CN103146648A - Animal source-free and serum-free culture medium of lymphocyte - Google Patents
Animal source-free and serum-free culture medium of lymphocyte Download PDFInfo
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Abstract
The invention relates to the biological field and discloses an animal source-free and serum-free culture medium of lymphocyte. The culture medium disclosed by the invention essentially consists of IMDM (Iscove Modified Dulbecco Medium), L-glutamine, sodium bicarbonate, recombinant human insulin, human transferrin, human serum albumin, 2-mercaptoethanol, N-acetyl-cysteine, lipid, amino acid, vitamin, microelement, ferric citrate, hydrocortisone, cholamine and non-essential amino acid. The serum-free culture medium disclosed by the invention has the advantages of clear chemical components, no animal source, no serum, safe and ideal culture cells; the instability caused by the doping of animal components and batches is avoided; the result of culturing lymphocyte shows that the total number of the cells and the cell phenotypes are normal; and the serum-free culture medium disclosed by the invention has a good industrial application prospect.
Description
Technical field
The present invention relates to biological field, particularly a kind of specific chemical components, non-animal derived lymphocyte serum free medium.
Background technology
Cellular immunization treatment is a kind of emerging, brand-new antitumour treatments with significant curative effect, make up the drawback of traditional operation, radiotherapy, chemotherapy, be considered to a kind for the treatment of means active, the most rising in 21st century combined therapy of tumour pattern.The cellular immunization treatment is to gather human immunocyte's (being mainly lymphocyte), through vitro culture, its quantity thousandfold is increased, the targeting killing ability strengthens, and then feed back to human body and kill pathogenic agent, the cancer cells in blood and tissue, the cell of sudden change, break immunological tolerance, the immunological competence of activation and enhancing body, the double effects of taking into account treatment and keeping healthy.
It is substratum that vitro culture, amplifying lymphocyte use maximum reagent; substratum provides basic environment for the growth amplification of cell; comprise suitable osmotic pressure; for lymphocytic amplification provides growth necessary nutritive ingredient; comprise amino acid, inorganic salt, trace element, VITAMIN etc., also protect simultaneously the infringement of the toxicant that lymphocyte avoids producing in metabolic process.Traditional lymphocytic amplification system is mainly that basic medium adds certain density foetal calf serum (FBS).Although Europe etc. allow to use FBS, China, the countries such as the U.S. forbid to use the animal source composition in cell therapy.Contain foreign protein matter in FBS, itself has the risk of carrying bacterium, virus, albumen communicable disease or Protein virus.In addition, there are some researches show that lymphocyte can engulf the albumen in substratum in culturing process, contain bovine serum albumin, can make the anti-bovine protein antibody of the interior generation of recipient's body cause immune response, thereby cause especially treatment inefficacy after repeating the infusion cell therapy of patient.Therefore the unfavorable factor of FBS in the clinical large scale culturing of cell comes out gradually, now the existing lymphocytic serum free medium of a lot of scholar's research.Some companies have also developed the lymphocyte serum free medium. as the LONZA exploitation X-VIVO of company serum free medium, the Austrian PAA exploitation QUANTUM-007 of company lymphocyte serum free medium etc.But these serum-free culture based components are indefinite, and expensive, and the cost that this has increased the immunocyte treatment greatly is unfavorable for the popularization of widespread use.And QUANTUM-007 lymphocyte serum free medium contains animal source composition (specification sheets).
We provide a kind of lymphocyte serum free medium, and this substratum specific chemical components is non-animal derived, serum-free.This type of substratum is present safe, the most ideal substratum, at first can guarantee the consistence between substratum batch, is secondly that the character of substratum is clear and definite, helps further to study lymphocytic mechanism of action.The exploitation of this substratum can greatly promote immunocyte treatment stdn and cultivate, and has also saved production cost.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of serum free medium, consist of IMDM 17.7 g/L, L-glutaminate 1-5 mM, sodium bicarbonate 3.024 g/L, recombinant human insulin 1-10 mg/L, human transferrin 5-20 mg/L, human serum albumin 4-10 g/L, 2 mercapto ethanol 55 uM, N-acetyl-halfcystine 100-300 mg/ml, lipid 0.1-0.5%, amino acid/11-5%, VITAMIN 1-5%, micro-0.1-0.5%, hydrocortisone 1-10ng/ml, thanomin 1-5mg/l, non-essential amino acid 1-5%, ironic citrate 1-10mg/L.
