A method for isolating and culturing umbilical cord blood-derived hematopoietic stem cells

Abstract

The present invention provides a method for isolating and culturing umbilical cord blood-derived hematopoietic stem cells, the method comprising preparing hematopoietic stem cells using a hydroxyethyl starch (HES) precipitation method, wherein a sodium selenite and phosphodipeptide are contained in a hydroxyethyl starch solution used. The hematopoietic stem cells obtained by the method of the present invention have increased recovery rate and increased removal rate of red blood cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hematopoietic stem cell preparation, in particular to a method for isolating and culturing hematopoietic stem cells from umbilical cord blood. BACKGROUND

[0002] As a kind of pluripotent stem cells, hematopoietic stem cells continuously supply red blood cells, white blood cells and platelets into blood. Hematopoietic stem cells have high self-renewal or self-replication capacity to maintain the number of stem cells at a certain level; at the same time, hematopoietic stem cells can further differentiate into progenitor cells and mature blood cells. Hematopoietic stem cells are usually divided into CD34 + cell population and CD34 - cell population, wherein CD34 + cell population accounts for more than 95%, while CD34 - cell population is less than 5%. Therefore, the hematopoietic stem cells we refer to are usually CD34 + cell population.

[0003] Hematopoietic stem cells have various sources, among which umbilical cord blood hematopoietic stem cells are a kind of cells with differentiation, self-renewal and proliferation capacity in umbilical cord blood. Moreover, umbilical cord blood hematopoietic stem cells can be specialized into various cells or tissues under the influence or induction of specific factors. In the medical field, umbilical cord blood hematopoietic stem cell transplantation can repair the hematopoietic function of the subject and improve the immune function. At present, umbilical cord blood hematopoietic stem cells have been widely used in the treatment of leukemia, autoimmune diseases, etc.

[0004] How to prepare high-quality hematopoietic stem cells from umbilical cord blood is a key factor for the therapeutic effect of hematopoietic stem cells. So far, many countries and regions have built standard umbilical cord blood banks. In the prior art, there are problems of low recovery rate and low red blood cell removal rate in isolating hematopoietic stem cells from umbilical cord blood. SUMMARY

[0005] The present application overcomes the above-mentioned shortcomings and provides a novel method for isolating and culturing hematopoietic stem cells from umbilical cord blood, which is simple to operate, has high yield and high purity.

[0006] Specifically, in a first aspect, the present application provides a novel method for preparing hematopoietic stem cells, characterized in that the method comprises using hydroxyethyl starch (HES) precipitation method to prepare hematopoietic stem cells, wherein the hydroxyethyl starch solution used contains sodium selenite and phosphoramidon.

[0007] The method for preparing hematopoietic stem cells of the present application comprises mixing hydroxyethyl starch solution and umbilical cord blood, centrifuging to collect white blood cell-rich plasma, and centrifuging to remove the plasma.

[0008] The novel method of preparing hematopoietic stem cells of the present invention further comprises one, two, three, four or five of the following steps:

[0009] a. obtaining cryopreserved and thawed umbilical cord blood;

[0010] b. preparing a hydroxyethyl starch solution and adding sodium selenite and phospho-dipeptide to the solution;

[0011] c. mixing the hydroxyethyl starch solution with the added sodium selenite and phospho-dipeptide with the umbilical cord blood;

[0012] d. centrifuging and collecting the white blood cell-enriched plasma;

[0013] e. centrifuging a second time to remove the plasma.

[0014] In an embodiment of the present invention, the concentration of the sodium selenite is 1-100 μM: the concentration of the phospho-dipeptide is 0.1-50 μM. Illustratively, the concentration of the sodium selenite is 10-50 μM, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 μM; the concentration of the phospho-dipeptide is 0.5-30 μM, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 μM.

[0015] Further preferably, the concentration of the sodium selenite is 10-20 μM, and the concentration of the phospho-dipeptide is 2-10 μM. Illustratively, when the concentration of the sodium selenite is 10-20 μM, the concentration of the phospho-dipeptide is 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. Alternatively, when the concentration of the phospho-dipeptide is 2-10 μM, the concentration of the sodium selenite is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 μM. The concentrations of the sodium selenite and the phospho-dipeptide can be combined in any manner, for example, the concentration of the sodium selenite is 10 μM and the concentration of the phospho-dipeptide is 2 μM; or the concentration of the sodium selenite is 20 μM and the concentration of the phospho-dipeptide is 10 μM.

