A method for in vitro sorting of target cells for cell therapy

Abstract

The present invention relates to a method for in vitro sorting of target cells for cell therapy, belonging to the technical field of cell therapy. The present invention utilizes a washing solution containing EGTA, EDTA, α-casein, and prostaglandins to achieve a sorting method with high viable cell recovery efficiency, thereby enabling better cell therapy.

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, and 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 isolating 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 subjecting the blood sample to flow cytometry sorting to obtain a second sorting product, and finally separating single cells using a mouth pipette method.

[0003] The isolation of hematopoietic stem cells is typically based on cell surface markers such as CD34, CD133, and CD90. CN103509753B describes a method for isolating CD34+ hematopoietic stem cells and their precursors from umbilical cord blood or bone marrow-derived blood samples. These cells are cultured in a specific culture medium and analyzed for cell differentiation using FACS flow cytometry to detect cell surface molecular markers. Furthermore, CN118064365A provides a method for isolating mononuclear cells from frozen and revived umbilical cord blood, then sorting CD34+ hematopoietic stem cells and expanding 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 use with CAR-T cells. By changing the cell antigen epitopes, 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 modified hematopoietic stem cells and / or progenitor cells with improved engraftment and homing properties, as well as a method for preparing the therapeutic composition.

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

[0006] Existing wash solution formulations typically contain only PBS and a single chelating agent (such as EDTA), which has limited effectiveness in removing platelets. Platelet adhesion to other cells involves multiple mechanisms, including calcium-dependent and calcium-independent pathways, and a single chelating agent is unlikely to effectively block these interactions. Furthermore, existing technologies lack understanding and application of the role of wash solution additives (such as α-casein and prostaglandins) in preventing cell aggregation and protecting cell viability. These factors can lead to low cell recovery rates, poor viability and purity, and incomplete impurity removal, ultimately compromising 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; and (c) sorting the washed blood sample based on cell surface proteins and culturing the washed blood sample. The washing solution comprises PBS (phosphate buffered saline), 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 (PGE1).

[0011] In certain embodiments, the wash 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 wash 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 heparin or ACD anticoagulant.

[0021] In certain embodiments, the method 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.

[0022] The beneficial effects of the present invention are as follows: by optimizing the components of the washing solution before sorting, especially replacing EDTA with EGTA, or combining EGTA and EDTA, the recovery rate of CD34+ viable cells can be significantly increased without affecting 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 viable 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 in the sorting method in this application before sorting on the machine do not affect subsequent cell culture, and can further ensure the quality and efficacy of the cell preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] none DETAILED DESCRIPTION

[0024] The technical solutions of the present invention are described clearly and completely below with the aid of embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0025] The present invention provides a method for in vitro sorting of target cells for cell therapy. During the sorting process, a washing solution with specific components is used to wash a blood sample (blood cells) before being sorted 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 ratio used herein is 3:1 to 1:1, preferably 3:1, 2:1, or 1:1. In some embodiments, the total concentration of EGTA+EDTA used herein does not exceed 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, or 2 mM.In the above optional embodiment, the washing solution of the present application further comprises α-casein and / or prostaglandin (PGE1): 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.

[0026] Phosphate-Buffered Saline (PBS) used in this application is a buffer solution widely used in cell sorting and biomedical experiments. It is 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 + ), thereby inhibiting cell-cell adhesion and preventing cell aggregation. Ethylene glycol bis(2-aminoethyl ether)-tetraacetic acid (EGTA) is a more selective chelating agent, especially for Ca² + It has stronger complexing ability. α-Casein is a naturally occurring protein that acts as a protective agent in cell sorting wash buffers. It interacts with the cell membrane to form a protective layer, reducing damage to cells from mechanical stress and shear forces, while inhibiting nonspecific binding, reducing cell loss and background noise. The primary role of prostaglandins (such as PGE1) in cell sorting is to maintain cell activity and functionality by regulating cell signaling pathways. For example, PGE1 can inhibit apoptosis caused by overactivation and reduce cell-cell interactions, thereby improving the quality of single-cell suspensions.

[0027] The introduced gene editing systems include but are not limited to CRISPR gene editing system, TALEN system, and ZFN system.

