CN105886465A - Serum substitute for immune cell suspension culture - Google Patents
Serum substitute for immune cell suspension culture Download PDFInfo
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Abstract
The invention relates to a serum substitute for immune cell suspension culture; the serum substitute is composed of pharmaceutical-grade human albumin, recombinant human transferrin, recombinant human insulin, lipids, trace elements, hormones, a shear force protective agent, resveratrol, D-glucosamine hydrochloride, R-848 and C3. The serum substitute and a base culture medium can be conveniently made into a serum-free culture medium through a certain process before use, and the serum-free culture medium can be used for immune cell suspension culture. The serum substitute has the following advantages: 1) the serum substitute contains substitutable small molecular compounds, reduces the protein content to a greatest extent, has clear composition, and has strong consistency; 2) the serum substitute can be prepared by using a conventional method, and is preserved at the temperature of -80 to -20 DEG C; and 3) the serum substitute can be preserved in a subpackaging manner, is conveniently mixed with the base culture medium for use before use, and avoids repeated freezing and thawing of the culture medium.
Description
Technical field
The present invention relates to a kind of serum substitute for immunocyte suspension culture, belong to cell engineering and biological medicine skill
Art field.
Background technology
Treated autologous cell refers to answer autologous, the allogeneic of employment or the somatic cell of xenogenesis (non-human), through manipulation in vitro
The Therapeutic Method of rear feedback (or implantation) human body.This manipulation in vitro includes cell passing in vitro, expands, screens
And medicine or other can change the process of cell behaviors, the somatic cell after manipulation in vitro can be used for disease
Treatment is it can also be used to the diagnosis of disease and prevention.Treated autologous cell has multiple different type, including internal feedback body
The mononuclearcell of outer activation such as Tumor-infiltrating lymphocytes (Lymphokine Activated Killer Cells,
LAK), tumor infiltrating lymphocyte (Tumor Infiltrative Lymphocytes, TIL), mononuclear cell, huge bite
The killing cell (IVS) etc. of cell or external sensitization.Domestic immune cell therapy more application is in treatment tumor and disease
Toxicity catches such as hepatitis, acquired immune deficiency syndrome (AIDS) etc..At present clinic is applied the more ripe autoimmune cell treatment technology to be
One of important method of tumor biotherapy, is under the effect of panimmunity active factors, and effective activation and amplification are from certainly
The immunologically competent cell (mononuclearcell) separated in peripheral body, is fed back in the patient, and direct killing or induction are exempted from
Epidemic disease effector lymphocyte's killing tumor cell or virus infected cell, or regulate and the immunologic function of enhancing body.Autoimmune is thin
Born of the same parents' treatment technology include cytokine induced kill cell (Cytokine Induced Killer Cells, CIK) immunization therapy,
Dendritic cell (Dendritic Cells, DC) immunization therapy, DC-CIK cellular immunotherapy, natural killer cell (Nature
Killer, NK) immunization therapy etc..
CIK cell be the earliest 1991 by reported first such as Stanford Univ USA Schmidt Wolf, they find exist
Under the common effect of cytokine profiles such as IFN-γ, CD3 monoclonal antibody, il-1 and interleukin-2 etc., periphery stranguria with blood
Bar cell can be directed to induce and breed in a large number becomes tumor-killing cell.Due to this kind of cell express simultaneously CD3 and
Two kinds of membrane protein molecules of CD56, therefore have the non-principal tissue of the powerful anti-tumor activity of T lymphocyte and NK cell
The restricted advantage killing tumor of histocompatibility complex.Compared with other immunization therapy cells, CIK cell have growth rate fast,
Kill that tumor activity is high, kill that tumor spectrum is wide, side effect is little, on advantages such as the impact of normal bone marrow hematogenesis are slight, therefore, apply CIK
Cell carries out the preferred option that autoimmune cell treatment is considered as a new generation's oncotherapy.
