WO2010000125A1 - The key technologies of isolation, purification, freeze-drying, anabiosis of fetal liver hemopoietic stem cell and the preparation methods - Google Patents

Abstract

A culture solution useful for enhancing expression of fetal liver hemopoietic stem cell CD34 and its preparation methods are provided. The culture solution comprises (wt.%): 56.5-64.7% of MEM, 29.4-30.4% of RPMI-1640, 5.88-13.0% of Hanks etc. The preparation methods of such culture solution comprise: (a) preparing the basic culture solution; (b) taking MEM, RPMI-1640, Hanks according to the proportion and adding to the basic culture solution, stirring and dissolving; (c) implanting purified fetal liver mesenchymal cells to culture flasks, culturing, washing, and mixing; continuously culturing and collecting all supernatant fluid, filtering and centrifuging the supernatant fluid to remove all the cell debris and insoluble substances to obtain conditioned culture solution; (d) mixing the conditioned culture solution with MEM, regulating pH value of the mixed culture solution to 7.40-7.45 with sodium bicarbonate, filtering to obtain the culture solution. The culture solution enhances expression of fetal liver hemopoietic stem cell CD34, and improves markable number of stem cell. It is safe and reliable, and meets the clinical application.

Description

 Description Key technologies and preparation methods for separation, purification, lyophilization and resuscitation of fetal stem cells

 The invention relates to a culture liquid in the field of bioengineering technology and a preparation method thereof. Specifically, it relates to a culture solution for enhancing CD34 expression of fetal liver hematopoietic stem cells and a preparation method thereof. Background technique

 Hematopoietic stem cells have been proposed at the beginning of the last century and have been shown to have therapeutic potential in clinical applications. Many blood system diseases have improved the clinical symptoms of patients after stem cell transplantation, and the survival of patients has been significantly prolonged. The main sources of hematopoietic stem cells include bone marrow-derived hematopoietic stem cells, cord blood hematopoietic stem cells, and fetal liver hematopoietic stem cells. Due to the different sources of hematopoietic stem cells, the antigenicity of stem cells is also different. The main manifestations are: Bone marrow-derived hematopoietic stem cells have strong antigenicity (eg, HLA), bone marrow-derived hematopoietic stem cells implanted in the body, if not matched, first host The immune system rejects hematopoietic stem cells implanted in the body. The input of antigenic stem cells also rejects the host, and strong rejection causes a series of systemic immune allergies and leads to dysfunction and depletion of human organs. Therefore, the implantation of bone marrow-derived hematopoietic stem cells must be completely compared with the antigenic type of the donor and the ligand, and the donor and host antigens can be found to be "identical" or "close" in order to be treated for cell implantation. Ultimately, patients have only 100,000 or one in a million chances to find a donor that meets their own cell requirements, and clinical application is extremely difficult.

Hematopoietic stem cells provided by cord blood are weaker than bone marrow-derived hematopoietic stem cells. Therefore, cord blood hematopoietic stem cells are ideal for stem cells with clinical therapeutic value, and their body rejection is lighter than bone marrow-derived hematopoietic stem cells. Due to the limited number of cord blood hematopoietic stem cells, the clinical requirements for implanting stem cells to repair the body in adults and older children cannot be met. Due to the proliferation of cord blood hematopoietic cells in vitro, many key technical problems remain unsolved, and clinical application is difficult. Compared with the former two, fetal liver hematopoietic stem cells have the following advantages. First, hematopoietic stem cell antigenicity is weaker than cord blood hematopoietic stem cells, so stem cell antigen matching is relatively simple and easy to find in small-scale human tissue donors. Second, fetal liver hematopoietic tissue contains a considerable number of stem cells (5Q) ~120xl0 ⁶ ), while cord blood hematopoietic stem cells only have 1~ 2xl0 ⁶ , the number of cord blood hematopoietic cells can only meet the requirements of patients with body weight ≤ 20 kg; Third, stem cells have high survival rate and strong differentiation . The cell colonies produced by unit cells are higher than those of bone marrow and umbilical cord blood-derived hematopoietic stem cells. The purified stem cells only need ABQ, HLA, and six-point matching, so they can be used in patients. The success rate of matching can be as high as 25%. .

