CN107304411A - A kind of serum substitute for the culture that suspended for immunocyte - Google Patents
A kind of serum substitute for the culture that suspended for immunocyte Download PDFInfo
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Abstract
The present invention relates to a kind of serum substitute for the culture that suspended for immunocyte, it is made up of pharmaceutical grade human albumin, restructuring human transferrin, rh-insulin, lipid, trace element, hormone, shearing force protective agent, resveratrol, D aminoglucose hydrochlorides, R 848 and C3.The serum substitute conveniently by certain technique can be prepared into serum free medium with basal medium using preceding, and the serum free medium can be used for immunocyte to suspend culture.The serum substitute of the present invention has the advantage that：1) serum substitute contains alternative micromolecular compound, and protein ingredient content is reduced to greatest extent, and definite ingredients, uniformity is strong；2) conventional method can be used to prepare for the serum substitute, and in 80~20 DEG C of preservations；3) serum substitute can dispense preservation, using it is preceding easily mixed with basal medium after use, it is to avoid culture medium multigelation.
Description
Technical field
The present invention relates to a kind of serum substitute for the culture that suspended for immunocyte, belong to cell engineering and biological medicine skill
Art field.
Background technology
Treated autologous cell refers to the body cell using the autologous of people, allogeneic or xenogenesis (non-human), through manipulation in vitro
The treatment method of (or implantation) human body is fed back afterwards.This manipulation in vitro includes passage, amplification, the screening of cell in vitro
And medicine or other can change the processing of cell behaviors, body cell after manipulation in vitro can be used for disease
Treatment, it can also be used to the diagnosis and prevention of disease.Treated autologous cell has a variety of different types, including feeds back body in vivo
The mononuclearcell of outer activation such as Tumor-infiltrating lymphocytes (Lymphokine Activated Killer Cells,
LAK), tumor infiltrating lymphocyte (Tumor Infiltrative Lymphocytes, TIL), monocyte, macrophage
Cell or the killing cell (IVS) of external sensitization etc..Domestic immune cell therapy more application is in treatment tumour and disease
Toxicity infectious disease such as hepatitis, AIDS.It is using more ripe autoimmune cell treatment technology in clinical at present
One of important method of tumor biotherapy, is that in the presence of panimmunity active factors, effective activation and amplification are from certainly
The immunocompetent cell (mononuclearcell) separated in peripheral body, is fed back in patient's body, and direct killing or induction are exempted from
Epidemic disease effector cell killing tumor cell or virus infected cell, or adjust and strengthen the immunologic function of body.Autoimmunity is thin
Born of the same parents' treatment technology include cytokine induced kill cell (Cytokine Induced Killer Cells, CIK) immunization therapy,
BMDC (Dendritic Cells, DC) immunization therapy, DC-CIK cellular immunotherapies, NK (Nature
Killer, NK) immunization therapy etc..
CIK cell is to be reported first by Stanford Univ USA Schmidt Wolf etc. for 1991 earliest, and they have found
Under the cytokine profiles such as collective effect of interferon, CD3 monoclonal antibodies, il-1 and proleulzin, periphery blood strangury
Bar cell, which can be directed to induce and largely breed, turns into tumor-killing cell.Due to this kind of cell simultaneously express CD3 and
Two kinds of membrane protein molecules of CD56, therefore the non-principal tissue with the powerful anti-tumor activity of T lymphocytes and NK cells
The restricted advantage for killing knurl of histocompatibility complex.Compared with other immunization therapy cells, CIK cell have growth rate it is fast,
The advantages of tumor activity is high, kill knurl composes wide, Small side effects, influences slight to normal marrow hemopoiesis is killed, therefore, using CIK
Cell carries out the preferred option that autoimmune cell treatment is considered as oncotherapy of new generation.
