Target cell in-vitro sorting method for cell therapy

Abstract

The invention relates to a target cell in-vitro sorting method for cell therapy, and belongs to the technical field of cell therapy. According to the method, the cleaning solution containing EGTA, EDTA, alpha-casein and prostaglandin is used, so that the sorting method with high living cell recovery efficiency is obtained; and cell therapy can be better realized.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to a method for in vitro sorting of target cells for cell therapy. Background Art

[0002] Cell therapy is a cutting-edge technology in the current medical field, among which the separation, purification and cultivation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are key steps for the success of cell therapy. These cells have the ability to self-renew and differentiate into various blood cell types, and play an important role in the treatment of blood system diseases. At present, the method for separating target cells from blood samples mainly includes techniques such as immunomagnetic bead sorting and flow cytometry sorting. For example, CN108473955B discloses a method for obtaining target cells from a blood sample, which includes subjecting the blood sample to immunomagnetic bead sorting to obtain a first sorting product, then performing flow cytometry sorting to obtain a second sorting product, and finally separating single cells using a mouth pipette method.

[0003] The separation of hematopoietic stem cells is usually based on cell surface markers, such as CD34, CD133, CD90, etc. CN103509753B describes a method for isolating CD34+ hematopoietic stem cells and their precursor cells from umbilical cord blood or bone marrow-derived blood samples, culturing these cells in a specific culture medium, and analyzing the differentiation of cells by detecting cell surface molecular markers using FACS flow cytometry analysis technology. In addition, CN118064365A provides a method for isolating mononuclear cells from frozen and resuscitated umbilical cord blood, and then sorting CD34+ hematopoietic stem cells and amplifying and culturing them.

[0004] With the development of gene editing technology, target cells for cell therapy have shown great potential in the field of cell therapy. WO2022228471A1 discloses a gene-edited hematopoietic stem cell and its combined application with CAR-T cells, which changes the cell antigen epitopes so that the cells are not killed by CAR-T or antibody drugs, thereby alleviating the side effects of CAR-T products or antibody drugs in tumor treatment. CN112516167A provides a therapeutic composition of altered hematopoietic stem cells and / or progenitor cells with improved implantation and homing properties, and a method for preparing the therapeutic composition.

[0005] However, there are some problems in the prior art. First, in the washing step before cell sorting, there is a lack of a washing solution formula specifically for platelet removal. The presence of platelets will interfere with the cell sorting process, reducing sorting efficiency and purity. Secondly, the existing washing solutions mainly focus on cell protection and optimization of the sorting process, and fail to fully consider the needs of platelet removal. More optimized washing and sorting methods are needed to ensure cell survival and live cell recovery efficiency.

[0006] The washing solution formula in the prior art usually only contains PBS and a single chelating agent (such as EDTA), which has limited effect in removing platelets. The adhesion between platelets and other cells involves multiple mechanisms, including calcium-dependent and calcium-independent pathways, and a single chelating agent is difficult to effectively block these interactions. In addition, the prior art lacks the understanding and application of the role of additives in washing solutions (such as α-casein and prostaglandins) in preventing cell aggregation and protecting cell activity. These factors may cause problems such as low cell recovery rate, poor viability and purity, and incomplete removal of impurities, ultimately affecting the quality and efficacy of cell preparations. Summary of the invention

[0007] The present invention provides an optimized method for sorting hematopoietic stem cells.

[0008] Specifically, the present invention provides a method for in vitro sorting of target cells for cell therapy, comprising: (a) collecting a blood sample; (b) washing the blood sample with a washing solution; (c) sorting and culturing the washed blood sample based on cell surface proteins. The washing solution comprises PBS (phosphate buffer), EGTA and EDTA.

[0009] In certain embodiments, the concentration of EGTA in the washing solution is 1-2 mM, and the concentration of EDTA is 0-0.5 mM.

[0010] In certain embodiments, the washing solution further comprises 0.1% to 0.5% α-casein and / or 10 to 100 ng / mL prostaglandin (PEG1).

[0011] In certain embodiments, the washing solution comprises PBS (pH 7.3), 1.5 mM EGTA and 0.5 mM EDAT.

