CN114058578A - Method for amplifying NK cells from stored umbilical cord blood - Google Patents
Method for amplifying NK cells from stored umbilical cord blood Download PDFInfo
- Publication number
- CN114058578A CN114058578A CN202111638979.1A CN202111638979A CN114058578A CN 114058578 A CN114058578 A CN 114058578A CN 202111638979 A CN202111638979 A CN 202111638979A CN 114058578 A CN114058578 A CN 114058578A
- Authority
- CN
- China
- Prior art keywords
- culture
- cells
- cord blood
- cunk
- centrifugal
- 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.)
- Granted
Links
- 210000004700 fetal blood Anatomy 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 40
- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 33
- 230000008569 process Effects 0.000 claims abstract description 18
- 230000003321 amplification Effects 0.000 claims abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 8
- 238000011084 recovery Methods 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims description 33
- 239000011259 mixed solution Substances 0.000 claims description 29
- 239000006285 cell suspension Substances 0.000 claims description 24
- 239000002609 medium Substances 0.000 claims description 21
- 238000000576 coating method Methods 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- 210000002966 serum Anatomy 0.000 claims description 11
- 239000011248 coating agent Substances 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 8
- 238000005138 cryopreservation Methods 0.000 claims description 7
- 239000008354 sodium chloride injection Substances 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims description 6
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims description 6
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 6
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 3
- 238000001802 infusion Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000003068 static effect Effects 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 14
- 210000004698 lymphocyte Anatomy 0.000 abstract description 9
- 230000002829 reductive effect Effects 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 210000002993 trophoblast Anatomy 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 229920001917 Ficoll Polymers 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000002458 cell surface marker Substances 0.000 description 3
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000034578 Multiple myelomas Diseases 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000003289 regulatory T cell Anatomy 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 208000027205 Congenital disease Diseases 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 206010050081 Neonatal hypoxia Diseases 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000024207 chronic leukemia Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 210000005008 immunosuppressive cell Anatomy 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000002660 stem cell treatment Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Images
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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Zoology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for amplifying NK cells from stored umbilical cord blood, which comprises a recovery process and an amplification process. The invention has the beneficial effects that: the lymphocyte is obtained by recovering and purifying the cryopreserved umbilical cord blood, and then the NK cells in the lymphocyte are selectively stimulated and amplified, so that the application direction of the cryopreserved umbilical cord blood is expanded, the activity of the purified lymphocyte is high, the cells are not required to be sorted, trophoblast cells are not required to be used, plasma is not required to be used, the operation complexity is reduced, the cost is reduced, the number of the amplified NK cells is large, the purity is high, and the clinical application requirements are met.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a method for amplifying NK cells from stored umbilical cord blood.
Background
Cord blood contains CD34+ hematopoietic stem cells, has strong differentiation capacity and low immunogenicity, brings the advantages of less immunological rejection among donors and the like, and the cord blood hematopoietic stem cell treatment technology is applied to the treatment of 11 major diseases: acute leukemia, chronic leukemia, myelodysplastic syndrome, multiple myeloma, severe radiation sickness, aplastic anemia, severe poverty, some congenital diseases of partial genetic diseases, partial metabolic diseases, lymphoma and other malignant tumors.
Therefore, in China, the cord blood is stored as an important biological resource, and the storage capacity of the cord blood in China reaches over 100 thousands of the current time, and is greatly increased year by year.
In recent years, as research on umbilical cord blood has been conducted, it has been found that umbilical cord blood contains abundant immune cells such as T cells, NK cells, regulatory T cells, monocytes and the like in addition to hematopoietic stem cells, and has been developed and used. Regulatory T cells are a class of immunosuppressive cells that can be used to treat autoimmune and inflammatory diseases; the monocyte is a type of immunoregulation cell and can be applied to neonatal hypoxia-ischemia encephalopathy and the like; the T cells are immune cells with the capability of killing cancer cells, and the CAR-T cells obtained by transforming the T cells from cord blood also have good application prospect; NK cells are natural killer cells independent of MHC molecules, the cells are more primitive, the natural killer cells can be used for treating various cancers such as breast cancer and multiple myeloma, and the CAR-NK modified by gene editing also has good application value.