After searching, the Chinese patent application number: 02134314.4, public number: CN1389566, name: nerve dry Cell culture medium and preparation method thereof, the technology self-reported as: a neural stem cell culture medium and a preparation method thereof, a basic culture solution prepared by mixing DMEM and F12 according to 1:1, insulin and butanediamine hydrochloride It is prepared by sodium selenide, hydrocortisone, L-glutamine, human transferrin and progesterone. It has the function of directional development and differentiation of seed cells from different sources such as bone marrow tissue into neural stem cells, which can be accurately and at any time. Medical research teaching and clinical application provide relevant neural stem cells. Since the CD34 expression of hematopoietic stem cells is greatly affected by the microenvironment, some stem cells have very weak CD34 expression in some cycles, while some stem cells have strong CD34 expression in the same cycle (this phenomenon is also called "drift". expression";). This biological property can only purify CD34-expressing stem cells (labeling CD34 cells by fluorescently labeled antibody or CD34 isolation kit) using current purification methods, while stem cells with weak CD34 expression in the same cycle are not effective. Markers are ignored, and the biological characteristics of stem cells (CD34 expression) allow current purification methods to purify only those identified thousand cells, making stem cells inefficiently purified and ineffective in purifying sufficient hematopoiesis Stem cells are used clinically to provide patients with multiple treatment opportunities.

 The technical function is to provide a cell culture medium having the function of directing and differentiation of seed cells of different origins such as bone marrow into neural stem cells. Due to the uncertainty of the cells in the process of directed development and differentiation of neural stem cells, whether the differentiated cells inherit and have the characteristics of primitive stem cells is still uncertain, so there is a greater risk in clinical application. Disclosure of invention

 SUMMARY OF THE INVENTION The object of the present invention is to provide a culture solution for enhancing the expression of CD34 of fetal liver hematopoietic stem cells and a preparation method thereof, in view of the deficiencies of the prior art. The culture solution of the present invention has the function of performing enhanced expression on the CD34 expression characteristics of the stem cells, achieving the range of identifiable and purified, thereby greatly increasing the number of stem cells identifiable; the preparation method of the present invention, the original hematopoietic stem cells Some CD34-expressing stem cells are re-appeared, which makes the original hematopoietic stem cells more visible, greatly improving the number of hematopoietic stem cell purification, safe and reliable, and meeting clinical applications.

 The above culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells according to the present invention, the composition and the weight percentage thereof are -

MEM: 56.5%-64.7%;

 RPMI-1640: 29.4%-30.4%;

 Hanks : 5.88% - 13.0%;

Human stem cell growth factor ^{_{-2: 470 X 10- 9% -}} 1740 X 1 (J- 9%;

Human stem cell growth factor: 1.9χ1 (Γ ⁹ % - 2.8x10-9 % ;

Human granulocyte - macrophage colony stimulating ^{factor: 2.0x10- 9% - 2.9χ1 (Γ} 9%;

CD135 ligand protein: 9.4x10- ^9% - 10.8 <10 a ^9%;

Human erythropoietin factors: 1.4x10- ^9% -2.4xl0_ ^9%; Human epithelial cell production factor: 9.4x10-9% - 10.8χ10_ ⁹ %;

Human contributes to fibroblast production factor-b _: 9.4 χΙ (Γ ⁹ % - 10.8 x l CT ⁹ %).

 The terms and symbols of the above components of the present invention are specifically described as follows:

 The MEM refers to: minimum essential medium, animal cell culture medium produced by GIBCO, USA, containing glutamine, free of ammonium bicarbonate, sodium pyruvate, stored at 2 ° C - 8 ° C, Suitable for supporting the growth of various mammalian cells. There are public records: 1, Zhang Yingchun, Liao Xiaomei, et al., "Effects of different media on hippocampal brain slice activity and microtubule-associated protein tau expression", Journal of the Chinese Academy of Medical Sciences, CLC number: R745.7 Document code: A Article ID: 1000-503X(2005)04-0513-05; 2, Han Ming, Dong Liping, Yuan Fang, Effects of Earle's solution and MEM medium on neuronal activity [J], Chinese Rehabilitation Theory and Practice, 2005, 11 (2) : 125 -126.