Cell culture medium as the main carriers in cell culture, to immunocyte activation in vitro and expanding effect and
Tumor cytotoxicity effect all plays vital effect, has great to the popularization and application of autoimmune cell treatment technology
Influence.The existing frequently-used complete medium in clinical CIK cell culture generally contains 10%AB type human serums
RPMI1640 culture mediums, because of its type serum of AB containing someone, though the detection through HBV, HCV and HIV, but still have
Other diseases may be propagated, security is low, and also have that differences between batches big, unsuitable Quality Control, composition be indefinite, price
Expensive the problems such as.Compared with traditional complete medium, serum free medium can reach biologic cleanliness requirement, definite ingredients,
Steady quality, be easy to Quality Control, the customer service shortcoming of people's AB type serum, can reduce the chance of patient's inadvertent contamination, for
The popularization and application of immunization therapy are significant.Majority research shows that serum free medium can replace the complete training containing serum
Base is supported, the number and function phase of the two gained culture are worked as, and Jiang Yongxin et al. is to serum free medium and complete medium body
The cell function of outer induced amplification CIK cell has carried out systematic analysis, by CIK cell multiplication capacity, thin
Born of the same parents' phenotypic analysis and the result of study killed in terms of tumor activity show serum free medium sertoli cell multiplication capacity more
It is reliable and stable, infringement is not received using cell function obtained by serum free medium culture.(Jiang Yongxin etc., Chinese tumour is prevented
Control magazine, 2006,13 [12]：900-903；Jiang Yongxin etc., treatment and prevention of tumour research, 2006,33 [11]：784-787)
The mode of blood serum substituting composition is added during free serum culture two kinds, and a kind of is in the free serum culture newly researched and developed
Various blood serum substituting compositions have been contained in based component, can directly have been cultivated；Another is that blood serum substituting is provided separately
Thing, replaces serum addition to enter in cultivating system in culture, also need separately to add part serum be replaced into point as grow because
Son, hormone etc..Commercially available serum substitute is largely imported product in the market, such as Gibco companies
The PuriQTM Serum Replacement of KnockOutTM SR and Amsbio companies are suitable for embryonic stem cell
Free serum culture, the SERUM REPLACEMENT 3 and Biowest companies that Sigma Aldriches are provided is carried
The FreeAdd of confession is then applied to the free serum culture of regular growth.
Although serum free medium has the incomparable advantage of many traditional serum-containing media, its still suffer from
Under it is not enough to be improved：A. cell is easily influenceed in serum free medium by some mechanical factors and chemical factor, training
The preservation and application of base are supported be not as convenient as traditional synthetic media；B. it is with strong points, different types of cell, not even
It is all different to the demand of medium nutrient content with cell line or cell line；C. primary cell is carried out using serum free medium
Cell yield is relatively low during separation；D. insulin and transferrins necessary to current most of serum free medium are still with animal
For source, the potential safety hazard that serum is brought is not eliminated；E. cost is higher.
The content of the invention
In order to solve the above technical problems, it is an object of the present invention to provide a kind of blood for the culture that suspended for immunocyte
Clear substitute, the wherein substitute can be trained using preceding with basal medium conveniently by certain technique composition serum-free
Base is supported, the culture medium can be used for immunocyte suspension culture.The serum substitute has specific chemical components, non-animal derived
Property composition, preserve it is easy, with strong points, safe and effective, simple and easy to apply, with low cost the advantages of.
Second object of the present invention is to provide a kind of serum substitute for including the culture that suspends for immunocyte
Composition, the composition typically by the serum substitute and basal medium using preceding conveniently by certain
Technique prepare, the culture medium be typically be used for immunocyte suspend culture serum free medium.
What first purpose of the present invention was realized in：
A kind of serum substitute for the culture that suspended for immunocyte, it is characterised in that：It is included with final concentration 50-200
Mg/L pharmaceutical grades human albumin, 50-500mg/L restructuring human transferrin, 10-100mg/L rh-insulins,
15-60mg/L lipids, 1.5-30mg/L trace elements, the protection of 10-100mg/L hormones, 500-3000mg/L shearing forces
Agent, 1-50mg/L resveratrols, 500-2000mg/L D-Glucosamine Hydrochlorides, 1-50mg/L R-848 and, 1-200
mg/L C3。
Preferably, it is included is turned with final concentration 100-200mg/L pharmaceutical grades human albumin, 100-300mg/L recombined humans
Ferritin, 30-80mg/L rh-insulins, 30-50mg/L lipids, 2-15mg/L trace elements, 20-50mg/L
Hormone, 1000-2000mg/L shearing forces protective agent, 10-20mg/L resveratrols, 1000-1500mg/L D- amino Portugal
Grape sugar hydrochloride, 10-30mg/L R-848 and, 10-150mg/L C3.
It is highly preferred that it is included turns iron egg with final concentration 100mg/L pharmaceutical grades human albumin, 100mg/L recombined humans
In vain, 100mg/L rh-insulins, 40mg/L lipids, 5mg/L trace elements, 30mg/L hormones, 1500mg/L
Shearing force protective agent, 15mg/L resveratrols, 1200mg/L D-Glucosamine Hydrochlorides, 20mg/L R-848 and
80mg/L C3。
Wherein, the lipid includes but is not limited to arachidonic acid, cholesterol, oleic acid, linoleic acid, lipoic acid, flax
Acid etc.；The trace element includes but is not limited to iron, copper, zinc, selenium, cobalt, nickel, manganese etc.；The hormone is included but not
It is limited to dexamethasone, progesterone, cholesterol etc.；The shearing force protective agent includes but is not limited to poloxamer etc..
What second object of the present invention was realized in：
A kind of composition for including the serum substitute cultivated that suspended for immunocyte, it is characterised in that：Described group
Compound is made up of 50%-95% basal medium and 5-50% serum substitute of the present invention；Preferably, the composition
It is made up of 70%-95% basal medium and 5-30% serum substitute of the present invention；It is highly preferred that the composition by
85%-95% basal medium and 5-15% serum substitute of the present invention are constituted；Most preferably, the composition is by 90%
Basal medium and 10% serum substitute of the present invention constitute.
Wherein, the basal medium includes but is not limited to RPMI1640 culture mediums, DMEM culture mediums etc..
The composition is usually serum free medium, and the serum free medium is generally used for immunocyte suspension culture.
Wherein, the composition of the basal medium and the serum substitute for immunocyte suspension culture is by conventional method
Prepare and (wear incubation et al., number of patent application 201110081988.5).
The serum substitute of the present invention has the advantage that：1) basic media components clearly, can use routine
Method is prepared, and is preserved in normal temperature；2) the serum substitute protein content cultivated that suspended for immunocyte is low,
Can be in -80~-20 DEG C of preservations；3) serum substitute cultivated that suspended for immunocyte can dispense preservation,
Using it is preceding easily mixed with basal medium after use, it is to avoid culture medium multigelation.
In preferred embodiments, the serum free medium comprising serum substitute of the present invention is bred available for CIK cell
Culture.By the culture medium comprising serum substitute of the present invention and complete medium (containing 10%FBS), commercially available import
Serum free medium, commercially available domestic serum free medium are compared.Periphery is isolated and purified according to the method for document report
Blood mononuclear cell (PBMC), and carry out external evoked preparation CIK cell.CIK cell growth curve result shows,
Serum free medium comprising clear substitute of the invention can be effectively promoted the in-vitro multiplication of CIK cell, its cultivation effect
It is significant to be better than complete medium and domestic serum free medium, and with commercially available import serum free medium X-VIVO 15
Effect it is suitable.The measurement result of CIK surface markers expressions shows, includes the nothing of serum substitute of the present invention
The quality for the CIK cell that blood serum medium is induced is better than traditional complete medium and domestic serum free medium, with
The CIK cell quality that import medium culture goes out is suitable.The evaluation of CIK cell in vitro killing efficiency shows, comprising this hair
CIK cell prepared by the serum free medium of bright serum substitute is significantly higher than complete culture to the killing-efficiency of target cell
Base and domestic serum free medium；At 20: 1, its killing activity is slightly above the serum free medium of import.
In another preferred embodiment, the serum free medium comprising serum substitute of the present invention can be used for NK thin
The in vitro culture of born of the same parents.By the culture medium comprising serum substitute of the present invention and complete medium (containing 10%FBS), city
Import serum free medium, the commercially available domestic serum free medium sold are compared.Separated according to the method for document report
Purifying peripheral blood PBMC simultaneously carries out external evoked preparation NK cells.NK growth curve results show, include the present invention
Effect and import serum free medium of the serum free medium of serum substitute for NK cell proliferation in vitro cultures
The effects of X-VIVO 15 are suitable, hence it is evident that better than complete medium and domestic serum free medium.NK cell surface molecule marks
The testing result of will thing shows, NK cells its table prepared using the serum free medium comprising serum substitute of the present invention
The expression of face related molecule sign is significantly higher than complete medium and domestic serum free medium, and slightly above import
Serum free medium, shows that the NK cells that the serum free medium comprising serum substitute of the present invention is turned out have very well
Cell purity.
Brief description of the drawings
Fig. 1 is the growth in vitro curve of the CIK cell of different culture media culture.
Fig. 2 is the CIK cell surface markers expression of different culture media induction.
When Fig. 3 is using human leukemia cell line K562 as target cell, the CIK cell of different culture media culture is with effect target
The cell toxicant percentage of ratio.
When Fig. 4 is using Human cervical cancer cell lines Hela as target cell, the CIK cell of different culture media culture is with effect target
The cell toxicant percentage of ratio.
Fig. 5 be and human lung adenocarcinoma cell line A549 be target cell when, the CIK cell of different culture media culture is with effect
Target than cell toxicant percentage.
Fig. 6 is the growth in vitro curve of the NK cells of different culture media culture.
Fig. 7 is the NK cell surface marker developed by molecule levels of different culture media induction.
Embodiment
In order to which technical characteristic, purpose and beneficial effect to the present invention are more clearly understood from, now to the technology of the present invention
Scheme carry out it is described further below, but it is not intended that to the present invention can practical range restriction.
Embodiment 1. is used for the preparation of the serum substitute of immunocyte suspension culture
The blood serum substituting composition formula A of the present invention is as shown in table 1.
Table 1：The formula (formula A) of serum substitute of the present invention
The compound method of the serum substitute is as follows：
1. prepare iron saturation human transferrin solution：
(1) by 40mg FeCl320ml 1mM HCl solutions are dissolved in, dispenses and freezen protective is in -20 DEG C, obtain 1
Number storing liquid；
(2) take 200mg people to turn iron albumin, be dissolved in 20ml RPMI1640 culture mediums, add the 1 of 300 μ l
Number storing liquid simultaneously stirs, and produces the human transferrin solution (10mg/ml) of iron saturation, in -30 DEG C of preservations.
2. prepare cholesterol solution：58mg cholesterol powder is weighed, is added in 20ml RPMI1640 culture mediums, is surpassed
Acoustic wave oscillator shakes 1 hour in 4 DEG C, is allowed to emulsification completely, cholesterol storing liquid (2.9mg/ml) is produced, in -30 DEG C
Preserve.
3. prepare Linoleic Acid solution：Weigh 80mg linoleic acid to be dissolved in 20ml graded alcohols, fully shaking dissolving is adopted
It is degerming with 0.45 μm of membrane filtration 2 times, linoleic acid storing solution (4mg/ml) is produced, in 8 DEG C of storages.
4. remaining composition is dissolved in RPMI1640 culture mediums, storing solution is obtained.Wherein, pharmaceutical grade human albumin is laid in
Liquid (10mg/ml), rh-insulin's storing solution (10mg/ml) are in -20 DEG C of storages；Ferrous sulfate (0.2mg/ml),
Copper sulphate (0.02mg/ml), zinc sulfate (0.1mg/ml), sodium selenite (0.01mg/ml), cobalt chloride (0.1mg/ml),
Nickel chloride (0.05mg/ml), manganese chloride (0.02mg/ml), dexamethasone (0.1mg/ml), PLURONICS F87
(150mg/ml), resveratrol (1.5mg/ml), R-848 (2mg/ml) and C3 (8mg/ml) its storing solution
In 8 DEG C of storages.
Uniform, addition RPMI1640 trainings are mixed and stirred in container 5. each 10ml of the storing solution of above-mentioned each component is added
Support base and cumulative volume be supplemented to 900ml, use each one of 0.45um and 0.22um filter membranes (upper strata for 0.45um, under
Layer is 0.22um) filtration sterilization is carried out, and pH is adjusted to 7.2 with 5%NaHCO3, add RPMI1640 trainings
Support base and volume is supplemented to 1000ml, produce serum substitute A of the present invention.
6. serum substitute A is dispensed and in -20 DEG C of storages.
The preparation of serum free medium of the embodiment 2. comprising serum substitute of the present invention
1. commercially available culture medium by specification is prepared, the basal mediums of RPMI 1640 (RPMI Medium 1640 are produced
Culture medium, Beijing Orient Hua Hui biological medicines Science and Technology Ltd.).The wherein basal medium formulations of RPMI 1640 such as table
Shown in 2.
2. the formula of the serum free medium comprising serum substitute of the present invention：Serum free medium (the culture medium of the present invention
A) include 90% the basal mediums of RPMI 1640 and 10% serum substitute A.
3. the preparation of serum free medium (culture medium A)：100ml serum substitutes A is slowly added into before use
In the basal mediums of 800ml RPMI 1640, after stirring, pH is adjusted to 7.2 with 5%NaHCO3, then add
Enter RPMI1640 culture mediums and volume is supplemented to 1000ml, produce the serum-free comprising serum substitute A of the present invention and train
Support base (culture medium A).
Table 2RPMI1640 basal medium formulations
The serum free medium A of embodiment 3. cultivates the evaluation of CIK cell multiplication capacity
Using healthy human peripheral blood, PMNC (PBMC) is isolated and purified according to the method for document report,
And carry out external evoked, preparation CIK cell.Experiment is divided into 4 groups (tables 3), is respectively adopted：1) nothing of the invention
Blood serum medium culture medium A (culture medium A), 2) complete medium (RPMI 1640+10%FBS), 3) import
Serum free medium (X-VIVO 15 (LONZA)) and 4) domestic serum free medium, carry out cell culture.
PBMC after purification adjusts cell number to 5 × 106/ ml, is seeded in T175 blake bottles.From induction the 1st day,
Carry out within every 3 days half amount and change liquid, and keep the final concentration of cell factor in culture medium constant.Sampling is refused using trypan blue simultaneously
Dye method is counted, until culture terminates on the 21st day, draws CIK cell growth curve.Wherein, using commercially available without blood
The experimental group of clear culture medium and traditional complete medium, CIK induction is carried out according to method reported in the literature, is used
Mouse anti human CD3 monoclonal antibodies including humanization, INF- γ and recombinant human il-2；Use the reality of culture medium of the present invention
Group is tested, recombinant human il-2 is only added during culture CIK.Specific experiment is grouped as follows shown in table：
The evaluation of table 3CIK ability of cell proliferation is grouped
| Numbering | Experiment packet |
| 1 | Complete medium |
| 2 | X-VIVO 15(LONZA) |
| 3 | Domestic culture medium |
| 4 | Culture medium A |
Experimental result as shown in figure 1, the serum free medium (culture medium A) of the present invention can be effectively promoted CIK thin
The in-vitro multiplication of born of the same parents, its cultivation effect is significantly better than traditional complete medium and domestic serum free medium；With it is commercially available
Serum free medium X-VIVO 15 effect it is suitable.
The serum free medium A of embodiment 4. induces the measure of CIK cell surface markers expression
Prepared CIK cell in embodiment 3, is harvested when culture was to 21 days.Cell is carried out using fluorescence antibody
Flow cytometry is carried out after mark, expression of the detection CIK cell surface markers before culture and after culture becomes
Change.The cell concentration of each sample mark is 1 × 106, the cell surface molecule of detection includes：CD3, CD4, CD8,
And CD56.After detection, by the different cell subsets and control group of four experimental groups, that is, the cell before inducing is compared；
The data before four groups after induction are compared simultaneously, after observation culture in harvesting the content of aim cell and
Induced efficiency.Experiment packet is as shown in table 4.
The measure packet of table 4CIK cell surface marker developed by molecule levels
Experimental result is as shown in Figure 2.Compared with control group (before inducing), CD3+ cell subsets in four experimental groups
Percentage increase, but without significant difference；The result compared between four experimental groups is also without significant difference.
Show in the ratio of two double positive cells subgroups of CD3+/CD56+ and CD3+/CD8+, four experimental groups after induction
Write and rise, wherein 15 groups of X-VIOV and the ratio using culture medium group of the present invention are significantly higher than complete medium group.
The ratio of CD3+/CD4+ double positive cells subgroups is sent out after induction in complete medium group and domestic serum-free experimental group
Slight up-regulation is given birth to；Declined in import culture medium and culture medium A group.CIK cell, which is mainly, to be passed through
Two T cell subgroups of CD3+/CD56+ and CD3+/CD8+ play broad spectrum activity antitumor actions, above-mentioned two subgroup it is thin
Born of the same parents' ratio determines the bioactivity of CIK cell, is the major criterion for judging CIK cell quality.This result shows,
The quality for the CIK that culture medium A is induced is better than complete medium and domestic serum free medium, with import culture medium
The CIK cell quality turned out is suitable.
The CIK cell in vitro of the serum free medium A of embodiment 5. cultures kills the evaluation of efficiency
The CIK target cells in vitro killing activities that different experiments group is obtained are determined using fluorescein based dye CFSE, the target used is thin
Born of the same parents include human leukemia cell line K562, Human cervical cancer cell lines Hela, and human lung adenocarcinoma cell line A549.CFSE
Staining kit is purchased from Life Technology companies (article No.：Cat.34554), propidium iodide (PI) is purchased from Sigma
Company.K562, Hela and A549 cell line are purchased from Institute of Basic Medical Sciences of China Concord Medical Science University.Experiment packet
It is same as Example 3, as shown in table 3.
The density of three kinds of target cells is adjusted to 6x104/ml, is added in the U-shaped well culture plate of bottom 96, per the μ l of hole 100.
The CIK cell of four experimental group cultures is harvested, CIK cell is marked according to kit specification, after mark
Cell according to effect ratio of the target than 5: 1,10: 1, and 20: 1 mixed in equal volume with target cell, setting 3 parallel control skies.
It is empty as control that individual effect cell and blank cultures are set simultaneously.Culture plate is at 37 DEG C after mixing, under the conditions of 5%CO2
It is incubated 4 hours.After incubation terminates, 25 μ l PI dye liquors (100 μ g/ μ l) are added per hole, after ice bath is incubated 5 minutes, with stream
Formula cell instrument is analyzed, and determines killing abilities of the CIK to different target cells.The calculating side of the killing percentage of target cell
Formula is：
Cell toxicant percentage=[target of the natural death of the target cell of the dead target cell-natural death of experimental group/100 1 is thin
Born of the same parents] × 100
Experimental result is as shown in Fig. 3, Fig. 4, Fig. 5.The CIK cell that same medium culture goes out with effect target than
Increase, its killing-efficiency to three kinds of target cells shows incremental trend；And in identical effect target ratio, different experiments
The result that group is compared shows, the killing-efficiency of CIK cell prepared by serum free medium culture medium A to target cell
It is significantly higher than complete medium group and domestic serum-free control group；At 20: 1, killing activity is slightly above the serum-free of import
Culture medium, but without significant difference.
The serum free medium A of embodiment 6. cultivates the evaluation of NK ability of cell proliferation
Using cell separation liquid (people's mononuclearcell separating liquid 1.077, article No. 25710, Beijing Orient China brightness biological medicine
Science and Technology Ltd.) PBMC is collected from 5-10ml healthy human peripheral bloods, PBMC is washed 3 times with sterile PBS,
And be resuspended in PBS, sampling is counted.Experiment is divided into 4 groups (tables 5), is respectively adopted：1) serum-free of the invention
Culture medium A, 2) complete medium (RPMI 1640+10%FBS), 3) import serum free medium (X-VIVO 15
(LONZA)) and 4) domestic serum free medium, carry out cell culture.
Use above culture medium dilutes isolated PBMC respectively, and adjustment cell density is in 1-3 × 106In the range of/ml.
The PBMC diluted is seeded in T175 blake bottles, while adding recombinant human il-2 to final concentration 10ng/ml, weight
Group human IL-15 is to final concentration 50ng/ml, and recombinant human serum albumin is put into 37 DEG C, 5%CO2 to final concentration 0.1% (W/V)
Cultivated in environment.Carry out within every 3 days half amount and change liquid, and maintain the concentration level of each cell factor in culture medium to keep constant,
Sampling is counted simultaneously, draws cell growth curve, incubation time totally 15 days.
The evaluation of table 5NK ability of cell proliferation is grouped
| Numbering | Experiment packet |
| 1 | Complete medium |
| 2 | X-VIVO 15 |
| 3 | Domestic culture medium |
| 4 | Culture medium A |
Experimental result is as shown in Figure 6.Cultivated by 15 days, serum free medium A is trained for NK cell proliferation in vitro
Foster effect is suitable with import serum free medium 15 effects of X-VIVO, hence it is evident that better than complete medium and domestic without blood
Clear culture medium.
The level of the serum free medium A induced NK cell surface molecular marks of embodiment 7.
Harvest uses fluorescent labeled antibody combination flow cytometry, detection using the NK cells of different culture media culture
The expression of NK cell surface markers, including CD3, CD56, CD16, CD8 and CD57, wherein CD3-/CD56+,
CD16+/CD56+ and CD8+/CD57+ are the significant molecular phenotypes of NK cells.Experiment packet is as shown in table 6.
Concrete operations are to collect cell before detection, 1000rpm, and 4 DEG C centrifuge 5 minutes.Cell is resuspended with PBS
And count, adjustment cell density is 1x106/ml.Take 5x105 cell as measuring samples, add dilute with 2%BSA
The fluorescent labeled antibody flag F ITC-CD3 (eBioscience, Cat.11-0038-41) released, APC-CD56
(eBioscience, Cat.17-0569-42), PerCP-CD8 (eBioscience, Cat.8043-0087-120), PE-CD16
(eBioscience, Cat.12-0167-42) and Pacific Blue-CD57 (eBioscience, Cat.322315) are carried out to cell
Mark, room temperature lucifuge is incubated 20 minutes.Washed after sample incubation with PBS 2 times, add 500 μ l PBS resuspensions, use
Flow cytometer is detected.
The measure packet of table 6NK cell surface marker developed by molecule levels
| Numbering | Experiment packet |
| 1 | Control group (PBMC before induction) |
| 2 | Complete medium |
| 3 | X-VIVO 15(LONZA) |
| 4 | Domestic culture medium |
| 5 | Culture medium A |
Experimental result is as shown in Figure 7.NK cells its surface associated molecule mark prepared using serum free medium A
Expression be significantly higher than complete medium group and domestic serum free medium group, slightly above import serum free medium
Group, shows that the NK cells that serum free medium A is turned out have good cell purity.
Claims (10)
1. a kind of serum substitute for the culture that suspended for immunocyte, it is characterised in that the serum substitute is included：
Pharmaceutical grade human albumin, restructuring human transferrin, rh-insulin, lipid, trace element, hormone, shearing are tried hard to keep
Protect agent, resveratrol, D-Glucosamine Hydrochloride, R-848 and C3.
