CN104450614A - Animal protein-free immune cell serum-free medium and using method thereof - Google Patents
Animal protein-free immune cell serum-free medium and using method thereof Download PDFInfo
- Publication number
- CN104450614A CN104450614A CN201410737699.XA CN201410737699A CN104450614A CN 104450614 A CN104450614 A CN 104450614A CN 201410737699 A CN201410737699 A CN 201410737699A CN 104450614 A CN104450614 A CN 104450614A
- Authority
- CN
- China
- Prior art keywords
- acid
- animal protein
- immunocyte
- vitamin
- free medium
- 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.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses the field of culture in vitro of immune cells and particularly relates to an animal protein-free immune cell serum-free medium and a culture method thereof, wherein the culture medium is a liquid culture medium. The culture medium per liter comprises the following components: 1400-1500mg of amino acids, 70-75mg of vitamins, 9800-10000mg of salts, 9500-9700mg of organic matters and 76-80mg of proteins. The culture medium prepared by the invention satisfies the special condition required by growth of immune cells well, so that the culture medium is more suitable for growth of immune cells, thereby remarkably improving the cell proliferation efficiency and the biological potency. The culture medium overcomes the defect that in the prior art, batches are different in quality caused by diversity of human or animal serum and component limited donors added into the culture medium, the quality is easy to control, the culture medium is not polluted due to the quality of serum, and the tumor-killing rate effect is good and the cost is low.
Description
Technical field
The present invention relates to vitro culture immunocyte field, be specifically related to a kind of immunocyte serum free medium and using method thereof of animal protein-free source.
Background technology
Adoptive cellular immunotherapy (the Adoptive cellular immunotherapy of tumour, ACI or AIT) be point to tumour patient to transfer immunocyte (specific and relative specificity) the direct killing tumour or excitating organism immune response killing tumor cell with anti-tumor activity, reach the object for the treatment of tumour.This cellular immunotherapy is a kind of emerging, brand-new antitumour treatments with significant curative effect of tumor recovering medical science, is called as a kind for the treatment of means active, the most rising in 21st century combined therapy of tumour pattern.
Cellular immunotherapy just be unable to do without mass propgation, amplification and activating immune cell in vitro effectively.The substratum of appropriate immune Growth of Cells is that cell provides suitable basic environment as nutritive substance, osmotic pressure, pH value etc., and cell by the product withdraw of metabolism in substratum.The existing substratum in market is not cultivated required condition for immunocyte and is prepared, and causes cell proliferation efficiency and biological efficacy not good, and a lot of substratum with the addition of human or animal's serum and component is supplemented completely.But human serum is expensive, be not suitable for extensive amplification, and due to the diversity of donor, cause Serology Quality often criticize between there are differences, especially by danger that hepatitis virus or HIV are polluted.Be added with the serum free medium of humanized's components such as human serum albumin, human transferrin, insulin human, though detect and sterilization through pathogeny, but still keep away unavoidably due to the difference of donor often criticize between there are differences, be unfavorable for the control of quality.If foetal calf serum, new-born calf serum etc. are from the serum component of ox material, although have enough amount supplies, low price, there is the risk disseminated infection, as the danger that mycoplasma, virus (as the BSE pathogeny factor) are polluted in animal serum.
Summary of the invention
The object of this invention is to provide a kind of immunocyte serum free medium of animal protein-free source, this substratum is especially for the cultivation of immunocyte, meet the specified conditions needed for immune cell growth, more be applicable to the growth of immunocyte, improve cell proliferation efficiency and biological efficacy, and can not be polluted because of Serology Quality problem, quality controllability is strong, and cost is low.
Another object of the present invention is to provide the cultural method of the immunocyte serum free medium in above-mentioned animal protein-free source.
Object of the present invention can be achieved through the following technical solutions:
The immunocyte serum free medium in animal protein-free source, is characterized in that: this substratum is liquid nutrient medium, and often liter of substratum comprises following component,
Amino acid is 1400-1500mg, and VITAMIN is 70-75mg, and salt is 9800-10000mg, and organic matter is 9500-9700mg, and protein is 76-80mg.Preferably, described amino acid is 1453mg, and VITAMIN is 72.31mg, and salt is 9841.1524mg, and organic matter is 9610.64mg, and protein is 78.5mg.
Described amino acid is the composition of glycine, L-arginine, L-amino-succinamic acid, L-Aspartic acid, L-hydrochloric acid Gelucystine, Pidolidone, L-Histidine, L-oxyproline, ILE, L-Leu, LYS, L-Methionine, L-Phe, L-PROLINE, Serine, L-threonine, L-Trp, TYR disodium salt dihydrate, Valine, Bipeptide of glutamate and alanine.