[0028] The cells obtained after sorting in the present invention can serve as target cells (target cells). In some embodiments, cells introduced with the CRISPR gene editing system (genetically modified cells) can be used in the field of cell therapy. In particular, after sorting blood cells from a patient (for example, including but not limited to patients with HBB gene deficiency), specific cells (including but not limited to CD34+ cells) are obtained. The target cells are then genetically modified by introducing the CRISPR gene editing system and then returned to the patient. The term "CRISPR gene editing system" herein refers to a system consisting of a CRISPR-Cas protein or its encoding nucleic acid, and a guide RNA (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, RNPs of Cas protein and gRNA, and nucleic acids encoding Cas protein and gRNA. CRISPR technology achieves site-specific cleavage, insertion, deletion, or replacement of target cell genes through complementary base pairing of guide RNA (gRNA) with target sequences, combined with Cas family nucleases. Furthermore, derivative technologies such as CRISPRi (CRISPR interference) and CRISPRa (CRISPR activation) can achieve transcriptional regulation of transgenes. TALEN technology achieves precise editing of target genes by combining specific DNA-binding modules with nucleases. ZFNs, on the other hand, use zinc finger domains to specifically bind to nucleic acids, guiding nucleases to complete sequence editing at the target site. In addition, vector technologies such as integrase, retroviral, or lentiviral vectors are also commonly used for delivery. Non-integrating systems such as mRNA delivery and electroporation offer highly efficient and low-risk methods.

[0029] "HBB gene deficiency" in this article refers to a class of inherited disorders caused by mutations or functional loss of the HBB gene (encoding the adult hemoglobin β chain). Typical clinical manifestations include thalassemia (β-thalassemia) and sickle cell anemia. These disorders typically result from HBB gene mutations, leading to insufficient or abnormal β-globin chain synthesis, resulting in hemoglobin functional defects, abnormal red blood cell destruction, and a range of associated pathophysiological changes. Common HBB gene mutations in patients include point mutations, deletion mutations, insertion mutations, and gene rearrangements, resulting in significantly reduced or complete loss of hemoglobin A production, or the production of abnormal hemoglobin structures (such as HbS). Treatments for patients with HBB gene deficiency have traditionally relied on blood transfusions and iron chelation therapy to alleviate symptoms, but these therapies are not curative. With the advancement of gene editing technologies, tools such as CRISPR / Cas9, TALENs, and ZFNs have been widely used to correct HBB gene mutations or to compensate for the 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 offer 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.

[0030] When cells are sorted from a blood sample according to the present invention, the target cells include, but are not limited to, CD34+ cells. In some embodiments, the sorting is based on antibodies and their specific recognition of the cell surface protein; preferably, the sorting is based on immunomagnetic bead technology; more preferably, the sorting is performed using a sorting instrument. The sorting technique utilizes commonly used sorting instruments, including but not limited to those listed below. Cell sorting technology is an important tool in modern biomedical research and clinical applications, used to isolate specific cell subpopulations to meet diverse experimental and therapeutic needs. Examples include CD34+ hematopoietic stem cells, CD133+ stem cells, CD90+ mesenchymal stem cells, and target cell populations associated with immune function, such as CD38, HLA-DR, CD25, and CD127 cells. Common cell sorting techniques include magnetic sorting (MACS), flow cytometry (FACS), and microfluidics- and physical property-based sorting methods. Magnetic separation involves binding antibodies to magnetic microbeads, allowing the isolation of target cells under the influence of a magnetic field. Representative instruments include Miltenyi Biotec's CliniMACS and autoMACS series, which offer rapid separation speeds and ease of use, making them suitable for initial isolation of clinical-grade cells. The CliniMACS Prodigy is used to isolate target cells, such as CD34+ cells. It also features a closed, GMP-grade workflow and integrates cell enrichment, genetic modification, and expansion, making it suitable for clinical applications such as CAR-T cell production or stem cell therapy. Flow cytometry uses fluorescently labeled antibodies to bind to specific antigens on the surface of target cells. Laser detection and charge deflection are used to precisely separate cells. Commonly used instruments include the BD Biosciences FACSAria, Beckman Coulter's MoFlo Astrios, and Sony's SH800. It is suitable for high-purity screening of immune subsets, such as CD38+ cells. In recent years, microfluidic separation technology has gained prominence due to its label-free and low-shear characteristics, making it suitable for experiments requiring high cell viability. 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.