 The RPMI-1640, referred to as: RPMI-1640 medium, by Roswell Park Memorial

The Institute developed a liquid medium for culturing normal human leukocytes, containing L-Glutamine, without ammonium bicarbonate (HEPES), and stored at 2 ° C to 8 ° C. There are public records in the literature: 1. Huang Wei, Li Zhengang, et al., “Preliminary observation of RPMI1640 medium on osmotic nutrition of leeches”, Journal of Zoology, CLC number: Q172, Document code: A, Article ID: 025023263 (2001) 06240203, 2001; 2. E Zheng et al., Tissue Culture Technology, Beijing: People's Medical Publishing House, 1985. 219.

 The Hanks, referred to as: GENMED Hanks, is the most commonly used solution for cleaning or histological pretreatment of cells and tissues. Its standardized composition is based on Hanks and Wallace in 1949. Published results, and in line with the provisions of the US Tissue Culture Standards Committee (In Vitro). Origin of the United States. The product is ready to use, strictly sterile and stable in performance. The standard Hanks Balanced Salt Solution (HBSS) contains potassium chloride, sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, D-glucose, etc., but does not contain calcium or magnesium ions. It has been published in the public literature: "National Standard of the People's Republic of China GB 15193-10-94 Non-Procedural DNA Synthesis Test".

 Human stem cell growth factor-2, full name: human Stem Cell Growth Factor-2; human stem cell growth factor, full name: human Stem Cell Growth Factor; human granulocyte-macrophage colony-stimulating factor, full name: human Granulocyte Macrophage- Colony Stimulation Factor; CD135 ligand protein, full name: human flt3 ligand; human erythropoietin, full name: human Erythropoietin; human epithelial cell production factor, full name: human Epithelial Growth Factor; human fibroblast production factor-b , Full name: human Fibroblast Growth Factor-b; The above terms are documented in the public literature.

 Preferred weight percentages of the components of the invention are:

 MEM : 59.36% - 61%;

RPMI-1640: 29.7%-30.14%; Hanks: 9.3% - 10.5%;

Human stem cell growth factor-2: 850xl (T ⁹ % - 1260 χ 10 ^_9 % _;

Human stem cell growth factor: 2.1χ10 ^_9% _2.5χ10- _^9%;

Human granulocyte - macrophage colony stimulating ^{factor: 2.2χ1 (Γ 9% -2.6χ10- 9} %;

CD135 ligand ^{protein: 9.8x10- 9% - 10.4x10- 9%} ;

Human erythropoietin: 1.7χ1 (Γ ⁹ %—2.1 <10 ^_9 %;

Human epithelial cell growth ^{factor: 9.7x10- 9% - 10.4x1 (Γ} 9%;

Human fibroblast production factor-b _: 9.6χ10 ^_9 % - 10.4χ1 (Γ ⁹ %. The preparation method of the culture medium for enhancing CD34 expression of fetal liver hematopoietic stem cells according to the present invention makes the expression of some CD34 in the original hematopoietic stem cells weak. The stem cells are re-appeared to promote the natural CD34 expression ability of stem cells, and the hematopoietic stem cells with weak CD34 expression in fetal liver can be expressed at a level that can be recognized by fluorescently labeled antibodies. However, stem cells with strong CD34 expression in stem cells are not affected. Affects, maintains its original CD34 expression level. Activates the inactive CP34 gene through the cellular "signal" regulatory system, causing cells to enhance CD34 protein expression. Therefore, the present invention does not cause differentiation and proliferation of hematopoietic stem cells, but rather The number of original hematopoietic stem cells is more apparent, which can greatly increase the amount of hematopoietic stem cells purified to meet clinical applications.

The culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells according to the present invention can enhance the expression of CD34-expressing stem cells in a specific cycle, and the expression of CD34 protein can reach the range that can be identified. Taking fetal liver hematopoietic stem cells as an example, the number of labeled CD34 fetal liver hematopoietic stem cells is 10~20xlQ ⁶ by conventional fluorescent antibody labeling method, and the "culture medium for enhancing expression of fetal liver hematopoietic stem cells CD34" prepared by the present invention is applied. workup and purification, fetal liver hematopoietic stem cells can markable amount more than 5 times, to tag the number of hematopoietic stem cells 80~100xl0 ^6.