The concentration of each component in described amino acid is glycine 17-19mg/L, L-arginine 185-195mg/L, L-amino-succinamic acid 40-45mg/L, L-Aspartic acid 15-20mg/L, L-hydrochloric acid Gelucystine 55-60mg/L, Pidolidone 10-15mg/L, L-Histidine 20-30mg/L, L-oxyproline 10-15mg/L, ILE 120-130mg/L, L-Leu 75-85mg/L, LYS 95-105mg/L, L-Methionine 20-25mg/L, L-Phe 30-35mg/L, L-PROLINE 20-30mg/L, Serine 40-50mg/L, L-threonine 25-35mg/L, L-Trp 10-20mg/L, TYR disodium salt dihydrate 40-45mg/L, Valine 40-45mg/L, Bipeptide of glutamate and alanine 480-520mg/L.Preferably, the concentration of each component in described amino acid is glycine 18mg/L, L-arginine 190mg/L, L-amino-succinamic acid 45mg/L, L-Aspartic acid 20mg/L, L-hydrochloric acid Gelucystine 60mg/L, Pidolidone 15mg/L, L-Histidine 25mg/L, L-oxyproline 15mg/L, ILE 130mg/L, L-Leu 80mg/L, LYS 100mg/L, L-Methionine 25mg/L, L-Phe 35mg/L, L-PROLINE 25mg/L, Serine 45mg/L, L-threonine 30mg/L, L-Trp 15mg/L, TYR disodium salt dihydrate 40mg/L, Valine 40mg/L, Bipeptide of glutamate and alanine 500mg/L.
Described VITAMIN is the composition of vitamin H, choline chloride 60, calcium pantothenate, folic acid, niacinamide, para-amino benzoic acid, pyridoxine hydrochloride, vitamin, vitamin B12 and inositol; The concentration of each component in described VITAMIN is respectively vitamin H 0.2-0.4mg/L, choline chloride 60 0.8-1.2mg/L, calcium pantothenate 2.5-3.5mg/L, folic acid 4-6mg/L, niacinamide 8-12mg/L, para-amino benzoic acid 0.4-0.6mg/L, pyridoxine hydrochloride 1.1-1.3mg/L, vitamin 1.1-1.3mg/L, vitamin B12 0.01mg/L, inositol 48-52mg/L.Preferably, the concentration of each component in described VITAMIN is respectively vitamin H 0.4mg/L, choline chloride 60 1mg/L, calcium pantothenate 3mg/L, folic acid 5mg/L, niacinamide 10mg/L, para-amino benzoic acid 0.5mg/L, pyridoxine hydrochloride 1.2mg/L, vitamin 1.2mg/L, vitamin B12 0.01mg/L, inositol 50mg/L.
Described organic matter is the composition of glucose, gsh, hydroxyethyl piperazine second thiosulfonic acid, phenol red, Sodium.alpha.-ketopropionate, xanthoglobulin, linolic acid, hydrochloric acid butanediamine, Thioctic Acid, thymidine and tween 80; The concentration of each component in described organic matter is glucose 3500mg/L, gsh 1-4mg/L, hydroxyethyl piperazine second thiosulfonic acid 6000mg/L, phenol red 5-7mg/L, Sodium.alpha.-ketopropionate 100mg/L, xanthoglobulin 1-3mg/L, linolic acid 0.04mg/L, hydrochloric acid butanediamine 0.1-0.2mg/L, Thioctic Acid 0.1-0.2mg/L, thymidine 0.4-0.6mg/L, tween 80 are 0-2mg/L.Preferably, the concentration of each component in described organic matter is glucose 3500mg/L, gsh 2mg/L, hydroxyethyl piperazine second thiosulfonic acid 6000mg/L, phenol red 6mg/L, Sodium.alpha.-ketopropionate 100mg/L, xanthoglobulin 2mg/L, linolic acid 0.04mg/L, hydrochloric acid butanediamine 0.1mg/L, Thioctic Acid 0.1mg/L, thymidine 0.4mg/L, tween 80 are 0-2mg/L.For increasing organic solubleness, solubility promoter tween 80 can be added.
Described protein is the composition of recombinant human serum albumin, recombinant human Transferrins,iron complexes, recombinant human insulin, recombinant human somatropin.Composition concentration in described protein is recombinant human serum albumin 2-4mg/L, recombinant human Transferrins,iron complexes 50-70mg/L, recombinant human insulin 12-16mg/L recombinant human somatropin 0.3-0.6mg/L.Preferably, the composition concentration in described protein is recombinant human serum albumin 3mg/L, recombinant human Transferrins,iron complexes 60mg/L, recombinant human insulin 15mg/L recombinant human somatropin 0.5mg/L.