[0012] In certain embodiments, the wash solution comprises PBS (pH 7.3), 2 mM EGTA and 1 mM EDAT.

[0013] In certain embodiments, the washing solution comprises PBS (pH 7.3), 1.5 mM EGTA, 0.5 mM EDTA and 0.3% α-casein.

[0014] In certain embodiments, the wash solution comprises PBS (pH 7.3), 1.5 mM EGTA, 0.5 mM EDTA, and 50 ng / mL prostate.

[0015] In certain embodiments, the washing solution comprises PBS (pH 7.3), 1.5 mM EGTA, 0.5 mM EDTA, 0.3% α-casein, and 50 ng / mL prostaglandin.

[0016] In certain embodiments, the target cells are hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs); preferably, the cell surface proteins are selected from the group consisting of one or more of the following: CD34, CD133, CD90, CD38, HLA-DR, CD25 and CD127.

[0017] In certain embodiments, the target cells are introduced into a gene editing system. In certain embodiments, the target cells are introduced into a CRISPR gene editing system. In certain embodiments, the target cells are introduced into a gene editing system after sorting. In certain embodiments, the target cells are introduced into a CRISPR gene editing system after sorting.

[0018] In certain embodiments, the sorting is performed based on the specific binding of an antibody against the cell surface protein and the cell surface protein; preferably, the sorting is based on immunomagnetic bead technology; more preferably, the sorting is performed based on a sorting instrument.

[0019] In certain embodiments, the blood sample is peripheral blood, umbilical cord blood, and / or an apheresis product.

[0020] In certain embodiments, the method further comprises anticoagulation treatment using an anticoagulant after step a; preferably, the anticoagulant is an anti-heparin anticoagulant or an ACD anticoagulant.

[0021] In certain embodiments, the method further comprises treating the sample with a lysis reagent before step b; preferably, the lysis reagent contains a fixative or does not contain a fixative.

[0022] The beneficial effect of the present invention is that by optimizing the components of the washing solution before sorting, especially using EGTA instead of EDTA, or using EGTA and EDTA for combination, the recovery rate of CD34+ live cells can be significantly increased without affecting the cell viability and purity. Experimental data show that after adding 0.3% α-casein and / or 50ng / mL prostaglandin to the washing solution, the purity of CD34+ cells after sorting can reach more than 80%, the cell viability can reach more than 80%, and the recovery rate of CD34+ cells after sorting can be significantly improved, specifically up to about 68~90%. The present invention obtains a sorting method with high live cell recovery efficiency by using a washing solution containing EGTA, EDTA, α-casein and prostaglandin; cell therapy, especially cell therapy based on the patient's own immune cells, can be better achieved. The washing solution components (α-casein and prostaglandin) used by the sorting method in the present application before sorting on the machine do not affect the subsequent cell culture, and can further ensure the quality and efficacy of the cell preparation. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described clearly and completely below with the help of embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The present invention provides a method for in vitro sorting of target cells for cell therapy. In the sorting process, a washing liquid with specific components is used to wash the blood sample (blood cells) before sorting on a machine. In some embodiments, the present application uses a washing solution containing PBS, EGTA and EDTA to wash the blood sample: wherein the concentration of EGTA is 1~5 mM; preferably, it can be 1~4 mM, 1~3 mM or 1~2 mM; more preferably, the concentration of EGTA is 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 mM; wherein the concentration of EDTA is 0.1~3 mM; preferably, it can be 0.1~2 In some embodiments, the concentration of EGTA is 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3 mM; the concentration of EDTA is 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 nM. In some embodiments, the EGTA:EDTA used in the present application is 3:1-1:1, preferably 3:1, 2:1, 1:1. In some embodiments, the total concentration of EGTA+EDTA used in the present application does not exceed 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2 mM.In the above optional embodiments, the washing liquid of the present application further comprises α-casein and / or prostaglandin (PEG1): wherein the concentration of α-casein is 0.1% to 0.5%, 0.1% to 0.4%, 0.1% to 0.3%, 0.2% to 0.4%, 0.2% to 0.3%; more preferably, the concentration of α-casein is 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%; optionally, the concentration of prostaglandin is 10 to 100%, 20 to 90%, 30 to 80%, 40 to 50%; ng / mL; preferably, the concentration of prostaglandins is 10, 15, 20, 25, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100 ng / mL.