[0031] In some embodiments, the sorting method of the present application further comprises anticoagulation treatment with an anticoagulant after step a; preferably, the anticoagulant is heparin or ACD anticoagulant. Anticoagulation treatment after blood sampling is a key step to ensure the stability of the blood sample and the accuracy of subsequent analysis. After sampling, the sample is usually anticoagulated by adding an anticoagulant to prevent the blood from coagulating during storage or transportation. Preferably, the anticoagulant includes a heparin anticoagulant or an ACD (Acid Citrate Dextrose) anticoagulant. Heparin anticoagulants provide a mild and highly effective anticoagulant effect by inhibiting thrombin activity and the effects of other coagulation factors. They are widely used in experiments that require maintaining blood cell activity or function, such as immune cell function analysis or cell culture. ACD anticoagulants, with citrate as the main component, inhibit the coagulation cascade by complexing calcium ions. They also contain an appropriate amount of glucose to maintain cellular metabolic function, making them suitable for long-term sample storage or applications where cell integrity needs to be preserved.

[0032] In some embodiments, the separation method of the present application further comprises lysing the sample using a lysis reagent prior to step b; preferably, the lysis reagent contains or does not contain a fixative. Lysis reagents used in blood cell sorting are crucial for lysing red blood cells (RBCs) and retaining target cells. They are used to remove RBC components from the sample to optimize sorting efficiency and downstream analysis. Lysis reagent formulations are generally categorized as either fixative-containing or fixative-free. Fixative-containing lysis reagents, such as FACS Lysing Solution, IQ Lyse, and VersaLyse, simultaneously lyse RBCs and fix them. They are commonly used in experiments requiring long-term storage or subsequent fluorescent staining analysis, offering advantages such as well-maintained cell morphology and stable antigenic epitopes. Fixative-free lysis reagents, such as Ortho-mune Lysing Reagent, Quicklysys, Ammonium Oxalate-Based Lysing Solution, NH4Cl-Based Lysing Solution, and ACKBuffer, prioritize rapid RBC lysis while preserving the integrity of target cells. These reagents are typically used in scenarios requiring high cell viability or for subsequent functional analysis.

[0033] In the following examples, mononuclear cells were collected from the femoral vein of mobilized β-thalassemia patients using a COM.TEC blood component separator. The collection method was as follows: 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 ratio 1:10-1:14, centrifugation speed 1400-1700 rpm, and processed blood volume / in vivo whole blood volume 2-4.

[0034] Example 1

[0035] Three batches of single-collected blood samples were collected. After rewarming, the single-collected blood samples were directly incubated with magnetic beads and sorted without manual pre-treatment. The specific steps are as follows:

[0036] (a) Collect blood samples; specifically, take three batches of single-collected blood samples, centrifuge them, remove the supernatant, and adjust the volume to 350 ml.

[0037] (b) The sample in (a) is not washed and is directly sorted on the machine;

[0038] (c) Washed blood samples were sorted using a MACS system (Miltenyi Biotec). The sorting recovery rate, CD34+ cell purity, and cell viability were then assessed to determine the suitability of different wash buffer components for CD34+ cell enrichment. The results 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 ranged from 40.03% to 43.79%, which is relatively low and requires process optimization to improve the recovery rate.

[0039] Table 1 Purity, cell viability, and recovery rate of unwashed CD34+ cells before and after sorting

[0040]

[0041] Note: Recovery rate of live CD34+ cells (%) = number of live CD34+ cells after sorting / number of live CD34+ cells before sorting.

[0042] Example 2

[0043] To improve cell recovery and optimize the composition of the wash buffer, a single-sample blood sample was divided into three equal parts. The experimental conditions were the same as in Example 1, except that a wash step was performed before sorting in step (b). The wash buffer components were designed into the following three groups. The wash procedure involved adding sorting buffer to the single-sample blood sample in the transfer bag, centrifuging at 200g for 15 minutes, removing as much of the supernatant as possible, and adding wash buffer for the first wash. The sample was then centrifuged at 200g for 15 minutes, removing as much of the supernatant as possible, and repeating the wash cycle. The final volume was then filled with sorting buffer to a volume of 200-300 mL. This experiment was repeated five times.

