CN117070453A - Culture medium for in vitro erythroid differentiation of CD34 positive hematopoietic stem cells of umbilical cord blood - Google Patents
Culture medium for in vitro erythroid differentiation of CD34 positive hematopoietic stem cells of umbilical cord blood Download PDFInfo
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- 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
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Abstract
The invention discloses a culture medium for in-vitro erythroid differentiation of umbilical cord blood CD34 positive hematopoietic stem cells, which comprises a first-stage culture medium and a second-stage culture medium, wherein the first-stage culture medium consists of the following components: SCF, 8-12 ng/ml; IL-3, 8-12 ng/ml; EPO, 1.5-2.5U/ml; the balance of Stem cell SFEM II culture medium; the second stage culture medium consists of the following components: human AB serum, 2% -5%; EPO, 1.5-2.5U/ml; the balance being Stem cell SFEM II medium. The experiment shows that the number of the living cells can be amplified by 200 times on the 18 th day of in vitro differentiation culture; the proportion of CD71 positive cells can reach 63% on day 14, the proportion of CD235a positive cells can reach 39% on day 25, and the proportion of HbF positive cells can reach 38%.
Description
Technical Field
The invention relates to a culture medium for in-vitro erythroid differentiation of umbilical cord blood CD34 positive hematopoietic stem cells, belonging to the technical field of in-vitro culture of hematopoietic stem cells.
Background
Hematopoietic stem/progenitor cells (HSCs) are adult stem cells in the blood system, a heterogeneous population with the potential for long-term self-renewal and differentiation into various mature blood cells. With the rapid development of technology, HSCs have been widely used in research in hematopoietic reconstitution, gene therapy, tumor purification, immunotherapy, and the like; also used in clinical treatment of diseases such as leukemia, thalassemia, hemophilia, etc. In terms of hematological disorders, a first affiliated hospital at university of guang western medicine, month 1, 2022, healed a child with beta thalassemia using hematopoietic stem cell-based gene therapy. A serious thalassemia patient (. Beta.0/. Beta.0 type) was cured by a gene therapy based on hematopoietic stem cells in a ninth two-good hospital of the national civil release army's co-work support army at 12.30.2021. The same applies to the ninth, second and third hospitals of the Hunan elegance hospital and the release army. Clinical trial data of Vor Bio, published in the company of Vor Bio, on the treatment of Acute Myeloid Leukemia (AML) patients by grinding CRISPR/Cas9 gene editing HSPCs drug Trem-cel (VOR 33) show that the effect is good and no side effect. It also has great effect in the treatment of many rare diseases. The Orchard Therapeutics company developed a pipeline OTL-200 for the treatment of metachromatic leukodystrophy using hematopoietic stem cells, a drug which has been approved in the european union. Meanwhile, the OTL-201 line is a treatment for type IIIA mucopolysaccharidosis (MPS-IIIA) using hematopoietic stem cells, and the N-sulfoglucosamine sulfohydrolase level in the cerebrospinal fluid of 5 patients has been restored after the treatment.
There are 3 current clinical approaches to hematopoietic stem cells, respectively from bone marrow, neonatal cord blood, and mobilized peripheral blood. Wherein the hematopoietic stem cells derived from neonatal umbilical cord blood perform better in terms of stem properties than the other two sources. During drug development, erythroid differentiation of hematopoietic stem cells in vitro is required to detect whether hematopoietic stem cells can be used for subsequent drug development after modification. However, in vitro, hematopoietic stem cells derived from neonatal umbilical cord blood tend to be more difficult to erythroid differentiate than other sources of hematopoietic stem cells, thereby affecting subsequent development.
The prior art methods for in vitro erythroid differentiation of peripheral blood and bone marrow derived hematopoietic stem cells generally do not allow for good erythroid differentiation of neonatal cord blood derived hematopoietic stem cells in vitro.
Disclosure of Invention
Aiming at the prior art, the invention provides a culture medium for in-vitro erythroid differentiation of CD34 positive hematopoietic stem cells of umbilical cord blood, and the culture medium can successfully realize erythroid differentiation of CD34 positive hematopoietic stem cells of umbilical cord blood sources. The culture medium has simple components and is easy to operate during culture.
