CN114591910A - Method for enriching cord blood hematopoietic stem cells by using mesenchymal stem cells cultured in hypoxic three-dimensional environment - Google Patents

Method for enriching cord blood hematopoietic stem cells by using mesenchymal stem cells cultured in hypoxic three-dimensional environment Download PDF

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CN114591910A
CN114591910A CN202210310427.6A CN202210310427A CN114591910A CN 114591910 A CN114591910 A CN 114591910A CN 202210310427 A CN202210310427 A CN 202210310427A CN 114591910 A CN114591910 A CN 114591910A
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李栋
付强
戴晓宇
刘抗
刘玲玲
邓恩杰
白希
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Chongqing Boerfei Cell Biotechnology Co ltd
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Abstract

The invention provides a method for enriching cord blood hematopoietic stem cells by utilizing mesenchymal stem cells cultured in a hypoxic three-dimensional environment. The method comprises the following steps: inoculating mesenchymal stem cells on a cell culture substrate with a micro-topological structure on the surface, and culturing under a low-oxygen condition; inoculating the cord blood mononuclear cells into mesenchymal stem cells for co-culture, thereby enriching hematopoietic stem cells. According to the invention, a specific hypoxia three-dimensional environment is utilized to enrich the mesenchymal stem cells in CD34 positive hematopoietic stem cells in cord blood mononuclear cells, and the hematopoietic stem cells in the cord blood mononuclear cells are directly separated out through the interaction between the mesenchymal stem cells and the hematopoietic stem cells. The method has low cost and simple experimental steps, and can maintain the dryness and activity of hematopoietic stem cells in vitro.

Description

Method for enriching cord blood hematopoietic stem cells by using mesenchymal stem cells cultured in hypoxic three-dimensional environment
Technical Field
The invention relates to the technical field of biology, in particular to a method for enriching cord blood hematopoietic stem cells by utilizing mesenchymal stem cells cultured in a hypoxic three-dimensional environment.
Background
Hematopoietic Stem Cells (HSCs) maintain a unique self-renewal and pluripotent differentiation capacity throughout life, with the potential to differentiate into all types of blood cells, including myeloid cells (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets and dendritic cells) and lymphoid lineage cells (T cells, B cells, NK cells). HSCs are critical for maintaining blood and immune function. In the past decades, hematopoietic stem cell transplantation has been used as a standard treatment for various hematological diseases such as severe combined immunodeficiency disease, congenital neutropenia, and malignancies.
There are some HSCs in bone marrow, mobilized peripheral blood and umbilical cord blood, because the success rate of matching Umbilical Cord Blood (UCB) collected from postpartum placenta and umbilical cord is higher, and the self-renewal rate, regeneration capacity and activity of HSC in umbilical cord blood are much higher than those of HSC derived from bone marrow and peripheral blood. Therefore, UCB is considered the best HSC source (Smith R A, Wagner E J. alternative hash cell sources for transplantation: place of the geometric code of the Br J Haematol.2009 Oct; 147(2): 246-61.). However, the limited number of HSCs in a single donor UCB tends to delay recovery of neutrophils and platelets in patients after transplantation, resulting in a limited success rate of transplantation. On the other hand, T cells in UCB mononuclear cells cause a certain degree of Graft Versus Host Disease (GVHD) despite low sequelae from UCB transplantation. Therefore, there is a need to develop an efficient HSC expansion method in vitro and an efficient separation means to obtain HSCs with high purity, thereby improving the therapeutic effect. In addition, the separated hematopoietic stem cells can be used as drug screening model cells in various aspects of graft-versus-tumor therapy, tolerance induction, gene therapy and the like.
The CD34 antigen is the most commonly used surface molecule for identifying hematopoietic stem cells in basic or clinical studies, and a flow sorting method or an immunomagnetic bead method is commonly used for sorting CD34 positive HSC from mononuclear cells in UCB. However, the flow sorting method is expensive, the amount of single separated cells is small, the operation procedure of the immunomagnetic bead method is time-consuming, and the long-time treatment can cause the dryness and activity of HSC to be reduced. In addition, it has been demonstrated that there are a small number of HSCs in UCB that have a more powerful megakaryocyte/erythrocyte differentiation potential, CD34 negative, and that this fraction of cells is lost by flow sorting and magnetic bead sorting (Sumide K, Matsuoka Y, Kawamura H, et al. A reviewed road map for the recommendation of human cord blood CD34-negative biochemical step cells. Nat Commun.2018Jun 6; 9(1): 2202.). Therefore, a low-cost and cell-friendly separation method for obtaining CD34 positive HSC in cord blood is urgently needed in clinic.
