CN103834615A - Serum-free medium suitable for culturing gamma delta T cells - Google Patents

Serum-free medium suitable for culturing gamma delta T cells Download PDF

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CN103834615A
CN103834615A CN201410120087.6A CN201410120087A CN103834615A CN 103834615 A CN103834615 A CN 103834615A CN 201410120087 A CN201410120087 A CN 201410120087A CN 103834615 A CN103834615 A CN 103834615A
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叶永清
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Shanghai Ke Laixun Bioisystech Co Ltd
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Abstract

The invention discloses a serum-free medium suitable for culturing gamma delta T cells. The serum-free medium consists of a basal culture medium component I, a gamma delta T cell specific culture component II and water. Experiments show that by adopting the serum-free medium suitable for culturing the gamma delta T cells, the gamma delta T cells obtained is greater in quantity, higher in purity and better in killing effect. The serum-free medium suitable disclosed by the invention has the advantages that 1) the risk of animal components which are used in animal serums in the cell culture with the gamma delta T cells on patients with cell therapy and uncertain impact of uncertain components in the animal serums on the result of the cell culture are avoided; 2) the yield of the gamma delta T cells is greatly improved; and 3) the final density of the gamma delta T cell culture is increased so as to lower the cost of cell culture.

Description

A kind of serum free medium that is applicable to cultivate gamma delta T cells
Technical field
The present invention relates to a kind of serum free medium that is applicable to cultivate gamma delta T cells.
Background technology
γ δ Τ cell is found in eighties of last century the mid-80, mainly contains two kinds of hypotypes: V γ 9/V δ 2 and V δ 1, γ δ Τ only accounts for 0.5%～5% in periphery blood T lymphocyte, be wherein greater than 90% for V γ 9/V δ 2 cells.V δ 1 is mainly distributed in epithelium, as enteron aisle, and spleen, skin, small intestine, esophagus, lung and sexual organ official rank.
Gamma delta T cells has quick response characteristic in vivo.In the time being upset, can discharge pro-inflammatory cytokine prior to α β-T cell, as IFN-γ and TNF-α; Tumour cell is had to High Fragmentation, thereby can start multiple killing tumor cell mode by the membranin such as MICA/B and DNAM-1 on surface of cell membrane molecule NKG2D tumor cell surface, as discharged pore-forming protein, granzyme, FasL, the modes such as Trail; γ δ Τ cell can also activate the Tumoricidal action of NK cell; Can stimulate mutually maturation with DC cell; In addition, γ δ Τ cell also has the function that APC cell antigen are offered, and activates the immunological effect of α β-T cell.The people such as Bouet-Toussaint have extracted 6 routine colorectal cancers and 4 routine Peripheral Blood of Patients with Hepatocellular Carcinomas and have carried out the vitro culture of γ δ Τ cell, find that colorectal cancer and the autologous tumour cell of liver cancer patient can be by V γ 9/V δ 2 lysises of corresponding cultivation separately, and autologous normal tissue cell is also insensitive to V γ 9/V δ 2 lysis effects, the γ δ Τ cell adoptive therapy liver cancer of this results suggest amplification in vitro is seemingly a kind of effective method.
