CN113046314B - Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells - Google Patents
Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells Download PDFInfo
- Publication number
- CN113046314B CN113046314B CN202110341346.8A CN202110341346A CN113046314B CN 113046314 B CN113046314 B CN 113046314B CN 202110341346 A CN202110341346 A CN 202110341346A CN 113046314 B CN113046314 B CN 113046314B
- Authority
- CN
- China
- Prior art keywords
- combination
- cells
- hematopoietic stem
- iii
- decidua
- 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.)
- Active
Links
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
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
-
- 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
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- 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
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- 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
- C12N2501/26—Flt-3 ligand (CD135L, flk-2 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
Abstract
The application discloses the in vitro induction and amplification of decidua-like autologous hematopoietic stem cellsA method of killing a cell comprising: will CD34 + Four-stage culture of hematopoietic stem cells in vitro, wherein stage I is CD34 + Expansion of hematopoietic Stem cells, stage II Induction of CD34 + Differentiation of hematopoietic stem cells into NK cell precursors, stage III for inducing NK cell precursors into NK cells, stage IV for inducing NK cell expansion and maturation, and collecting CD3 ‑ CD56 + NK cells, thereby obtaining decidua-like natural killer cells, wherein the culturing is started with fresh basal medium containing cytokine combination I, half-liquid change culture is performed at stages I, II, III and IV of culturing using fresh basal medium containing cytokine combination I, combination II, combination III and combination IV, respectively, wherein cytokine combination I is Flt3L and SCF; combination II was Flt3L, SCF and IL-15; combination III was Flt3L, SCF and IL-15; combination IV is IL-15.
Description
Technical Field
The application belongs to the technical field of biomedicine, and particularly relates to a Cord Blood (CB) or autologous Bone Marrow (BM) derived CD34 + Method for inducing and expanding Hematopoietic Stem Cells (HSC) in vitro to obtain decidua-like Natural Killer (NK) cells.
Background
The fetus is used as a semi-homograft of a mother, and the rejection reaction of the embryo capable of avoiding the immune system of the mother in the process of pregnancy is a precondition for the establishment of pregnancy. With the invasion of trophoblast cells, the molting of endometrium is gradually completed, and a maternal-fetal interface of maternal and fetal interaction is formed. In the early pregnancy period, the immune cells account for about 30% of decidual tissue of the maternal-fetal interface, wherein decidual natural killer (dNK) cells exist in large quantity, and the proportion is as high as 70%.
NK cells are used as immune cells of a maternal-fetal interface main body and play an important role in physiology. dNK cells have a unique phenotype and function, i.e., they are characterized by a phenotype of peripheral blood NK (pNK) cellsCD56 bright CD16 - And high expression Tissue resident NK cell (trNK) marker molecule CD49a, and has the characteristics of strong secretion function, low killing property and the like. The NK cells on the maternal-fetal interface participate in inducing immune tolerance, can promote the invasion of trophoblast cells, participate in the process of reconstructing spiral arteries, promote the development of placenta and fetus, and the like. Abnormal decidua NK cell activity of maternal-fetal interface can cause pathological pregnancy.
Decidua NK cells have limited sources and numbers, limiting their clinical use. At present, a method for obtaining decidua NK cells in large quantity does not exist. On the one hand, decidua NK cells isolated from decidua tissue of early pregnancy are fewer in number. On the other hand, cord blood and bone marrow are important sources of HSC, under certain conditions, the cord blood and bone marrow can be differentiated into NK cells in vivo and in vitro, and no report is reported to completely explain the developmental and differentiation pathway of decidua NK cells, and no method for inducing and amplifying decidua NK cells in vitro in large quantities by hematopoietic stem cells is provided. The decidua-like NK cells induced and amplified are applied to clinic, and a new method for thinking and a new method for treating pregnancy diseases such as recurrent abortion can be provided.
Disclosure of Invention
The application provides a method for inducing and amplifying hematopoietic stem cells from cord blood or autologous bone marrow into decidua-like NK cells in vitro so as to solve the problems that the decidua NK cells in the prior art are insufficient in source and difficult to industrially produce. Specifically, the present application provides the following technical solutions.
1. A method for inducing and expanding decidua-like natural killer cells in vitro from hematopoietic stem cells, comprising:
will CD34 + Hematopoietic stem cells are cultured in vitro in four stages, wherein stage I is CD34 + Expansion of hematopoietic Stem cells, stage II Induction of CD34 + Differentiation of hematopoietic stem cells into NK cell precursors, induction of differentiation of NK cell precursors into NK cells in stage III, induction of NK cell expansion and maturation in stage IV,
collection of CD3 - CD56 + NK cells, thereby obtaining decidua-like natural killer cells,
wherein the culturing is started with fresh basal medium containing cytokine combination I, and half-replenisher culturing is performed at stages I, II, III and IV of culturing using fresh basal medium containing cytokine combination I, combination II, combination III and combination IV, respectively, wherein the cytokine combination I is Flt3L and SCF; combination II was Flt3L, SCF and IL-15; combination III was Flt3L, SCF and IL-15; the combination IV is IL-15.
2. The method of clause 1, wherein the basal medium is SCGM medium supplemented with fetal bovine serum, e.g., 10% fetal bovine serum.
3. The method of clauses 1 or 2, wherein the respective cytokine concentrations in the fresh basal medium containing cytokine combinations I, II, III and IV are combination I:30ng/ml Flt3L,20ng/ml SCF; combination II:30-50ng/ml Flt3L,20-40ng/ml SCF, and 20-40ng/ml IL-15; combination III:10-30ng/ml Flt3L,5-15ng/ml SCF,30-50ng/ml IL-15; and combination IV: IL-15 at 30-50 ng/ml.
4. The method of any of clauses 1-3, wherein phase I is on days 0-3, phase II is on days 4-14, phase III is on days 15-20, and phase IV is on days 21-35.
5. The method of any one of items 1 to 4, wherein the culturing is effected at a constant temperature of 37 ℃,5% CO 2 Culturing under the conditions of relative saturation humidity of 95% and pH value of 7.2-7.4 of cell culture medium.
6. The method of item 1, wherein the CD34 + Hematopoietic stem cells are derived from cord blood or bone marrow.
7. An decidua-like natural killer cell obtained by the method of any one of items 1 to 6.
8. Use of hematopoietic stem cells, preferably CD34, in the preparation of decidua-like natural killer cells + Hematopoietic stem cells.
9. A kit for the in vitro induced expansion of decidua-like natural killer cells from hematopoietic stem cells comprising cytokine combination I, combination II, combination III and combination IV, wherein the cytokine combination I is Flt3L and SCF; combination II was Flt3L, SCF and IL-15; combination III was Flt3L, SCF and IL-15; combination IV is IL-15, and instructions for use.
The invention uses cord blood and marrow-derived hematopoietic stem cells with higher amplification and differentiation potential, so that the method can obtain a large amount of decidua-like NK cells in vitro. The invention finely regulates and controls the HSC differentiation process, uses the differentiation cell factor with definite composition and stable quality, realizes stable and efficient differentiation, and finally cultures CD3 in a system - CD56 + The proportion of NK cells can reach more than 90 percent. The culture system has no feeder cells, reduces the pollution of exogenous cells, solves the problem of source of decidua NK cells for drug research and cell therapy, and has wide application prospect and value.
Drawings
FIG. 1 shows the detection of CD34 by flow cytometry + HSC sort purity results. The left panel shows a representative flow cytometry assay with CD34 circled + The HSC of (1). The right panel shows the statistical results. As shown in the right figure, CD34 + The sorting purity of the HSC reaches more than 95 percent.
FIG. 2 is a flow chart of the method for inducing and amplifying decidua-like NK cells.
FIG. 3 shows the proportion of NK cells in the induction end-point culture system. The left panel shows a representative flow cytometry assay with CD3 circled - CD56 + The NK cell of (1). The right panel shows the statistical results. It can be seen that CD3 - CD56 + The proportion of NK cells reaches more than 90 percent.
Figure 4 shows the decidua-like NK cell phenotypic identification. The left panel shows a representative diagram of the detection results of flow cytometry, and the expression conditions of pNK, dNK and idNK surface molecules CD49a, CD151, CD39, CD9 and CD16 are analyzed respectively. The right panel shows the statistical results. And (4) conclusion: the expression conditions of idNK surface molecules CD49a, CD151, CD39, CD9 and CD16 are similar to those of dNK and have obvious difference with pNK.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments. It should be noted that the specific embodiments are only illustrative of the present invention and should not be construed as limiting the present invention. Unless otherwise defined, terms herein have the meanings commonly understood by those skilled in the art.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: isolation of cord blood (or bone marrow) mononuclear cells
Cord blood and bone marrow samples were obtained from healthy blood donors at the first hospital affiliated with the university of science and technology in china. Collecting fresh human umbilical cord blood (or bone marrow) in an anticoagulation tube according to the following ratio: mononuclear cells were isolated after dilution with sterile 1 × PBS =1 ratio. The sample was aspirated by a 10ml pipette and extended to a position 0.5cm above the liquid surface of a human peripheral blood lymphocyte separation medium (Tianjin-bearing ocean company, cat. LTS1077, hereinafter referred to as Ficoll), and the sample was naturally dropped and spread on the Ficoll liquid surface, and then the whole sample was gently applied to the Ficoll. 600g, centrifuge for 25 min at 20 ℃. After the centrifugation is completed, obvious layering appears in the centrifuging tube, and from bottom to top respectively: red blood cell layer, ficoll layer, mononuclear cell layer (i.e., leukocyte layer) and plasma layer. The leukocyte membrane layer was aspirated, transferred to a new centrifuge tube, diluted with sterile 1 × PBS (PBS to cell suspension volume ratio should be greater than 1), and mixed well. 600g, centrifuge at 4 ℃ for 10 minutes and discard the supernatant. Cells were resuspended using sterile 1 × PBS, mixed well and counted. The cell suspension is centrifuged at 300g and 4 ℃ for 10 minutes, the supernatant is discarded, and the cell sediment is the cord blood (or bone marrow) mononuclear cells.
Example 2: CD34 + HSC isolation and purification
CD34 magnetic bead sorting kit (Miltenyi Co., ltd., cat # 130-046-702) was used for CD34 + The sorting of HSC includes the following steps. Based on the counting results, the cell pellet obtained in example 1 was resuspended every 1X 10 using MACS buffer (see sorting kit instructions for the preparation method) 7 Cells were resuspended by adding 30. Mu.l of MACS buffer to each cell. Each 1 × 10 7 For each cell, 10. Mu.l of FcR Blocking Reagent was added and mixed well. Next, every 1 × 10 7 Add 10. Mu.l of CD34Micro Beads to each cell and mix well. The mixture is placed in a refrigerator at the temperature of 4 ℃,incubate for 30 minutes. After the incubation was completed, 10ml of MACS buffer was added and mixed well, and then centrifuged at 300g and 4 ℃ for 10 minutes. The supernatant was discarded and the cells were resuspended using 2ml of MACS buffer. The MS separation column (Miltenyi, inc., cat # 130-042-201) was placed in a magnet, 500. Mu.l of MACS buffer was added to rinse the column, and the cell suspension was added to the separation column. Immediately after the cell suspension had drained off, 1.5ml of MACS buffer was added to the column in three portions. And (3) when the liquid is about to drain, removing the separation column from the magnetic field, adding 1.5ml of MACS buffer solution, filling a plug, lightly beating the liquid out to obtain a target cell suspension, supplementing the total volume to 10ml by using sterile 1 XPBS, counting the cells, taking part of the cells, detecting the sorting purity by using flow cytometry (see example 3), and culturing the rest cells.
Example 3: flow cytometry for CD34 detection + HSC sorting purity
Collecting cell suspension in 1.5ml EP tube, adding 1 × PBS to volume of 1ml, mixing, centrifuging at 500g,4 deg.C for 5 min, and discarding supernatant; 100. Mu.l of 1 XPBS (phosphate buffered saline) resuspended cells were added to each tube, 2. Mu.l of a fluorescein-conjugated flow antibody CD34-PE (BD Co., ltd.; cat. No.: 555822) was added thereto, and after mixing, the cells were incubated in a refrigerator at 4 ℃ for 30 minutes; adding 1ml of 1 XPBS into a 1.5ml EP tube, mixing uniformly, centrifuging at 500g and 4 ℃ for 5 minutes, removing supernatant, and adding 200 mu l of 1 XPBS heavy suspension cells into each tube; the cell suspension was transferred to a flow tube through a 200 mesh nylon mesh and examined using a multi-laser flow cytometer (BD corporation). The experimental results were analyzed using FlowJo _ V10 software. And (3) displaying a detection result: among the cells obtained by sorting, CD34 + The cell ratio of (A) is more than 95%, and the analysis and statistical results are shown in figure 1.