The existing methods for effectively proliferating and differentiating natural killer cells from umbilical cord blood mostly require the use of fresh umbilical cord blood, and mainly select CD3 negative cells for cell differentiation and expand.
Therefore, in order to expand the application of cord blood, it is necessary to develop a method for amplifying NK cells in large quantities against stored cord blood to meet the market demand in the future.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, the method for expanding the NK cells from the stored umbilical cord blood without sorting the cells, using trophoblast cells and plasma is provided.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for amplifying NK cells from stored umbilical cord blood comprises a resuscitation process and an amplification process,
the resuscitation process comprises the following steps:
a1, performing resuscitation treatment on the cryopreserved bag storing the umbilical cord blood;
a2, slowly injecting resuscitation solution of 5% -10% human serum albumin into a cryopreservation bag and standing to obtain cell suspension;
a3, transferring the cell suspension into a first centrifuge tube;
a4, injecting a 0.9% sodium chloride injection into a freezing storage bag for washing, and transferring all liquid in the freezing storage bag into a first centrifuge tube;
a5, centrifuging the first centrifuge tube to obtain the required cell suspension;
the amplification process comprises the following steps:
b1, adding the required cell suspension obtained in the recovery step and the mixed solution of CUNK-2 and the serum substitute into a culture flask;
and B2, placing the culture bottle in a constant-temperature incubator for static culture, and supplementing liquid every 2-3 days in the culture process.
Further, the centrifugation process comprises:
a51, performing primary centrifugation treatment on the first centrifuge tube;
a52, removing supernatant in the first centrifugal tube, and carrying out primary dilution on the first centrifugal tube to obtain diluted mixed solution;
a53, transferring the diluted mixed solution into a second centrifugal tube, wherein the second centrifugal tube contains Ficoll;
a54, performing secondary centrifugal treatment on the second centrifugal tube;
a55, sucking the tunica albuginea layer to a third centrifuge tube, and performing secondary dilution on the third centrifuge tube;
a56, carrying out third centrifugation treatment on a third centrifuge tube;
a57, removing the supernatant, and diluting a third centrifuge tube in a constant volume manner;
a58, performing fourth centrifugation treatment on the third centrifuge tube;
and A59, removing the supernatant, and carrying out heavy suspension and uniform mixing by using NK medium to obtain the required cell suspension.
Further, the parameter of the first centrifugal treatment is centrifugation for 15min under the centrifugal force of 400 g;
the parameter of the second centrifugal treatment is centrifugation for 30min under the centrifugal force of 400 g;
the parameter of the third centrifugal treatment is centrifugation for 10min under the centrifugal force of 400 g;
the parameter of the fourth centrifugal treatment is centrifugation for 10min under the centrifugal force of 200 g.
Further, in step B2, the fluid infusion specifically includes:
b21, on the 4 th day of culture, adding a mixed solution of 20mL of NK medium, 0.25 cuNK-2 and 3mL of serum substitute;
b22, on the 6 th day of culture, adding a mixed solution of 40mL of NK medium, 0.5 cuNK-2 and 6mL of serum substitute;
b23, culturing for 8 days, transferring the culture suspension in the culture bottle into a cell culture bag, and adding 100mL of mixed solution of NK culture medium and 0.1 count of CUNK-3;
b24, on the 10 th day of culture, adding 200mL of mixed solution of NK medium and 0.2 branch of CUNK-3;
b25, on the 12 th day of culture, adding 400mL of mixed solution of NK medium and 0.4 branch of CUNK-3;
b26, on the 14 th day of culture, adding 600mL of mixed solution of NK medium and 0.6 branch of CUNK-3;
b27, on day 16 of culture, 600mL of a mixture of NK medium and 0.6 cuNK-3 was added.
Further, before step B1, the method further comprises a coating process for the culture bottle, wherein the coating process comprises:
b01, adding CUNK-1 and DPBS into a culture bottle, shaking and uniformly mixing to obtain a coating solution, and standing in a thermostat at 37 +/-1 ℃;
b02, removing the supernatant of the coating liquid in the culture flask to obtain the culture flask with the coating.