Clinically at present, γ δ Τ cell is generally used for the various tumours for the treatment of, to respiratory system, Digestive tract, reproductive system and part Hematological Malignancies good effect.Gamma delta T cells and NK cell form complementation, can kill and wound the insensitive target cell of NK cell, support antineoplastic natural basic defence line thereby jointly build up body with NK cell.And the method that obtains clinically γ δ Τ cell generally increases from peripheral blood mononuclear cell, in culturing process, generally need to add animal serum to improve the effect of cell cultures, animal serum can provide Growth of Cells required basic nutrition material: amino acid, VITAMIN, inorganics, lipid material, nucleic acid derivative etc., can also provide hormone and various somatomedin: Regular Insulin, adrenocortical hormone (hydrocortisone, dexamethasone), steroid hormone (estradiol, testosterone, progesterone), fibroblast growth factor, Urogastron, PDGF etc.In animal serum, existing for of these materials obtains clinically cellular product safely and effectively strong guarantee is provided.But, owing to containing animal component in animal serum, if it is for clinical cell therapy system, may cause some potential risks, this is mainly due to animal serum complicated component, though containing many to the favourable composition of cell, but also there is the factor of many unfavorable Growth of Cells to exist: 1) animal serum contains some to the toxigenous material of cell, as polyamine oxidase, can with react (as spermine from the polyamines of height propagated cell, spermidine) be formed with the poly-spermine of cytotoxic effect, complement, antibody, bacteriotoxins etc. all can affect Growth of Cells, even cause necrocytosis, 2) animal individual difference, the serum place of production, lot number difference, every quality of lot difference is very large, and its composition can not be consistent, 3) may bring mycoplasma, virus in drawing materials, cell is produced to potential impact, may cause the failure of an experiment or experimental result unreliability, 4) in scale operation, serum source is more and more difficult, expensive, is to form one of animal cell culture major portion to production cost.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of serum free medium that is applicable to cultivate gamma delta T cells, ingredient is chemically defined composition, not containing any animal serum composition, thereby avoided using the problem of the security that animal serum may cause, and cost is lower.
The present invention is achieved by the following technical solutions:
Be applicable to cultivate a serum free medium for gamma delta T cells, be made up of basic medium component I, gamma delta T cells specificity cultivation component II and water, wherein, described basic medium component I is made up of the component of following concentration:
Calcium Chloride Powder Anhydrous 28～33.82mg/L, D-VB5 calcium 2.24～3.19mg/L, TYR 4～5.9mg/L, L-Methionine 3～5mg/L, Valine 6～13.7mg/L, L-Phe 4～7mg/L, D-Glucose 1789～1802mg/L, pyridoxine hydrochloride (vitamin B6) 0.055～0.066mg/L, xanthoglobulin 3～5mg/L, Serine 7～15mg/L, sodium-chlor 7200～7599mg/L, riboflavin 0.022～0.048mg/L, linolic acid 0.077～0.094mg/L, L-threonine 10～13.9mg/L, Sodium phosphate dibasic 120～149mg/L, thymidine 0.22～0.39mg/L, Thioctic Acid 0.12～0.29mg/L, L-asparagine 7～18.01mg/L, L-arginine hydrochloride 198～213mg/L, ASPARTIC ACID 6～19.3mg/L, 1, 4-butanediamine dihydrochloride 0.11～0.19mg/L, Cys hydrochloride 22～39.12mg/L, L-glutaminate 113～149mg/L, Pidolidone 2～17.7mg/L, Sodium.alpha.-ketopropionate 100～110mg/L, vitamin B12 0.312～0.657mg/L, glycine 6～8mg/L, L-Leu 11～13.4mg/L, vitamin H 0.004～0.008mg/L, L lysine HCL 34～39.5mg/L, L-Histidine hydrochloride 9～25mg/L, L-PROLINE 25～34.5mg/L, as shown in table 1.
Described gamma delta T cells specificity cultivation component II is made up of the component of following concentration:
Transferrins,iron complexes 20～100mg/L, TCR γ δ monoclonal antibody 20～50mg/L, nerve growth factor 1～10mg/L, IL-450～100mg/L, become Mierocrystalline cellulose growth factor-21～100mg/L, insulin-like growth factor 50～200mg/L, IL-1250～500mg/L, Pamidronic Acid disodium 150～500mg/L.As shown in table 2.The present invention nerve growth factor, one-tenth Mierocrystalline cellulose cell growth factor, insulin-like growth factor, IL-4, IL-12 used all purchases the company from R & D, TCR γ δ monoclonal antibody is purchased from Zhejiang Rui Shun biotech company, and the buying of Pamidronic Acid disodium is from sea, Shenzhen king's medicine company.