[0016] Further, the concentration of the hydroxyethyl starch solution is 6%.

[0017] The ratio of the hydroxyethyl starch solution to the umbilical cord blood is 1:4-1:6, preferably 1:5.

[0018] In step a, the cord blood is from a cord blood bank or from a cord blood sample that is in a collection bag within 12 hours of collection.

[0019] In step b, the sodium selenite and phosphodipeptide are added to the hydroxyethyl starch solution such that the sodium selenite and phosphodipeptide are thoroughly mixed with the hydroxyethyl starch solution.

[0020] In step c, the hydroxyethyl starch solution is mixed with the cord blood by means of a shaker or by hand, for example by shaking on a shaker for 5-10 minutes or by hand inversion.

[0021] In step d, the centrifugation is the first centrifugation and is at a temperature of less than 15°C, for example at 5-15°C, at 40-60g for 5-10 minutes.

[0022] In step e, the centrifugation is the second centrifugation and is at a temperature of less than 15°C, for example at 5-15°C, at 300-500g for 5-10 minutes.

[0023] Optionally, the mononuclear cells isolated from the cord blood are subjected to adherent culture such that the mononuclear cells are expanded and then subjected to adherent purification, i.e. purified hematopoietic stem cells.

[0024] The method of preparing hematopoietic stem cells according to the present application is well tolerated by the subject as the method does not cause any harm to the subject's body as compared to other approaches.

[0025] It has been found that the hematopoietic stem cells isolated from cord blood samples are more viable. Moreover, a large amount of cord blood is discarded if not collected. Therefore, isolation of hematopoietic stem cells from cord blood is a desirable way of converting waste into a valuable resource.

[0026] In a second aspect, the present application provides a hematopoietic stem cell prepared by the isolation method of the present application.

[0027] Optionally, the stem cell is a hematopoietic stem cell prepared by isolation which is further expanded in vitro and purified.

[0028] In a third aspect, the present application provides the use of sodium selenite and / or phosphodipeptide in the preparation of a kit for isolating hematopoietic stem cells from cord blood.

[0029] Exemplarily, the present application provides the use of sodium selenite in the preparation of a kit for isolating hematopoietic stem cells from cord blood.

[0030] Further, the present application provides use of phosphodipeptide in the preparation of a kit for isolating hematopoietic stem cells from umbilical cord blood.

[0031] Alternatively, the present application provides use of a combination of sodium selenite and phosphodipeptide in the preparation of a kit for isolating hematopoietic stem cells from umbilical cord blood.

[0032] Illustratively, the present application also provides a kit comprising a combination of sodium selenite and phosphodipeptide, wherein the ratio (mass) of sodium selenite to phosphodipeptide can be about 1:0.01 to about 1:99, preferably about 1:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, and the ratio range between each of the above ratios; or about 0.1:1 to about 100:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 1:1, about 1:1.1, about 1.2:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and the ratio range between each of the above ratios.

[0033] Further, the present application also provides a kit comprising sodium selenite and phospho-dipeptide provided separately, wherein the ratio (mass) of sodium selenite and phospho-dipeptide can be about 1:0.01 to about 1:99, preferably about 1:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, and the ratio range between each of the above ratios; or about 0.1:1 to about 100:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 1:1, about 1:1.1, about 1.2:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and the ratio range between each of the above ratios.

[0034] In a fourth aspect, the present application provides a hydroxyethyl starch solution comprising sodium selenite and phospho-dipeptide.

[0035] In an embodiment of the present application, the concentration of sodium selenite is 1-100 μM, and the concentration of phospho-dipeptide is 0.1-50 μM. Illustratively, the concentration of sodium selenite is 10-50 μM, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 μM; and the concentration of phospho-dipeptide is 0.5-30 μM, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 μM.

[0036] Further preferably, the concentration of sodium selenite is 10-20 μM, and the concentration of phospho-dipeptide is 2-10 μM. Illustratively, when the concentration of sodium selenite is 10-20 μM, the concentration of phospho-dipeptide is 2, 3, 4, 5, 6, 7, 8, 9, or 10 μM. Alternatively, when the concentration of phospho-dipeptide is 2-10 μM, the concentration of sodium selenite is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM. The concentrations of sodium selenite and phospho-dipeptide can be combined in any manner, for example, the concentration of sodium selenite is 10 μM and the concentration of phospho-dipeptide is 2 μM; or the concentration of sodium selenite is 20 μM and the concentration of phospho-dipeptide is 10 μM.