Further, in step B01, the number of CUNK-1 is 1, and the DPBS is 13 mL.
Further, in step B01, the standing time is at least 2 hours.
Further, in the step B2, the temperature of the culture environment of the constant temperature incubator is 37. + -. 1 ℃ and the humidity is 5.0. + -. 0.2%.
Further, in step A2, 25mL of resuscitating fluid containing 5% human albumin was injected into the cryopreserved bag containing umbilical cord blood for 30. + -. 1 seconds.
Further, the step A59 is followed by counting the desired cell suspension.
The invention has the beneficial effects that: the lymphocyte is obtained by recovering and purifying the cryopreserved umbilical cord blood, and then the NK cells in the lymphocyte are selectively stimulated and amplified, so that the application direction of the cryopreserved umbilical cord blood is expanded, the activity of the purified lymphocyte is high, the cells are not required to be sorted, trophoblast cells are not required to be used, plasma is not required to be used, the operation complexity is reduced, the cost is reduced, the number of the amplified NK cells is large, the purity is high, and the clinical application requirements are met.
Drawings
The specific effects of the invention are detailed below with reference to the accompanying drawings:
FIG. 1 shows the results of expression of surface markers of cells obtained by the resuscitation protocol of the present invention;
FIG. 2 shows the expression of surface markers of cells after proliferation according to the amplification protocol of the present invention;
FIG. 3 is a graphic representation of NK cell proliferation of cord blood during the whole culturing process of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more features. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be connected or detachably connected or integrated; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, "above" or "below" a first feature means that the first and second features are in direct contact, or that the first and second features are not in direct contact but are in contact with each other via another feature therebetween. Also, the first feature being "on," "above" and "over" the second feature includes the first feature being directly on and obliquely above the second feature, or merely indicating that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature includes the first feature being directly under and obliquely below the second feature, or simply meaning that the first feature is at a lesser elevation than the second feature.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Examples
A method for amplifying NK cells from stored umbilical cord blood comprises a resuscitation process and an amplification process,
the resuscitation process comprises the following steps:
a1, performing resuscitation treatment on the cryopreserved bag storing the umbilical cord blood;
the volume of the cryopreservation bag storing the umbilical cord blood is 25mL, and the cryopreservation bag is transferred to an operation area by using a dry ice transport box. Setting the electric heating constant-temperature water bath kettle to be 37 +/-1 ℃, after the temperature is stable, putting the frozen bag into the water bath kettle for resuscitation for 4min to completely dissolve the frozen umbilical cord blood, taking the frozen bag out of the water bath kettle after the dissolution is finished, disinfecting the frozen bag by using 75% alcohol, and putting the sterilized bag into a biological safety cabinet.
A2, slowly injecting resuscitation solution of 5% -10% human serum albumin into a cryopreservation bag and standing to obtain cell suspension;
and taking out a 50mL syringe, slowly injecting 25mL resuscitation solution containing 5% human albumin into the cryopreservation bag containing the umbilical cord blood for 30 +/-1 seconds, and standing the cryopreservation bag.
A3, transferring the cell suspension into a first centrifuge tube;
after standing for 5min, the cell suspension was completely aspirated by syringe and transferred to a 250mL first centrifuge tube.
A4, injecting a 0.9% sodium chloride injection into a freezing storage bag for washing, and transferring all liquid in the freezing storage bag into a first centrifuge tube;
in order to collect as many cells as possible from the cord blood, 50mL of 0.9% sodium chloride injection was again drawn by syringe and injected into the cryopreserved bag for washing, and after washing was completed, the entire liquid was drawn and transferred to the first centrifuge tube.