Table 1
Figure BDA0000483384550000031
Table 2
Figure BDA0000483384550000041
Preferably, described basic medium component I is made up of the component of following concentration: Calcium Chloride Powder Anhydrous 29mg/L, D-VB5 calcium 2.5mg/L, TYR 4.5mg/L, L-Methionine 4mg/L, Valine 13.5mg/L, L-Phe 6mg/L, D-Glucose 1800mg/L, pyridoxine hydrochloride 0.055mg/L, xanthoglobulin 5mg/L, Serine 8mg/L, sodium-chlor 7500mg/L, riboflavin 0.028mg/L, linolic acid 0.079mg/L, L-threonine 13mg/L, Sodium phosphate dibasic 135mg/L, thymidine 0.25mg/L, Thioctic Acid 0.19mg/L, L-asparagine 14.4mg/L, L-arginine hydrochloride 200mg/L, ASPARTIC ACID 13.5mg/L, 1, 4-butanediamine dihydrochloride 0.18mg/L, Cys hydrochloride 29mg/L, L-glutaminate 124mg/L, Pidolidone 13.9mg/L, Sodium.alpha.-ketopropionate 105mg/L, vitamin B12 0.35mg/L, glycine 7mg/L, L-Leu 13.4mg/L, vitamin H 0.005mg/L, L lysine HCL 37.5mg/L, L-Histidine hydrochloride 14mg/L, L-PROLINE 25.6mg/L,
Described gamma delta T cells specificity cultivation component II is made up of the component of following concentration:
Transferrins,iron complexes 50mg/L, TCR γ δ monoclonal antibody 50mg/L, nerve growth factor 10mg/L, IL-4100mg/L, becomes Mierocrystalline cellulose cell growth factor 50mg/L, Pamidronic Acid disodium 250mg/L, insulin-like growth factor 100mg/L, IL-12250mg/L; As shown in table 3.
Table 3
Figure BDA0000483384550000042
Figure BDA0000483384550000051
The described preparation method who is applicable to the serum free medium of cultivating gamma delta T cells, step is as follows:
(1) get each raw material of component I, mixing mixes, under 60 DEG C of vacuum dry 15 hours, for subsequent use; Get each raw material of component II, under 30 DEG C of vacuum, be dried 18 hours, mixing mixes, for subsequent use;
(2) above-mentioned dried component I and component II are mixed, pin mill 3 hours, crosses 30 mesh sieves, obtains solid state powder, and production process temperature is controlled at 20～25 DEG C, and humidity is controlled at 15～20%, and nitrogen pressure is controlled at 150～300psi; When use, add water, mix, 0.22 μ m membrane filtration, must be applicable to cultivate the serum free medium of gamma delta T cells.
The serum free medium that is applicable to cultivate gamma delta T cells of the present invention, some components that some can promote gamma delta T cells proliferated specifically and differentiation in component II, are comprised, mainly comprise and can and can promote the cytokine of gamma delta T cells propagation and differentiation as TCR γ δ monoclonal antibody with NK cell surface receptor specific binding, Pamidronic Acid disodium, IL-12 and IL-4 etc., the experiment proved that, adopt the serum free medium cultivation gamma delta T cells that is applicable to cultivate gamma delta T cells of the present invention, can obtain more, higher and the better gamma delta T cells of fragmentation effect of purity.The serum free medium that is applicable to cultivate gamma delta T cells of the present invention, has the following advantages: in the animal serum of 1) having avoided being used in cell cultivation process by gamma delta T, animal component is on uncertain impact that in the risk of accepting cell therapy patient and bringing and animal serum, uncertain composition causes the result of cell cultures; 2) greatly improved gamma delta T cells yield; 3) increase the whole density that gamma delta T cells is cultivated, thereby reduced the cost of cell cultures.
Brief description of the drawings
Fig. 1: use serum free medium of the present invention to cultivate the auspicious Ji's Albert'stain Albert of gamma delta T cells figure, visible typical comet sample tail.
Fig. 2: use serum free medium of the present invention and the contrast of other substratum gamma delta T cells propagation multiple, wherein, M1 is serum free medium of the present invention, and M2 is substratum as a control group, is Optimizer substratum (LifeTechnology company).
Fig. 3: use serum free medium of the present invention and the contrast of other substratum gamma delta T cells culture densities, wherein, M1 is serum free medium of the present invention, and M2 is control medium.
Fig. 4: use serum free medium gamma delta T cells of the present invention to cultivate streaming result, wherein, left upper quadrant reaction is CD3+CD γ δ+cell content.