Mesenchymal Stem Cells (MSCs) are capable of Cell-to-Cell direct interaction with HSCs via N-cadherin, integrins and the SDF-1/CXCR4 axis, and the niches provided by MSCs help to maintain the dryness and activity of HSCs (Walnda T, Bork S, Horn P, et al. Co-culture with Mesenchymal cells secretion and maintenance of hematopoietic promoter cells. J. Cell Mol. Med.201Jan; 14(1-2): 337-50.). Therefore, the co-culture of MSC and cord blood mononuclear cells becomes a potential method for enriching HSC. The SDF-1/CXCR4 axis has been shown to be a major participant in the interaction between MSCs and HSCs. However, when cultured in vitro, the expression level of CXCR4 in MSCs is significantly reduced, affecting the translocation of HSCs to niches (Ponte L A, Marais E, Gallay N, et al. the in vitro hybridization capacity of human bone marrow mesenchymal stem cells: comprison of chemokines and growth factor chemoactivity. Stem cells.2007 Jul; 25(7): 1737-45.). In addition, the Cell volume gradually increases and the proliferation rate becomes slow with the increase of the number of passages of MSC in the conventional Two-Dimensional (2D) Culture process, which is not favorable for the enrichment of HSC (Mo M, Zhou Y, Li S, et al, three-Dimensional Culture Cell Size By inclusion of vector expression. Stem cells.2018 Feb; 36(2): 286-.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for enriching cord blood hematopoietic stem cells by using mesenchymal stem cells cultured in a hypoxic three-dimensional environment, which solves the problems of high cost and reduced activity of the cells in the enrichment process in the prior art.
The invention provides a method for enriching hematopoietic stem cells by utilizing mesenchymal stem cells cultured in a hypoxic three-dimensional environment, which comprises the following steps:
s1: inoculating mesenchymal stem cells on a cell culture substrate with a micro-topological structure on the surface, and culturing under a low-oxygen condition;
s2: inoculating the cord blood mononuclear cells into mesenchymal stem cells for co-culture, thereby enriching hematopoietic stem cells.
Further, the micro-topological structure comprises a plurality of micro-islands which are arranged at intervals.
Has the advantages that: the micron island structure is more beneficial to the adhesion, proliferation, cytoskeleton development and expression of extracellular matrix protein genes of the mesenchymal stem cells, thereby being more beneficial to promoting the growth of the mesenchymal stem cells. The arrangement mode of the micron islands comprises regular arrangement, semi-random arrangement and completely random arrangement.
Furthermore, the diameter of the micron island is 5-100 μm, and the height is 20-200 μm.
Has the advantages that: the size of the micro island is in the range, and the adhesion, the extension and the gene expression of the mesenchymal stem cells contacted with the micro island can be effectively regulated, so that the enrichment of the mesenchymal stem cells on the hematopoietic stem cells is more facilitated, and the culture and the proliferation of the hematopoietic stem cells are facilitated.
Preferably, the micro-islands have a diameter of 50 μm and a height of 100 μm.
Further, the coverage rate of the micro-island on the cell culture substrate is more than 90%.
Further, the material of the cell culture substrate is polystyrene.
Further, in step S1, the concentration of oxygen under the hypoxic condition is 1% to 5%, and the culture time is 12 to 48 hours. Preferably, the concentration of oxygen is 1% and the incubation time is 24 hours.
Further, the method can be used for preparing a novel materialIn step S2, the density of the cord blood mononuclear cell inoculation is 1-5 multiplied by 106And (2) one cell per mL, wherein the co-culture time is 2-6 hours.
Preferably, the density of the cord blood mononuclear cell inoculation is 1 × 106one/mL, the co-cultivation time is 2 hours.
Further, the method also includes step S3: digesting the cord blood mononuclear cells and the mesenchymal stem cells which are co-cultured, wherein the digestive juice is pancreatin containing 0.025-0.1% of EDTA; preferably, the digestive juice is pancreatin with 0.1% EDTA.
In addition, in the invention, the mesenchymal stem cells are extracted by a conventional tissue block method, the cord blood mononuclear cells are prepared by a conventional method, and the preparation method of the micro-topological structure is the conventional method.
In the invention, the method for establishing the low oxygen environment is a conventional method, and specifically comprises the following steps:
(1) introducing two gases of nitrogen and carbon dioxide into the incubator at the same time;
(2) the oxygen concentration in the incubator is measured by an oxygen meter, and the oxygen concentration in the incubator is controlled by adjusting a pressure reducing valve of nitrogen while maintaining the concentration of carbon dioxide.
In the present invention, the term "3D" means culture under three-dimensional conditions; "2D" means culture under two-dimensional conditions; "micron island" means a block with three dimensions on the micron scale, and the shape of the block includes a cube, a cylinder and the like.
It is known in the art that the concentration of a gas, such as oxygen, and carbon dioxide, refers to volume percent, such as 1% oxygen, and the volume of oxygen is air volume percent. The concentration unit related to EDTA is g/ml.