[0025] Phosphate-Buffered Saline (PBS) used in this application is a buffer solution widely used in cell sorting and biomedical experiments, composed of phosphate and sodium chloride, with a pH between 7.2 and 7.4 (preferably 7.3). Ethylenediaminetetraacetic acid (EDTA) is a commonly used chelating agent used to complex divalent metal ions (such as Ca²⁺ and Mg²⁺) in the solution, thereby inhibiting cell-to-cell adhesion and avoiding cell aggregation. Ethylene glycol bis(2-aminoethyl ether)-tetraacetic acid (EGTA) is a more selective chelating agent, especially with stronger complexing ability for Ca²⁺. α-Casein is a naturally occurring protein that acts as a protective agent in the washing solution of cell sorting. It interacts with the cell membrane to form a protective layer, reducing mechanical stress and shear force damage to cells, while inhibiting nonspecific binding, reducing cell loss and background noise. The main role of prostaglandins (such as PGE1) in cell sorting is to maintain their activity and functionality by regulating the cell's signaling pathways. For example, PGE1 can inhibit apoptosis caused by over-activation of cells and reduce cell-cell interactions, thereby improving the quality of single-cell suspensions.

[0026] The introduced gene editing systems include but are not limited to CRISPR gene editing system, TALEN system, and ZFN system.

[0027] The cells obtained after sorting in the present invention can be used as target cells (target cells). In some embodiments, the cells (genetically modified cells) introduced into the CRISPR gene editing system can be used in the field of cell therapy; in particular, after sorting out specific cells (including but not limited to CD34+ cells) based on the blood cells of patients (for example, including but not limited to patients with HBB gene defect diseases), the target cell genes are modified by introducing the CRISPR gene editing system, and then transported back into the patient. The "CRISPR gene editing system" herein refers to a system composed of CRISPR-Cas protein or its encoding nucleic acid, and gRNA or its encoding nucleic acid, including but not limited to: a mixture of Cas protein and gRNA, a complex of Cas protein and gRNA, RNP of Cas protein and gRNA, and encoding nucleic acid of Cas protein and gRNA. CRISPR technology achieves site-specific cutting, insertion, deletion or replacement of target cell genes by complementary base pairing of guide RNA (gRNA) with target sequences and combining with Cas family nucleases, and at the same time achieves transcriptional regulation of transgenes through derivative technologies such as CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation). TALEN technology achieves precise editing of target genes through the combination of specific DNA binding modules and nucleases; ZFN guides nucleases to complete sequence editing at the target site through the specific binding of zinc finger domains to nucleic acids. In addition, vector technologies such as integrase, retrovirus or lentivirus vectors are also often used for delivery, and non-integration systems such as mRNA delivery and electroporation technology provide efficient and low-risk operation methods.

[0028] The term "HBB gene defect disease" in this article refers to a class of genetic diseases caused by mutations or functional loss of the HBB gene (gene encoding the β chain of adult hemoglobin), with typical clinical manifestations including thalassemia (β thalassemia) and sickle cell anemia. These diseases usually result from insufficient or abnormal synthesis of β globin chains due to mutations in the HBB gene, which leads to hemoglobin functional defects, abnormal destruction of red blood cells, and a series of related pathophysiological changes. In patients, common types of HBB gene mutations include point mutations, deletion mutations, insertion mutations, and gene rearrangements, which lead to a significant reduction or complete loss of hemoglobin A production, or the production of abnormally structured hemoglobin molecules (such as HbS). Treatments for patients with HBB gene defects traditionally rely on blood transfusions and iron chelation therapy to relieve symptoms, but these therapies cannot fundamentally cure the disease. With the development of gene editing technology, tools including CRISPR / Cas9, TALEN, and ZFN have been widely used to repair HBB gene mutations or to compensate for defective β globin chains by activating the production of fetal hemoglobin (HbF). In addition, lentiviral vector-mediated HBB gene replacement therapy and hematopoietic stem cell transplantation-based strategies also provide potential cures for patients. Innovative approaches combining these technologies not only improve treatment options for HBB gene deficiency diseases, but also lay the foundation for the development of precision medicine for inherited blood diseases.