[0044] Group 1: Wash twice with PBS (pH 7.3) + 2 mM EDTA (available on the market) before sorting.

[0045] Group 2: Washed twice with PBS (pH 7.3) + 2 mM EGTA before sorting;

[0046] Group 3: Wash twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA before sorting.

[0047] Table 2 Optimization of metal ion chelating agents in washing liquid

[0048]

[0049] 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, and both achieved 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.

[0050] Example 3

[0051] To further improve cell recovery, the wash solution composition was optimized. One batch of single-sample blood samples was divided equally into four aliquots. The experimental conditions were the same as in Example 2, except that a wash step was performed before sorting in step (b). The wash solution composition was designed into the following four groups. Five experiments were repeated. The experimental results are shown in Table 3, and the average values are used.

[0052] Group 4: Washed twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDAT before sorting;

[0053] Group 5: Washed twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA + 0.3% α-casein before sorting;

[0054] Group 6: Wash twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA + 50 ng / mL prostaglandin before sorting;

[0055] Group 7: Washed twice with PBS (pH 7.3) + 1.5 mM EGTA + 0.5 mM EDTA + 0.3% α-casein + 50 ng / mL prostaglandin before sorting;

[0056] Table 3 Purity, cell viability, and recovery rate of CD34+ cells washed with different components

[0057]

[0058] The results showed that adding 0.3% α-casein and / or 50 ng / mL prostaglandin (PGE1) to the washing solution resulted in a purity of CD34+ cells >80% and a cell viability >80% after sorting, both meeting sorting requirements. This significantly increased the recovery rate of CD34+ cells after sorting, reaching a specific range of 67.12-83.33%.

[0059] The recovery rates of live CD34+ cells in groups 5, 6, and 7 were significantly higher than those in group 4, and the differences were statistically significant (P<0.05).

[0060] Especially when α-casein and / or prostaglandin are added at the same time, the CD34+ recovery rate can reach more than 80%, indicating that the use of the washing solution with the above components for washing can effectively remove platelets and has a significant effect on improving the recovery rate.

[0061] 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 they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. 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 various 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) Take a single blood sample, centrifuge it, and remove the supernatant; (b) washing the blood sample with washing solution; (c) The washed blood samples are sorted based on cell surface proteins and cultured; The washing solution consists of PBS with a pH of 7.2-7.4, 1-3 mM EGTA, 0.3-0.8 mM EDTA, and 0.1%-0.5% α-casein, or PBS with a pH of 7.2-7.4, 1-3 mM EGTA, 0.3-0.8 mM EDTA, and 10-100 ng / mL prostaglandin PGE1, or PBS with a pH of 7.2-7.4, 1-3 mM EGTA, 0.3-0.8 mM EDTA, 0.1%-0.5% α-casein, and 10-100 ng / mL prostaglandin PGE1; The cell surface protein is CD34.

2. The method according to claim 1, characterized in that The washing solution consisted of PBS (pH 7.3), 1.5 mM EGTA, 0.5 mM EDTA, and 0.3% α-casein.

3. The method according to claim 1, characterized in that The washing solution consisted of PBS (pH 7.3), 1.5 mM EGTA, 0.5 mM EDTA, and 50 ng / mL prostaglandin PGE1.

4. The method according to claim 1, wherein The washing solution consisted of PBS (pH 7.3), 1.5 mM EGT A, 0.5 mM EDTA, 0.3% α-casein, and 50 ng / mL prostaglandin PGE1.

5. The method according to claim 1, wherein The sorting is performed based on the specific binding of the antibody against the cell surface protein to the cell surface protein.

6. The method according to claim 1, wherein The method further comprises performing anticoagulation treatment using an anticoagulant after step (a).

7. The method according to claim 6, characterized in that The anticoagulant is heparin or ACD anticoagulant.

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