The technical principle of the invention is as follows:
when the mesenchymal stem cells are separated from the body to the surface of the monolayer cell culture substrate for culture, the original signal transmission path, mechanism and the like are damaged, thereby influencing the growth behavior of the mesenchymal stem cells. The inventor finds that the growth of the mesenchymal stem cells and the expression of cytokines can be effectively promoted when the mesenchymal stem cells are cultured under the hypoxia condition on the cell culture substrate with the micro-topological structure through repeated research and screening, thereby being beneficial to the direct contact of the mesenchymal stem cells and the hematopoietic stem cells. The inventor further finds that when the topological structure is a micron island topological structure, the mesenchymal stem cells can effectively enrich the hematopoietic stem cells, promote the proliferation of the hematopoietic stem cells, keep good dryness and activity and reduce differentiation. Further, the results of the proliferation and the enrichment of the hematopoietic stem cells of the mesenchymal stem cells are obviously superior to the effects in the normoxic three-dimensional environment when the mesenchymal stem cells are cultured in the hypoxic three-dimensional environment, and the effects in the normoxic three-dimensional environment are obviously superior to the effects in the normoxic 2D environment, so that the effects of the hypoxia and the three-dimensional environment synergistically promoting the proliferation of the mesenchymal stem cells and the enrichment of the hematopoietic stem cells are shown. The reason for this is probably that the hypoxic three-dimensional environment simulates the local microenvironment for the survival of stem cells, and realizes the regulation and control of the morphology and function of mesenchymal stem cells through the interaction of cells and extracellular matrix, the secretion of cytokines including N-cadherin, integrin, CXCR4 and the like, and the activation of signal pathways.
Compared with the prior art, the invention has the following beneficial effects:
(1) the expression of cell adhesion molecules is improved by treating the mesenchymal stem cells with hypoxia, so that the adsorption efficiency of the mesenchymal stem cells to CD34 positive cells is improved; MSC is cultured in a three-dimensional culture environment to promote the proliferation capacity of cells, and the proliferation capacity of MSC is further improved in a hypoxic three-dimensional culture environment, so that the collection rate of HSC is improved.
(2) The hypoxia and the three-dimensional environment have a synergistic effect on the culture of the mesenchymal stem cells, promote the proliferation of the mesenchymal stem cells, the secretion of cell factors and the proliferation of hematopoietic stem cells, and maintain the dryness and activity of the hematopoietic stem cells, thereby improving the success rate of stem cell transplantation and improving the cure rate of diseases.
(3) According to the invention, a specific hypoxic three-dimensional environment is utilized to enable the mesenchymal stem cells to enrich the CD34 positive hematopoietic stem cells in the cord blood mononuclear cells, and the hematopoietic stem cells in the cord blood mononuclear cells are directly separated through the interaction between the mesenchymal stem cells and the hematopoietic stem cells.
(4) The three-dimensional cell culture enables the cell culture to have intuitiveness and condition controllability.
(5) In conjunction with 3D printing techniques to prepare the micro-topology of the cell culture substrate, the chemical, mechanical or optical properties of the cell culture substrate do not occur, thereby eliminating uncontrollable variables.
(6) The cell mixed solution of the mesenchymal stem cells and the hematopoietic stem cells obtained by the invention does not influence the immune rejection after the hematopoietic transplantation, and can reduce the occurrence of graft-versus-host disease (GVHD) after allogeneic hematopoietic transplantation.
Drawings
FIG. 1 is a schematic and microscopic view of a 2D group culture dish and a 3D group culture dish in example 1 of the present invention.
FIG. 2 is a flow identification diagram of umbilical cord mesenchymal stem cells in example 1 of the present invention.
FIG. 3 is a proportion of CD34 positive cells of cord blood mononuclear cells in example 1 of the present invention.
Fig. 4 is a morphological diagram of the mesenchymal stem cells of the 2D group and the hypoxic 3D group in example 7 of the present invention after 24 hours of culture.
FIG. 5 is a light microscope photograph of the cord blood mononuclear cells cultured for 2 hours after the inoculation of the cord blood mononuclear cells in the 2D group and the hypoxic 3D group in example 7 of the present invention.
FIG. 6 is a graph showing the proliferation of CFSE cells after 48 hours of culture of mesenchymal stem cells among the 2D group, the 3D group and the hypoxic 3D group in example 7 of the present invention.
FIG. 7 is a CD34 flow chart of the control group, 2D group, 3D group and hypoxic 3D group in example 7 of the present invention.
FIG. 8 is a statistical graph of the collection rate of CD34 positive cells in the 2D group and the hypoxic 3D group in example 7 of the present invention.
FIG. 9 is a graph of CFU-GM colonies formed by 14 days in the 2D and hypoxic 3D groups of cells in example 7 of the present invention.