A5, centrifuging the first centrifuge tube to obtain the required cell suspension;
the centrifugation treatment specifically comprises:
a51, carrying out first centrifugation treatment on a first centrifuge tube, wherein the first centrifugation treatment parameter is centrifugation for 15min under 400g of centrifugal force, and the acceleration and deceleration of the centrifuge are the slowest gears;
a52, removing supernatant in the first centrifugal tube, and carrying out primary dilution on the first centrifugal tube to obtain diluted mixed solution;
and after the first centrifugation treatment is finished, removing the supernatant in the first centrifuge tube, carrying out first dilution on the first centrifuge tube by using 0.9% sodium chloride injection, and diluting the precipitate to 60mL so as to obtain a diluted mixed solution.
A53, transferring the diluted mixed solution into a second centrifuge tube, wherein the second centrifuge tube contains Ficoll (polysucrose);
the diluted mixture was layered into a second centrifuge tube containing 15mL of Ficoll (Ficoll).
A54, performing secondary centrifugation treatment on a second centrifuge tube, wherein the parameters of the secondary centrifugation treatment are centrifugation for 30min under the centrifugal force of 400g, and the acceleration and deceleration of the centrifuge are the slowest gears;
a55, sucking the tunica albuginea layer to a third centrifuge tube, and performing secondary dilution on the third centrifuge tube;
and after the second centrifugation treatment is finished, sucking the white membrane layer in the second centrifuge tube to a third centrifuge tube, performing second dilution on the third centrifuge tube by using 0.9% sodium chloride injection, and diluting the precipitate to 45 mL.
A56, carrying out third centrifugation treatment on a third centrifugal tube, wherein the third centrifugation treatment parameter is centrifugation for 10min under the centrifugal force of 400g, and the acceleration and deceleration of the centrifuge are the slowest gear;
a57, removing the supernatant, and diluting a third centrifuge tube in a constant volume manner;
after the third centrifugation, the supernatant in the third centrifuge tube was removed, the third centrifuge tube was diluted with 0.9% sodium chloride injection to a third dilution, and the precipitate was diluted to 45 mL.
A58, performing fourth centrifugation treatment on a third centrifuge tube, wherein the fourth centrifugation treatment parameter is centrifugation for 10min under 200g of centrifugal force, and the acceleration and deceleration of the centrifuge are the slowest gear;
and A59, removing the supernatant, and carrying out heavy suspension and uniform mixing by using NK medium to obtain the required cell suspension.
And after the fourth centrifugation treatment is finished, removing the supernatant in the third centrifuge tube, and carrying out heavy suspension and uniform mixing by using 20mL of NK medium to obtain the required cell suspension.
Further comprising the step of counting the desired cell suspension to a total WBC cell count of 4.8X 107And taking out 0.5mL of required cell suspension to detect the cell surface marker expression and the cell survival rate, wherein the cell surface marker expression result is shown in figure 1, and the survival rate is 93%.
The amplification process comprises the following steps:
before step B1, the method further comprises a coating process of the culture bottle, wherein the coating process comprises the following steps:
b01, adding 1 CUNK-1 and 13mL of DPBS into a culture bottle, shaking left and right for 3-5 times, mixing uniformly to obtain a coating solution, placing the culture bottle in a thermostat at 37 +/-1 ℃ and standing for at least 2 hours;
b02, removing the supernatant of the coating liquid in the culture flask to obtain the culture flask with the coating.
B1, adding the required cell suspension obtained in the recovery step and the mixed solution of CUNK-2 and the serum substitute into a culture flask;
adding 20mL of required cell suspension obtained in the recovery step into a culture flask with the coating completed, and adding 0.25 CUNK-2 and 3mL of serum substitute for culture.
And B2, placing the culture bottle in a constant-temperature incubator for static culture, and supplementing liquid every 2-3 days in the culture process, wherein the temperature of the culture environment of the constant-temperature incubator is 37 +/-1 ℃, and the humidity is 5.0 +/-0.2%.