Described salt is the composition of calcium nitrate tetrahydrate, magnesium sulfate, Repone K, sodium bicarbonate, sodium-chlor, Sodium phosphate dibasic, Sodium Selenite pentahydrate, copper sulfate pentahydrate, FeSO47H2O, nickelous chloride, stannous chloride dihydrate, ZINC SULFATE HEPTAHYDRATE.Described magnesium sulfate and Sodium phosphate dibasic are nodeless mesh water.
The concentration of each component in described salt is calcium nitrate tetrahydrate 70-85mg/L, magnesium sulfate 58-62mg/L, Repone K 450-500mg/L, sodium bicarbonate 2450-2550mg/L, sodium-chlor 6000mg/L, Sodium phosphate dibasic 700mg/L, Sodium Selenite pentahydrate 0.13-0.16mg/L, copper sulfate pentahydrate 0.002-0.003mg/L, FeSO47H2O 0.3-0.6mg/L, nickelous chloride 0.0001-0.0002mg/L, stannous chloride dihydrate 0.0001-0.0002mg/L, ZINC SULFATE HEPTAHYDRATE 0.3-0.5mg/L.Preferably, the concentration of each component in salt is calcium nitrate tetrahydrate 80mg/L, magnesium sulfate 60mg/L, Repone K 500mg/L, sodium bicarbonate 2500mg/L, sodium-chlor 6000mg/L, Sodium phosphate dibasic 700mg/L, Sodium Selenite pentahydrate 0.15mg/L, copper sulfate pentahydrate 0.002mg/L, FeSO47H2O 0.5mg/L, nickelous chloride 0.0002mg/L, stannous chloride dihydrate 0.0002mg/L, ZINC SULFATE HEPTAHYDRATE 0.5mg/L.
The pH value of described substratum is 6.8-7.2, and the pH value of preferred substratum is 7.0.PH value can be regulated with 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide.Described substratum is degerming by 0.22um membrane filtration twice.
The preservation temperature of described substratum is 3-5 DEG C, preferably 4 DEG C.Described substratum needs lucifuge preservation.
The using method of the immunocyte serum free medium in above-mentioned animal protein-free source, its step comprises: the immunocyte physiological saline cultivated by needs is centrifugal, removes supernatant liquor; Counting cells, with the immunocyte serum free medium in animal protein-free source, cell being mixed with concentration is 1 × 10
6the cell suspension of individual/mL; Add in 6 orifice plates by every hole 2mL substratum cell suspension, add the INF-r Interferon, rabbit of 1000IU/mL, cultivate 24 hours for 37 DEG C; Add the IL-2 of 50ng/mL CD3 monoclonal antibody and 500IU/mL; Add the immunocyte serum free medium in animal protein-free source and the IL-2 of 500IU/mL every 48 hours, count up to 1 × 10
6individual/mL cell concn, Dual culture 14 days, observation of cell form.
Immunocyte serum free medium prepared by the present invention is particularly applicable to lymphocyte and NK cell.
Compared with prior art, beneficial effect of the present invention is:
(1) substratum of the present invention's preparation cultivates for immunocyte especially, meets the specified conditions needed for immune cell growth, be more applicable to the growth of immunocyte, significantly improve cell proliferation efficiency and biological efficacy.
(2) diversity that the substratum of the present invention's preparation overcomes human or animal's serum and the limited donor of component added in substratum of the prior art cause quality often criticize between the defect that there are differences, and can not to be polluted because of Serology Quality problem.
(3) substratum quality controllable of the present invention's preparation, it is effective to kill ratio of outflow, and cost is low.
Accompanying drawing explanation
Fig. 1 is the cell proliferation rate comparison diagram of four groups of samples.
Fig. 2 is the cell survival rate comparison diagram of four groups of samples.
Fig. 3 is the phenotype comparison diagram of the cell of four groups of samples.
Fig. 4 be the cell of four groups of samples kill ratio of outflow comparison diagram.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment 1
Using tri-distilled water as solvent, and be dissolved with amino acid, VITAMIN, salt, organic matter and protein, be mixed with substratum, solubility promoter tween 80 can be added for increasing organic solubleness, regulate pH value with 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide, make the pH value of substratum be 6.8-7.2.Substratum is degerming through 0.22um membrane filtration twice, and 4 degrees Celsius of refrigerators keep in Dark Place.