Fig. 5: use serum free medium of the present invention and other substratum gamma delta T cells to cultivate and kill the contrast of knurl experimental result, wherein, M1 is serum free medium of the present invention, and M2 is control medium.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 preparation is applicable to cultivate the serum free medium of gamma delta T cells
Preparation substratum 100L, calculates according to table 3, and the quality of the needed each component of configuration 100L gamma delta T cells serum-free medium is in table 4.
Table 4
Figure BDA0000483384550000061
Figure BDA0000483384550000071
Preparation method is:
(1) get each raw material of component I, mixing mixes, under 60 DEG C of vacuum dry 15 hours, for subsequent use; Get each raw material of component II, under 30 DEG C of vacuum, be dried 18 hours, mixing mixes, for subsequent use;
(2) above-mentioned dried component I and component II are mixed, pin mill 3 hours, crosses 30 mesh sieves, obtains solid state powder, and production process temperature is controlled at 20～25 DEG C, and humidity is controlled at 15～20%, and nitrogen pressure is controlled at 150～300psi; In solid state powder, add water, mix, 0.2 μ m membrane filtration, must be applicable to cultivate the serum free medium of gamma delta T cells.
Embodiment 2 uses gamma delta T cells serum free medium to cultivate gamma delta T cells and compares culture effect with control group substratum
Step is as follows:
(1) machine adopts at least 1 × 10 9individual patient's mononuclearcell (PBMC) (setting program is the autoMNC in Leukocyte-Aphersis for blood cell separator model C OM.TEC, the Fresenius Kabi of producer);
(2) blood sample of collection is proceeded to 50ml centrifuge tube, centrifugal 400g, 10min; Get supernatant blood plasma frozen, remaining blood dilutes with PBS;
(3) 30ml dilute blood is slowly added on the Ficoll-Hypaque of 15ml, moderating process is set as after brake off, centrifugal 900g, 20min;
(4) after centrifugal end, flat mouth Pasteur dropper is inserted into mononuclearcell layer gently, carefully draw accurately this confluent monolayer cells and transfer in another new 50ml centrifuge tube along tube wall, every pipe adds physiological saline 50ml, blow gently even and fine born of the same parents, 2 (room temperature 1300r/min, 7min for the first time, of centrifuge washing; For the second time, room temperature 1200r/min, 10min), remove thrombocyte and separating medium, last centrifugal front counting;
(5) supernatant discarded, with 2 kinds of different gamma delta T substratum (substratum of the present invention and optimizer substratum) with 1 × 10 6the concentration of/ml is resuspended, and spreads the culturing bottle into T175, and 50ml/ bottle, is placed in 37 DEG C, 5%CO 2in incubator;
(6) fluid infusion every other day, adds IL-2500U/L;
(7) the 0th, 10,15,20 days to gamma delta T cells counting, staining analysis.
Result: the gamma delta T cells being obtained by gamma delta T cells serum-free medium, endochylema is abundant, contain more granular material, there is comet sample tail, meet gamma delta T cells morphocytology feature (Fig. 1), with control group substratum contrast, can learn that gamma delta T cells serum free medium of the present invention cultivates gamma delta T cells propagation multiple obviously raise (Fig. 2), in addition, use gamma delta T cells serum free medium of the present invention to obtain the whole density of NK cell and can reach 3.15*10 6/ ml is apparently higher than the whole density of gamma delta T cells that control group obtains (Fig. 3).
To sum up, use gamma delta T cells serum free medium of the present invention can under equal conditions obtain more cell proliferation multiple, the whole density of higher cell cultures, therefore used gamma delta T cells serum free medium of the present invention can reduce cell cultures cost.
Embodiment 3 gamma delta T cells flow cytometer detections
Step is as follows:
(1) will approximately contain 1x10 6cell suspension to be checked adds the streaming pipe of reference numeral, 1500rpm, 5min, supernatant discarded;
(2) add 2mL streaming dyeing buffer, re-suspended cell, 1500rpm, 5min, supernatant discarded;
(3) add 300 μ L streaming dyeing buffer, re-suspended cell, is divided into three parts, and portion is made as negative control (not adding antibody), and other two parts of pipes add respectively fluorescently-labeled monoclonal antibody (noting adding corresponding amount according to specification sheets);
(4) normal temperature lucifuge is hatched 30min; Add 1mL streaming dyeing buffer, 1500rpm, 5min, supernatant discarded; Repeat to wash one time;
(5) with 300 μ L streaming dyeing buffer re-suspended cells, wait to be analyzed.
Result: two kinds of different culture medias are obtained to gamma delta T cells on the 15th day in cultivation and carry out flow cytometer detection, use gamma delta T cells serum free medium of the present invention to obtain gamma delta T cells ratio and can reach 87.6%(Fig. 4), and use control group cultural method to obtain gamma delta T cells ratio at 65%(table 5), obvious significant difference there is.
Table 5
Type of culture medium Cultivate 15 days gamma delta T cells per-cent (%)
M1 87.6
M2 65
Embodiment 4 gamma delta T cells kill tumor activity and detect
The gamma delta T cells action effect cell of getting cultivation, tumour cell is target cell, and effect target is than being 10:1, and 20:1 adds 96 orifice plates, separately establishes individual effect cell hole and independent target cell hole in contrast, establishes 3 multiple holes for every group.37 DEG C, 5%CO 2hatch 24 hours, with MTT tetramethyl-azo azoles salt development process, 570nm place in microplate reader surveys OD value.
It is all to have and significantly kill tumor activity (Fig. 5) in 10:1 or 20:1 situation at effect target than (E/T) that two kinds of different cultural methods obtain gamma delta T cells, it kills tumor activity between 65%～85%, uses gamma delta T cells serum free medium of the present invention to cultivate acquisition NK cell and can serve as an effective effector cell for clinical.

Claims (4)

1. a serum free medium that is applicable to cultivate gamma delta T cells, is characterized in that: be made up of basic medium component I, gamma delta T cells specificity cultivation component II and water, wherein, described basic medium component I is made up of the component of following concentration:
Calcium Chloride Powder Anhydrous 28～33.82mg/L, D-VB5 calcium 2.24～3.19mg/L, TYR 4～5.9mg/L, L-Methionine 3～5mg/L, Valine 6～13.7mg/L, L-Phe 4～7mg/L, D-Glucose 1789～1802mg/L, pyridoxine hydrochloride (vitamin B6) 0.055～0.066mg/L, xanthoglobulin 3～5mg/L, Serine 7～15mg/L, sodium-chlor 7200～7599mg/L, riboflavin 0.022～0.048mg/L, linolic acid 0.077～0.094mg/L, L-threonine 10～13.9mg/L, Sodium phosphate dibasic 120～149mg/L, thymidine 0.22～0.39mg/L, Thioctic Acid 0.12～0.29mg/L, L-asparagine 7～18.01mg/L, L-arginine hydrochloride 198～213mg/L, ASPARTIC ACID 6～19.3mg/L, 4-butanediamine dihydrochloride 0.11～0.19mg/L, Cys hydrochloride 22～39.12mg/L, L-glutaminate 113～149mg/L, Pidolidone 2～17.7mg/L, Sodium.alpha.-ketopropionate 100～110mg/L, vitamin B12 0.312～0.657mg/L, glycine 6～8mg/L, L-Leu 11～13.4mg/L, vitamin H 0.004～0.008mg/L, L lysine HCL 34～39.5mg/L, L-Histidine hydrochloride 9～25mg/L, L-PROLINE 25～34.5mg/L,
Described gamma delta T cells specificity cultivation component II is made up of the component of following concentration:
Transferrins,iron complexes 20～100mg/L, TCR γ δ monoclonal antibody 20～50mg/L, nerve growth factor 1～10mg/L, IL-450～100mg/L, become Mierocrystalline cellulose growth factor-21～100mg/L, Pamidronic Acid disodium 150～500mg/L, insulin-like growth factor 50～200mg/L, IL-1250～500mg/L.
2. the serum free medium that is applicable to cultivate gamma delta T cells according to claim 1, it is characterized in that: described basic medium component I is made up of the component of following concentration: Calcium Chloride Powder Anhydrous 29mg/L, D-VB5 calcium 2.5mg/L, TYR 4.5mg/L, L-Methionine 4mg/L, Valine 13.5mg/L, L-Phe 6mg/L, D-Glucose 1800mg/L, pyridoxine hydrochloride 0.055mg/L, xanthoglobulin 5mg/L, Serine 8mg/L, sodium-chlor 7500mg/L, riboflavin 0.028mg/L, linolic acid 0.079mg/L, L-threonine 13mg/L, Sodium phosphate dibasic 135mg/L, thymidine 0.25mg/L, Thioctic Acid 0.19mg/L, L-asparagine 14.4mg/L, L-arginine hydrochloride 200mg/L, ASPARTIC ACID 13.5mg/L, 1, 4-butanediamine dihydrochloride 0.18mg/L, Cys hydrochloride 29mg/L, L-glutaminate 124mg/L, Pidolidone 13.9mg/L, Sodium.alpha.-ketopropionate 105mg/L, vitamin B12 0.35mg/L, glycine 7mg/L, L-Leu 13.4mg/L, vitamin H 0.005mg/L, L lysine HCL 37.5mg/L, L-Histidine hydrochloride 14mg/L, L-PROLINE 25.6mg/L,
Described gamma delta T cells specificity cultivation component II is made up of the component of following concentration:
Transferrins,iron complexes 50mg/L, TCR γ δ monoclonal antibody 50mg/L, nerve growth factor 10mg/L, IL-4100mg/L, becomes Mierocrystalline cellulose cell growth factor 50mg/L, Pamidronic Acid disodium 250mg/L, insulin-like growth factor 100mg/L, IL-12 250mg/L.
3. being applicable to described in claim 1 or 2 cultivated the preparation method of the serum free medium of gamma delta T cells, it is characterized in that: step is as follows:
(1) get each raw material of component I, mixing mixes, under 60 DEG C of vacuum dry 15 hours, for subsequent use; Get each raw material of component II, under 30 DEG C of vacuum, be dried 18 hours, mixing mixes, for subsequent use;
(2) above-mentioned dried component I and component II are mixed, pin mill 3 hours, crosses 30 mesh sieves, obtains solid state powder, adds water, and mixes, and 0.2 μ m membrane filtration, must be applicable to cultivate the serum free medium of gamma delta T cells.
4. preparation method according to claim 3, is characterized in that: in described step (2), in pin mill production process, temperature is controlled at 20～25 DEG C, and humidity is controlled at 15～20%, N 2gaseous tension is controlled at 150～300psi.
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CN105886468A (en) * 2015-05-22 2016-08-24 北京康爱瑞浩生物科技股份有限公司空港分公司 Method for efficiently inducing [gamma][delta]T cells and application of [gamma][delta]T cells
CN106190972A (en) * 2015-04-30 2016-12-07 广州复大医疗股份有限公司 A kind of induced amplification cultural method of peripheral blood gamma delta T cells
CN108025023A (en) * 2015-08-25 2018-05-11 Uab研究基金会 Method for stem cell transplantation
CN109234236A (en) * 2018-09-29 2019-01-18 吉林大学第医院 Preparation method of chimeric antigen receptor gamma delta T cell
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

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106190972A (en) * 2015-04-30 2016-12-07 广州复大医疗股份有限公司 A kind of induced amplification cultural method of peripheral blood gamma delta T cells
CN105886468A (en) * 2015-05-22 2016-08-24 北京康爱瑞浩生物科技股份有限公司空港分公司 Method for efficiently inducing [gamma][delta]T cells and application of [gamma][delta]T cells
CN105886468B (en) * 2015-05-22 2019-09-06 北京康爱瑞浩生物科技股份有限公司空港分公司 A kind of method and application of efficient induction gamma delta T cells
CN108025023A (en) * 2015-08-25 2018-05-11 Uab研究基金会 Method for stem cell transplantation
CN109234236A (en) * 2018-09-29 2019-01-18 吉林大学第医院 Preparation method of chimeric antigen receptor gamma delta T cell
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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