The fluid infusion specifically comprises:
b21, on the 4 th day of culture, adding 20mL of NK culture medium, 0.25 branch of CUNK-2 and 3mL of mixed solution of serum substitute, and continuously standing and culturing in a constant-temperature incubator with the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%;
b22, on the 6 th day of culture, adding a mixed solution of 40mL of NK culture medium, 0.5 CUNK-2 and 6mL of serum substitute, and continuing to perform standing culture in a constant-temperature incubator at the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%;
b23, culturing for 8 days, transferring the culture suspension in the culture bottle into a cell culture bag, adding 100mL of mixed solution of NK culture medium and 0.1 count of CUNK-3, and continuously standing and culturing in a constant-temperature incubator with the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%;
b24, on the 10 th day of culture, adding 200mL of mixed solution of NK medium and 0.2 branch of CUNK-3, and continuously standing and culturing in a constant-temperature incubator with the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%;
b25, on the 12 th day of culture, adding 400mL of mixed solution of NK medium and 0.4 branch of CUNK-3, and continuously standing and culturing in a constant-temperature incubator with the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%;
b26, on the 14 th day of culture, adding 600mL of mixed solution of NK medium and 0.6 branch of CUNK-3, and continuing to perform standing culture in a constant-temperature incubator at the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%;
b27, on the 16 th day of culture, adding 600mL of a mixed solution of NK medium and 0.6 branch of CUNK-3, and continuing to perform standing culture in a constant-temperature incubator at the temperature of 37 +/-1 ℃ and the humidity of 5.0 +/-0.2%.
Culturing till the day 19, finishing culturing to obtain the proliferated cell suspension, taking out 0.5mL of the proliferated cell suspension to detect the cell surface marker expression, and obtaining the result as shown in figure 2, wherein the purity of the amplified umbilical cord blood NK cells reaches 90.49%.
The proliferation of NK cells in cord blood during the whole culture process is shown in FIG. 3, and the number of the expanded NK cells reaches 4.66X 109And meets the requirement of clinical application.
In the above, the reagents used were purchased from the local union laboratories of the national individualized cell therapy technology, including NK medium, CUNK-1, CUNK-2 and CUNK-3, serum replacement was purchased from Gibco, DPBS was purchased from tianjin tertiary sea biologicals ltd, and other consumables were purchased from Thermo.
From the above description, the beneficial effects of the present invention are: the lymphocyte is obtained by recovering and purifying the cryopreserved umbilical cord blood, and then the NK cells in the lymphocyte are selectively stimulated and amplified, so that the application direction of the cryopreserved umbilical cord blood is expanded, the activity of the purified lymphocyte is high, the cells are not required to be sorted, trophoblast cells are not required to be used, plasma is not required to be used, the operation complexity is reduced, the cost is reduced, the number of the amplified NK cells is large, the purity is high, and the clinical application requirements are met.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method of expanding NK cells from stored cord blood, comprising: comprises a recovery process and an amplification process,
the resuscitation process comprises the following steps:
a1, performing resuscitation treatment on the cryopreserved bag storing the umbilical cord blood;
a2, slowly injecting resuscitation solution of 5% -10% human serum albumin into a cryopreservation bag and standing to obtain cell suspension;
a3, transferring the cell suspension into a first centrifuge tube;
a4, injecting a 0.9% sodium chloride injection into a freezing storage bag for washing, and transferring all liquid in the freezing storage bag into a first centrifuge tube;
a5, centrifuging the first centrifuge tube to obtain the required cell suspension;
the amplification process comprises the following steps:
b1, adding the required cell suspension obtained in the recovery step and the mixed solution of CUNK-2 and the serum substitute into a culture flask;
and B2, placing the culture bottle in a constant-temperature incubator for static culture, and supplementing liquid every 2-3 days in the culture process.
2. The method of expanding NK cells from stored cord blood according to claim 1, wherein: the centrifugation treatment comprises:
a51, performing primary centrifugation treatment on the first centrifuge tube;
a52, removing supernatant in the first centrifugal tube, and carrying out primary dilution on the first centrifugal tube to obtain diluted mixed solution;
a53, transferring the diluted mixed solution into a second centrifugal tube, wherein the second centrifugal tube contains Ficol l;
a54, performing secondary centrifugal treatment on the second centrifugal tube;
a55, sucking the tunica albuginea layer to a third centrifuge tube, and performing secondary dilution on the third centrifuge tube;
a56, carrying out third centrifugation treatment on a third centrifuge tube;
a57, removing the supernatant, and diluting a third centrifuge tube in a constant volume manner;
a58, performing fourth centrifugation treatment on the third centrifuge tube;
and A59, removing the supernatant, and carrying out heavy suspension and uniform mixing by using NK medium to obtain the required cell suspension.