Be as the criterion with the substratum that 1L prepares, the component containing following concentration in its component:
Aminoacid component: glycine concentration is 18mg/L, L-arginine concentration is 190mg/L, L-amino-succinamic acid 45mg/L, L-Aspartic acid 20mg/L, L-hydrochloric acid Gelucystine 60mg/L, Pidolidone 15mg/L, L-Histidine 25mg/L, L-oxyproline 15mg/L, ILE 130mg/L, L-Leu 80mg/L, LYS 100mg/L, L-Methionine 25mg/L, L-Phe 35mg/L, L-PROLINE 25mg/L, Serine 45mg/L, L-threonine 30mg/L, L-Trp 15mg/L, TYR disodium salt dihydrate 40mg/L, Valine 40mg/L, glutamine & phenylalanine dipeptide 500mg/L, amount to 1453mg/L.
Vitamin ingredients: vitamin H 0.4mg/L, choline chloride 60 1mg/L, calcium pantothenate 3mg/L, folic acid 5mg/L, niacinamide 10mg/L, para-amino benzoic acid 0.5mg/L, pyridoxine hydrochloride 1.2mg/L, vitamin 1.2mg/L, vitamin B12 0.01mg/L, inositol 50mg/L, amounts to 72.31mg/L
Salt constituents: the anhydrous 60mg/L of calcium nitrate tetrahydrate 80mg/L, magnesium sulfate, Repone K 500mg/L, sodium bicarbonate 2500mg/L, sodium-chlor 6000mg/L, the anhydrous 700mg/L of Sodium phosphate dibasic, Sodium Selenite pentahydrate 0.15mg/L, copper sulfate pentahydrate 0.002mg/L, FeSO47H2O 0.5mg/L, nickelous chloride 0.0002mg/L, stannous chloride dihydrate 0.0002mg/L, ZINC SULFATE HEPTAHYDRATE 0.5mg/L, amounts to 9841.1524mg/L.
Organic composition: glucose 3500mg/L, gsh 2mg/L, hydroxyethyl piperazine second thiosulfonic acid 6000mg/L, phenol red 6mg/L, Sodium.alpha.-ketopropionate 100mg/L, xanthoglobulin 2mg/L, linolic acid 0.04mg/L, hydrochloric acid butanediamine 0.1mg/L, Thioctic Acid 0.1mg/L, thymidine 0.4mg/L, amounts to 9610.64mg/L.
Protein component: recombinant human serum albumin 3mg/L, recombinant human Transferrins,iron complexes 60mg/L, recombinant human insulin 15mg/L recombinant human somatropin 0.5mg/L, amounts to 78.5mg/L.
Solubility promoter tween 80 is 2mg/L.
Embodiment 2
The substratum of preparation in embodiment 1 is carried out quality test
1) Sterility testing
Use flat band method detects, and gets SBA dull and stereotyped 1 piece and dull and stereotyped 1 piece of Sabouraud's agar, balances to room temperature.In Example 1 substratum of preparation be sample after the centrifugal 15min of 4000rpm from pipette samples bottom sample hose, the dull and stereotyped 100 μ l of every block, rule with inoculating needle.Flat board is put into 37 DEG C of incubators, after cultivating 48h, observations is negative, sample passes.
2) intracellular toxin detects
Use gel method detects, and in Example 1, the substratum of preparation is sample after dilution, and same negative control, positive control, trial-product positive control add in the tachypleus amebocyte lysate after sterility test water melts again respectively, often kind of parallel two pipes.Result is negative control pipe is feminine gender, and positive control pipe is positive, and trial-product positive control pipe is positive and sample hose is negative, sample passes.
3) detection of mycoplasma is that PCR method detects
By in embodiment 1 preparation substratum be use after the centrifugal 3min of 12000rpm after sample carries out boiling water bath 10min, adopt 25 μ L systems to carry out pcr amplification to testing sample, positive control, negative control; Amplified production carries out agarose gel electrophoresis, and detected result observed by gel imaging instrument.Detected result is negative, sample passes.
Embodiment 3
Use lymphocyte separation medium density gradient centrifugation separating peripheral blood mononuclear cells, or frozen mononuclearcell of recovering.Centrifugal 5 minutes of physiological saline 500g, removes supernatant liquor.Counting cells is 1 × 10 with in embodiment 1, the substratum of preparation is made into concentration
6individual/mL cell suspension.Add in 6 orifice plates by every hole 2mL substratum cell suspension, often organize 3 parallel holes.First day adds the INF-r of 1000IU/mL, is put in CO2gas incubator and cultivates, and adds 50ng/mL CD3 monoclonal antibody and 500IU/mL IL-2 after 24 hours.Later every 48 hours, add described substratum and 500IU/mL IL-2, count up to 1 × 10
6individual/mL cell concn.Dual culture 18 days observation of cell forms.Cultivation results is: cell shape is Polygons, and form is full, well-grown.