[0037] Further, the concentration of hydroxyethyl starch solution is 6%.

[0038] In a fifth aspect, the present application provides a composition comprising sodium selenite and phospho-dipeptide.

[0039] Illustratively, the ratio (mass) of sodium selenite and phospho-dipeptide can be about 1:0.01 to about 1:99, preferably about 1:1, about 1:1.5, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, and the ratio range between each of the above ratios; or about 0.1:1 to about 100:1, for example, about 0.1:1, about 0.2:1, about 0.3:1, about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about 1:1, about 1:1.1, about 1.2:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and the ratio range between each of the above ratios.

[0040] In a sixth aspect, the present application provides the use of hematopoietic stem cells isolated or cultured by the method of the present application in the treatment of leukemia or immunological diseases.

[0041] In specific embodiments, the leukemia includes acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, and mixed cell leukemia, etc.

[0042] An immunological disease refers to a pathological reaction caused by damage to the immune system. For example, infectious diseases, hypersensitivity diseases, autoimmune diseases, immune proliferative diseases, immune deficiency diseases, and immune-related diseases, etc. Illustratively, the immunological disease is lupus erythematosus, ankylosing spondylitis, rheumatoid arthritis, psoriasis, erythoderma, glomerulonephritis, ANCA-associated vasculitis, scleroderma, primary systemic amyloidosis, autoimmune hepatitis, autoimmune pancreatitis, autoimmune gastritis, Crohn's disease, ulcerative colitis, erythema nodosum, thyroiditis, alopecia areata, diabetes-related.

[0043] The hematopoietic stem cell population of the present application can be formulated and administered as a pharmaceutical composition. Illustratively, the pharmaceutical composition is in any form known in the medical field, for example, an injection (e.g., an injection solution, a lyophilized powder). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution (e.g., an isotonic saline solution or physiological saline), dispersion, suspension, or emulsion.

[0044] In a seventh aspect, the present application provides a medicament for treating leukemia or an immunological disease, comprising the hematopoietic stem cell isolated or cultured by the method of the present application.

[0045] Illustratively, the growth curve of the isolated hematopoietic stem cell is determined by a WST method, an MTT method, an ATP detection method, a DNA content detection method, etc. to evaluate the growth activity of the umbilical cord hematopoietic stem cell. In addition, the isolated and cultured umbilical cord hematopoietic stem cell can be identified by detecting cell surface markers by flow cytometry, detecting cell expressed genes by a PCR method.

[0046] In an eighth aspect, the present application provides a pharmaceutical composition comprising the hematopoietic stem cell isolated or cultured by the present application. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient, or other possible required adjuvants.

[0047] In this context, the term "adjuvant" refers to substances other than the active ingredient (hematopoietic stem cell) that are necessary for the manufacture or formulation of a pharmaceutical preparation. In general, these substances are not physiologically active and do not affect the drug efficacy, content determination, or stability of the pharmaceutical composition in the pharmaceutical preparation. The main purpose of adding adjuvants is to facilitate the preparation of the pharmaceutical composition and facilitate clinical application. Preferably, the adjuvant used in the pharmaceutical composition of the present application is pharmaceutical and also does not conflict with the active ingredient (hematopoietic stem cell).

[0048] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the hematopoietic stem cell.

[0049] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable biomaterial, including but not limited to collagen, gelatin, aminated gelatin, chitosan, cellulose, fibrin, polylactic acid, cellulose polylactic acid, polyurethane, tropoelastin, hyaluronic acid, sodium alginate, polyethylene glycol, poly(lactic-co-glycolic acid), polycaprolactone, silicate, silicone rubber, extracellular matrix, or any combination thereof, and the like. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solution (e.g., isotonic saline or physiological saline), dispersion, emulsion, or suspension.

[0050] In certain embodiments, the pharmaceutical composition can be in any form known in the medical field. For example, the pharmaceutical composition can be in the form of a tablet, pill, suspension, emulsion, solution, gel, capsule, powder, granule, elixir, lozenge, suppository, injection (e.g., injection solution, lyophilized powder), and the like. In certain preferred embodiments, the pharmaceutical composition is an injection (including injection solution, lyophilized powder) or an aerosol.