3. The method of claim 2 for expanding NK cells from stored cord blood, wherein: the parameter of the first centrifugal treatment is centrifugation for 15min under the centrifugal force of 400 g;
the parameter of the second centrifugal treatment is centrifugation for 30min under the centrifugal force of 400 g;
the parameter of the third centrifugal treatment is centrifugation for 10min under the centrifugal force of 400 g;
the parameter of the fourth centrifugal treatment is centrifugation for 10min under the centrifugal force of 200 g.
4. The method of claim 3 for expanding NK cells from stored cord blood, wherein: in step B2, the fluid infusion specifically includes:
b21, on the 4 th day of culture, adding a mixed solution of 20mL of NK medium, 0.25 cuNK-2 and 3mL of serum substitute;
b22, on the 6 th day of culture, adding a mixed solution of 40mL of NK medium, 0.5 cuNK-2 and 6mL of serum substitute;
b23, culturing for 8 days, transferring the culture suspension in the culture bottle into a cell culture bag, and adding 100mL of mixed solution of NK culture medium and 0.1 count of CUNK-3;
b24, on the 10 th day of culture, adding 200mL of mixed solution of NK medium and 0.2 branch of CUNK-3;
b25, on the 12 th day of culture, adding 400mL of mixed solution of NK medium and 0.4 branch of CUNK-3;
b26, on the 14 th day of culture, adding 600mL of mixed solution of NK medium and 0.6 branch of CUNK-3;
b27, on day 16 of culture, 600mL of a mixture of NK medium and 0.6 cuNK-3 was added.
5. The method of claim 4 for expanding NK cells from stored cord blood, wherein: before step B1, the method further comprises a coating process of the culture bottle, wherein the coating process comprises the following steps:
b01, adding CUNK-1 and DPBS into a culture bottle, shaking and uniformly mixing to obtain a coating solution, and standing in a thermostat at 37 +/-1 ℃;
b02, removing the supernatant of the coating liquid in the culture flask to obtain the culture flask with the coating.
6. The method of claim 5 for expanding NK cells from stored cord blood, wherein: in step B01, the number of CUNK-1 is 1, and the DPBS is 13 mL.
7. The method of claim 6 for expanding NK cells from stored cord blood, wherein: in step B01, the standing time is at least 2 hours.
8. The method of claim 7 for expanding NK cells from stored cord blood, wherein: in the step B2, the temperature of the culture environment of the constant temperature incubator is 37 +/-1 ℃ and the humidity is 5.0 +/-0.2%.
9. The method of claim 8 for expanding NK cells from stored cord blood, wherein: in step A2, 25mL of resuscitating fluid containing 5% human albumin was injected into the cord blood-containing cryopreserved bag for 30. + -.1 seconds.
