8 immunocyte recovery culture solution of embodiment to DC cell phenotype positive ratio
Embodiment CD40 Phenotype positive ratio (%) CD68 Phenotype positive ratio (%)
Embodiment 5 20.2 30.3
Embodiment 6 21.5 31.3
Embodiment 7 22.7 32.5
Embodiment 8 26.3 36.5
By table 3,4 it is found that embodiment 8 is apparently higher than other embodiments, embodiment 8 to the amplification rate of DC cell culture CD40 and CD86 cell phenotype positive ratio be apparently higher than other embodiments, illustrate the present embodiment 8 culture solution be the present invention Most preferred embodiment.
Comparative example 1
The culture solution of the embodiment of the present invention 8 and commercially available culture medium are compared, detect them to DC cell culture Amplification rate and impact effect to DC cell phenotype positive ratio.
The main component of the cell culture medium of comparative example 1 has: amino acid includes alanine, arginine, asparagine, asparagus fern Propylhomoserin, cysteine, glutamic acid, glycine, histidine, isoleucine；Vitamin includes biotin, choline chloride, D-VB5 Calcium；Salt includes sodium bicarbonate, calcium chloride, potassium chloride, magnesium chloride, magnesium sulfate, sodium chloride, sodium dihydrogen phosphate；Lipid includes ground Sai meter Song, oleic acid, cholesterol, ethanol amine, linoleic acid, lipoic acid, lipid；Polypeptide and/or cell factor include fine adhesion Albumen, laminin, glass table Fibronectin, fibroblast growth factor 1, transferrins, human serum albumins, colony-stimulating The factor 1, tumor necrosis factor, macrophage colony stimulating factor, granulocyte colony stimulating factor, proleulzin, interleukin- 4, IL-5, IL-12, interleukin-15, IL-23；Antibody includes anti-cd 3 antibodies.
Comparative example 2
The main component of the cell culture medium of comparative example 2 has: l-Alanine, L-arginine hydrochloride, L-Aspartic acid, L- Aspartic acid hydrate, l-cysteine hydrochloride, Pidolidone, L-Glutamine, L-Histidine hydrochloride, l-Isoleucine； Calcium chloride, potassium chloride, bitter salt, sodium selenite, sodium chloride, sodium dihydrogen phosphate, sodium bicarbonate；Biotin, chlorination gallbladder Alkali, D-VB5 calcium, folic acid, inositol, niacinamide, riboflavin, thiamine hydrochloride, vitamin, pyridoxal hydrochloride；D-Glucose, 4- hydroxyl Ethyl piperazidine ethanesulfonic acid, insulin, phenol red, Sodium Pyruvate, transferrins.
The culture solution of the embodiment of the present invention 8 and the culture medium of comparative example 1,2 are compared, detect them to DC cell The amplification rate of culture and impact effect to DC cell phenotype positive ratio, the results are shown in Table 5,6.
Table 5: the effect of 8 immunocyte recovery culture solution of embodiment and the amplification rate of 1,2 pair of DC cell culture of comparative example
Table 6: the influence of 1,2 pair of DC cell phenotype positive ratio of 8 immunocyte recovery culture solution of embodiment and comparative example
Embodiment CD40 Phenotype positive ratio (%) CD68 Phenotype positive ratio (%)
Embodiment 8 26.3 36.5
Comparative example 1 16.2 22.1
Comparative example 2 17.3 20.9
By table 5,6 it is found that embodiment 8 is significantly higher than comparative example 1,2, amplification rate to the amplification rate of DC cell culture It is 11 times of comparative example 1 respectively, is 12.3 times of comparative example 2.CD40 the and CD86 cell phenotype positive ratio of embodiment 8 is significant Higher than comparative example 1,2.Compared with comparative example 1, the CD40 Phenotype positive ratio of embodiment 8 is 1.6 times of comparative example 1, CD68 table Type positive ratio is 1.6 times of comparative example 1；Compared with comparative example 2, the CD40 Phenotype positive ratio of embodiment 8 is comparative example 1 1.5 times, CD68 Phenotype positive ratio is 1.7 times of comparative example 1.