Cell culture medium cultivate as cell in main carriers, to immunocyte activation in vitro and expanding effect and
Tumor cytotoxicity effect all plays vital effect, and the popularization and application to autoimmune cell treatment technology have great
Impact.The existing frequently-used complete medium cultivated in clinical CIK cell is generally containing 10%AB type human serum
RPMI1640 culture medium, because it contains people's AB type serum, though through the detection of HBV, HCV and HIV, but still have
May propagate other diseases, safety is low, and also has that differences between batches Quality Control big, unsuitable, composition be indefinite, price
The problems such as costliness.Compared with traditional complete medium, serum-free medium can reach biologic cleanliness requirement, definite ingredients,
Steady quality, it is simple to Quality Control, the customer service shortcoming of people's AB type serum, the chance of patient's inadvertent contamination can be reduced, for
The popularization and application of immunization therapy are significant.Most researchs show, serum-free medium can replace the training completely containing serum
Supporting base, the number of the two gained culture and function suitable, 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' phenotype analytical and kill the result of study of the aspects such as tumor activity and show that serum-free medium supports ability of cell proliferation more
Reliable and stable, use serum-free medium to cultivate gained cell function and do not receive infringement.(Jiang Yongxin etc., China's tumor 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 adding blood serum substituting composition during serum-free culture has two kinds, and a kind of is the serum-free culture in new research and development
Based component contains various blood serum substituting composition, can directly cultivate；Another kind is that blood serum substituting is provided separately
Thing, replaces when cultivating serum to add and enters in cultivating system, also need to add separately part serum be replaced into point such as grow because of
Son, hormone etc..The most commercially available serum substitute major part is imported product, such as Gibco company
The PuriQTM Serum Replacement of KnockOutTM SR and Amsbio company is all applicable to embryonic stem cell
Serum-free culture, SERUM the REPLACEMENT 3 and Biowest company that Sigma Aldrich provides carries
The FreeAdd of confession is then applicable to the serum-free culture of regular growth.
Although serum-free medium has many traditional advantages incomparable containing blood serum medium, but its still suffer from
Under deficiency to be improved: a. cell is easily affected by some mechanical factor and chemical factor in serum-free medium, training
Preservation and the application of supporting base are not so good as traditional synthetic medium conveniently；The most with strong points, different types of cell, not even
The most different to the demand of medium nutrient content with cell line or cell strain；C. serum-free medium is used to carry out primary cell
During separation, cell yield is on the low side；D. insulin necessary to the most most of serum-free medium and transferrins are still with animal
For source, do not eliminate the potential safety hazard that serum brings；The most relatively costly.
Summary of the invention
For solving above-mentioned technical problem, it is an object of the present invention to provide a kind of blood for immunocyte suspension culture
Clear substitute, wherein this substitute can be trained conveniently by certain technique composition serum-free with basal medium before use
Supporting base, this culture medium can be used for immunocyte suspension culture.This serum substitute has specific chemical components, non-animal derived
Property composition, preserve the advantages such as easy, with strong points, safe and effective, simple, with low cost.
Second object of the present invention is that providing a kind of comprises the described serum substitute for immunocyte suspension culture
Compositions, described compositions typically by described serum substitute and basal medium before use conveniently by necessarily
Technique prepare, described culture medium is typically for the serum-free medium of immunocyte suspension culture.
First purpose of the present invention is achieved in that
A kind of serum substitute for immunocyte suspension culture, it is characterised in that: it comprises with final concentration 50-200
Mg/L pharmaceutical grade human albumin, 50-500mg/L recombined human transferrins, 10-100mg/L recombinant human insulin,
15-60mg/L lipid, 1.5-30mg/L trace element, 10-100mg/L hormone, 500-3000mg/L shearing force are protected
Agent, 1-50mg/L resveratrol, 500-2000mg/L D-Glucosamine Hydrochloride, 1-50mg/L R-848 and, 1-200
mg/L C3。
Preferably, it comprises and turns with final concentration 100-200mg/L pharmaceutical grade human albumin, 100-300mg/L recombined human
Ferritin, 30-80mg/L recombinant human insulin, 30-50mg/L lipid, 2-15mg/L trace element, 20-50mg/L
Hormone, 1000-2000mg/L shearing force protective agent, 10-20mg/L resveratrol, 1000-1500mg/L D-amino Portugal
Grape sugar hydrochlorate, 10-30mg/L R-848 and, 10-150mg/L C3.
It is highly preferred that it comprises turns ferrum egg with final concentration 100mg/L pharmaceutical grade human albumin, 100mg/L recombined human
In vain, 100mg/L recombinant human insulin, 40mg/L lipid, 5mg/L trace element, 30mg/L hormone, 1500mg/L
Shearing force protective agent, 15mg/L resveratrol, 1200mg/L D-Glucosamine Hydrochloride, 20mg/L R-848 and
80mg/L C3。
Wherein, described lipid includes but not limited to arachidonic acid, cholesterol, oleic acid, linoleic acid, thioctic acid, Caulis et Folium Lini
Acid etc.；Described trace element includes but not limited to ferrum, copper, zinc, selenium, cobalt, nickel, manganese etc.；Described hormone include but not
It is limited to dexamethasone, progesterone, cholesterol etc.；Described shearing force protective agent includes but not limited to poloxamer etc..