Embodiment 4
Adopt the cell culture processes in embodiment 3, the substratum of immunocyte is replaced with PRMI1640+10%FBS, be labeled as A, PRMI 1640+ humanized albumin, Transferrins,iron complexes, Regular Insulin and tethelin, be labeled as B, adopt the substratum of this patent, be labeled as C, protein in the component of this patent substratum is replaced with humanized's albumin, Transferrins,iron complexes, Regular Insulin and tethelin, is labeled as D.When the 15th day, Trypan Blue calculates cell proliferation multiple and motility rate, and result respectively as depicted in figs. 1 and 2.As can be seen from Figure, the immunocyte appreciation rate adopting the culture medium culturing of the present invention's preparation to go out and survival rate are all higher than employing other several groups.
Embodiment 5
When getting cultivation the 15th day, immunocyte suspension is sample, 2000rpm, centrifugal 10min.Abandon supernatant, add appropriate physiological saline, mixing.Get appropriate EP to manage, carry out mark, the cell suspension of centrifugal rear mixing is divided and is filled in 1.5mL EP pipe, often pipe 0.5mL.Often add homotype corresponding to phenotype to be measured and antibody 5ul in pipe, vortex mixer vibration 30S, lucifuge hatches 30min.Take out the test tube of hatching, the centrifugal 10min of 2000rpm.Abandon supernatant, again add 500ul physiological saline, after mixing, upper machine testing, the results are shown in Figure shown in 3, as can be seen from Figure, the Phenotypic examination result of the immunocyte adopting culture medium culturing of the present invention to go out is apparently higher than other several groups, and the cultivation of substratum of the present invention to immunocyte has specificity.
Embodiment 6
In embodiment 4, the immunocyte of separation and Culture to 15 day is effector cell, with preparation in embodiment 1 substratum wash 1 time, be mixed with 1 × 10
6individual/mL cell suspension.With K562 cell for target cell, wash 1 time with the substratum of preparation in embodiment 1, being mixed with density is 1 × 10
5individual/mL suspension.By effect, target cell is inoculated in 96 orifice plates than 10:1,37 degrees Celsius, 5% carbonic acid gas cultivates 24 hours.Use CCK-8 test kit to survey absorbance, calculate and kill ratio of outflow.
Kill ratio of outflow %=1-(test holes-effector cell hole)/(Target cell wells-blank well), the results are shown in Figure shown in 4, as seen from the figure, the immunocyte adopting culture medium culturing of the present invention to go out kill ratio of outflow apparently higher than other several groups.
The above is preferred embodiment of the present invention, but the present invention should not be confined to the content disclosed in this embodiment.The equivalence completed under not departing from spirit disclosed in this invention so every or amendment, all fall into the scope of protection of the invention.
Claims (10)
1. the immunocyte serum free medium in animal protein-free source, is characterized in that: this substratum is liquid nutrient medium, and often liter of substratum comprises following component,
Amino acid is 1400-1500mg, and VITAMIN is 70-75mg, and salt is 9800-10000mg, and organic matter is 9500-9700mg, and protein is 76-80mg.
2. the immunocyte serum free medium in animal protein-free source according to claim 1, is characterized in that: described amino acid is the composition of glycine, L-arginine, L-amino-succinamic acid, L-Aspartic acid, L-hydrochloric acid Gelucystine, Pidolidone, L-Histidine, L-oxyproline, ILE, L-Leu, LYS, L-Methionine, L-Phe, L-PROLINE, Serine, L-threonine, L-Trp, TYR disodium salt dihydrate, Valine, Bipeptide of glutamate and alanine.
3. the immunocyte serum free medium in animal protein-free source according to claim 2, it is characterized in that: the concentration of each component in described amino acid is glycine 17-19mg/L, L-arginine 185-195mg/L, L-amino-succinamic acid 40-45mg/L, L-Aspartic acid 15-20mg/L, L-hydrochloric acid Gelucystine 55-60mg/L, Pidolidone 10-15mg/L, L-Histidine 20-30mg/L, L-oxyproline 10-15mg/L, ILE 120-130mg/L, L-Leu 75-85mg/L, LYS 95-105mg/L, L-Methionine 20-25mg/L, L-Phe 30-35mg/L, L-PROLINE 20-30mg/L, Serine 40-50mg/L, L-threonine 25-35mg/L, L-Trp 10-20mg/L, TYR disodium salt dihydrate 40-45mg/L, Valine 40-45mg/L, Bipeptide of glutamate and alanine 480-520mg/L.