The invention is realized by the following technical scheme:
a medium for in vitro erythroid differentiation of cord blood CD34 positive hematopoietic stem cells comprising a first stage medium and a second stage medium, wherein the first stage medium is comprised of the following concentrations of components: SCF, 8-12 ng/ml; IL-3, 8-12 ng/ml; EPO (human erythropoietin) 1.5-2.5U/ml; the balance of Stem cell SFEM II culture medium; the second stage culture medium consists of the following components in concentration: human AB serum, 2% -5% (volume percentage); EPO, 1.5-2.5U/ml; the balance being Stem cell SFEM II medium.
Preferably, the first stage medium is composed of the following concentrations of components: SCF,10ng/ml; IL-3, 10ng/ml; EPO,2U/ml; the balance being Stem cell SFEM II medium.
Preferably, the second stage medium is composed of the following concentrations of components: human AB serum, 3%; EPO,2U/ml; the balance being Stem cell SFEM II medium.
The Stem cell SFEM II culture medium is a hematopoietic Stem cell serum-free culture medium, is a commercial culture medium in the prior art, and can be purchased in the conventional market. The SCF, IL-3, EPO, human AB serum and the like are all common growth factors in the field and can be purchased in the conventional market.
The culture medium for in-vitro erythroid differentiation of the cord blood CD 34-positive hematopoietic stem cells is applied to culture of the cord blood CD 34-positive hematopoietic stem cells. In specific application, the first-stage culture medium is used for culturing the CD34 positive hematopoietic stem cells for 14 days, and then the second-stage culture medium is used for continuous culture until the enucleation is finished, and the liquid is changed every 3-4 days.
A culture method for in vitro erythroid differentiation of cord blood CD34 positive hematopoietic stem cells comprises the following steps: culturing the CD34 positive hematopoietic stem cells for 14 days by using a first-stage culture medium, and then continuously culturing by using a second-stage culture medium until the enucleation is finished, and changing the liquid every 3-4 days.
Further, the CD 34-positive hematopoietic stem cells are isolated from umbilical cord blood using density gradient centrifugation and magnetic bead sorting.
The culture medium for in vitro erythroid differentiation of the CD34 positive hematopoietic Stem cells of the umbilical cord blood is prepared by adding specific concentration of SCF, IL-3, EPO, human AB serum and the like on the basis of a Stem cell SFEM II culture medium, and the CD34 positive hematopoietic Stem cells of the umbilical cord blood source can be successfully differentiated by using the culture medium, and experiments show that the number of living cells can be amplified by about 200 times on the 18 th day of in vitro differentiation culture; the proportion of CD71 positive cells can reach about 63% on day 14, about 39% on day 25, and about 38% on HbF positive.
Drawings
Fig. 1: living cell count results are schematically shown.
Fig. 2: cell diameter measurement results are schematically shown.
Fig. 3: flow cytometry detection red differentiation marker expression results are schematically shown.
Fig. 4: schematic of positive cell ratios on days 14, 18, 25.
Fig. 5: flow cytometry is used for detecting the proportion of the fetal hemoglobin positive cells.
Fig. 6: comparative schematic of the proportion of positive cells in commercial and experimental groups.
Fig. 7: erythroid differentiation 18 days cell photograph.
Fig. 8: schematic representation of cell morphology during erythroid differentiation in vitro.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.
Example 1 in vitro erythroid differentiation culture of cord blood CD 34-positive hematopoietic Stem cells
The method comprises the following steps:
isolation of mononuclear cells
(1) The blood sample with anticoagulant added is transferred to a centrifuge tube.
(2) Centrifugation was carried out at 1800rpm (750 g) for 15 minutes, 9 at an ascending speed and 7 at a descending speed.
(3) After centrifugation, the upper plasma layer was transferred to a new centrifuge tube and sealed with a sealing film.
(3) The lower blood cells were diluted by adding PBS buffer at a volume ratio of 1:1 and gently mixed.
(4) A clean 50mL centrifuge tube was taken and 20mL room temperature lymphocyte separation solution was added.
(5) The diluted blood sample (25 mL) was removed by pipetting with a 25mL pipette, and slowly added by adherence, and the blood sample was placed on top of the lymphocyte separator (non-whippable lymphocyte separator).
(6) Put carefully into a centrifuge, centrifuge 700g for 30 minutes, speed up 3, speed down 3.
(7) The centrifuge tube was carefully removed and the record was not allowed to shake.