 The method for preparing the culture medium for enhancing CD34 expression of fetal liver hematopoietic stem cells according to the present invention comprises the following steps:

 1 After mixing MEM, RPMI 1640> Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basic culture solution, and is uniformly stirred and dissolved;

3 Take 19-20 weeks of standard human liver test, separate fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 1Q ⁷ fetal liver mesenchymal cells into T- Type 75 flask, add DMEM medium

(DMEM medium, Dulbecco's modified Eagle's medium) and 2% human serum were cultured. After 10-14 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were treated with Hanks. Rinse twice. Then, DMEM medium and 5% human serum were added and mixed with fetal liver mesenchymal cells in a T-75 culture flask, and cultured continuously for 48 hours (condition control requirement: temperature 37 ° C, C0 ₂ concentration 5 ° /.) , Collect all the supernatant. The supernatant was filtered through a 0.2 micron filter, and then all cell debris and non-soluble substances were removed by high-speed centrifugation at 2,500 g to prepare a conditioned medium.

4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.40 to 7.45 with sodium bicarbonate (0.1 μmol NaHC0 ₃ ), and filter through two 0.2 micron filters. A stem cell CD34 enhanced expression culture medium can be prepared.

 The culture solution of the present invention is characterized in that the CD34 expression characteristic of the thousand cells is enhanced, and the CD34 expression characteristic of the stem cells originally having the CD34 expression characteristic is enhanced, and the range of labeling and purification is achieved, thereby The earth has increased the number of stem cells that can be identified. Since there is no cell differentiation process, stem cell identification and purification are safe and reliable, and overcome the shortcomings of cell differentiation uncertainty existing in the prior art.

 The preparation method of the culture liquid of the invention reproduces some stem cells with weak expression of CD34 in the original hematopoietic cells, and promotes the natural CD34 expression ability of the stem cells, and expresses the hematopoietic stem cells with weak CD34 expression in the fetal liver to be fluorescently labeled. The level at which antibodies can be identified. Stem cells with strong CD34 expression in stem cells were not affected, and their original CD34 expression levels were maintained. The inactive CD34 gene-expressing cells are activated by the cell "signal" regulatory system. Therefore, the present invention does not cause differentiation and proliferation of hematopoietic stem cells, but allows the number of original hematopoietic stem cells to be more revealed, thereby greatly increasing hematopoiesis. The amount of stem cells purified meets clinical application. The best way to implement the invention

 The embodiments of the present invention are described in detail below. The present embodiment is implemented on the premise of the technical solution of the present invention, and detailed embodiments are given, but the scope of protection of the present invention is not limited to the following embodiments. Embodiment 1

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a total weight ratio of 1 liter (L):

 RPMI-1640 250 ml;

 Hanks 110 ml;

Human stem cell growth factor - 2 4 μ _§ ;

 Human stem cell growth factor 16 ng;

 Human granulocyte-macrophage colony-stimulating factor 17 ng;

 CD135 ligand protein 92 ng;

 Human erythropoietin 12 ng;

Human epithelial cell production factor 92 ng; Human pro-fibroblast production factor-b 92 ng _;

 MEM margin.

 1 After mixing MEM, RPMI-1640 and Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basic culture solution, and is uniformly stirred and dissolved;

3 Take the 19-week standard test human fetal liver, separate the fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. Culture flask, add DMEM culture solution

(DMEM medium, Dulbecco's modified Eagle's medium) and 2% human serum were cultured. After 13 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Then add DMEM medium and 5 ° /. Human serum and fetal liver mesenchymal cells were mixed in a T-75 culture flask, and cultured continuously for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), and the supernatant was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

4 After mixing the conditioned medium with MEM, mix well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.42 with sodium bicarbonate (0.1 mol NaHCQ ₃ ), and filter through two 0.2 μm filters. A stem cell CD34 enhanced expression culture medium can be prepared.

Effect embodiment: the preparation of the above-described method of "fetal liver hematopoietic stem cell CD34 expression was enhanced in culture" can fetal liver hematopoietic stem cells may increase the number of markers to 80xl0 ^6. Embodiment 2

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic cells has a total weight of 1 L.