Example 4: NK cell induced expansion
The cell suspension obtained in example 2 was centrifuged at 300g at 4 ℃ for 10 minutes, and after discarding the supernatant, the cell suspension was cultured using a medium according to the counting result. CD34 + HSC culture initial density was 1X 10 per 1ml medium 6 And (4) cells. The basic medium used was SCGM medium (CellGenix, cat # 20802-0500) supplemented with 10% fetal bovine serum (Gibco, cat # 10091-148). The cells were co-cultured for 35 days,the culture conditions were: constant temperature of 37 ℃ and CO 2 The concentration is 5%, the relative saturation humidity is 95%, and the pH value of the cell culture medium is 7.2-7.4. The culture process is divided into four stages, and the flow chart is shown in figure 2: stage I is the 0-3 days of culture, and is mainly for CD34 + Expanding the HSC; stage II, which is about day 4-14 of culture, mainly induces differentiation of HSCs to NK cell precursors; stage III, which is about day 15-20 of culture, mainly induces NK cell precursors to differentiate into NK cells; stage IV, which is about day 21-35 of culture, induces NK cell expansion and maturation. On day 0 of culture, culture was carried out using a basal medium containing cytokine combination 1 (30 ng/ml of Flt3L (PeproTech Co., ltd., cat # AF-300-19), 20ng/ml of SCF (PeproTech Co., cat # AF-300-07), half-volume fluid changes were carried out every week for 2 times during the culture, and at the stages I, II, III and IV of culture, media used for fluid changes were fresh basal media containing cytokine combination I (30 ng/ml of SCF, 20ng/ml of SCF), combination II (30-50 ng/ml of Flt3L,20-40ng/ml of SCF, and 20-40ng/ml of IL-15), combination III (10-30 ng/ml of Flt3L,5-15ng/ml of SCF,30-50ng/ml of IL-15) and combination IV (30-50 ng/ml of IL-15), respectively.
Example 5: isolation of peripheral blood mononuclear cells
Fresh peripheral blood samples were obtained from healthy blood donors in the province of blood center of Anhui province. Peripheral blood of volunteer donors is collected in an anticoagulation tube, according to the peripheral blood: mononuclear cells were isolated after dilution with sterile 1 × PBS =1 ratio. All samples were gently applied to Ficoll. 600g, centrifuge for 25 min at 20 ℃. After the centrifugation is completed, obvious layering appears in the centrifuging tube, and from bottom to top respectively: red blood cell layer, ficoll layer, mononuclear cell layer (i.e., leukocyte layer) and plasma layer. And (3) sucking the leucocyte layer, transferring the leucocyte layer into a new centrifuge tube, diluting the leucocyte layer by using 1 XPBS, and uniformly mixing the leucocyte layer and the cell suspension in a volume ratio of more than 1. 600g, centrifuge at 4 ℃ for 10 minutes and discard the supernatant. Resuspending the cells with 1 × PBS, and mixing to obtain a cell suspension, i.e., peripheral blood mononuclear cells.
Example 6: isolation of decidua tissue mononuclear cells
Fresh decidua tissue samples were taken from the affiliated first hospital of Chinese science and technology university for voluntary termination of pregnancyHealthy donors and informed consent was obtained prior to surgery. Placing collected decidua tissue into cell culture dish, cleaning with 1 × PBS, removing specimen with blood clot, discarding liquid, and cutting tissue to 1mm with scissors 3 The size was determined by adding the mixture to a 50ml centrifuge tube, adding 10ml of collagenase IV (Sigma; cat # C-5138) at a concentration of 2mg/ml, and digesting the mixture at 37 ℃ for 40 to 50 minutes on a shaker at 180 rpm. The digested tissue suspension was filtered using a 70 μm filter to remove undigested tissue mass, the cell suspension was collected into a new 50ml centrifuge tube, 1 × PBS was added to 50ml, after thoroughly mixing by inversion, 300g was centrifuged at 4 ℃ for 10 min, the supernatant was discarded, and the cell pellet was resuspended and mixed using 25ml1 × PBS. The cell suspension was slowly added to the Ficoll. 600g, centrifuge for 25 min at 20 ℃. After the centrifugation is completed, obvious layering appears in the centrifuge tube, and the centrifuge tube is respectively from bottom to top: red blood cell layer, ficoll layer, mononuclear cell layer (i.e., leukocyte layer) and plasma layer. The leukocyte membrane layer was aspirated, transferred to a new centrifuge tube, diluted with 1 × PBS (volume ratio to cell suspension should be greater than 1), and mixed well. 600g, centrifuge at 4 ℃ for 10 minutes and discard the supernatant. Resuspending the cells by using 1 XPBS, and mixing uniformly to obtain the cell suspension, namely the decidua mononuclear cells.