[0029] When the present invention sorts cells of a blood sample, the target cells include but are not limited to CD34+ cells. In some embodiments, the sorting is based on antibodies to the cell surface protein and its specific recognition; preferably, the sorting is based on immunomagnetic bead technology; more preferably, the sorting is based on a sorting instrument. The sorting technology uses conventionally used sorting instruments, including but not limited to the sorting instruments listed below. Cell sorting technology is an important tool for modern biomedical research and clinical applications, used to separate specific cell subpopulations to meet different experimental and therapeutic needs, such as CD34+ hematopoietic stem cells, CD133+ stem cells, CD90+ mesenchymal stem cells, and target cell populations such as CD38, HLA-DR, CD25 and CD127 cells related to immune function. Common cell sorting techniques include magnetic sorting (MACS), flow cytometry sorting (FACS), and sorting methods based on microfluidics and physical properties. Magnetic sorting separates target cells under the action of a magnetic field by combining antibodies with magnetic microbeads. Representative instruments include Miltenyi Biotec's CliniMACS and autoMACS series, which are fast and easy to operate, and are suitable for the initial separation of clinical-grade cells. CliniMACS Prodigy is used to separate target cells such as CD34+. It also has a closed GMP-level operation process, integrated cell enrichment, gene modification and amplification functions, and is suitable for clinical applications such as CAR-T cell preparation or stem cell therapy. Flow cytometry sorting uses fluorescently labeled antibodies to bind to specific antigens on the surface of target cells, and sorts cells with high precision through laser detection and charge deflection. Commonly used instruments include BD Biosciences' FACSAria, Beckman Coulter's MoFlo Astrios, and Sony's SH800, which are suitable for high-purity screening of immune subpopulations such as CD38+. In recent years, microfluidic sorting technology has gradually emerged with its advantages such as label-free and low shear force, and is suitable for experiments with high requirements for cell activity. In addition, analytical instruments such as Cytek Biosciences' Aurora hyperspectral flow analyzer, Beckman Coulter's CytoFLEX, and BD's LSR series perform excellently in multi-parameter analysis, providing important support for the optimization of sorting strategies.

[0030] In some embodiments, the sorting method of the present application further comprises anticoagulation treatment with an anticoagulant after step a; preferably, the anticoagulant is an antiheparin anticoagulant or an ACD anticoagulant. Anticoagulation treatment after blood sampling is a key step to ensure the stability of blood samples and the accuracy of subsequent analysis. After sampling, the sample is usually anticoagulated by adding an anticoagulant to prevent blood from coagulating during storage or transportation. Preferably, the anticoagulant includes an antiheparin anticoagulant or an ACD (Acid Citrate Dextrose) anticoagulant. Antiheparin anticoagulants provide a mild and efficient anticoagulant effect by inhibiting the activity of thrombin and the action of other coagulation factors, and are widely used in experiments that require the maintenance of blood cell activity or function, such as immune cell function analysis or cell culture. ACD anticoagulant uses citrate as the main component, inhibits the coagulation cascade reaction by complexing calcium ions, and contains an appropriate amount of glucose to maintain cell metabolic function. It is suitable for long-term storage of samples or application scenarios where cell integrity needs to be retained.