(8) After centrifugation, the liquid in the centrifuge tube is divided into 4 layers from top to bottom: the uppermost layer is the plasma layer, the second layer is the lymphocyte layer, the third layer is the lymphocyte separation liquid layer, and the lowest layer is the red cell layer (red precipitate).
(9) The white annular layer was gently pipetted away with a 10mL pipette to remove more than two-thirds of the blood slurry and was not agitated.
(10) The second layer of lymphocytes was transferred to a clean 50mL centrifuge tube, with as little of the plasma layer and lymphocyte separation layer aspirated.
(11) To the lymphocyte suspension, PBS buffer was added to a volume of 50mL, and gently mixed upside down.
(12) Placing the centrifugal tube in a centrifugal machine, setting the rotating speed to be 300g, setting the lifting speed to be 6, setting the centrifugal time to be 10 minutes, and distributing lymphocytes on the bottom layer of the centrifugal tube after the centrifugal is finished.
(13) The centrifuged supernatant was carefully aspirated off and 5mL PBS was added to the centrifuge tube to re-suspend the lymphocytes. The centrifuge tube was then continuously added with 40mL PBS and mixed upside down.
(14) And (3) placing the centrifugal tube into a centrifugal machine for centrifugation, wherein the rotating speed is set to be 200g, the lifting speed is set to be 6, the centrifugation time is 10 minutes, and after the centrifugation is finished, lymphocytes are distributed on the bottom layer of the centrifugal tube.
Isolation of CD 34-Positive hematopoietic Stem cells from magnetic beads in mononuclear cells
(1) Preparing Buffer solution: 44.2mL of PBS (pH 7.2) +0.25g BSA+5.8mL 5% EDTA, and 0.22 μm.
(2) PBMC were resuspended in 300. Mu.L Buffer solution. 10 8 Each cell was resuspended with a maximum of 300. Mu.L Buffer solution.
(3) 100 mu L FcR Blocking Reagent was added.
(4) mu.L of CD34 microblades was added.
(5) After mixing evenly, incubating for 30min at the temperature of 2-8 ℃.
(6) Cells were washed by adding 10mL Buffer. 300g,10min,4 ℃. The supernatant was discarded.
(7) Add 500. Mu.L Buffer to resuspend cells.
(8) MS column magnetic rack. The MS column was washed once with 500. Mu.L Buffer solution.
(9) The cell suspension was applied to an MS column and the effluent was collected.
(10) The column was washed 3 times with Buffer. 500 mu L each time. And collecting effluent.
(11) The MS column was left from the magnet holder and placed above the sterile tube.
(12) 1mL of buffer solution is added to the column, and the cells are pushed into a sterile tube by a piston, and the cells in the tube are CD34 positive hematopoietic stem cells.
(III) cell culture
(1) Will be 1X 10 4 The CD34 positive hematopoietic stem cells/well were inoculated into a culture plate and differentiated using 3 hematopoietic stem cell in vitro erythroid differentiation methods. 3 technical replicates per method.
The first group is a commodity group. The first stage medium is: stemSpan TM SFEM II addition stem cell StemSpan TM Erythroid Expansion Supplement (100X). The second stage culture medium is: human AB serum, 3%; EPO,2U/ml; the balance being Stem cell SFEM II medium.
The second group is the experimental group. The first stage medium is: SCF,10ng/ml; IL-3, 10ng/ml; EPO,2U/ml; the balance being Stem cell SFEM II medium. The second stage culture medium is: human AB serum, 3%; EPO,2U/ml; the balance being Stem cell SFEM II medium.
The third group is the article group. Reference is made to the method for in vitro differentiation of hematopoietic stem cells in article Highly efficient therapeutic gene editing of human hematopoietic stem cells.
Basal Medium EDM Medium 330. Mu.g/ml human transferrin, 10. Mu.g/ml human recombinant insulin, 2IU/ml heparin, 5% human AB plasma, 3IU EPO were added from IMDM.
The first stage medium is: the basal medium was further supplemented with 1. Mu.M cortisol, 100ng/ml SCF,5ng/ml IL-3. The second stage culture medium is: the basal medium was supplemented with 100ng/ml SCF. The third stage medium is the basal medium.
(2) On day 3 of culture, the experimental and commercial groups were added with the same amount of first stage medium as the initial amount. The article group is completely changed, and the culture is continued.