More preferably, its necessity of serum free medium of the present invention consists of IMDM 17.7 g/L, L-glutaminate 5 mM, sodium bicarbonate 3.024 g/L, N-acetyl-halfcystine 160mg/L, recombinant human insulin 10 mg/L, human transferrin 10 mg/L, human serum albumin 5 g/L, 2 mercapto ethanol 55 uM, hydrocortisone 40ug/L, lipid 0.1%, amino acid 2%, VITAMIN 1%, trace element 1%, non-essential amino acid 1%, ironic citrate 1-10mg/L.
Compare with QUANTUM-007 lymphocyte serum free medium, X-VIVO serum free medium training lymphocyte with non-animal derived, serum free medium provided by the invention, result shows, its cells expanded, Cell viability are better than this two kinds of substratum, the cell phenotype result shows that also the ratio of helper T cell and regulatory T cells is higher than these two kinds of substratum, under certain proportion, the CIK cell of serum free medium of the present invention amplification kills ratio of outflow higher than these two kinds of substratum, and avoided animal component and batch unstable.
The invention provides a kind of specific chemical components, non-animal derived, serum-free lymphocytes culture medium, this substratum is safe, the most ideal present substratum, at first can guarantee the consistence between substratum batch, next is that the character of substratum is clear and definite, helps further to study lymphocyte amplification situation.The lymphocyte of transplanting interacts to reach each other by cytokine profiles and microenvironment and adapts to, microenvironment mesostroma cell produces cytokine to transplanting rear lymphocytic chemotactic, going back to the nest, activate and survive and all play an important role, and the non-animal derived composition of substratum, can avoid introducing animal component in the process of cell cultures the interference cell growth.
Description of drawings
Fig. 1 is that A, B in embodiment, C serum free medium are cultivated bleeding of the umbilicus CIK cellular form figure;
Fig. 2 is that A, B in embodiment, C serum free medium are cultivated peripheral blood CIK cellular form figure;
Fig. 3 is that A, B in embodiment, C serum free medium are cultivated bleeding of the umbilicus CIK amplification times;
Fig. 4 is that A, B in embodiment, C serum free medium are cultivated peripheral blood CIK amplification times;
Fig. 5 is that A, B in embodiment, C serum free medium are cultivated bleeding of the umbilicus CIK cell viability;
Fig. 6 is that A, B in embodiment, C serum free medium cultivate bleeding of the umbilicus cell phenotype after CIK21 days;
Fig. 7 is that A, B in embodiment, C serum free medium cultivate peripheral blood cell phenotype after CIK21 days;
Fig. 8 be A, B in embodiment, C serum free medium cultivate bleeding of the umbilicus after CIK21 days cell kill ratio of outflow;
Embodiment
The invention discloses a kind of non-animal derived lymphocyte serum free medium, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.Non-animal derived lymphocyte serum free medium of the present invention and application are described by preferred embodiment, the related personnel obviously can change methods and applications as herein described within not breaking away from content of the present invention, spirit and scope or suitably change and combination, realizes and use the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, the present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1: the preparation of substratum
The preparation of recombinant human insulin's storage liquid: take the 100mg recombinant human insulin and be dissolved in 40mL water for injection, drip 2M HCl and dissolve fully to the recombinant human insulin, be settled to 50mL, be mixed with the storage liquid of 20mg/mL, 4 ℃ of preservations.
The preparation of human transferrin storage liquid: take the 100mg human transferrin and be dissolved in 40mL water for injection, to being settled to 50mL after dissolving fully, be mixed with the storage liquid of 20mg/mL, 4 ℃ of preservations.
Medium component of the present invention is as follows:
| Component | Addition |
| IMDM | 17.7 g/L |
| L-glutaminate | 1-5 mM |
| Sodium bicarbonate | 3.024 g/L |
| The recombinant human insulin | 1-10 mg/L |
| Human transferrin | 5-20 mg/L |
| Human serum albumin | 1-10 g/ |
| 2 mercapto ethanol | 55 uM |
| N-acetyl-halfcystine | 100-300mg/L |
| Lipid | 0.1-0.5% |
| Amino acid | 1-5% |
| VITAMIN | 1-5% |
| Trace element | 0.1-0.5% |
| Non-essential amino acid | 1-5% |
| Hydrocortisone | 1-10ng/L |
| Ironic citrate | 1-10 mg/L |
More preferably, medium component of the present invention is as follows:
| Component | Addition |
| IMDM | 17.7 g/L |
| L- |
5 mM |
| Sodium bicarbonate | 3.024 g/L |
| The recombinant |
10 mg/L |
| |
10 mg/L |
| Human serum albumin | 5g/ |
| 2 mercapto ethanol | 55 uM |
| N-acetyl-halfcystine | 160mg/L |
| Lipid | 0.1% |
| |
1% |
| VITAMIN | 2% |
| Trace element | 0.1% |
| Non-essential |
1 |
| Hydrocortisone | |
| 40 ug/L | |
| |
2 mg/L |
Amino acid described in upper table is the mixture of L-arginine, CYSTINE, L-Histidine, ILE, L-Leu, 1B, METHIONINE, L-Phe, L-threonine, L-Trp, TYR, Valine, ALANINE, L-Aspartic acid, altheine, Pidolidone, glycine, L-PROLINE, Serine;
Described VITAMIN comprises calcium pantothenate, choline chloride 60, folic acid, meso-inositol, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride.