Second object of the present invention is achieved in that
A kind of compositions comprising the described serum substitute for immunocyte suspension culture, it is characterised in that: described group
Compound is made up of the serum substitute of the present invention of the basal medium of 50%-95% with 5-50%；Preferably, described compositions
It is made up of the serum substitute of the present invention of the basal medium of 70%-95% with 5-30%；It is highly preferred that described compositions by
The basal medium of 85%-95% forms with the serum substitute of the present invention of 5-15%；Most preferably, described compositions is by 90%
Basal medium and 10% serum substitute of the present invention composition.
Wherein, described basal medium includes but not limited to RPMI1640 culture medium, DMEM culture medium etc..
Described compositions is usually serum-free medium, and described serum-free medium is generally used for immunocyte suspension culture.
Wherein, the compositions of described basal medium and the described serum substitute for immunocyte suspension culture is by conventional method
Preparation (wearing incubation et al., number of patent application 201110081988.5).
The serum substitute of the present invention has the advantage that 1) described basic media components is clear and definite, it is possible to use conventional
Method is prepared, and preserves in room temperature；2) the described serum substitute protein content for immunocyte suspension culture is low,
Can be in-80～-20 DEG C of preservations；3) the described serum substitute for immunocyte suspension culture can preserve with subpackage,
Use after mixing with basal medium easily before using, it is to avoid culture medium multigelation.
In preferred embodiments, the serum-free medium comprising serum substitute of the present invention can be used for CIK cell propagation
Cultivate.Culture medium and complete medium (containing 10%FBS), the commercially available import of serum substitute of the present invention will be comprised
Serum-free medium, commercially available domestic serum-free medium compare.The isolated and purified periphery of method according to document report
Blood mononuclear cell (PBMC), and carry out external evoked preparation CIK cell.CIK cell growth curve result shows,
The serum-free medium comprising the clear substitute of the present invention can be effectively promoted the in-vitro multiplication of CIK cell, its cultivation effect
Significantly be better than complete medium and domestic serum-free medium, and with commercially available import serum-free medium X-VIVO 15
Effect suitable.The measurement result of CIK surface markers expression shows, comprises the nothing of serum substitute of the present invention
The quality of the CIK cell that blood serum medium induces is better than traditional complete medium and domestic serum-free medium, with
The CIK cell quality that import culture medium culturing goes out is suitable.CIK cell in vitro kills the evaluation of usefulness and shows, comprises this
The killing-efficiency of target cell is significantly higher than and cultivates completely by CIK cell prepared by the serum-free medium of bright serum substitute
Base and domestic serum-free medium；When 20: 1, its killing activity is slightly above the serum-free medium of import.
In another preferred embodiment, it is thin that the serum-free medium comprising serum substitute of the present invention can be used for NK
The In vitro culture of born of the same parents.Culture medium and complete medium (containing 10%FBS), the city of serum substitute of the present invention will be comprised
Import serum-free medium, the commercially available domestic serum-free medium sold compare.Separate according to the method for document report
Purification peripheral blood PBMC also carries out external evoked preparation NK cell.NK growth curve result shows, comprises the present invention
The effect that the serum-free medium of serum substitute is cultivated for NK cell proliferation in vitro and import serum-free medium
X-VIVO 15 effect is suitable, hence it is evident that be better than complete medium and domestic serum-free medium.NK cell surface molecule mark
The testing result of will thing shows, uses its table of NK cell prepared by 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 cell that the serum-free medium comprising serum substitute of the present invention is turned out has very well
Cell purity.
Accompanying drawing explanation
Fig. 1 is the growth in vitro curve of the CIK cell that different culture media is cultivated.
Fig. 2 is the CIK cell surface markers expression of different culture media induction.
When Fig. 3 is with human leukemia cell line K562 for target cell, the CIK cell that different culture media is cultivated is along with effect target
The cell toxicant percentage ratio of ratio.
When Fig. 4 is with Human cervical cancer cell lines Hela for target cell, the CIK cell that different culture media is cultivated is along with effect target
The cell toxicant percentage ratio of ratio.