[0031] In some embodiments, the sorting method of the present application further comprises lysing the sample using a lysis reagent before step b; preferably, the lysis reagent contains a fixative or does not contain a fixative. The lysis reagent used in the blood cell sorting process is an important reagent for achieving red blood cell lysis and retaining target cells, and can be used to remove red blood cell components in the sample to optimize sorting efficiency and downstream analysis. The formulations of lysis reagents are generally divided into two categories: those containing a fixative and those not containing a fixative. Lysis reagents containing fixatives, such as FACS Lysing Solution, IQ Lyse, and VersaLyse, can fix cells while lysing red blood cells, and are often used in experiments that require long-term storage or subsequent fluorescent staining analysis, and have the advantages of maintaining good cell morphology and stable antigenic epitopes. Lysis reagents without fixatives focus more on quickly lysing red blood cells in a short time while retaining the integrity of target cells, such as Ortho-mune Lysing Reagent, Quicklysys, Ammonium Oxalate-Based Lysing Solution, NH 4 Cl-Based Lysing Solution and ACKBuffer, these reagents are usually used in scenarios that require high cell activity or subsequent functional analysis.

[0032] The following examples use a COM.TEC blood component separator to collect mononuclear cells from peripheral blood from the femoral vein of mobilized β-thalassemia patients. The collection method is: circulating volume 250-350 ml, pumped buffy coat volume 10-20 ml, collected buffy coat volume 4-10 ml, whole blood flow rate 20-60 ml / min, anticoagulant: whole blood 1:10-1:14, centrifugal speed 1400-1700 r / min, processed blood volume / body whole blood volume 2-4.

[0033] Example 1 Take 3 batches of single blood samples, and directly incubate and sort the single blood samples without manual pre-treatment. The specific steps are as follows: (a) Collect blood samples; specifically, take three batches of single blood samples, centrifuge them, remove the supernatant, and then adjust the volume to 350 ml; (b) The sample in (a) is not washed and is directly sorted on the machine; (c) The washed blood samples were sorted, and the sorting instrument used was the MACS system (Miltenyi Biotec). Then the sorting recovery rate, CD34+ cell purity, and cell viability were investigated to determine the applicability of different washing solution components to CD34+ cell enrichment. The results are shown in Table 1. The results of CD34+ cell purity before and after sorting, cell viability, and CD34+ cell recovery rate after sorting are shown in Table 1. The data showed that the purity of CD34+ cells after sorting was >80%, and the cell viability was >80%. However, the recovery rate of CD34+ cells after sorting was 40.03~43.79%, which was low, and the process needs to be optimized to improve the recovery rate.

[0034] Table 1 Purity, cell viability and recovery rate of unwashed CD34+ cells before and after sorting Note: Recovery rate of live CD34+ cells (%) = number of live CD34+ cells after sorting / number of live CD34+ cells before sorting.

[0035] Example 2 In order to improve the cell recovery rate and optimize the composition of the washing solution, a batch of single blood samples was divided into 3 parts. The experimental conditions were the same as in Example 1, but in step (b), a washing step was performed before sorting, and the components of the washing solution were designed into the following 3 groups. The specific washing operation was to add the sorting buffer to the single blood in the transfer bag, centrifuge at 200g for 15min, remove the supernatant as much as possible, add the washing solution for the first wash; then centrifuge at 200g for 15min, remove the supernatant as much as possible, repeat the wash once, and then add the sorting buffer to 200~300 mL. Repeat the experiment 5 times.

[0036] Group 1: Wash twice with PBS (pH 7.3) + 2 mM EDTA currently used on the market before sorting; Group 2: Washed twice with PBS (pH 7.3) + 2 mM EGTA before sorting; Group 3: Wash twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA before sorting.

[0037] Table 2 Optimization of metal ion chelating agents in washing liquid The experimental results are shown in Table 2. The average value of the result data was taken. It was found that the recovery rate of live CD34+ cells in Group 3 was significantly higher than that in Group 1 and Group 2, both reaching statistically significant differences (P<0.05), indicating that EGTA can significantly increase the recovery rate of CD34+ cells when it partially replaces EDTA for washing.

[0038] Example 3 In order to further improve the cell recovery rate, the components of the washing solution were optimized again, and a batch of single blood samples were divided into 4 parts. The experimental conditions were the same as in Example 2, but in step (b), a washing step was performed before sorting. The components of the washing solution were designed into the following 4 groups, and the experiment was repeated 5 times. The experimental results are shown in Table 3, and the result data were averaged.

