A bone marrow preservation solution for efficient acquisition of hematopoietic stem cells and methods of use

Abstract

The application provides a bone marrow storage solution for efficiently obtaining hematopoietic stem cells, which comprises a phosphate buffer solution of non-essential amino acids and EDTA-Na2. The bone marrow tissue storage solution prepared by using the non-essential amino acids and the EDTA-Na2 buffer solution not only maintains the activity of the bone marrow tissue and the hematopoietic stem cells, but also prevents the bone marrow tissue from being anticoagulated, so that the hematopoietic stem cells are efficiently separated.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell biology, and particularly relates to a bone marrow storage solution for efficiently obtaining hematopoietic stem cells and a use method. BACKGROUND

[0002] Hematopoietic stem cells are adult stem cells in the blood system, have the ability of long-term self-renewal and the potential of differentiating into various mature blood cells. It is a kind of adult stem cell with the longest research history and the most in-depth research, and has important guiding significance for the research of various stem cells, and is widely used in blood system diseases and autoimmune diseases. Hematopoietic stem cells mainly exist in bone marrow, umbilical cord blood, placenta and peripheral blood.

[0003] The essence of bone marrow transplantation, i.e. hematopoietic stem cell transplantation, is to reconstruct the normal hematopoiesis and immune system of patients by intravenous infusion of hematopoietic stem cells, progenitor cells and the like, so as to treat a series of diseases. It is the most effective method for treating leukemia at present, and is also applied to the treatment or auxiliary treatment of more than 40 diseases such as autoimmune diseases and hemoglobin.

[0004] Among all tissues, the content of hematopoietic stem cells in bone marrow is the highest, but the extraction of hematopoietic stem cells from bone marrow needs to pass through bone marrow puncture technology, which causes great harm to the donor; at the same time, hematopoietic stem cells are difficult to be massively expanded in vitro, so the acquisition of the number of hematopoietic stem cells is one of the key links in the process of bone marrow transplantation.

[0005] After the bone marrow is extracted by the bone marrow puncture technology, it needs to be transported to a professional laboratory for extraction of hematopoietic stem cells, so it is particularly important to maintain the activity of bone marrow tissue when the bone marrow is preserved. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a bone marrow storage solution for efficiently obtaining hematopoietic stem cells. The bone marrow tissue storage solution provided by the present application not only maintains the activity of bone marrow tissue and hematopoietic stem cells, but also prevents the bone marrow tissue from being anticoagulated, so as to achieve the purpose of efficiently separating hematopoietic stem cells.

[0007] The present application provides a bone marrow storage solution for efficiently obtaining hematopoietic stem cells, comprising: a non-essential amino acid and an EDTA-Na2 phosphate buffer.

[0008] Further, the bone marrow storage solution for efficiently obtaining hematopoietic stem cells comprises:

[0009] 0.1 mM of a non-essential amino acid solution;

[0010] 10-20 mg / ml of an EDTA-Na2 phosphate buffer.

[0011] The bone marrow storage solution for efficiently obtaining hematopoietic stem cells provided by the application comprises a 0.1 mM non-essential amino acid solution, wherein the 0.1 mM non-essential amino acid solution is obtained by diluting MEM NEAA non-essential amino acid solution 10 mM (100X) by 100 times.

[0012] The bone marrow storage solution provided by the application further comprises 10-20 mg / ml EDTA-Na2 phosphate buffer.

[0013] In some specific embodiments of the application, the bone marrow storage solution comprises:

[0014] 0.1 mM non-essential amino acid solution;

[0015] 10 mg / ml EDTA-Na2 phosphate buffer.

[0016] In some specific embodiments of the application, the bone marrow storage solution comprises:

[0017] 0.1 mM non-essential amino acid solution;

[0018] 15 mg / ml EDTA-Na2 phosphate buffer.

[0019] In some specific embodiments of the application, the bone marrow storage solution comprises:

[0020] 0.1 mM non-essential amino acid solution;

[0021] 20 mg / ml EDTA-Na2 phosphate buffer.

[0022] The application further provides a use method of the bone marrow storage solution for efficiently obtaining hematopoietic stem cells, comprising the following steps:

[0023] mixing the bone marrow and the bone marrow storage solution in equal volume and then storing at 2-8 DEG C for 24 hours to separate hematopoietic stem cells.

[0024] Compared with the prior art, the application provides a bone marrow storage solution for efficiently obtaining hematopoietic stem cells, which comprises non-essential amino acid and EDTA-Na2 phosphate buffer. The bone marrow tissue storage solution prepared by using non-essential amino acid and EDTA-Na2 buffer not only maintains the activity of bone marrow tissue and hematopoietic stem cells, but also prevents the bone marrow tissue from being anticoagulated, thereby achieving the purpose of efficiently separating hematopoietic stem cells.