4. the immunocyte serum free medium in animal protein-free source according to claim 1, is characterized in that: described VITAMIN is the composition of vitamin H, choline chloride 60, calcium pantothenate, folic acid, niacinamide, para-amino benzoic acid, pyridoxine hydrochloride, vitamin, vitamin B12 and inositol; Described organic matter is the composition of glucose, gsh, hydroxyethyl piperazine second thiosulfonic acid, phenol red, Sodium.alpha.-ketopropionate, xanthoglobulin, linolic acid, hydrochloric acid butanediamine, Thioctic Acid, thymidine and tween 80; Described protein is the composition of recombinant human serum albumin, recombinant human Transferrins,iron complexes, recombinant human insulin, recombinant human somatropin.
5. the immunocyte serum free medium in animal protein-free source according to claim 4, is characterized in that: the concentration of each component in described VITAMIN is respectively vitamin H 0.2-0.4mg/L, choline chloride 60 0.8-1.2mg/L, calcium pantothenate 2.5-3.5mg/L, folic acid 4-6mg/L, niacinamide 8-12mg/L, para-amino benzoic acid 0.4-0.6mg/L, pyridoxine hydrochloride 1.1-1.3mg/L, vitamin 1.1-1.3mg/L, vitamin B12 0.01mg/L, inositol 48-52mg/L; The concentration of each component in described organic matter is glucose 3500mg/L, gsh 1-4mg/L, hydroxyethyl piperazine second thiosulfonic acid 6000mg/L, phenol red 5-7mg/L, Sodium.alpha.-ketopropionate 100mg/L, xanthoglobulin 1-3mg/L, linolic acid 0.04mg/L, hydrochloric acid butanediamine 0.1-0.2mg/L, Thioctic Acid 0.1-0.2mg/L, thymidine 0.4-0.6mg/L, tween 80 are 0-2mg/L.
6. the immunocyte serum free medium in animal protein-free source according to claim 4, is characterized in that: described salt is the composition of calcium nitrate tetrahydrate, magnesium sulfate, Repone K, sodium bicarbonate, sodium-chlor, Sodium phosphate dibasic, Sodium Selenite pentahydrate, copper sulfate pentahydrate, FeSO47H2O, nickelous chloride, stannous chloride dihydrate, ZINC SULFATE HEPTAHYDRATE.
7. the immunocyte serum free medium in animal protein-free source according to claim 4, it is characterized in that: the concentration of each component in described salt is calcium nitrate tetrahydrate 70-85mg/L, magnesium sulfate 58-62mg/L, Repone K 450-500mg/L, sodium bicarbonate 2450-2550mg/L, sodium-chlor 6000mg/L, Sodium phosphate dibasic 700mg/L, Sodium Selenite pentahydrate 0.13-0.16mg/L, copper sulfate pentahydrate 0.002-0.003mg/L, FeSO47H2O 0.3-0.6mg/L, nickelous chloride 0.0001-0.0002mg/L, stannous chloride dihydrate 0.0001-0.0002mg/L, ZINC SULFATE HEPTAHYDRATE 0.3-0.5mg/L.
8. the immunocyte serum free medium in animal protein-free source according to claim 4, is characterized in that: the composition concentration in described protein is recombinant human serum albumin 2-4mg/L, recombinant human Transferrins,iron complexes 50-70mg/L, recombinant human insulin 12-16mg/L, recombinant human somatropin 0.3-0.6mg/L.
9. the immunocyte serum free medium in animal protein-free source according to claim 1, is characterized in that: the pH value of described substratum is 6.8-7.2, and preservation temperature is 3-5 DEG C.