Fig. 5 be and human lung adenocarcinoma cell line A549 for target cell time, different culture media cultivate CIK cell along with effect
The cell toxicant percentage ratio of target ratio.
Fig. 6 is the growth in vitro curve of the NK cell that different culture media is cultivated.
Fig. 7 is the NK cell surface marker developed by molecule level of different culture media induction.
Detailed description of the invention
In order to the technical characteristic of the present invention, purpose and beneficial effect are more clearly understood from, the existing technology to the present invention
Scheme carries out described further below, but it is not intended that to the present invention can the restriction of practical range.
Embodiment 1. is 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 described serum substitute is as follows:
1. prepare ferrum saturated human transferrin solution:
(1) by 40mg FeCl3It is dissolved in 20ml 1mM HCl solution, subpackage freezen protective in-20 DEG C, obtains 1
Number store liquid；
(2) take 200mg people and turn ferrum albumin, be dissolved in 20ml RPMI1640 culture medium, add the 1 of 300 μ l
Number store liquid stirring, obtain the human transferrin solution (10mg/ml) that ferrum is saturated, in-30 DEG C of preservations.
2. prepare cholesterol solution: weigh 58mg cholesterol powder, add in 20ml RPMI1640 culture medium, super
Acoustic wave oscillator shakes 1 hour in 4 DEG C, is allowed to complete emulsifying, obtains cholesterol and stores liquid (2.9mg/ml), in-30 DEG C
Preserve.
3. prepare Linoleic Acid solution: weigh 80mg linoleic acid and be dissolved in 20ml graded alcohols, fully shake dissolving, adopt
Degerming with the membrane filtration 2 times of 0.45 μm, obtain linoleic acid storing solution (4mg/ml), in 8 DEG C of storages.
4., during remaining composition is all dissolved in RPMI1640 culture medium, obtain storing solution.Wherein, pharmaceutical grade human albumin deposit
Liquid (10mg/ml), recombinant human insulin's storing solution (10mg/ml) are in-20 DEG C of storages；Ferrous sulfate (0.2mg/ml),
Copper sulfate (0.02mg/ml), zinc sulfate (0.1mg/ml), sodium selenite (0.01mg/ml), cobaltous chloride (0.1mg/ml),
Nickel dichloride. (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.
5. being added by each 10ml of storing solution of above-mentioned each component is mixed and stirred for uniformly in container, add RPMI1640 training
Support base and cumulative volume be supplemented to 900ml, use each one of 0.45um and 0.22um filter membrane (upper strata is 0.45um, under
Layer is 0.22um) carry out filtration sterilization, and regulate pH to 7.2 with 5%NaHCO3, add RPMI1640 training
Support base and volume is supplemented to 1000ml, obtain serum substitute A of the present invention.
6. by serum substitute A subpackage and in-20 DEG C of storages.
Embodiment 2. comprises the preparation of the serum-free medium of serum substitute of the present invention
1., by commercially available culture medium by specification preparation, obtain RPMI 1640 basal medium (RPMI Medium 1640
Culture medium, Beijing Orient Hua Hui biological medicine Science and Technology Ltd.).Wherein RPMI 1640 basal medium formulation such as table
Shown in 2.
2. comprise the formula of the serum-free medium of serum substitute of the present invention: the serum-free medium (culture medium of the present invention
A) RPMI 1640 basal medium of 90% and the serum substitute A of 10% are comprised.
3. the preparation of serum-free medium (culture medium A): before use 100ml serum substitute A is slowly added into
In 800ml RPMI 1640 basal medium, after stirring, regulate pH to 7.2 with 5%NaHCO3, then add
Enter RPMI1640 culture medium and volume is supplemented to 1000ml, the serum-free training of serum substitute A of the present invention must be comprised
Support base (culture medium A).
Table 2 RPMI1640 basal medium formulation
Embodiment 3. serum-free medium A cultivates the evaluation of CIK cell multiplication capacity
Utilize healthy human peripheral blood, according to the isolated and purified PERIPHERAL BLOOD MONONUCLEAR CELL of the method (PBMC) of document report,
And carry out external evoked, prepare CIK cell.Experiment is divided into 4 groups (tables 3), is respectively adopted: 1) nothing of the present 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 cultivation.
Cell number is adjusted to 5 × 10 by PBMC after purification6/ ml, is seeded in T175 culture bottle.From induction the 1st day,
Within every 3 days, carry out half amount and change liquid, and keep the final concentration of cytokine in culture medium constant.Sampling simultaneously utilizes trypan blue to refuse
Dye method counts, and within the 21st day, terminates until cultivating, and draws CIK cell growth curve.Wherein, commercially available depletion of blood is used
Clear culture medium and the experimental group of traditional complete medium, the induction of CIK carries out according to the method for report in document, uses
Including humanized mouse anti human CD3 monoclonal antibody, INF-γ and recombinant human il-2；Use the reality of culture medium of the present invention
Test group, when cultivating CIK, only add recombinant human il-2.Specific experiment is grouped as follows shown in table:
Table 3 CIK cell multiplication capacity evaluation is grouped
| Numbering | Experiment packet |
| 1 | Complete medium |
| 2 | X-VIVO 15(LONZA) |
| 3 | Domestic culture medium |
| 4 | Culture medium A |
Experimental result as it is 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 commercially available
The effect of serum-free medium X-VIVO 15 suitable.
The mensuration of embodiment 4. serum-free medium A induction CIK cell surface markers expression
CIK cell prepared in embodiment 3, the results when cultivating to 21 days.Use fluorescent antibody that cell is carried out
Carrying out flow cytometry after labelling, detection CIK cell surface markers expression before cultivation and after cultivation becomes
Change.The cell concentration of each sample labelling is 1 × 106, the cell surface molecule of detection includes: CD3, CD4, CD8,
And CD56.After detection, different cell subsets and the matched group of four experimental grouies, i.e. cell before induction are compared；
Data before four groups after induction are compared simultaneously, observe purpose cell in harvesting after cultivating content and
Induced efficiency.Experiment packet is as shown in table 4.
The mensuration packet of table 4 CIK cell surface markers expression
| Numbering | Experiment packet |
| 1 | Matched 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 2.Compared with matched group (i.e. before induction), CD3+ cell subsets in four experimental grouies
Percentage ratio all increase, but there is no significant difference；The result compared between four experimental grouies does not has significant difference yet.
The ratio of the double positive cell subgroup of CD3+/CD56+ and CD3+/CD8+ two, the most aobvious in four experimental grouies after induction
Writing and rise, wherein X-VIOV 15 groups and the use present invention cultivate basis set ratio and are significantly higher than complete medium group.
The ratio of the double positive cell subgroup of CD3+/CD4+ is sent out after induction in complete medium group and domestic serum-free experimental group
Give birth to slight rise；Decline is there occurs in import culture medium and culture medium A group.CIK cell is mainly passed through
Two T cell subgroups of CD3+/CD56+ and CD3+/CD8+ play broad spectrum activity antitumor actions, above-mentioned two subgroup thin
Born of the same parents' ratio determines the biological activity of CIK cell, is the major criterion judging CIK cell quality.This result shows,
The quality of the CIK that culture medium A induces 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 that embodiment 5. serum-free medium A cultivates kills the evaluation of usefulness
Utilizing fluorescein based dye CFSE to measure the CIK target cells in vitro killing activity that different experiments group obtains, the target of use 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 company (article No.: Cat.34554), and propidium iodide (PI) is purchased from Sigma
Company.K562, Hela and A549 cell line is purchased from Institute of Basic Medical Sciences of China Concord Medical Science University.Experiment packet
Same as in Example 3, as shown in table 3.
The density of three kinds of target cells is all adjusted to 6x104/ml, joins in well culture plate of the U-shaped end 96, every hole 100 μ l.
Gather in the crops the CIK cell that four experimental grouies are cultivated, according to test kit description, CIK cell is marked, after labelling
Cell according to effect target ratio 5: 1,10: 1, and the ratio of 20: 1 mixes with target cell equal-volume, arranges 3 parallel control skies.
Set individual effect cell and blank cultures as comparison sky simultaneously.After mixing, culture plate is at 37 DEG C, under the conditions of 5%CO2
Hatch 4 hours.After hatching end, every hole adds 25tl PI dye liquor (100 μ g/ μ l), after ice bath hatches 5 minutes, with stream
Formula cell instrument is analyzed, and measures the CIK killing ability to different target cells.The calculating side killing percentage ratio of target cell
Formula is:
Cell toxicant percentage ratio=[target of the target cell/100-natural death of target cell-natural death that experimental group is dead 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 culture medium culturing goes out is along with effect target ratio
Increasing, it all shows incremental trend to the killing-efficiency of three kinds of target cells；And in identical effect target ratio, different experiments
The result that group compares shows, CIK cell prepared by the serum-free medium culture medium A killing-efficiency to target cell
It is significantly higher than complete medium group and domestic serum-free matched group；When 20: 1, killing activity is slightly above the serum-free of import
Culture medium, but there is no significant difference.
Embodiment 6. serum-free medium A cultivates the evaluation of NK ability of cell proliferation
Use cell separation liquid (people's mononuclearcell separation liquid 1.077, article No. 25710, Beijing Orient China brightness biological medicine
Science and Technology Ltd.) from 5-10ml healthy human peripheral blood, collect PBMC, PBMC aseptic PBS washing 3 times,
And be resuspended in PBS, sampling counting.Experiment is divided into 4 groups (tables 5), is respectively adopted: 1) serum-free of the present 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, carries out cell cultivation.
Use the PBMC of above culture medium dilution isolated respectively, adjust cell density in 1-3 × 106In the range of/ml.
The PBMC diluted is seeded in T175 culture bottle, adds recombinant human il-2 to final concentration 10ng/ml, weight simultaneously
Group human IL-15 to final concentration 0.1% (W/V), puts into 37 DEG C, 5%CO2 to final concentration 50ng/ml, recombinant human serum albumin
Environment is cultivated.Within every 3 days, carry out half amount and change liquid, and maintain the concentration level of each cytokine in culture medium to keep constant,
Sampling simultaneously counts, and draws cell growth curve, incubation time totally 15 days.
Table 5 NK ability of cell proliferation evaluation 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.Cultivating through 15 days, serum-free medium A trains for NK cell proliferation in vitro
The effect supported is suitable with import serum-free medium X-VIVO 15 effect, hence it is evident that be better than complete medium and domestic depletion of blood
Clear culture medium.
The level of embodiment 7. serum-free medium A induced NK cell surface molecular mark
Results use the NK cell that different culture media is cultivated, and use fluorescent-labeled antibody to combine flow cytometry, detection
The expression of NK cell surface marker, including CD3, CD56, CD16, CD8 and CD57, wherein CD3-/CD56+,
CD16+/CD56+ and CD8+/CD57+ is the significant molecular phenotype of NK cell.Experiment packet is as shown in table 6.
Concrete operations are, are collected by cell, 1000rpm before detection, and 4 DEG C are centrifuged 5 minutes.By resuspended for cell PBS
And count, adjustment cell density is 1x106/ml.Taking the cell of 5x105 as measuring samples, addition 2%BSA is dilute
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
Cell is carried out by (eBioscience, Cat.12-0167-42) and Pacific Blue-CD57 (eBioscience, Cat.322315)
Labelling, room temperature lucifuge hatches 20 minutes.Wash 2 times with PBS after sample incubation, add 500 μ l PBS resuspended, use
Flow cytometer detects.
The mensuration packet of table 6 NK cell surface marker developed by molecule level
| Numbering | Experiment packet |
| 1 | Matched 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.Use its surface associated molecule mark of NK cell prepared by serum-free medium A
Expression be significantly higher than complete medium group and domestic serum-free culture is basis set, slightly above import serum-free medium
Group, shows that the NK cell that serum-free medium A turns out has good cell purity.
Claims (10)
1. the serum substitute for immunocyte suspension culture, it is characterised in that this serum substitute comprises:
Pharmaceutical grade human albumin, recombined human transferrins, recombinant human insulin, lipid, trace element, hormone, shearing are tried hard to keep
Protect agent, resveratrol, D-Glucosamine Hydrochloride, R-848 and C3.
Serum substitute the most according to claim 1, it is characterised in that: it comprises with final concentration 50-200
Mg/L pharmaceutical grade human albumin, 50-500mg/L recombined human transferrins, 10-100mg/L recombinant human insulin,
15-60mg/L lipid, 1.5-30mg/L trace element, 10-100mg/L hormone, 500-3000mg/L shearing force are protected
Agent, 1-50mg/L resveratrol, 500-2000mg/L D-Glucosamine Hydrochloride, 1-50mg/L R-848 and, 1-200
mg/L C3。
Serum substitute the most according to claim 1, it is characterised in that: preferably, it comprises with final concentration
Meter 100-200mg/L pharmaceutical grade human albumin, 100-300mg/L recombined human transferrins, 30-80mg/L recombined human pancreas
Island element, 30-50mg/L lipid, 2-15mg/L trace element, 20-50mg/L hormone, 1000-2000mg/L shearing force
Protective agent, 10-20mg/L resveratrol, 1000-1500mg/L D-Glucosamine Hydrochloride, 10-30mg/L R-848
With, 10-150mg/L C3.
Serum substitute the most according to claim 1, it is characterised in that: it is highly preferred that it comprises with the denseest
Degree meter 100mg/L pharmaceutical grade human albumin, 100mg/L recombined human transferrins, 100mg/L recombinant human insulin,
40mg/L lipid, 5mg/L trace element, 30mg/L hormone, 1500mg/L shearing force protective agent, 15mg/L are white
Veratryl alcohol, 1200mg/L D-Glucosamine Hydrochloride, 20mg/LR-848 and 80mg/L C3.
Serum substitute the most as claimed in any of claims 1 to 4, wherein: described lipid include but
It is not limited to arachidonic acid, cholesterol, oleic acid, linoleic acid, thioctic acid, linolenic acid etc.；Described trace element include but
It is not limited to ferrum, copper, zinc, selenium, cobalt, nickel, manganese etc.；Described hormone includes but not limited to that dexamethasone, progesterone, gallbladder are solid
Alcohol etc.；Described shearing force protective agent includes but not limited to poloxamer etc..
6. comprising a compositions for serum substitute as claimed in any of claims 1 to 5, it is special
Levy and be: described compositions can be by serum substitute as claimed in any of claims 1 to 5 and basis training
Foster base is prepared by certain technique the most before use.Wherein, described basal medium includes but not limited to RPMI1640
Culture medium, DMEM culture medium etc..
Compositions the most according to claim 6, it is characterised in that: described compositions is by the base of 50%-95%
Basal culture medium forms with the serum substitute as claimed in any of claims 1 to 5 of 5-50%；Preferably,
Described compositions is as claimed in any of claims 1 to 5 by the basal medium of 70%-95% and 5-30%'s
Serum substitute forms；It is highly preferred that described compositions by the basal medium of 85%-95% and 5-15% according to right
Require the serum substitute composition described in any one in 1 to 5；Most preferably, described compositions is trained by the basis of 90%
Support the serum substitute as claimed in any of claims 1 to 5 composition of base and 10%.
Compositions the most according to claim 6, it is characterised in that: described compositions is serum-free medium.
Compositions the most according to claim 6, it is characterised in that: described compositions can be used for immunocyte and hangs
Floating cultivation.
Compositions the most according to claim 6 is in immunocyte application in suspension, it is characterised in that: described
Immunocyte include but not limited to cytokine induced kill cell (Cytokine Induced Killer Cells, CIK),
Natural killer cell (Nature Killer, NK) cell etc..
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410645405.0A CN105886465A (en) | 2014-11-15 | 2014-11-15 | Serum substitute for immune cell suspension culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410645405.0A CN105886465A (en) | 2014-11-15 | 2014-11-15 | Serum substitute for immune cell suspension culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105886465A true CN105886465A (en) | 2016-08-24 |
Family
ID=56698201
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410645405.0A Pending CN105886465A (en) | 2014-11-15 | 2014-11-15 | Serum substitute for immune cell suspension culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105886465A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107304411A (en) * | 2016-04-22 | 2017-10-31 | 康思葆（北京）生物技术有限公司 | A kind of serum substitute for the culture that suspended for immunocyte |
| CN107574150A (en) * | 2017-09-06 | 2018-01-12 | 万向东方生物科技有限公司 | A kind of serum-free immune cell media and preparation method thereof |
| 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 |
| CN112424342A (en) * | 2018-05-08 | 2021-02-26 | 生命科技公司 | Compositions and methods for culturing and expanding 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 |
-
2014
- 2014-11-15 CN CN201410645405.0A patent/CN105886465A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107304411A (en) * | 2016-04-22 | 2017-10-31 | 康思葆（北京）生物技术有限公司 | A kind of serum substitute for the culture that suspended for immunocyte |
| CN107574150A (en) * | 2017-09-06 | 2018-01-12 | 万向东方生物科技有限公司 | A kind of serum-free immune cell media and preparation method thereof |
| CN112424342A (en) * | 2018-05-08 | 2021-02-26 | 生命科技公司 | Compositions and methods for culturing and expanding cells |
| 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 |
| 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 |
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Application publication date: 20160824 |