2. serum substitute according to claim 1, it is characterised in that：It is included with final concentration 50-200
Mg/L pharmaceutical grades human albumin, 50-500mg/L restructuring human transferrin, 10-100mg/L rh-insulins,
15-60mg/L lipids, 1.5-30mg/L trace elements, the protection of 10-100mg/L hormones, 500-3000mg/L shearing forces
Agent, 1-50mg/L resveratrols, 500-2000mg/L D-Glucosamine Hydrochlorides, 1-50mg/L R-848 and, 1-200
mg/L C3。
3. serum substitute according to claim 1, it is characterised in that：Preferably, it is included with final concentration
Count 100-200mg/L pharmaceutical grades human albumin, 100-300mg/L restructuring human transferrin, 30-80mg/L recombined human pancreases
Island element, 30-50mg/L lipids, 2-15mg/L trace element, 20-50mg/L hormones, 1000-2000mg/L shearing forces
Protective agent, 10-20mg/L resveratrols, 1000-1500mg/L D-Glucosamine Hydrochlorides, 10-30mg/L R-848
With, 10-150mg/L C3.
4. serum substitute according to claim 1, it is characterised in that：It is highly preferred that it is included with dense eventually
Degree meter 100mg/L pharmaceutical grades human albumin, 100mg/L restructuring human transferrin, 100mg/L rh-insulins,
40mg/L lipids, 5mg/L trace elements, 30mg/L hormones, 1500mg/L shearing forces protective agent, 15mg/L are white
Veratryl alcohol, 1200mg/L D-Glucosamine Hydrochlorides, 20mg/L R-848 and 80mg/L C3.
5. serum substitute as claimed in any of claims 1 to 4, wherein：The lipid include but
It is not limited to arachidonic acid, cholesterol, oleic acid, linoleic acid, lipoic acid, leukotrienes etc.；The trace element include but
It is not limited to iron, copper, zinc, selenium, cobalt, nickel, manganese etc.；It is solid that the hormone includes but is not limited to dexamethasone, progesterone, courage
Alcohol etc.；The shearing force protective agent includes but is not limited to poloxamer etc..
6. a kind of composition for including serum substitute as claimed in any of claims 1 to 5, it is special
Levy and be：The composition can be trained by serum substitute as claimed in any of claims 1 to 5 with basis
Foster base is easily prepared using preceding by certain technique.Wherein, the basal medium includes but is not limited to RPMI1640
Culture medium, DMEM culture mediums etc..
7. composition according to claim 6, it is characterised in that：The composition by 50%-95% base
Basal culture medium and 5-50% serum substitute as claimed in any of claims 1 to 5 composition；Preferably,
The composition by 70%-95% basal medium and 5-30% it is as claimed in any of claims 1 to 5
Serum substitute is constituted；It is highly preferred that the composition by 85%-95% basal medium and 5-15% according to right
It is required that the serum substitute composition in 1 to 5 described in any one；Most preferably, the composition is trained by 90% basis
Support base and 10% serum substitute as claimed in any of claims 1 to 5 is constituted.
8. composition according to claim 6, it is characterised in that：The composition is serum free medium.
9. composition according to claim 6, it is characterised in that：The composition is outstanding available for immunocyte
Floating culture.
10. application of the composition according to claim 6 in immunocyte in suspension, it is characterised in that：It is described
Immunocyte include but is not limited to cytokine induced kill cell (Cytokine Induced Killer Cells, CIK),
NK (Nature Killer, NK) cell etc..
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|---|---|---|---|---|
| CN109234223A (en) * | 2018-11-21 | 2019-01-18 | 南京基蛋生物医药有限公司 | Low albumen serum-free cell culture medium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886465A (en) * | 2014-11-15 | 2016-08-24 | 赵宇 | Serum substitute for immune cell suspension culture |
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2016
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105886465A (en) * | 2014-11-15 | 2016-08-24 | 赵宇 | Serum substitute for immune cell suspension culture |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234223A (en) * | 2018-11-21 | 2019-01-18 | 南京基蛋生物医药有限公司 | Low albumen serum-free cell culture medium |
| CN109234223B (en) * | 2018-11-21 | 2021-01-19 | 南京基蛋生物医药有限公司 | Low-protein serum-free cell culture medium |
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