Example 7: decidua-like NK cell identification and comparison
The cells cultured up to day 35 in example 4, the peripheral blood mononuclear cells in example 5, and the decidua tissue mononuclear cells in example 6 were obtained, respectively. The cells were labeled with fluorescein-conjugated monoclonal flow antibodies CD45-APCCy7 (BD, cat # 557833), CD3-BV605 (Biolegged, cat # 344836), CD56-BV421 (Biolegged, cat # 318327), CD49a-Alexa Fluor647 (Biolegged, cat # 328310), CD9-PE (BD, cat # 555372), CD151-PE (BD, cat # 556057), CD39-PECy7 (Biolegged, cat # 328211), CD16-PerCP5.5 (Biolegged, cat # 302028), respectively. Detection was performed using a multi-laser flow cytometer (BD). The results of the experiment were analyzed using FlowJo _ V10 software. And detecting and analyzing the proportion of NK cells in the induction end-point culture system, analyzing and counting, wherein the result is shown in figure 3. Can find CD3 in the induction system - CD56 + The proportion of NK cells can reach more than 90 percent. Detecting and identifying phenotype of decidua-like NK cells, and analyzing and counting the result as shown in FIG. 4, first, the peripheral blood mononuclear cells, decidua mononuclear cells and CD45 in the induction system are circled + CD3 - CD56 + The induced decidua-like NK cells (idNK) and decidua NK cells (dNK) have higher similarity and are obviously different from peripheral blood NK cells (pNK) by analyzing the phenotypic characteristics of the NK cells: idNK and dNK both highly express tissue resident molecule CD49a and decidua NK cell characteristic molecules CD151, CD39 and CD9, while pNK hardly expresses these molecules. pNK expresses CD16 at a high level, while dNK expresses little CD16, idNK is similar to dNK with CD16 expression at a significantly lower level than pNK. CD16 is generally considered to be indicative of NK cell killing, so pNK has a strong killing function, whereas dNK and idNK killing functions are weak.
From the results, the invention successfully induces and amplifies decidua-like natural killer cells in vitro from human umbilical blood or bone marrow hematopoietic stem cells, thereby realizing the large-scale preparation of decidua-like NK cells in vitro.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for inducing and expanding decidua-like natural killer cells in vitro from hematopoietic stem cells, comprising:
will CD34 + Hematopoietic stem cells are cultured in vitro in four stages, wherein stage I is CD34 + Expansion of hematopoietic Stem cells, stage II Induction of CD34 + Differentiation of hematopoietic stem cells into NK cell precursors, induction of differentiation of NK cell precursors into NK cells in stage III, induction of NK cell expansion and maturation in stage IV,
collection of CD3 - CD56 + NK cells, thereby obtainingTo decidua-like natural killer cells,
wherein the culturing is initiated with fresh basal medium containing cytokine combination I, half-life broth culturing is performed at stages I, II, III and IV of culturing using fresh basal medium containing cytokine combination I, combination II, combination III and combination IV, respectively, wherein cytokine combination I is Flt3L and SCF; combination II is Flt3L, SCF and IL-15; combination III was Flt3L, SCF and IL-15; the combination IV is IL-15, and,
wherein the cytokine concentrations in the fresh basal medium containing cytokine combinations I, II, III and IV are combination I:30ng/ml Flt3L,20ng/ml SCF; combination II:30-50ng/ml Flt3L,20-40ng/ml SCF, and 20-40ng/ml IL-15; combination III:10-30ng/ml Flt3L,5-15ng/ml SCF,30-50ng/ml IL-15; and combination IV:30-50ng/ml of IL-15,
wherein phase I is on days 0-3, phase II is on days 4-14, phase III is on days 15-20, and phase IV is on days 21-35.
2. The method of claim 1, wherein the basal medium is SCGM medium supplemented with fetal bovine serum.
3. The method of claim 2, wherein the basal medium is SCGM medium supplemented with 10% fetal bovine serum.
4. The method of any one of claims 1-3, wherein the culturing is done at a constant temperature of 37 ℃,5% CO 2 And culturing under the conditions that the relative saturation humidity is 95 percent and the pH value of a cell culture medium is 7.2-7.4.