[0051] Further exemplarily, in the case where the pharmaceutical composition is an injection formulation, in certain embodiments, the mesenchymal stem cells are administered at a dose of at least 1 x 10 5 cells / mL (e.g., at least 2 x 10 5 cells / ml, at least 3 x 10 5 cells / ml, at least 5 x 10 5 cells / ml, at least 7 x 10 5 cells / ml, at least 1 x 10 6 cells / ml, at least 3 x 10 6 cells / ml, at least 5 x 10 6 cells / ml, at least 7 x 10 6 cells / ml, at least 1 x 10 7 cells / ml, at least 3 x 10 7 cells / ml, at least 5 x 10 7 cells / ml, at least 7 x 10 7 cells / ml, at least 1 x 10 8 cells / ml, at least 3 x 10 8 cells / ml, at least 5 x 10 8 cells / ml, at least 7 x 10 8 cells / ml, at least 1 x 10 9 cells / ml, at least 3 x 10 9 cells / ml, at least 5 x 10 9 cells / ml, at least 7 x 10 9 cells / ml.

[0052] In certain embodiments, the subject to which the pharmaceutical composition is directed is a mammal, e.g., a human.

[0053] In a ninth aspect, the present application provides a method of treating a disease, comprising administering to a subject a pharmaceutical composition comprising hematopoietic stem cells of the present application.

[0054] In some embodiments, the medicament is in unit dosage form, and the unit dosage (e.g., per milliliter) of medicament contains at least 1 x 10 4 (e.g., at least 1 x 10 4 (e.g., at least 3 x 10 4 (e.g., at least 5 x 10 4 (e.g., at least 7 x 10 4 (e.g., at least 1 x 10 5 (e.g., at least 3 x 10 5 (e.g., at least 5 x 10 5 (e.g., at least 7 x 10 5 (e.g., at least 1 x 10 6 (e.g., at least 3 x 10 6 (e.g., at least 5 x 10 6 (e.g., at least 7 x 10 6 (e.g., at least 1 x 10 7 (e.g., at least 3 x 10 7 (e.g., at least 5 x 10 7 (e.g., at least 7 x 10 7 (e.g., at least 1 x 10 8 (e.g., at least 3 x 10 8 (e.g., at least 5 x 10 8 (e.g., at least 7 x 10 8 (e.g., at least 1 x 10 9 (e.g., at least 3 x 10 9 (e.g., at least 5 x 10 9 (e.g., at least 7 x 10 9 (e.g., at least 1 x 10 10 (e.g., at least 3 x 10 10 (e.g., at least 5 x 10 10 (e.g., at least 7 x 10 10 (e.g., 3-6 x 10 6 (e.g., at least 1 x 10

[0055] In certain embodiments, the subject is a mammal, e.g., a human.

[0056] In certain embodiments, the prophylactically and / or therapeutically effective amount of hematopoietic stem cells is administered to the subject by injection, mucosally, enterally, orally, by the respiratory tract, or dermally.

[0057] Advantages

[0058] The present inventors surprisingly found that the hematopoietic stem cells obtained by the method of the present application and then cultured have strong uniformity and high cell viability, mainly manifested in fast cell expansion speed and high passage number, compared with the mainstream separation methods of the prior art. Unexpectedly, the addition of sodium selenite and phosphodipeptide increases the recovery rate of hematopoietic stem cells obtained by the method of the present application and the removal rate of red blood cells. These two compounds may promote the recovery of nucleated cells and CD34 cells by affecting the hematopoietic system.

[0059] Sodium selenite and phosphodipeptide are two different compounds, and the reason why their combination has an impact on the recovery rate of nucleated cells and the removal rate of red blood cells may be related to their mechanisms of action in the body.

[0060] Sodium selenite is a selenium-containing compound, and selenium has an antioxidant effect in the body, which can promote the proliferation and differentiation of hematopoietic stem cells, and thus may lead to an increase in the recovery rate of nucleated cells and CD34 cells.

[0061] Phosphodipeptide is a phosphorus-containing compound, and phosphorus participates in many biochemical processes in cells, including energy metabolism and cell signaling. Phosphodipeptide may affect the function of hematopoietic stem cells by regulating cell metabolism and signaling pathways, thereby improving the recovery rate of nucleated cells and CD34 cells. DETAILED DESCRIPTION

[0062] In order to make the objects, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below.