10. the using method of the immunocyte serum free medium in animal protein-free source according to claim 1, its step comprises: the immunocyte physiological saline cultivated by needs is centrifugal, removes supernatant liquor; Counting cells, with the immunocyte serum free medium in animal protein-free source, it is 1 × 10 that concentration is made in cell mixing
6the cell suspension of individual/mL; Add in 6 orifice plates by every hole 2mL substratum cell suspension, add the INF-r Interferon, rabbit of 1000IU/mL, cultivate 24 hours for 37 DEG C; Add the IL-2 of 50ng/mL CD3 monoclonal antibody and 500IU/mL; Add the immunocyte serum free medium in animal protein-free source and the IL-2 of 500IU/mL every 48 hours, count up to 1 × 10
6individual/mL cell concn, Dual culture 13-15 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410737699.XA CN104450614B (en) | 2014-12-05 | 2014-12-05 | The immunocyte serum free medium and its application method in a kind of animal protein-free source |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410737699.XA CN104450614B (en) | 2014-12-05 | 2014-12-05 | The immunocyte serum free medium and its application method in a kind of animal protein-free source |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104450614A true CN104450614A (en) | 2015-03-25 |
| CN104450614B CN104450614B (en) | 2017-07-07 |
Family
ID=52897373
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410737699.XA Active CN104450614B (en) | 2014-12-05 | 2014-12-05 | The immunocyte serum free medium and its application method in a kind of animal protein-free source |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450614B (en) |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219708A (en) * | 2015-07-21 | 2016-01-06 | 中山大学 | Immunocyte cultivates test kit, immunocyte cultural method and application |
| CN105713880A (en) * | 2016-04-20 | 2016-06-29 | 广东艾时代生物科技有限责任公司 | Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof |
| CN105713873A (en) * | 2016-04-20 | 2016-06-29 | 广东艾时代生物科技有限责任公司 | Serum-free medium for vitro amplification culture of immune cells and application thereof |
| CN106666675A (en) * | 2016-12-26 | 2017-05-17 | 弎西艾斯（苏州）生物科技有限公司 | Complex animal protein substitute |
| CN106754648A (en) * | 2017-01-19 | 2017-05-31 | 弎西艾斯（苏州）生物科技有限公司 | A kind of non-animal derived culture medium |
| CN106754644A (en) * | 2016-12-26 | 2017-05-31 | 弎西艾斯（苏州）生物科技有限公司 | A kind of serum-free fibrous bud mother cell culture medium |
| CN106834225A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of immune cell media |
| CN107119014A (en) * | 2017-05-12 | 2017-09-01 | 青岛赛欧凯普图乐生物医药有限公司 | A kind of non-animal derived culture medium of NK cells |
| CN107674860A (en) * | 2017-07-14 | 2018-02-09 | 上海源培生物科技股份有限公司 | NK92 cell non-serum culture mediums |
| CN108949696A (en) * | 2018-08-21 | 2018-12-07 | 苏州米苏生物技术有限公司 | Immune cell media is applied with it |
| CN112041430A (en) * | 2017-12-08 | 2020-12-04 | 朱诺治疗学股份有限公司 | Serum-free medium formulations for culturing cells and methods of use thereof |
| CN113481101A (en) * | 2021-08-20 | 2021-10-08 | 北京三药科技开发公司 | Redissolution of cryopreserved strain and preparation method 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 |
| CN114621123A (en) * | 2022-04-11 | 2022-06-14 | 昕嘉生物技术(长沙)有限公司 | Porous particle calcium hydroxymethionine and application thereof in serum-free culture of immune cells |
| CN115044548A (en) * | 2022-08-11 | 2022-09-13 | 北京原生元生物科技有限公司 | Serum-free medium and application thereof |
Citations (4)
| 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 |
| CN102604891A (en) * | 2012-02-29 | 2012-07-25 | 天津美德太平洋科技有限公司 | High-amplification multifunctional serum-free medium for immunocyte treatment and preparation method thereof |
| US20130130373A1 (en) * | 2011-11-11 | 2013-05-23 | Essential Pharmaceuticals, Llc | Kit Comprising Serum Replacement and Labile Factors |
| CN103849600A (en) * | 2014-03-27 | 2014-06-11 | 叶永清 | Serum-free culture medium suitable for culturing NK (Natural Killer) cells |
-
2014
- 2014-12-05 CN CN201410737699.XA patent/CN104450614B/en active Active
Patent Citations (4)
| 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 |
| US20130130373A1 (en) * | 2011-11-11 | 2013-05-23 | Essential Pharmaceuticals, Llc | Kit Comprising Serum Replacement and Labile Factors |
| CN102604891A (en) * | 2012-02-29 | 2012-07-25 | 天津美德太平洋科技有限公司 | High-amplification multifunctional serum-free medium for immunocyte treatment and preparation method thereof |
| CN103849600A (en) * | 2014-03-27 | 2014-06-11 | 叶永清 | Serum-free culture medium suitable for culturing NK (Natural Killer) cells |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219708A (en) * | 2015-07-21 | 2016-01-06 | 中山大学 | Immunocyte cultivates test kit, immunocyte cultural method and application |