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 immunocyte recovery culture solution, which is characterized in that including amino acid, phosphonic acids compounds, vitamin, salt, Albumen or polypeptide, enzyme or coenzyme, cell factor, plant extracts.
2. immunocyte recovery culture solution as described in claim 1, which is characterized in that the amino acid includes the sweet egg ammonia of S- gland Acid, arginine, asparagine, aspartic acid, cysteine, hippuroyl-histidyl--leucine, homocysteinic acid, D- hydroxyl One of proline, DL- homocystine, DL-3- aminobutyric acid, poly arginine are a variety of.
3. immunocyte recovery culture solution as described in claim 1, which is characterized in that the phosphonic acids compounds include azoles One of phosphonic acids, Etidronic Acid, ibandronic acid, pamidronic acid, alendronic acid, Risedronic Acid, rice sieve phosphoric acid are a variety of.
4. immunocyte recovery culture solution as described in claim 1, which is characterized in that the vitamin includes vitamin E, dimension Raw element B12, hydrochloric tiamide, riboflavin, retinoic acid, puridoxine hydrochloride, D-VB5 calcium, niacin, menadione, N- hydroxysuccinimidyl acyl are sub- One of amine biotin, folic acid, D-Biotin, sodium ascorbate, ascorbic acid are a variety of；The salt include calcium chloride, One of potassium chloride, bitter salt, sodium selenite, sodium chloride, sodium dihydrogen phosphate, sodium bicarbonate are a variety of.
5. immunocyte recovery culture solution as described in claim 1, which is characterized in that the albumen or polypeptide include collagen egg One of white, histone, poly-D-lysine, fibronectin, laminin, actrapid monotard, γ-human immunoglobulin are more Kind；The enzyme or coenzyme include one of galactose oxidase, glutamine transaminage, Co-Q10, L-Asparaginasum or more Kind；The cell factor includes epidermal growth factor, platelet derived growth factor, insulin-like growth factor, blood vessel endothelium Porcine HGF, human serum albumins, colony stimulating factor, tumor necrosis factor, macrophage colony stimulating factor, Bai Jie One of element, interferon are a variety of.
6. immunocyte recovery culture solution as described in claim 1, which is characterized in that the plant extracts includes dragon's blood Element, rhodioside, Herba Dendrobii extract, Moringa seed extract it is one or more.
7. immunocyte recovery culture solution as claimed in claim 6, which is characterized in that the dragon's blood element, rhodioside, dendrobium nobile The mass ratio of extract, Moringa seed extract are as follows: dragon's blood element: rhodioside: Herba Dendrobii extract: Moringa seed extract=1~3:1 ~3:2~4:1~4.
8. immunocyte recovery culture solution as claimed in claim 7, which is characterized in that the dragon's blood element, rhodioside, dendrobium nobile The mass ratio of extract, Moringa seed extract are as follows: dragon's blood element: rhodioside: Herba Dendrobii extract: Moringa seed extract=3:1:3: 4。
9. immunocyte recovery culture solution as claimed in claim 8, which is characterized in that the dragon's blood element be 3-(2,4-Dimethoxy-phenyl)-1-(4-hydroxy-phenyl)-propan-1-one or/and Lourerin B.
10. a kind of preparation method of immunocyte recovery culture solution as described above, which comprises the following steps:
(1) said vitamin is added in the deionized water of certain volume, is stirred evenly；
(2) above-mentioned Chinese medical extract, albumen or polypeptide, enzyme or coenzyme, salt are sequentially added into the solution in step (1), 30~ 35 DEG C of 30~40min of water-bath, stir evenly；
(3) above-mentioned amino acid, phosphonic acids compounds, cell factor are sequentially added into the solution in step (2), 30~35 DEG C of water-baths 30~40min is stirred evenly, and stands 30~40min to get immunocyte recovery culture solution of the invention.
CN201910641433.8A 2019-07-16 2019-07-16 A kind of immunocyte recovery culture solution and preparation method thereof Pending CN110438074A (en)

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