[0063] Example 1

[0064] a. Thaw the cryopreserved umbilical cord blood, and mix 6% hydroxyethyl starch solution and umbilical cord blood at a ratio of 1:5;

[0065] b. Slowly shake the mixture prepared in step a on a shaker for 10 min;

[0066] c. First centrifugation: centrifuge at 50g and 10°C for 7 min, and then collect the upper white blood cell-enriched plasma;

[0067] d. Second centrifugation: centrifuge at 400g and 10°C for 15 min to remove the plasma and obtain hematopoietic stem cells,

[0068] wherein the hydroxyethyl starch solution contains phosphodipeptide at a concentration of 2 μM and sodium selenite at a concentration of 5 μM.

[0069] Example 2

[0070] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood in a ratio of 1 :5;

[0071] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0072] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, then the upper layer of white blood cell enriched plasma was collected;

[0073] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, the plasma was removed to obtain hematopoietic stem cells,

[0074] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 2 μM and sodium selenite at a concentration of 10 μM.

[0075] Example 3

[0076] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood in a ratio of 1 :5;

[0077] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0078] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, then the upper layer of white blood cell enriched plasma was collected;

[0079] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, the plasma was removed to obtain hematopoietic stem cells,

[0080] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 2 μM and sodium selenite at a concentration of 15 μM.

[0081] Example 4

[0082] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood in a ratio of 1 :5;

[0083] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0084] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, then the upper layer of white blood cell enriched plasma was collected;

[0085] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, the plasma was removed to obtain hematopoietic stem cells,

[0086] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 2 μM and sodium selenite at a concentration of 20 μM.

[0087] Example 5

[0088] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood in a ratio of 1 :5;

[0089] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0090] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, then the upper layer of white blood cell enriched plasma was collected;

[0091] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, the plasma was removed to obtain hematopoietic stem cells,

[0092] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 2 μΜ and sodium selenite at a concentration of 50 μΜ.

[0093] Example 6

[0094] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood in a ratio of 1 :5;

[0095] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0096] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, then the upper layer of white blood cell enriched plasma was collected;

[0097] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, the plasma was removed to obtain hematopoietic stem cells,

[0098] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 3 μΜ and sodium selenite at a concentration of 10 μΜ.

[0099] Example 7

[0100] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood in a ratio of 1 :5;

[0101] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0102] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, then the upper layer of white blood cell enriched plasma was collected;

[0103] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, the plasma was removed to obtain hematopoietic stem cells,

[0104] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 5 μΜ and sodium selenite at a concentration of 10 μΜ.

[0105] Example 8

[0106] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood at a ratio of 1 :5;

[0107] b. The mixture prepared in step a was shaken slowly on a shaker for 10 min;

[0108] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, and then the upper layer of white blood cell-enriched plasma was collected;

[0109] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, and the plasma was removed to obtain hematopoietic stem cells,

[0110] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 10 μΜ and sodium selenite at a concentration of 10 μΜ.

[0111] Example 9

[0112] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood at a ratio of 1 :5;

[0113] b. The mixture prepared in step a was shaken slowly on a shaker for 10 min;

[0114] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, and then the upper layer of white blood cell-enriched plasma was collected;

[0115] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, and the plasma was removed,

[0116] wherein the hydroxyethyl starch solution contains phosphorodipeptide at a concentration of 15 μΜ and sodium selenite at a concentration of 10 μΜ.

[0117] Example 10

[0118] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood at a ratio of 1 :5;

[0119] b. The mixture prepared in step a was shaken slowly on a shaker for 10 min;

[0120] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, and then the upper layer of white blood cell-enriched plasma was collected;

[0121] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, and the plasma was removed to obtain hematopoietic stem cells,

[0122] wherein the hydroxyethyl starch solution contains phospho dipeptide at a concentration of 20 μΜ and sodium selenite at a concentration of 10 μΜ.

[0123] Comparative Example 1

[0124] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood at a ratio of 1 :5;

[0125] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0126] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, and then the upper layer of white blood cell enriched plasma was collected;

[0127] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, and the plasma was removed to obtain hematopoietic stem cells.

[0128] Comparative Example 2

[0129] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood at a ratio of 1 :5;

[0130] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0131] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, and then the upper layer of white blood cell enriched plasma was collected;

[0132] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, and the plasma was removed to obtain hematopoietic stem cells, wherein the hydroxyethyl starch solution contains phospho dipeptide at a concentration of 2 μΜ.