Described lipid comprises arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linolic acid, linolenic acid, tetradecanoic acid, oleic acid, Zoomeric acid, palmitinic acid, stearic acid;
Described trace element is the mixture of Cu, Zn, Se, Fe, Sn, Ni, Ag, Al, Cr, Ge, Zr, Rb, Co, Cd, Ba, K.
Regulate substratum between 7.2-7.4 with pH meter, osmotic pressure between 260-320 mOsm/kg, intracellular toxin detections＜0.25EU/mL, 0.22 μ m filter filtration sterilization seals, 4 ℃ keep in Dark Place.
Embodiment 2: bleeding of the umbilicus separates
Derived from cord blood: bleeding of the umbilicus is from the eutocous puerpera of Beijing armed police hospital obstetrics full-term pregnancy, " the Donors Physical examination standard " of promulgating by the Ministry of Health is up to the standards, in the 3rd stages of labor totally-enclosed collection bleeding of the umbilicus after the umbilical vein sterilization, anticoagulant heparin, the on average amount of adopting 80～130 mL.Sepn process is as follows:
1: after fresh bleeding of the umbilicus and damping fluid PBS are pressed the dilution of 1:1 volume ratio, slowly add to parting liquid Ficoll upper strata along test tube wall.Note keeping two interfaces clear, blood is sneaked in parting liquid.
The centrifugal 15min of 2:1800rpm inserts white nepheloid layer (from the top down count the second layers) gently with capillary pipet, and along the tube wall mononuclearcell of sucking-off this layer gently, Sheng enters in another centrifuge tube.
3: the mononuclearcell suspension of gained is washed with the long-pending PBS of monoploid, and the centrifugal 10min of 1500rpm washs 3 times.
4: counting cells, adjust cell to required concentration, according to 2～5 * 10
6The density of/mL is inoculated, and cultivates in orifice plate or culturing bottle.
Embodiment 3: peripheral blood separates
Derived from peripheral blood: peripheral blood is from Beijing armed police hospital, and " the Donors Physical examination standard " of promulgating by the Ministry of Health is up to the standards, totally-enclosed collection peripheral blood after the vein sterilization, anticoagulant heparin, the on average amount of adopting 50mL.Sepn process is as follows:
1: after fresh peripheral blood and damping fluid PBS are pressed the dilution of 1:1 volume ratio, slowly add to parting liquid Ficoll upper strata along test tube wall.Note keeping two interfaces clear, blood is sneaked in parting liquid.
The centrifugal 15min of 2:1800rpm inserts white nepheloid layer (from the top down count the second layers) gently with capillary pipet, and along the tube wall mononuclearcell of sucking-off this layer gently, Sheng enters in another centrifuge tube.
3: the mononuclearcell suspension of gained is washed with the long-pending PBS of monoploid, and the centrifugal 10min of 1500rpm washs 3 times.
4: counting cells, adjust cell to required concentration, according to 2～5 * 10
6The density of/mL is inoculated, and cultivates in orifice plate or culturing bottle.
Embodiment 4: cell cultures
Respectively following several groups of A: embodiment 1 preparation serum-free lymphocytes culture medium, B:QUANTUM-007 lymphocyte serum free medium, C:X-VIVO serum free medium.Add the substratum of 6mL to join in the culturing bottle of T25 by every bottle, and add connector (IFN-γ 1000IU/mL, CD3 50ng/mL, IL-2 2000IU/mL, IL-1a 100IU/mL), culturing bottle is placed in 37 ℃, 5%CO
2Cultivate under the incubator condition.Every 3 days later on countings, adding serum-free lymphocyte culture fluid and factor adjustment cell density is 2 * 10
6/ mL.Microscopically observation of cell form is seen Fig. 1 and Fig. 2.Trypan Blue calculates cell proliferating number, sees Fig. 3 and Fig. 4.
Microscopically observation of cell form, the result demonstration, serum free medium of the present invention is similar with the CIK cellular form that X-VIVO serum free medium, QUANTUM-007 lymphocyte serum free medium are cultivated, and cell outline is clear.