 RPMI-1640 300 ml;;

 Hanks 70 ml;

Human stem cell growth factor-2 20 μ _§ ;

Human stem cell growth factor 32 ng _;

Human granulocyte-macrophage colony stimulating factor 33 ng _;

 CD135 ligand protein 108 ng;

Human erythropoiesis factor 28 n _g;

 Human epithelial cell production factor 108 ng;

 Human stimulating fibroblast production factor -b 108 ng;

MEM margin. 1 Take MEM, RPMI-1640, Hanks mixed according to the above ratio, and stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basal medium, and is fully stirred and dissolved;

3 Take the 19-week standard test human fetal liver, separate the fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. The flask was cultured, added to DMEM medium (DMEM medium, Dulbecco's modified Eagle's medium) and 2% human serum. After 13 days of culture, the supernatant was extracted, and Hanks was used to treat the fetal liver in the T-75 flask. Mesenchymal cells were washed twice. Then add DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), will The supernatant was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

 4 After mixing the conditioned medium with MEM, mix well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.42 with sodium bicarbonate (0.1 mol aHCOa), and filter through two 0.2 μm filters. A thousand cell CD34 enhanced expression culture can be prepared.

Effect of the implementation: The "culture medium for enhanced expression of fetal liver hematopoietic stem cells CD34" prepared by the above method can increase the labelable quantity of fetal liver hematopoietic stem cells to 85χ10 ⁶ . Embodiment 3

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a group distribution ratio of 1 L in total weight:

 PMI-1640 301 ml;

 Hanks 98ml;

Human stem cell growth factor-2 13 μ _§;

 Human stem cell growth factor 25 ng;

 Human granulocyte-macrophage colony-stimulating factor 30 ng;

 CD135 ligand protein 101 ng;

 Human erythropoietin 21 ng;

Human epithelial cell production factor 101 ng _;

 Human pro-fibroblast production factor -b 101 ng;

 MEM margin.

 1 After mixing MEM, RPMI-1640 and Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage finely according to the ratio Cell colony stimulating factor, CD135 ligand protein, human erythropoietin, human epithelial cell production factor, human fibroblast production factor-b are sequentially added to the basal medium, and uniformly stirred and dissolved;

3 Take the 19-week standard test human fetal liver, separate the fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. Culture flask, add DMEM culture solution

(DMEM culture medium, DulbeccQ's modified Eagle's medium) and 2% human serum were cultured. After 13 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Then add DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), will The supernatant was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

 4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, and adjust the pH of the mixed culture solution to 7.42 with sodium bicarbonate (0.1 μ ιηο1 ΝαΗΰθ3), and pass through two Q.2 micron (μΜ) filtration. Stem cell CD34 enhanced expression culture medium can be prepared by membrane filtration.

Effect embodiment: the preparation of the above-described method of "fetal liver hematopoietic stem cell CD34 expression was enhanced in culture" can fetal liver hematopoietic stem cells may increase the number of markers to 90xl0 ^6. Embodiment 4

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a group distribution ratio of -1 L in total weight -

RPMI-1640 285 ml;

 Hanks 130 ml;

Human stem cell growth factor - 2 6 μ _Ε ;

 Human stem cell growth factor 19 ng;

 Human granulocyte-macrophage colony-stimulating factor 21 ng;

 CD135 ligand protein 96 ng;

 Human erythropoietin 16 ng;

 Human epithelial cell production factor 96 ng;

Human pro-fibroblast production factor-b 96 ng _;

 MEM margin.

 1 According to the above ratio, take MEM, RPMI-1640, Hanks, mix well, and prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-1 is sequentially added to the basic culture solution, and is uniformly stirred and dissolved; 3 Take 20 weeks of standard human liver test, separate fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 1Q ⁷ fetal liver mesenchymal cells into T-75 type. Culture flask, add DMEM culture solution

(DMEM solution, DuIbeccQ's modified Eagle's medium) and 2% human serum were cultured. After 12 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Then add DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), will The supernatant was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.43 with sodium bicarbonate (0.1 μmol NaHC0 ₃ ), and pass through two 0.2 μm filters. A thousand cell CD34 enhanced expression culture medium can be prepared by filtration.

Effect of the implementation: The "culture medium for enhanced expression of fetal liver hematopoietic stem cells CD34" prepared by the above method can increase the labelable quantity of fetal liver hematopoietic stem cells to 89 χ 10 ^δ . Embodiment 5

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a group distribution ratio of 1 L in total weight:

 RPMI-1640 325 ml;

 Hanks 50 ml;

 Human stem cell growth factor -2 16 ;

Human thousand cell growth factor 27 ng _;

Human granulocyte-macrophage colony-stimulating factor 29 ng _;

 C0135 ligand protein 104 ng;

 Human erythropoiesis factor 24 ng;

 Human epithelial cell production factor 104 ng;

 Human pro-fibroblast production factor -b 104 ng;

 MEM margin.