(3) On day 7, the experimental and commercial groups, 1500rpm×5min, cells were collected, and the cells were continued to be cultured using the first stage medium, and resuspended using the first stage medium. The cell density is kept at 1-2X 10 4 /ml. The article group was cultured using a second stage medium.
(4) On days 10-11, the experimental and commercial groups, 1500rpm X5 min, cells were collected and the cells were resuspended using the first stage medium for continued culture. The cell density was kept at 1X 10 5 And/ml or less. After complete liquid exchange of the paper group, the culture was performed using a third-stage medium.
(5) The cells were collected after culturing for 14 days at 1500rpm X5 min in the experimental group and the commercial group. The culture was resuspended using the second stage medium by washing once with IMDM. The cell density is 5-10 multiplied by 10 5 Individual cells/ml. After the complete liquid exchange of the article group, the culture is continued by using the medium of the third stage. Until the end of the experiment.
(6) In vitro differentiation cultures were used for viable cell count and cell diameter measurements on days 7, 11, 14, 18, 21, 25.
(7) In vitro differentiation culture day 14, 18, 25, flow cytometry examined the expression of erythroid differentiation markers.
(8) On day 25 of in vitro differentiation culture, flow cytometry examined the expression of fetal hemoglobin (HbF).
(9) Cells were stained with giemsa on days 11 and 18 of in vitro differentiation culture.
Results: the living cell count results are shown in FIG. 1, and the cell diameter measurements are shown in FIG. 2.
The results of flow cytometry detection of erythroid differentiation marker expression are shown in FIG. 3. Positive cell ratios at days 14, 18, 25 are shown in fig. 4, for example.
The results of flow cytometry for detecting the proportion of fetal hemoglobin-positive cells are shown in FIG. 5, and the comparison between commercial and experimental groups is shown in FIG. 6. The cell photograph of the erythroid differentiated 18 days is shown in FIG. 7.
Cell morphology during in vitro erythroid differentiation is shown in FIG. 8, wherein the upper and middle portions are stained with Gimssa and the lower portion is microscopically bright field observed.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (7)
1. A culture medium for in vitro erythroid differentiation of cord blood CD34 positive hematopoietic stem cells, characterized in that: comprising a first stage culture medium and a second stage culture medium, wherein the first stage culture medium is composed of the following components with the following concentrations: SCF, 8-12 ng/ml; IL-3, 8-12 ng/ml; EPO, 1.5-2.5U/ml; the balance of Stem cell SFEM II culture medium; the second stage culture medium consists of the following components in concentration: human AB serum, 2% -5%; EPO, 1.5-2.5U/ml; the balance being Stem cell SFEM II medium.
2. The medium for in vitro erythroid differentiation of cord blood CD34 positive hematopoietic stem cells according to claim 1, characterized in that said first stage medium is composed of the following concentrations of components: SCF,10ng/ml; IL-3, 10ng/ml; EPO,2U/ml; the balance being Stem cell SFEM II medium.
3. The medium for in vitro erythroid differentiation of cord blood CD34 positive hematopoietic stem cells according to claim 1, characterized in that said second stage medium is composed of the following concentrations of components: human AB serum, 3%; EPO,2U/ml; the balance being Stem cell SFEM II medium.
4. Use of the medium according to any one of claims 1 to 3 for culturing cord blood CD 34-positive hematopoietic stem cells.
5. The use according to claim 44, wherein: in specific application, the first-stage culture medium is used for culturing the CD34 positive hematopoietic stem cells for 14 days, and then the second-stage culture medium is used for continuous culture until the enucleation is finished, and the liquid is changed every 3-4 days.
6. A method for culturing in vitro erythroid differentiation of cord blood CD34 positive hematopoietic stem cells, which is characterized by comprising the following steps: culturing CD 34-positive hematopoietic stem cells using the first stage medium of the medium of any one of claims 1-3 for 14 days, and then continuing the culture using the second stage medium until the enucleation is completed, changing the liquid every 3-4 days.
7. The method for culturing in vitro erythroid differentiation of cord blood CD 34-positive hematopoietic stem cells of claim 6, wherein: the CD34 positive hematopoietic stem cells are isolated from umbilical cord blood using density gradient centrifugation and magnetic bead sorting.
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