[0039] Group 4: Wash twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDAT before sorting; Group 5: Washed twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA + 0.3% α-casein before sorting; Group 6: Wash twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA + 50 ng / mL prostaglandin before sorting; Group 7: Wash twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA + 0.3% α-casein + 50 ng / mL prostaglandin before sorting; Table 3 Purity, cell viability and recovery rate of CD34+ cells washed with different components The results showed that by adding 0.3% α-casein and / or 50 ng / mL prostaglandin (PGE1) to the washing solution, the purity of CD34+ cells after sorting was >80%, and the cell viability was >80%, both of which met the sorting requirements; it could significantly improve the recovery rate of CD34+ cells after sorting, specifically reaching 67.12~83.33%.

[0040] The recovery rates of live CD34+ cells in groups 5, 6, and 7 were significantly higher than those in group 4, reaching statistically significant differences (P<0.05).

[0041] Especially when α-casein and / or prostaglandin are added at the same time, the CD34+ recovery rate can reach more than 80%, indicating that washing with the washing solution of the above components can effectively remove platelets and has a significant effect on improving the recovery rate.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for in vitro sorting of target cells for cell therapy, characterized in that: The method comprises: (a) Collecting blood samples; (b) washing the blood sample with a washing solution; (c) sorting the washed blood samples based on cell surface proteins and culturing them; The washing solutions include PBS, EGTA and EDTA.

2. The method according to claim 1, characterized in that The concentration of EGTA in the washing solution is 1~3mM, and the concentration of EDTA is 0.3~0.8mM.

3. The method according to claim 1 or 2, characterized in that: The washing solution also includes 0.1%~0.5% α-casein and / or 10~100 ng / mL prostaglandin.

4. The method according to claim 1 or 2, characterized in that: The wash buffer included PBS, pH 7.3, 2 mM EGTA, and 1 mM EDAT.

5. The method according to claim 1 or 2, characterized in that: The washing solution included PBS, pH 7.3, 1.5 mM EGTA and 0.5 mM EDAT.

6. The method according to claim 3, characterized in that The washing solution included PBS, pH 7.3, 1.5 mM EGTA, 0.5 mM EDTA, and 0.3% α-casein.

7. The method according to claim 3, characterized in that The wash solution included PBS pH 7.3, 1.5 mM EGTA, 0.5 mM EDTA, and 50 ng / mL prostate.

8. The method according to claim 3, characterized in that The wash buffer contained PBS, pH 7.3, 1.5 mM EGTA, 0.5 mM EDTA, 0.3% α-casein, and 50 ng / mL prostaglandins.

9. The method according to claim 1, characterized in that: The target cells are hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs).

10. The method according to claim 9, characterized in that The cell surface protein is selected from the group consisting of one or more of the following: CD34, CD133, CD90, CD38, HLA-DR, CD25 and CD127.

11. The method according to claim 10, characterized in that The target cells are introduced into the gene editing system after sorting.

12. The method according to claim 11, characterized in that The sorting is performed based on the specific binding of the antibody against the cell surface protein to the cell surface protein.

13. The method according to claim 12, characterized in that The sorting is based on immunomagnetic bead technology.

14. The method according to claim 13, characterized in that: The sorting is performed based on a sorting instrument.

15. The method according to claim 1, characterized in that The blood sample is peripheral blood, umbilical cord blood, and / or apheresis product.

16. The method according to claim 1, characterized in that It also includes anticoagulation treatment using an anticoagulant after step a.

17. The method according to claim 16, characterized in that The anticoagulant is an anti-heparin anticoagulant or an ACD anticoagulant.

18. The method according to claim 1, characterized in that It also includes performing a lysis treatment on the sample using a lysis reagent before step b.

19. The method according to claim 18, characterized in that The lysis reagent is a fixative; the fixative is FACS Lysing Solution, IQ Lyse, or VersaLyse.

20. The method according to claim 18, characterized in that The lysis reagent is a non-fixing agent; the non-fixing agent is Ortho-mune Lysing Reagent, Quicklysys, Ammonium Oxalate-Based Lysing Solution, NH4Cl-Based Lysing Solution, or ACK Buffer.

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