 1 After mixing MEM and RPMI-1640^ Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony factor, CD135 ligand protein, human erythropoietin, human epithelial cell production factor, human The fibroblast production factor-b is sequentially added to the basic culture solution, and is uniformly stirred and dissolved;

3 Take 20 weeks of standard human liver test, separate fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. Culture flask, add DMEM culture solution (DMEM culture medium, Dulbecco's modified Eagle's medium) and 2% human serum were cultured. After 12 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Then, DMEM medium and 5% human serum were added to the fetal liver mesenchymal cells and mixed in a T-75 culture flask, and cultured continuously for 48 hours (condition control requirement: temperature ³ 7 ° C, CQ ₂ concentration 5%). Collect all the supernatant. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 250Qg to obtain a conditioned medium;

4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.43 with sodium bicarbonate (0.1 mol NaHC0 ₃ ), and filter twice through 0.2 μm. Stem cell CD34 enhanced expression culture medium can be prepared by membrane filtration.

Effect embodiment: the preparation of the above-described method of "fetal liver hematopoietic stem cell CD34 expression was enhanced in culture" can fetal liver hematopoietic stem cells may increase the number of markers to 91xl0 ^6. Embodiment 6

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a group distribution ratio of 1 L in total weight:

 RPMI-1640 299 ml;

 Hanks 102 ml;

Human stem cell growth factor - 2 12 μ _§ ;

 Human stem cell growth factor 23 ng;

 Human granulocyte-macrophage colony-stimulating factor 28 ng;

 CD135 ligand protein 99 ng;

 Human erythropoietin 19 ng;

 Human epithelial cell production factor 99 ng;

 Human pro-fibroblast production factor -b 99 ng;

 MEM margin.

 1 After mixing MEM, RPMI-1640 and Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basic culture solution, and is uniformly stirred and dissolved;

3 Take 20 weeks of standard human liver test, separate fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. Culture flask, add DMEM culture solution

(DMEM culture medium, Dulbecco's modified Eagle's medium) and 2% human serum were cultured. After 12 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Of course After adding DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), will The supernatant was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

 4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.43 with sodium bicarbonate (0.1 μmol NaHCOs), and filter through two 0.2 μm filters. A stem cell CD34 enhanced expression culture medium can be prepared.

Effect embodiment: the preparation of the above-described method of "fetal liver hematopoietic stem cell CD34 expression was enhanced in culture" can fetal liver hematopoietic stem cells may increase the number of markers to 93x l0 ^6. Example 7

 In the present embodiment, the culture medium for enhancing the expression of C034 of fetal liver hematopoietic stem cells has a group distribution ratio of 1 L in total weight -

RPMI-1640 293 ml;

 Hanks 114 ml;

Human stem cell growth factor - 2 9 μ _§ ;

 Human stem cell growth factor 21 ng;

 Human granulocyte-macrophage colony-stimulating factor 24 ng;

 CD135 ligand protein 98 ng;

 Human erythropoietin 18 ng;

 Human epithelial cell production factor 98 ng;

Human pro-fibroblast production factor-b 98 ng _;

 MEM margin.

 1 After mixing MEM and RPMI-1640^ Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basic culture solution, and is uniformly stirred and dissolved;

3 Take the 19-week standard test human fetal liver, separate the fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. Culture flask, add DMEM culture solution

(DMEM medium, Dulbecco's modified Eagle's medium) and 2% human serum were cultured. After 13 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Then add DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), will The supernatant was collected. The supernatant passed 0.2 After filtering through a micrometer (μΜ) filter, all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, and adjust the pH of the mixed culture solution to 7.45 with sodium bicarbonate (Q.1 mol NaHC0 ₃ ), after two times of 0.2 μm (μΜ). The filter cell CD34 can be used to prepare a stem cell CD34 enhanced expression culture solution.

Effect embodiment: the preparation of the above-described method of "fetal liver hematopoietic stem cell CD34 expression was enhanced in culture" can fetal liver hematopoietic stem cells can be to increase the number of standard ¾ 98xl0 ^6. Example eight

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a group distribution ratio of 1 L in total weight:

 RPMI-1640 313 ml;

 Hanks 74 ml;

 Human thousand cell growth factor -2 11 ;

 Human stem cell growth factor 23 ng;

 Human granulocyte-macrophage colony-stimulating factor 26 ng;

 CD 135 ligand protein 102 ng;

 Human erythropoietin 22 ng;

Human epithelial cell production factor 102 n _g;

 Human pro-fibroblast production factor -b 102 ng;

 MEM margin. '

 1 After mixing MEM, RPMI-1640 and Hanks according to the above ratio, stir well to prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoiesis, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basal medium, and is fully stirred and dissolved;

3 Take the 19-week standard test human fetal liver, separate the fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. The flask was cultured, added to DMEM medium (DMEM medium, Dulbecco's modified Eagle's medium) and 2% human serum. After 13 days of culture, the supernatant was extracted, and Hanks was used to treat the fetal liver in the T-75 flask. Mesenchymal cells were washed twice. Then add DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 Ό, C0 ₂ concentration 5%), the supernatant The liquid was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium; 4 After mixing the conditioned medium with MEM, stir well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.45 with sodium bicarbonate (0.1 mol NaHC0 ₃ ), and filter through two 0.2 μm filters. A stem cell CD34 enhanced expression culture medium can be prepared.

Effect of the implementation: The "culture medium for enhanced expression of fetal liver hematopoietic stem cells CD34" prepared by the above method can increase the labelable quantity of fetal liver hematopoietic stem cells to 99 χ 10 ^δ . Example nine

 In the present embodiment, the culture medium for enhancing the expression of CD34 of fetal liver hematopoietic stem cells has a group distribution ratio of -1 L in total weight -

RPMI-1640 300 ml;

 Hanks 100 ml;

 Human stem cell growth factor - 2 11 μg ;

 Human stem cell growth factor 24 ng;

 Human granulocyte-macrophage colony stimulating factor 29 ng/l

 CD135 ligand protein 100 ng;

 Human erythropoietin. 20 ng;

 Human epithelial cell production factor 100 ng;

Human stimulating fibroblast production factor-b 100 ng _;

 MEM margin.

 1 According to the above ratio, take MEM, RPMI-1640, Hanks, mix well, and prepare the basic culture solution;

2 Take human stem cell growth factor-2, human stem cell growth factor, human granulocyte-macrophage colony-stimulating factor, CP135 ligand protein, human erythropoietin, human epithelial cell-producing factor, human fibroblast The cell-producing factor-b is sequentially added to the basic culture solution, and is uniformly stirred and dissolved;

3 Take the 19-week standard test human fetal liver, separate the fetal liver mesenchymal cells, and purify the mesenchymal cells, according to the counter reading, take 10 ⁷ fetal liver mesenchymal cells into the T-75 type. Culture flask, add DMEM culture solution

(DMEM medium, Dulbecco's modified Eagle's medium) and 2% human serum were cultured. After 13 days of culture, the supernatant was extracted, and the fetal liver mesenchymal cells in the T-75 flask were washed with Hanks. Times. Then add DMEM medium and 5% human serum and fetal liver mesenchymal cells mixed in T-75 culture flask, continuous culture for 48 hours (condition control requirements: temperature 37 ° C, C0 ₂ concentration 5%), will The supernatant was collected. The supernatant is filtered through a 0.2 micron (μΜ) filter, and then all cell debris and non-soluble substances are removed by high-speed centrifugation at 2500 g to prepare a conditioned medium;

4 After mixing the conditioned medium with MEM, mix well to form a mixed culture solution, adjust the pH of the mixed culture solution to 7.45 with sodium bicarbonate (0.1 μmol NaHC0 ₃ ), and pass through two 0.2 μm filters. Filter it A stem cell CD34 enhanced expression culture medium was prepared.

 Effect of the implementation: The culture solution prepared by the above method can increase the labelable quantity of fetal liver hematopoietic stem cells to

Claims

Claim  A culture medium for enhancing expression of fetal liver hematopoietic stem cell CP34, characterized in that the composition and weight percentage are:  MEM: 56.5%-64.7%;  RPMI-1640: 29.4%-30.4%;  Hanks : 5.88% - 13.0%; Human stem cell growth factor-2: 470xl0 ^_9 % - 1740xl0~ ⁹ %; Human stem cell growth factor: 1.9χ1 (Γ ⁹ %-2.8xl0 ^_9 %; Human granulocyte - macrophage colony stimulating ^{factor: 2.0xl0- 9% - 2.9xl (T} 9%; CD135 ligand ^{protein: 9.4x10- 9% - 10.8x10- 9%} ; Human erythropoietin ^{factors: 1.4χ1 (Γ 9% - 2.4x10-} 9%;. Human epithelial cell growth ^{factor: 9.4χ1 (Γ 9% - 10.8X} 10- 9%; Human stimulating fibroblast production factor-b _: 9.4 χ ¹⁰ _ ⁹ % - 10.8 χ ¹⁰ _ ⁹ %. 2. The culture medium for enhancing CD34 expression of fetal liver hematopoietic stem cells according to claim 1, wherein the percentage of the component and the halo is - MEM : 59.36% - 61%;  RPMI-1640: 29.7%-30.14%;  Hanks: 9.3% - 10.5%; Human stem cell growth factor ^{-2: 850x10- 9% - 1260χ1 (} Γ 9%; Human stem cell growth ^{factor: 2.1x10- 9% -2.5><10-}9%; Human granulocyte - macrophage colony stimulating ^{factor: 2.2χ10- 9% - 2.6χ1 (Γ} 9%; CD135 ligand ^{protein: 9.8xl0 _9% - 10.4x10- 9%} ; Human erythropoietin: 1.7χ1 (Γ ⁹ %-2.1 χ1 (Γ ⁹ % _; Human epithelial cell production factor: 9.7χ1 (Γ ⁹ % - 10.4χ1 (Γ ⁹ %; Human Fibroblast growth factor _{^{-b: 9.6xl0- 9% - 10.4xl0 -9}} %. 3. A method for preparing a culture medium for enhancing CD34 expression of fetal liver hematopoietic stem cells according to claim 1, comprising the steps of:  1 After mixing MEM, R?MI-1640, and Hanks according to the above ratio, stir well to prepare a basic culture solution; 2 Take human stem cell growth factor-2, human thousand cell growth factor, human granulocyte-macrophage colony-stimulating factor, CD135 ligand protein, human erythropoietin, human epithelial cell production factor, human The fibroblast-producing factor-1 is sequentially added to the basic culture solution, and is uniformly stirred and dissolved; 3 Isolation of fetal liver mesenchymal cells, the purified mesenchymal cells, 10 ⁷ fetal liver mesenchymal cells were placed in the culture flask according to the counter reading, and added to DMEM medium and 2% human serum for culture. Then the supernatant is withdrawn and rinsed.  Further, DMEM medium and 5% human serum were mixed with fetal liver mesenchymal cells in a culture flask, and the supernatant was collected continuously.  After the supernatant is filtered, it is centrifuged to remove all cell debris and non-soluble substances to obtain a conditioned medium;  4 After mixing the conditioned medium with MEM, the mixture was thoroughly stirred to form a mixed culture solution, and the pH of the mixed culture solution was adjusted to 7.4Q to 7.45 by sodium hydrogencarbonate, and the CD34-enhanced culture medium for stem cells was obtained by filtration. 4. The method for preparing a culture medium for enhancing CD34 expression of fetal liver hematopoietic stem cells according to claim 3, wherein the fetal liver according to step 3 is: a human fetus after a standard test of 19-20 weeks. liver. The method for preparing a culture medium for enhancing CD34 expression of fetal liver hematopoietic stem cells according to claim 3, wherein the culture of 2% human serum as described in step 3 means: culture for 10-14 days. , The method for preparing a culture medium for enhancing fetal liver hematopoietic cell CD34 expression according to claim 3, wherein after the supernatant is extracted in step 3, the fetal liver in the culture flask is treated with Hanks. Mesenchymal cells were washed twice. 7. The method according to claim 3, wherein the supernatant is cultured in a continuous manner, and the continuous culture is continued for 48 hours before the supernatant is collected in the step 3. Conditional control requirements: Temperature 37 ° C, C0 ₂ concentration 5%.  The method according to claim 3, wherein the filtering comprises: the supernatant is filtered through a 0.2 micron filter. A method for preparing a culture solution for enhancing CD34 expression of fetal liver hematopoietic stem cells according to claim 3, wherein the centrifugation in the step 3 means: high-speed centrifugation at 2500 g. The method according to claim 3, wherein the sodium hydrogencarbonate in the step 4 is: 0.1 mol of NaHCO ₃ .