[0025] The bone marrow preservation solution provided by the present invention has no side effects on bone marrow tissue, cells, or the human body, and can be used in subsequent scientific research and clinical settings. This preservation solution formula can significantly increase the number and viability of hematopoietic stem cells without affecting their phenotype, making it suitable for clinical transplantation therapy. DETAILED DESCRIPTION

[0026] To further understand the present invention, the bone marrow preservation fluid for efficiently obtaining hematopoietic stem cells and the method of use provided by the present invention are described below in conjunction with examples. The scope of protection of the present invention is not limited by the following examples.

[0027] In the following examples, the non-essential amino acids used were commercial solutions, MEM NEAA non-essential amino acid solution 10 mM (100X).

[0028] Example

[0029] 1. Bone marrow tissue preservation solution formula:

[0030] Experimental group:

[0031] The bone marrow tissue preservation solution was 1*non-essential amino acids, 10-20 mg / ml EDTA-Na2 phosphate buffer (the concentrations of EDTA-Na2 phosphate buffer in experimental groups 1-3 were 10 mg / ml, 15 mg / ml and 20 mg / ml, respectively).

[0032] Comparison group:

[0033] 1× PBS phosphate buffer

[0034] 2. Bone marrow collection and storage

[0035] One donor aged 20 to 35 years without infectious diseases was selected and signed an informed consent form and a surgical consent form. In the operating room, under epidural anesthesia, a closed bone marrow aspiration device was used to extract bone marrow from both iliac bones. The amount of marrow collected was 60 ml, and sodium heparin was used for anticoagulation during marrow collection.

[0036] The bone marrow was transported to a professional laboratory at low temperature and divided into four portions, each 15 ml. An equal volume of bone marrow tissue preservation fluid was added to the experimental group, and the same volume of phosphate buffer was added to the control group. The samples were stored at 2 to 8 degrees Celsius and hematopoietic stem cells were separated after 24 hours of storage.

[0037] 3. Hematopoietic Stem Cell Isolation

[0038] Bone marrow tissue was transferred to 150ml storage bottle after 24h preservation, 6% hydroxyethyl starch was added in the ratio of 4:1, and the upper liquid was separated after 20-30min natural settlement. The separated cells were centrifuged at 1500r / min for 10min at 4℃, and then washed twice with normal saline for detection of cell number, cell viability and cell surface markers.

[0039] 4. Detection of cell number and cell viability

[0040] The separated cells of each group were subjected to automatic cell counting for calculation and comparison of cell number and cell viability.

[0041] 5. Detection of cell surface markers

[0042] The separated hematopoietic stem cells of each group were washed twice with PBS, and the cell density was adjusted to 5*10 5 / ml. The cells were incubated with CD34-APC flow cytometry antibody in the dark for 20min. After incubation, the cells were washed twice with PBS to remove unbound antibodies, and the cell samples were subjected to flow cytometry detection.

[0043] Analysis of experimental results:

[0044] 1. Comparison of cell number and cell viability

[0045] Table 1 Comparison of cell number and cell viability

[0046]

[0047] The results of cell number and cell viability showed that there was a significant difference in cell number and cell viability between the experimental group and the control group (P<0.01), indicating that the formula of the experimental group could significantly improve the number and viability of the separated cells (P<0.01).

[0048] 2. Comparison of cell surface marker detection

[0049] Table 2 Comparison of cell surface marker detection

[0050]

[0051] The results of cell surface marker detection showed that there was a significant difference in cell surface marker between the experimental group and the control group (P<0.05), indicating that the formula of the experimental group could significantly improve the number and viability of the separated cells without affecting the characteristics of the cells.

[0052] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. Use of a bone marrow preservation solution composition for efficiently obtaining hematopoietic stem cells in preparing a bone marrow preservation solution for hematopoietic stem cells, characterized in that: The bone marrow preservation solution composition consists of non-essential amino acids and EDTA-Na2 phosphate buffer, and the non-essential amino acids are MEM NEAA non-essential amino acid solution 10 mM (100X).

2. The use according to claim 1, characterized in that The bone marrow preservation solution is composed of the following raw materials: 0.1 mM non-essential amino acid solution; 10-20 mg / ml EDTA-Na2 phosphate buffer.

3. The use according to claim 1, characterized in that The 0.1 mM non-essential amino acid solution is obtained by diluting a 10 mM (100X) MEMNEAA non-essential amino acid solution 100 times.

4. The use according to claim 1, characterized in that include: 0.1 mM non-essential amino acid solution; 10 mg / ml EDTA-Na2 in phosphate buffer.

5. The use according to claim 1, characterized in that The bone marrow preservation solution is composed of the following raw materials: 0.1 mM non-essential amino acid solution; 15 mg / ml EDTA-Na2 in phosphate buffer.

6. The use according to claim 1, characterized in that The bone marrow preservation solution is composed of the following raw materials: 0.1 mM non-essential amino acid solution; 20 mg / ml EDTA-Na2 in phosphate buffer.

7. The use according to claim 1, characterized in that The method for using the bone marrow preservation solution for efficiently obtaining hematopoietic stem cells comprises the following steps: After mixing equal volumes of bone marrow and bone marrow preservation fluid, store them at 2-8°C for 24 hours for hematopoietic stem cell isolation.

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