10. The method of claim 9 for expanding NK cells from stored cord blood, wherein: the counting of the desired cell suspension is also included after step a 59.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111638979.1A CN114058578B (en) | 2021-12-29 | 2021-12-29 | Method for amplifying NK cells from stored umbilical cord blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111638979.1A CN114058578B (en) | 2021-12-29 | 2021-12-29 | Method for amplifying NK cells from stored umbilical cord blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114058578A true CN114058578A (en) | 2022-02-18 |
| CN114058578B CN114058578B (en) | 2023-12-12 |
Family
ID=80230462
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111638979.1A Active CN114058578B (en) | 2021-12-29 | 2021-12-29 | Method for amplifying NK cells from stored umbilical cord blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114058578B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261323A (en) * | 2022-08-04 | 2022-11-01 | 河南省遗传资源细胞库有限公司 | Method for removing bacteria in umbilical cord blood cells |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120316079A1 (en) * | 2011-06-13 | 2012-12-13 | Indevr, Inc. | Low Density Microarrays for Vaccine Related Protein Quanitification, Potency Determination and Efficacy Evaluation |
| CN110144323A (en) * | 2019-05-29 | 2019-08-20 | 江苏省北科生物科技有限公司 | A kind of settle and separate method of peripheral blood immunocyte |
| CN112501117A (en) * | 2020-12-04 | 2021-03-16 | 深圳市北科生物科技有限公司 | Recovery method of umbilical cord blood mononuclear cells frozen at low temperature |
| CN113430168A (en) * | 2021-08-12 | 2021-09-24 | 山东省齐鲁干细胞工程有限公司 | Method for culturing cord blood NK cells in serum-free manner and kit thereof |
| CN114891744A (en) * | 2022-07-13 | 2022-08-12 | 山东省齐鲁干细胞工程有限公司 | Freezing umbilical cord blood NK cell in-vitro amplification method |
-
2021
- 2021-12-29 CN CN202111638979.1A patent/CN114058578B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120316079A1 (en) * | 2011-06-13 | 2012-12-13 | Indevr, Inc. | Low Density Microarrays for Vaccine Related Protein Quanitification, Potency Determination and Efficacy Evaluation |
| CN110144323A (en) * | 2019-05-29 | 2019-08-20 | 江苏省北科生物科技有限公司 | A kind of settle and separate method of peripheral blood immunocyte |
| CN112501117A (en) * | 2020-12-04 | 2021-03-16 | 深圳市北科生物科技有限公司 | Recovery method of umbilical cord blood mononuclear cells frozen at low temperature |
| CN113430168A (en) * | 2021-08-12 | 2021-09-24 | 山东省齐鲁干细胞工程有限公司 | Method for culturing cord blood NK cells in serum-free manner and kit thereof |
| CN114891744A (en) * | 2022-07-13 | 2022-08-12 | 山东省齐鲁干细胞工程有限公司 | Freezing umbilical cord blood NK cell in-vitro amplification method |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115261323A (en) * | 2022-08-04 | 2022-11-01 | 河南省遗传资源细胞库有限公司 | Method for removing bacteria in umbilical cord blood cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114058578B (en) | 2023-12-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4294316B2 (en) | Method for collecting placental stem cells | |
| US11965175B2 (en) | Cell expansion | |
| CN104726405A (en) | Separation and database establishing method for cord blood hematopoietic stem cells | |
| US20200385665A1 (en) | Fluid system for producing extracellular vesicles and associated method | |
| CN112251406A (en) | Exosome sorting method for NK cell activation stage | |
| CN114058578A (en) | Method for amplifying NK cells from stored umbilical cord blood | |
| CN110982779B (en) | Method for improving utilization rate of umbilical cord blood | |
| CN111518763B (en) | Method for separating high-purity umbilical cord stem cells | |
| CN110857435B (en) | Culture medium for culturing immune cells separated from cord blood and culture method thereof | |
| Campbell et al. | Isolation and generation of clinical-grade dendritic cells using the CliniMACS system | |
| CN104195107B (en) | Purposes of the microcapsule bubble in induction stem cell macronucleus differentiation | |
| CN114891744B (en) | Freezing umbilical cord blood NK cell in-vitro amplification method | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN214654995U (en) | Cell autosegregation instrument | |
| CN112300992B (en) | NK cell culture solution and multistage activated NK cell culture method | |
| CN108103018B (en) | Method for enriching human blood MNCs | |
| CN116162659B (en) | In vitro gene expression intervention method of primary MDSC | |
| CN219930047U (en) | Preparation system of chimeric antigen receptor cells | |
| CN113475500B (en) | Full-medicine-grade raw material tumor tissue cryopreservation liquid and application thereof | |
| TWI780516B (en) | Cytotoxic t cell culture method | |
| Dannull et al. | Transfecting Human Monocytes with RNA | |
| CN116515736A (en) | Umbilical cord blood-derived endothelial progenitor cells and culture method thereof | |
| Svensson | Isolation and culture of human hematopoietic progenitors for studies of dendritic cell biology | |
| CN116814371A (en) | Preparation system and method of chimeric antigen receptor cells | |
| CN115651893A (en) | Construction method and application of rainbow trout ovary granular cell line |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |