CN109486760A - The cultural method of placental blood CIK cell improvement - Google Patents

The cultural method of placental blood CIK cell improvement Download PDF

Info

Publication number
CN109486760A
CN109486760A CN201910045747.1A CN201910045747A CN109486760A CN 109486760 A CN109486760 A CN 109486760A CN 201910045747 A CN201910045747 A CN 201910045747A CN 109486760 A CN109486760 A CN 109486760A
Authority
CN
China
Prior art keywords
culture
cell
antibody
placental blood
cik
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN201910045747.1A
Other languages
Chinese (zh)
Inventor
周海涛
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yaoding (beijing) International Cell Medicine Technology Co Ltd
Original Assignee
Yaoding (beijing) International Cell Medicine Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yaoding (beijing) International Cell Medicine Technology Co Ltd filed Critical Yaoding (beijing) International Cell Medicine Technology Co Ltd
Priority to CN201910045747.1A priority Critical patent/CN109486760A/en
Publication of CN109486760A publication Critical patent/CN109486760A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a kind of cultural methods of placental blood CIK cell improvement.It is cultivated this method comprises: 1) will be inoculated in the culture vessel for being coated with CD3 antibody and CD28 antibody by the isolated monocyte of placental blood；Cell factor in cultivating system is IFN-γ；2) IL-2, IL-12, IL-15, CD16 antibody are added into cultivating system；3) liquid is changed, the cell factor in new cultivating system is IL-2, IL-12, IL-15；Basal medium in the cultivating system is the GT-T581 culture medium containing 4%~6% autologous plasma；4) IL-2 is added, culture harvested cell after 2~4 days.This method CIK expanding effect is good, and the CIK accounting activated is higher, T-reg cell accounting is lower.