[0133] Comparative Example 3

[0134] a. Thawed cord blood was resuscitated and mixed with 6% hydroxyethyl starch solution and cord blood at a ratio of 1 :5;

[0135] b. The mixture prepared in step a was mixed on a shaker at a slow speed for 10 min;

[0136] c. First centrifugation: centrifuged at 50g, 10°C for 7 minutes, and then the upper layer of white blood cell enriched plasma was collected;

[0137] d. Second centrifugation: centrifuged at 400g, 10°C for 15 min, and the plasma was removed to obtain hematopoietic stem cells, wherein the hydroxyethyl starch solution contains phospho dipeptide at a concentration of 10 μΜ.

[0138] Comparative Example 4

[0139] a. Thaw the cryopreserved cord blood and mix the 6% hydroxyethyl starch solution and the cord blood at a ratio of 1:5;

[0140] b. Slowly mix the mixture prepared in step a on a shaker for 10 min;

[0141] c. First centrifugation: centrifuge at 50g, 10°C for 7 min, and then collect the upper white blood cell-enriched plasma;

[0142] d. Second centrifugation: centrifuge at 400g, 10°C for 15 min, remove the plasma, and obtain the hematopoietic stem cells, wherein the concentration of sodium selenite is 10 μM.

[0143] Comparative Example 5

[0144] a. Thaw the cryopreserved cord blood and mix the 6% hydroxyethyl starch solution and the cord blood at a ratio of 1:5;

[0145] b. Slowly mix the mixture prepared in step a on a shaker for 10 min;

[0146] c. First centrifugation: centrifuge at 50g, 10°C for 7 min, and then collect the upper white blood cell-enriched plasma;

[0147] d. Second centrifugation: centrifuge at 400g, 10°C for 15 min, remove the plasma, and obtain the hematopoietic stem cells, wherein the concentration of sodium selenite is 30 μM.

[0148] The nucleated cells prepared in the above examples were subjected to nucleated cell recovery rate, CD34 cell recovery rate, and red blood cell removal rate analysis, and the results are as follows:

[0149] Analysis results of cord blood-derived hematopoietic stem cells

[0150] Example Nucleated cell recovery (%) CD34 cell recovery (%) Red cell depletion (%) Example 1 91.2 95.3 96.8 Example 2 92.6 96.8 97.3 Example 3 91.7 94.5 96.5 Example 4 92.3 96.1 98.1 Example 5 93.7 97.3 96.9 Example 6 93.4 95.2 97.3 Example 7 92.7 94.6 97.1 Example 8 92.1 94.9 97.6 Example 9 91.6 93.2 98.3 Example 10 92.1 94.2 95.9 Comparative Example 1 85.3 88.3 79.5 Comparative Example 2 82.9 83.4 81.2 Comparative Example 3 86.8 82.5 80.4 Comparative Example 4 83.6 81.2 81.1 Comparative Example 5 82.5 83.5 82.6

[0151] As can be seen, the hydroxyethyl starch solution added with both the combination of phosphorodipeptide and sodium selenite can improve the nucleated cell recovery rate, CD34 cell recovery rate, and red blood cell removal rate in the separation of hematopoietic stem cells from cord blood, and is not sensitive to the concentration, and can achieve significantly better results above 2 μM.

[0152] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for isolating cord blood stem cells from cord blood, comprising the steps of: a. obtaining cord blood; b. preparing a hydroxyethyl starch solution and adding sodium selenite and phospho-dipeptide to the solution; c. mixing the hydroxyethyl starch solution with the addition of sodium selenite and phospho-dipeptide with the cord blood; d. first centrifugation: centrifuging and collecting the white blood cell-rich plasma; e. second centrifugation: centrifuging to remove the plasma, wherein the concentration of sodium selenite is 10-20 μM; and the concentration of phospho-dipeptide is 2-10 μM.

2. The method according to claim 1, wherein the concentration of sodium selenite is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM; and the concentration of phospho-dipeptide is 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM.

3. The method according to claim 1 or 2, wherein the concentration of the hydroxyethyl starch solution is 6%.

4. The method according to claim 1 or 2, wherein the ratio of the hydroxyethyl starch solution to cord blood is 1:4-1:

6.

5. The method according to claim 4, wherein the ratio of the hydroxyethyl starch solution to cord blood is 1:

5.

6. The method according to claim 1 or 2, further comprising further culturing the isolated hematopoietic stem cells in vitro.