The count results demonstration, the CIK multiple of serum free medium amplification of the present invention will be higher than X-VIVO serum free medium and QUANTUM-007 lymphocyte serum free medium amplification times.
Embodiment 5: cell viability detects
Getting 1mL concentration is 10
6The CIK cell suspension of/ml, the Propidium iodide dye liquor that adds 10 μ L mixes, effect 10min; Add 5mL PBS, the centrifugal 5min of 1500r/min abandons supernatant, repeated washing 2 times, and fluidic cell detects cell viability, sees Fig. 5.
The result demonstration, the CIK cell viability of serum free medium amplification of the present invention is higher than the vigor of X-VIVO serum free medium and QUANTUM-007 lymphocyte serum free medium amplifying cells.
Lymphocytic phenotype detects
The CD3+ mark positive is the T lymphocyte; CD3+CD8+ is T effector cell; CD3+CD4+ is helper T cell; CD3+CD56-is the T cell; CD3-CD56+ is the NK cell; CD4+CD25+ is regulatory T cells; CD3+ CD56+ is the NKT cell.
Adopt flow cytometer to measure respectively the expression of CD3+ in amplifying cells, CD56+CD3-, CD3+CD4+, CD3+CD8+, CD4+CD25+, CD56+CD3+.Get the lymphocyte after amplification cultivation, add fluorescence antibody 20 μ L, detect with flow cytometer after mixing, with Cell Quest software analysis, establish a pipe contrast, the results are shown in Figure 6 and Fig. 7.
The result demonstration, in the CIK cell of serum free medium amplification of the present invention, the ratio of helper T cell and regulatory T cells is higher than the ratio in X-VIVO serum free medium and QUANTUM-007 lymphocyte serum free medium.
Embodiment 6: lymphocyte effect target kills and wounds detection
The CIK cell of getting amplification is the effector cell, and take the A549 cell as target cell, ratio is pressed 5:1,10:1, and 20:1 is inoculated in 96 orifice plates, 37 ℃, 5% CO
2Cultivated 24 hours.Survey absorbance with mtt assay, calculate and kill ratio of outflow.Kill ratio of outflow %=1-(experimental port-effect hole)/the target cell hole, see Fig. 8.
Result shows, the effector cell: in target cell=5: 1 situation, the CIK cell of serum free medium amplification of the present invention kills ratio of outflow higher than the cell in X-VIVO serum free medium and QUANTUM-007 lymphocyte serum free medium.
The above is only the preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. serum free medium, its necessity consists of IMDM 17.7 g/L, L-glutaminate 1-5 mM, sodium bicarbonate 3.024 g/L, recombinant human insulin 1-10 mg/L, human transferrin 5-20 mg/L, human serum albumin 1-10 g/L, 2 mercapto ethanol 55 uM, N-acetyl-halfcystine 100-300 mg/mL, lipid 0.1-0.5%, amino acid/11-5%, VITAMIN 1-5%, micro-0.1-0.5%, hydrocortisone 1-10ng/mL, thanomin 1-5mg/L, non-essential amino acid 1-5%, ironic citrate 1-10mg/L.
2. serum free medium according to claim 1, it is characterized in that, its necessity consists of IMDM 17.7 g/L, L-glutaminate 5 mM, sodium bicarbonate 3.024 g/L, N-acetyl-halfcystine 160mg/L, recombinant human insulin 10 mg/L, human transferrin 10 mg/L, human serum albumin 5 g/L, 2 mercapto ethanol 55 uM, hydrocortisone 40ug/L, lipid 0.1%, amino acid 2%, VITAMIN 1%, trace element 1%, non-essential amino acid 1%, ironic citrate 2mg/L.
3. the described serum free medium of claim 1-2 any one is cultivated lymphocyte.
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| CN105039251A (en) * | 2015-07-27 | 2015-11-11 | 广州市达晖生物技术有限公司 | Lymphocyte serum-free medium with stable pH value as well as preparation method and application of lymphocyte serum-free medium |
| CN105524882A (en) * | 2016-01-18 | 2016-04-27 | 华东理工大学 | Serum substitution combination for immune killer cell amplification in vitro |
| CN105524882B (en) * | 2016-01-18 | 2019-05-28 | 华东理工大学 | Serum substitute for immunologic cytotoxicity cell expansion ex vivo combines |
| CN105567634A (en) * | 2016-01-27 | 2016-05-11 | 上海润泉生物技术有限公司 | Culture medium and method for NK cell expansion in vitro |
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Application publication date: 20130612 |