5. The method of claim 1, wherein the CD34 + Hematopoietic stem cells are derived from cord blood or bone marrow.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110341346.8A CN113046314B (en) | 2021-03-30 | 2021-03-30 | Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110341346.8A CN113046314B (en) | 2021-03-30 | 2021-03-30 | Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113046314A CN113046314A (en) | 2021-06-29 |
| CN113046314B true CN113046314B (en) | 2022-11-25 |
Family
ID=76516528
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110341346.8A Active CN113046314B (en) | 2021-03-30 | 2021-03-30 | Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113046314B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104321425A (en) * | 2012-05-07 | 2015-01-28 | 高丽大学校产学协力团 | Method for inducing and proliferating natural killer cells derived from peripheral blood mononuclear cells |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8518397B2 (en) * | 2009-08-14 | 2013-08-27 | Case Western Reserve University | Notch induced natural killer cell generation and therapeutic uses |
| CN103194428A (en) * | 2013-04-18 | 2013-07-10 | 中国科学技术大学 | Culture method for in vitro differentiating and amplifying human natural killer cells through insulin-like growth factors and various cell factors |
| NZ733213A (en) * | 2014-12-31 | 2022-10-28 | Celularity Inc | Natural killer cells and uses thereof |
| US20170121683A1 (en) * | 2015-11-03 | 2017-05-04 | Regents Of The University Of Minnesota | Aryl hydrocarbon receptor disruption and enhancement |
| CN109777773A (en) * | 2019-02-26 | 2019-05-21 | 北京呈诺医学科技有限公司 | It is a kind of to break up the method and its culture medium for generating NK cell from candidate stem cell |
-
2021
- 2021-03-30 CN CN202110341346.8A patent/CN113046314B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104321425A (en) * | 2012-05-07 | 2015-01-28 | 高丽大学校产学协力团 | Method for inducing and proliferating natural killer cells derived from peripheral blood mononuclear cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113046314A (en) | 2021-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3070865B2 (en) | Subset of human progenitor cells | |
| US5965436A (en) | Method of isolating mesenchymal stem cells associated with isolated megakaryocytes by isolating megakaryocytes | |
| Petroff et al. | Isolation and culture of term human trophoblast cells | |
| Tomer et al. | Purification of human megakaryocytes by fluorescence-activated cell sorting | |
| WO2001079851A1 (en) | NON-INVASIVE PRENATAL DIAGNOSIS USING CD45-ve FETAL CELLS | |
| WO1998020731A9 (en) | Msc-megakaryocyte precursor composition and method of isolating mscs associated with isolated megakaryocytes by isolating megakaryocytes | |
| CN104877965B (en) | A kind of method for preparing mature erythrocyte | |
| Tanaka et al. | Isolation of human megakaryocytes by immunomagnetic beads | |
| JP2996513B2 (en) | Differential expansion of fetal stem cells in maternal circulation for use in prenatal genetic analysis | |
| CN112662631B (en) | CAR-T cell perfusion culture method | |
| CN108486039B (en) | Method for inducing human adipose-derived stem cells to differentiate into testicular interstitial cells by using small molecules | |
| CN113046314B (en) | Method for in vitro induced amplification of decidua-like natural killer cells of human umbilical blood or bone marrow hematopoietic stem cells | |
| CN110055219B (en) | Method for preparing heterogeneous hematopoietic stem and progenitor cells by using non-mobilized peripheral blood | |
| Beaujean | Methods of CD34+ cell separation: comparative analysis | |
| CN113046315B (en) | Method for obtaining decidua-like natural killer cells by in vitro induction of peripheral blood natural killer cells | |
| CN112300992B (en) | NK cell culture solution and multistage activated NK cell culture method | |
| Dunstan | The expression of ABH antigens during in vitro megakaryocyte maturation: origin of heterogeneity of antigen density | |
| CN108949686B (en) | Method for obtaining hematopoietic stem cells from placenta in hypoxic environment | |
| CN113106058A (en) | Screening and identifying method of human umbilical cord-derived Muse cells | |
| CN115896015B (en) | In-vitro culture method of myeloid-derived suppressor cells | |
| CN114107201B (en) | CD3+TCRValpha 7.2+T cell culture medium and expansion culture method | |
| CN113502263B (en) | Differentiation-promoting culture medium and method for promoting differentiation of CD34 positive cells into platelets | |
| CN114574438A (en) | Separation method of human decidua MDSCs | |
| Mohamad et al. | ISOLATION AND CHARACTERIZATION OF RETICULOCYTES DIFFERENTIATED FROM HUMAN PERIPHERAL BLOOD-DERIVED CD34+ HEMATOPOIETIC STEM CELLS AND THE POTENTIAL OF RETICULOCYTE INVASION BY PLASMODIUM FALCIPARUM | |
| CN110628715B (en) | Method for in vitro amplification of natural killer cells and natural killer T cells and pharmaceutical composition thereof |
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 |