Description

The cultural method of placental blood CIK cell improvement
Technical field
The present invention relates to technical field of cell culture, in particular to a kind of culture side of placental blood CIK cell improvement Method.
Background technique
Placenta is the vitals of mass exchange between fetus and parent, by embryo's embryophoric membrane and parent during being human pregnancy Endometrium organizes colligator official between combining the mothers and sons grown up to.Fetus develops in uterus, obtains nutrition from parent by placenta, And both sides keep comparable independence.
The collection capacity of placental blood can at most acquire 250mL generally in 150mL or more, can collection capacity be higher than Cord blood.Tire Primitive hematopoietic stem cell is rich in disk blood, quality and quantity matches in excellence or beauty with marrow, and quantity is higher than candidate stem cell in Cord blood and contains Amount.
Leucocyte content 11~19 × 109, it is much higher than peripheral blood, slightly above Cord blood.Monocyte content therein is high In peripheral blood and Cord blood content, 2 times of the content of monocyte nearly bleeding of the umbilicus.
Cytokine induced kill cell (cytokine-induced killer cells, CIK) be a group in vitro Through CD4+ based on CD3+CD56+ cell made of the induction such as IL-1 α, IL-2, IFN-γ and CD 3-resisting monoclonal antibody and CD8+ effector T cell group, with the powerful non-principal histocompatibility complex for killing tumor activity and NK cell of T cell (MHC) restricted the advantages of killing tumor.
Compared with other adoptive immunotherapy cells, CIK have that growth rate is fast, killing activity is high, it is wide to kill tumor spectrum and It is the features such as Small side effects, little on normal marrow hematopoiesis function influence, a kind of effective immunotherapy of tumors is considered by clinic Means.It is widely used in the treatment of the Several Kinds of Malignancy such as kidney, melanoma, liver cancer, gastric cancer and leukaemia.
CIK can play antitumor action through a variety of ways.CIK has direct killing effect to tumour cell, passes through secretion Granzyme and perforin penetrate target cell membrane, directly result in tumour cell rupture；CIK has stronger cytotoxic activity, can divide The cytotoxicity that the cytokine profiles such as IL-2, IFN-γ, TNF-α and GM-CSF further increase immune effector cell is secreted, Not only there is direct repression to tumour cell, can also kill indirectly oncocyte by adjusting immune system, induce tumour cell Apoptosis.The problems such as CIK breeding method in the prior art, generally existing amplification multiplying power is low, cell activity is poor.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of novel placental blood cytokine induced kill cell (cytokine- Induced killer cells, CIK) cultural method, this method is easy to operate, and it is preferable that culture obtains CIK mass, can be used for A large amount of preparations of CIK, make this method be more conducive to promotion and application.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention relates to a kind of cultural methods of placental blood CIK cell improvement, comprising:
1) culture vessel for being coated with CD3 antibody and CD28 antibody will be inoculated in by the isolated monocyte of placental blood In cultivated；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；
2) the final concentration of 900U/mL~1100U/mL of IL-2, the final concentration of 1.5ng/mL of IL-12 are added into cultivating system Final concentration of 8ng/mL~the 12ng/mL of~2.5ng/mL, IL-15, the final concentration of μ of 1.5 μ g/mL~2.5 g/mL of CD16 antibody；
3) liquid is changed, the cell factor in new cultivating system is IL-2 900U/mL~1100U/mL, IL-15 4ng/mL ~6ng/mL, IL-12 1.5ng/mL~2.5ng/mL；Basal medium in the cultivating system is self containing 4%~6% The GT-T581 culture medium of blood plasma；
4) IU/400ml of IL-2 45~550,000 is added, culture harvested cell after 2~4 days.
Compared with prior art, the invention has the benefit that
CIK expanding effect is good, and the CIK accounting activated is higher, T-reg cell accounting is lower.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the result detected to the CIK cell surface marker that one embodiment of the invention culture obtains.
Specific embodiment
The present invention relates to a kind of cultural methods of placental blood CIK cell improvement, comprising:
1) culture vessel for being coated with CD3 antibody and CD28 antibody will be inoculated in by the isolated monocyte of placental blood In cultivated；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；
2) the final concentration of 900U/mL~1100U/mL of IL-2, the final concentration of 1.5ng/mL of IL-12 are added into cultivating system Final concentration of 8ng/mL~the 12ng/mL of~2.5ng/mL, IL-15, the final concentration of μ of 1.5 μ g/mL~2.5 g/mL of CD16 antibody；
3) liquid is changed, the cell factor in new cultivating system is IL-2 900U/mL~1100U/mL, IL-15 4ng/mL ~6ng/mL, IL-12 1.5ng/mL~2.5ng/mL；Basal medium in the cultivating system is self containing 4%~6% The GT-T581 culture medium of blood plasma；
4) IU/400ml of IL-2 45~550,000 is added, culture harvested cell after 2~4 days.
In some embodiments, in step 1), cell factor in cultivating system be IFN-γ 950U/mL~ 1050U/mL。
In some embodiments, in step 1), the cell factor in cultivating system is IFN-γ 1000U/mL.
In some embodiments, in step 2), the final concentration that IL-2 is added in cultivating system is 1000U/mL, The IL-12 final concentration of 10ng/mL of final concentration of 2ng/mL, IL-15, the final concentration of 2 μ g/mL of CD16 antibody.
In some embodiments, in step 3), cell factor in new cultivating system be IL-2 950U/mL~ 1050U/mL, IL-15 4.5ng/mL~5.5ng/mL, IL-12 1.7ng/mL~2.3ng/mL.
In some embodiments, in step 3), the cell factor in new cultivating system is IL-2 1000U/mL, IL-15 5ng/mL, IL-12 2ng/mL；.
In some embodiments, in step 4), 500,000 IU/400ml of IL-2 is added.
In some embodiments, in step 1), method that the culture vessel is coated with CD3 antibody and CD28 antibody Are as follows:
It is 25cm with the culture area2Meter, be added 2.5~3.5mL contain CD3 antibody 400ng/mL~600ng/mL and The coating buffer of CD28 antibody 400ng/mL~600ng/mL is coated with 1.5h~2.5h under cell culture condition.
If non-specifically emphasized, the cell culture condition that herein described method is related to is between 30 to 45 DEG C and 1 To 10% CO2Between, preferably between 36 to 38 DEG C and 4 to 6% CO2Between.
Add CD3, it is to make T cell living that the antibody of CD28, which is the TCR activation and costimulatory signal in order to simulate T cell, Change the state for entering proliferation.
In some embodiments, the concentration of CD3 antibody is 500ng/mL.
In some embodiments, the concentration of CD28 antibody is 500ng/mL.
In some embodiments, the solvent of the coating buffer is D-PBS.
In some embodiments, in step 1), basal medium in the cultivating system be containing 4%~6% from The VIVO culture medium of body blood plasma.
In some embodiments, in step 1), the basal medium in the cultivating system is containing 5% autologous plasma VIVO culture medium.
It in some embodiments, is 25cm with the culture area in step 1)2Meter, the additional amount of culture medium are 8ml~12ml, cell inoculation amount are 0.8 × 107~1.2 × 107。
It in some embodiments, is 25cm with the culture area in step 1)2Meter, the additional amount of culture medium are 10ml, cell inoculation amount are 1.0 × 107。
In some embodiments, the incubation time of step 1) is 20h~28h.
In some embodiments, the incubation time of step 1) is for 24 hours.
In some embodiments, the incubation time of step 2) is 2d~4d.
In some embodiments, the incubation time of step 2) is 3d.
In some embodiments, in step 3), the basal medium in the cultivating system is containing 5% autologous plasma GT-T581 culture medium.
In some embodiments, in step 3), cell is passed on 2~3 times.
In some embodiments, the incubation time of step 3) is 8d~10d.
In some embodiments, the incubation time of step 3) is 9d.
In some embodiments, step 3) specifically includes:
It a) is 75cm with culture area215~25ml culture medium culture 3d is added in meter；
B) liquid is changed, is 175cm with culture area260ml~80ml culture medium culture 3d is added in meter；
C) liquid is changed, is cultivated using culture bag, 300ml~400ml culture medium culture 3d is added.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.
Embodiment 1
The present embodiment is related to a kind of cultural method of placental blood CIK improvement.
1. placental blood CIK cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 400ng/mL, CD28 antibody concentration 600ng/mL.
2) CIK initial medium: VIVO culture medium (contains 4% autologous plasma), IFN-γ 900U/mL.
3) CIK culture medium: GT-T581 culture medium (contains 4% autologous plasma), IL-2 concentration 1100U/mL, IL-15 4ng/ ML, IL-12 2.5ng/mL.
2. placental blood CIK cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 2.5mL coating buffer is added, is placed in incubator and is coated with 2.5h, it is spare.It takes The T25 culture bottle being coated with out, goes coating buffer, takes the placenta blood monocyte 0.8 × 10 prepared7, it is inoculated into T25 culture In bottle, adds 10mLCIK initial medium, be placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-2, the final concentration of 1.5ng/ of final concentration of 900U/mL, IL-12 is added The final concentration of 12ng/mL of mL, IL-15, the final concentration of 1.5 μ g/mL of CD16 antibody；Culture bottle is replaced in 37 DEG C, 5%CO2 In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CIK culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CIK culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCIK culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 13rd day, adds IL-2,450,000 IU.
7) it cultivates the 16th day, harvests cell.
Embodiment 2
The present embodiment is related to a kind of cultural method of placental blood CIK improvement.
1. placental blood CIK cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 600ng/mL, CD28 antibody concentration 400ng/mL.
2) CIK initial medium: VIVO culture medium (contains 6% autologous plasma), IFN-γ 1100U/mL.
3) CIK culture medium: GT-T581 culture medium (contains 6% autologous plasma), IL-2 concentration 900U/mL, IL-15 6ng/ ML, IL-12 1.5ng/mL.
2. placental blood CIK cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3.5mL coating buffer is added, is placed in incubator and is coated with 1.5h, it is spare.It takes The T25 culture bottle being coated with out, goes coating buffer, takes the placenta blood monocyte 1.2 × 10 prepared7, it is inoculated into T25 culture In bottle, adds 10mLCIK initial medium, be placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-2, the final concentration of 2.5ng/ of final concentration of 1100U/mL, IL-12 is added The final concentration of 8ng/mL of mL, IL-15, the final concentration of 2.5 μ g/mL of CD16 antibody；Culture bottle is replaced in 37 DEG C, 5%CO2Training It supports in case.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CIK culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CIK culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCIK culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 13rd day, adds IL-2,550,000 IU.
7) it cultivates the 16th day, harvests cell.
Embodiment 3
The present embodiment is related to a kind of cultural method of placental blood CIK improvement.
1. placental blood CIK cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 450ng/mL, CD28 antibody concentration 550ng/mL.
2) CIK initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 950U/mL.
3) CIK culture medium: GT-T581 culture medium (contains 5% autologous plasma), IL-2 concentration 1050U/mL, IL-15 4.5ng/mL, IL-12 2.2ng/mL.
2. placental blood CIK cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 2.8mL coating buffer is added, is placed in incubator and is coated with 2.2h, it is spare.It takes The T25 culture bottle being coated with out, goes coating buffer, takes the placenta blood monocyte 1.1 × 10 prepared7, it is inoculated into T25 culture In bottle, adds 10mLCIK initial medium, be placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-2, the final concentration of 1.8ng/ of final concentration of 1050U/mL, IL-12 is added The final concentration of 11ng/mL of mL, IL-15, the final concentration of 2.2 μ g/mL of CD16 antibody；Culture bottle is replaced in 37 DEG C, 5%CO2 In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CIK culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CIK culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCIK culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 13rd day, adds IL-2,480,000 IU.
7) it cultivates the 16th day, harvests cell.
Embodiment 4
The present embodiment is related to a kind of cultural method of placental blood CIK improvement.
1. placental blood CIK cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL.
2) CIK initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
3) CIK culture medium: GT-T581 culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL, IL-15 5ng/ ML, IL-12 2ng/mL.
2. placental blood CIK cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h, it is spare.Take out packet By good T25 culture bottle, coating buffer is gone, takes the placenta blood monocyte 1.0 × 10 prepared7, it is inoculated into T25 culture bottle It is interior, add 10mLCIK initial medium, is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-2, the final concentration of 2ng/mL of final concentration of 1000U/mL, IL-12 is added, The final concentration of 10ng/mL of IL-15, the final concentration of 2 μ g/mL of CD16 antibody；Culture bottle is replaced in 37 DEG C, 5%CO2Incubator In.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CIK culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CIK culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCIK culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 13rd day, adds IL-2,500,000 IU.
7) it cultivates the 16th day, harvests cell, do flow cytometer detection；Its result is as shown in Figure 1.
Comparative example 1
Placental blood CIK cultivates cellular processes
1) it cultivates the 0th day, takes the placenta blood monocyte 1.0 × 10 prepared7, T25 culture bottle is inoculated into (without packet Quilt) in, add 10mLCIK initial medium, is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, addition and IL-2, final concentration of 1000U/mL；CD3 monoclonal antibody concentration is added 500ng/mL, CD28 antibody concentration 500ng/mL；Culture bottle is replaced in 37 DEG C, 5%CO2In incubator.
Remaining step is the same as embodiment 4.
Comparative example 2
The replacement of CIK cell culture medium are as follows:
1) CIK initial medium: RPM1-1640 culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
2) CIK culture medium: RPM1-1640 culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
Remaining condition of culture is the same as embodiment 4.
Comparative example 3
A kind of cultural method of placental blood CIK.
1. placental blood CIK cell culture medium
1) coating buffer: D-PBS, CD3 monoclonal antibody concentration 500ng/mL, CD28 antibody concentration 500ng/mL.
2) CIK initial medium: VIVO culture medium (contains 5% autologous plasma), IFN-γ 1000U/mL.
3) CIK culture medium: VIVO culture medium (contains 5% autologous plasma), IL-2 concentration 1000IUmL.
2. placental blood CIK cultivates cellular processes
1) it cultivates the 0th day, takes T25 culture bottle, 3mL coating buffer is added, is placed in incubator and is coated with 2h, it is spare.Take out packet By good T25 culture bottle, coating buffer is gone, takes the placenta blood monocyte 1.0 × 10 prepared7, it is inoculated into T25 culture bottle It is interior, add 10mLCIK initial medium, is placed in 37 DEG C, 5%CO2In incubator.
2) it cultivates the 1st day, takes out culture bottle, IL-2 is added, culture bottle is replaced in 37 by final concentration of 1000U/mL DEG C, 5%CO2In incubator.
3) it cultivates the 4th day, cell is transferred completely into T75, adds CIK culture medium 20mL, is placed in 37 DEG C, 5%CO2Incubator In.
4) it cultivates the 7th day, cell is transferred in T175 culture bottle, adds CIK culture medium 70mL, is placed in 37 DEG C, 5%CO2 In incubator.
5) it cultivates the 10th day, cell is transferred to addition 400mLCIK culture medium in culture bag, is placed in 37 DEG C, 5%CO2Training It supports in case.
6) it cultivates the 14th day, harvests cell, do flow cytometer detection；
Experimental example
After the cell for collecting embodiment 4 and comparative example 1,2,3, cell count is carried out, and by flow cytometry to table Type is identified.Every group repetition six times, it is as a result as follows:
Group Cell number
Embodiment 4 1.02×109
Comparative example 1 9.8×108
Comparative example 2 5.4×108*
Comparative example 3 6.2×108*
*P < 0.05, vs embodiment 4.
*P < 0.05, vs embodiment 4.
It can be seen from the results above that RPM1-1640 culture medium can not promote well under the cultivating system of the application Into the proliferation of cell, but it has no significant effect the differentiation of CIK；
And it is coated with culture bottle in advance, the cell of post incoulation can be allowed to completely attach to the activation-inducing factor, reach more preferable Activation-inducing effect, it is thus possible to obtain more CD3+CD56+ cells；And by control differentiation opportunity, it is thin to reduce T-reg The ratio of born of the same parents.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution The range of art scheme.

Claims (9)

1. a kind of cultural method of placental blood CIK cell improvement characterized by comprising
1) by by the isolated monocyte of placental blood be inoculated in the culture vessel for being coated with CD3 antibody and CD28 antibody into Row culture；Cell factor in cultivating system is IFN-γ 900U/mL~1100U/mL；
2) into cultivating system be added the final concentration of 900U/mL~1100U/mL of IL-2, the final concentration of 1.5ng/mL of IL-12~ Final concentration of 8ng/mL~the 12ng/mL of 2.5ng/mL, IL-15, the final concentration of μ of 1.5 μ g/mL~2.5 g/mL of CD16 antibody；
3) change liquid, the cell factor in new cultivating system be IL-2 900U/mL~1100U/mL, IL-15 4ng/mL~ 6ng/mL, IL-12 1.5ng/mL~2.5ng/mL；Basal medium in the cultivating system is containing 4%~6% self blood The GT-T581 culture medium of slurry；
4) IU/400ml of IL-2 45~550,000 is added, culture harvested cell after 2~4 days.
2. the cultural method of placental blood CIK cell according to claim 1, which is characterized in that in step 1), the training The method that feeding container is coated with CD3 antibody and CD28 antibody are as follows:
It is 25cm with the culture area2Meter is added 2.5~3.5mL and contains CD3 antibody 400ng/mL~600ng/mL and CD28 The coating buffer of antibody 400ng/mL~600ng/mL is coated with 1.5h~2.5h under cell culture condition.
3. the cultural method of placental blood CIK cell improvement according to claim 2, which is characterized in that the coating buffer Solvent is D-PBS.
4. the cultural method of placental blood CIK cell improvement according to claim 1, which is characterized in that in step 1), institute Stating the basal medium in cultivating system is the VIVO culture medium containing 4%~6% autologous plasma.
5. the cultural method of placental blood CIK cell improvement according to claim 4, which is characterized in that in step 1), with The culture area is 25cm2Meter, the additional amount of culture medium are 8ml~12ml, and cell inoculation amount is 0.8 × 107~1.2 × 107。
6. the cultural method of placental blood CIK cell improvement according to claim 5, which is characterized in that the culture of step 1) Time is 20h~28h.
7. the cultural method of placental blood CIK cell improvement according to claim 1, which is characterized in that the culture of step 2) Time is 2d~4d.
8. the cultural method of placental blood CIK cell improvement according to claim 1, which is characterized in that, will in step 3) Cell passes on 2~3 times.
9. the cultural method of placental blood CIK cell improvement according to claim 8, which is characterized in that the culture of step 3) Time is 8d~10d.
CN201910045747.1A 2019-01-17 2019-01-17 The cultural method of placental blood CIK cell improvement Withdrawn CN109486760A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910045747.1A CN109486760A (en) 2019-01-17 2019-01-17 The cultural method of placental blood CIK cell improvement

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910045747.1A CN109486760A (en) 2019-01-17 2019-01-17 The cultural method of placental blood CIK cell improvement

Publications (1)

Publication Number Publication Date
CN109486760A true CN109486760A (en) 2019-03-19

Family

ID=65714680

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910045747.1A Withdrawn CN109486760A (en) 2019-01-17 2019-01-17 The cultural method of placental blood CIK cell improvement

Country Status (1)

Country Link
CN (1) CN109486760A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104357390A (en) * 2014-10-15 2015-02-18 深圳源正细胞医疗技术有限公司 Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells
CN105886469A (en) * 2016-05-04 2016-08-24 深圳瑞祥元科技有限公司 CIK (cytokine induced killer) cell and culture method and application thereof
WO2017142187A1 (en) * 2016-02-15 2017-08-24 신동혁 Medium addition kit for culturing nk cells and nk cell culturing method using kit
CN107488630A (en) * 2017-10-09 2017-12-19 天津长和生物技术有限公司 The amplification cultivation method of natural killer T cells culture matrix and natural killer T cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104357390A (en) * 2014-10-15 2015-02-18 深圳源正细胞医疗技术有限公司 Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells
WO2017142187A1 (en) * 2016-02-15 2017-08-24 신동혁 Medium addition kit for culturing nk cells and nk cell culturing method using kit
CN105886469A (en) * 2016-05-04 2016-08-24 深圳瑞祥元科技有限公司 CIK (cytokine induced killer) cell and culture method and application thereof
CN107488630A (en) * 2017-10-09 2017-12-19 天津长和生物技术有限公司 The amplification cultivation method of natural killer T cells culture matrix and natural killer T cells

Similar Documents

Publication Publication Date Title
CN107460168B (en) The amplification cultivation method of natural killer cells culture substrate and natural killer cells
CN108220239B (en) A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells
CN107475196A (en) The amplification cultivation method of NK culture matrix and NK
CN109294985A (en) A method of culture medium system and NK cell expansion ex vivo for NK cell expansion ex vivo
CN102676454B (en) Preparation method for CIK (cytokine induced killer) cell of umbilical cord blood source
CN107177548B (en) Culture system for in vitro lymphocyte amplification, amplification method and application
CN107488631A (en) The amplification cultivation method of NK culture matrix and NK
CN109456941A (en) A kind of cultural method for the DC-CIK cell loading tumour cell excretion body
CN109337869B (en) The cultural method of peripheral blood CIK cell improvement
CN115558641B (en) High-purity effector immune cell population, culture method, reagent composition and application thereof
CN101580817A (en) Method for preparing cell group containing cytokine-induced killing cell
CN109504658A (en) A kind of cultural method of placental blood NK cell
CN105112371A (en) Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation
CN109402057A (en) A kind of cultural method for the DC-CTL cell loading tumour cell excretion body
CN112410294A (en) Amplification culture method of peripheral blood CIK cells
CN109486759A (en) A kind of cultural method of Cord blood NK cell
CN109370987A (en) The cultural method of peripheral blood CTL cell improvement
CN105441390A (en) In-vitro three-dimensional amplification culture method for NK cells
CN109486761A (en) The cultural method of peripheral blood CTL cell
CN107475194A (en) NKT cell culture mediums, cultural method and the application of the two
CN109957543A (en) Utilize the method for Cord blood massive amplification Cord Blood Natural Killer Cells: Impact
CN109486760A (en) The cultural method of placental blood CIK cell improvement
CN104109653A (en) Method of large-scale amplification of human peripheral blood DNT cell by utilization of animal-serum-free culture system
CN109536445A (en) The cultural method of placental blood CIK cell
CN106085960A (en) A kind of culture medium cultivating DC cell and cultural method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20190319

WW01 Invention patent application withdrawn after publication