| CN105713880A (en) * | 2016-04-20 | 2016-06-29 | 广东艾时代生物科技有限责任公司 | Serum-free culture medium for hematopoietic stem cell in vitro expansion culture and application thereof |
| CN105713873A (en) * | 2016-04-20 | 2016-06-29 | 广东艾时代生物科技有限责任公司 | Serum-free medium for vitro amplification culture of immune cells and application thereof |
| CN106834225A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of immune cell media |
| CN106666675A (en) * | 2016-12-26 | 2017-05-17 | 弎西艾斯（苏州）生物科技有限公司 | Complex animal protein substitute |
| CN106754644A (en) * | 2016-12-26 | 2017-05-31 | 弎西艾斯（苏州）生物科技有限公司 | A kind of serum-free fibrous bud mother cell culture medium |
| CN106754648A (en) * | 2017-01-19 | 2017-05-31 | 弎西艾斯（苏州）生物科技有限公司 | A kind of non-animal derived culture medium |
| CN107119014A (en) * | 2017-05-12 | 2017-09-01 | 青岛赛欧凯普图乐生物医药有限公司 | A kind of non-animal derived culture medium of NK cells |
| CN107674860A (en) * | 2017-07-14 | 2018-02-09 | 上海源培生物科技股份有限公司 | NK92 cell non-serum culture mediums |
| CN112041430A (en) * | 2017-12-08 | 2020-12-04 | 朱诺治疗学股份有限公司 | Serum-free medium formulations for culturing cells and methods of use thereof |
| CN108949696A (en) * | 2018-08-21 | 2018-12-07 | 苏州米苏生物技术有限公司 | Immune cell media is applied with it |
| CN113481101A (en) * | 2021-08-20 | 2021-10-08 | 北京三药科技开发公司 | Redissolution of cryopreserved strain and preparation method 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 |
| CN114621123A (en) * | 2022-04-11 | 2022-06-14 | 昕嘉生物技术(长沙)有限公司 | Porous particle calcium hydroxymethionine and application thereof in serum-free culture of immune cells |
| CN115044548A (en) * | 2022-08-11 | 2022-09-13 | 北京原生元生物科技有限公司 | Serum-free medium and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450614B (en) | 2017-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104450614A (en) | Animal protein-free immune cell serum-free medium and using method thereof | |
| CN104371972B (en) | A kind of non-animal derived property and humanized's composition T lymphocytes culture medium and preparation method thereof | |
| CN102352343B (en) | Lymphocyte culture medium and use method thereof | |
| Cross et al. | Cultivation of Trypanosoma brucei sspp. in semi-defined and defined media | |
| CN101519646B (en) | CIK cell, as well as preparation method and cell preparation thereof | |
| CN105087487B (en) | A kind of method of efficient amplification CIK | |
| CN105462924B (en) | NK cell culture method and serum-free medium combination | |
| CN105039253A (en) | Serum-free culture medium suitable for immune cell large-scale culture | |
| Slivac et al. | Influence of different ammonium, lactate and glutamine concentrations on CCO cell growth | |
| CN108220235A (en) | A kind of Activated in Vitro expands people's natural kill（NK）The method of cell and its special cultivating system | |
| CN104513805B (en) | A method of producing anti-CD 20 antibodies | |
| CN105524882B (en) | Serum substitute for immunologic cytotoxicity cell expansion ex vivo combines | |
| CN104073463A (en) | Serum-free protein-free culture medium supporting CHO (Chinese Hamster Ovary Cell) high density suspension culture | |
| CN105296421B (en) | The T cell and preparation method of a kind of activation of bispecific antibody and application | |
| CN108707579A (en) | The serum free medium and preparation method and cultural method of a kind of human T lymphocyte's culture | |
| CN108949696A (en) | Immune cell media is applied with it | |
| CN102604891A (en) | High-amplification multifunctional serum-free medium for immunocyte treatment and preparation method thereof | |
| CN106834225A (en) | A kind of immune cell media | |
| CN104017770A (en) | Method for preparing CIK cell by using glycolipid | |
| Trigona et al. | Could heme-oxygenase-1 have a role in modulating the recipient immune response to embryonic stem cells? | |
| CN107988154B (en) | Composition and preparation method and application thereof | |
| AU2011202080B2 (en) | Biological medium for preserving a preparation of insulin-secreting cells | |
| CN115044548B (en) | Serum-free medium and application thereof | |
| JP2800338B2 (en) | Preparation method of serum-free medium for cell culture | |
| CN103468746A (en) | Method for constructing tumor cell line |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 201318 Shanghai City, Pudong New Area Road No. 588 building 3 indigo Applicant after: SHANGHAI ANGECON BIOTECHNOLOGY CO., LTD. Address before: 201318 Shanghai City, Pudong New Area Road No. 588 building 3 indigo Applicant before: Shanghai Angecon Biotechnology Co., Ltd. |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210112 Address after: 201306 building 10, No. 860, Xinyang Road, Lingang New District, Pudong New Area Free Trade Zone, Shanghai Patentee after: SHANGHAI ANKU SHENGYI BIOTECHNOLOGY Co.,Ltd. Address before: Building 3, 588 Qingdai Road, Pudong New Area, Shanghai, 201318 Patentee before: SHANGHAI ANGECON BIOTECHNOLOGY Co.,Ltd. |