Detailed Description
The technical solution of the present invention is further explained with reference to the drawings and the embodiments. In the following examples, umbilical cord mesenchymal stem cells are taken as an example for illustration, and the same principle can be applied to mesenchymal stem cells from other tissues.
Example 1 enrichment of cord blood hematopoietic Stem cells
1. Preparation of three-dimensional cell culture substrate
(1) A cylindrical micron island structure 3D model with the diameter of 5-100 microns and the height of 20-200 microns is designed by utilizing SolidWorks software, then 3D data of the model are converted into 3D printing codes, and the 3D printing codes are input into a 3D printer. The designed micro island structure is printed layer by layer on a substrate by using a 3D photoetching system (Photonic Professional GT, Nanoscube GmbH) provided with a 780nm femtosecond laser and an objective lens (25 x, 0.8NA, Zeiss, Plan Apochromat) and taking negative photoresist IP-S as a raw material. In the printing process, the movement of a laser focus is controlled by a current scanner, and the movement of a substrate is controlled by a high-resolution xyz platform, so that the positions of the micro island structures are randomly arranged and cover more than 90% of a cell culture substrate; the diameter and height of the micro-island structures are controlled by varying the laser power, voxel distance, and scan-line spacing so that the diameter and height of each micro-island structure are not exactly the same. After laser direct writing, the substrate was developed with 25mL propylene glycol methyl ether acetate for 10 minutes and then rinsed with 25mL isopropanol for 5 minutes.
(2) And putting the printed substrate and the polystyrene film into an embossing machine. The chamber was heated to 145 ℃ after removing air from the chamber with a vacuum pump, and the substrate was pressed into the polystyrene film by a normal downward force. After the substrate and the polystyrene membrane are separated, the polystyrene membrane is cut according to the size of a cell culture dish, finally fixed on the surface of the cell culture dish, dried in an oven at 56 ℃ overnight, sterilized by 75% alcohol and sterilized by an ultraviolet lamp to obtain the three-dimensional cell culture dish. The microstructure of the three-dimensional cell culture dish was observed under a light microscope, and a conventional 2D cell culture dish was used as a control, and the results are shown in FIG. 1.
From the results, it can be seen that the 2D cell culture dish has a smooth surface, while the three-dimensional cell culture dish has an uneven surface, which microscopically forms a large number of mountain shapes with different diameters and heights.
2. Extraction of mesenchymal stem cells: taking umbilical cord aseptically, removing adventitia and arteriovenous, cutting into 1mm3Tissue blocks, tissue blocks were placed in complete medium (purchased from Proxel) at 37 ℃ in 5% CO2Culturing in cell culture box, allowing adherent cells to climb out from tissue edge after 3 days, spreading to bottle bottom after 7 days, digesting with trypsin solution at a ratio of 3 × 104/cm2The density subculture of (2) and taking the third generation cells for flow identification and subsequent experiments. The flow assay results are shown in FIG. 2.
The results show that the obtained positive surface markers CD44, CD73, CD90 and CD105 of the mesenchymal stem cells are all higher than 99%, the endothelial cell marker CD34, the leukocyte marker CD45 and the lymphocyte marker HLA-DR are all lower than 0.05%, and the mesenchymal stem cells meet the international standard.
3. Extraction of cord blood mononuclear cells: freshly collected cord blood was transferred from the blood collection bag to a centrifuge tube and centrifuged at 1800rpm for 30 minutes at 20 ℃. The lower layer of blood cells were collected and diluted with an equal volume of physiological saline and transferred to a Ficoll tube. After centrifugation at 800g for 20 min at 20 ℃, the middle tunica albuginea layer was carefully aspirated and washed with physiological saline, centrifuged at 2000rpm for 10 min at 20 ℃, and the cells were subjected to CD34 flow assay. The flow assay results are shown in FIG. 3.
From the results, it was found that the CD 34-positive cells of cord blood mononuclear cells were 1.01% of the cord blood mononuclear cells.
4. Enrichment of cord blood hematopoietic stem cells: inoculation 106And (3) filling the mesenchymal stem cells into the three-dimensional cell culture dish prepared in the step 1. After the cells were attached to the wall, the three-dimensional cell culture dish was placed at 37 ℃ with 5% CO2And 1% of O2Was cultured in an incubator for 24 hours. When the cell confluence rate reaches about 80%, adding 1 × 10 of the mixture into the mesenchymal stem cells6Cord blood mononuclear cells were seeded at a density of one/mL. The cells were placed at 37 ℃ in 5% CO2After co-culturing for 2 hours in the incubator, the medium was discarded and washed once with PBS, and finally adherent cells were digested with pancreatin containing 0.1% EDTA for detection.
Example 2 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, except that: the diameter of the micro-islands was 50 μm and the height was 100 μm.
EXAMPLE 3 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, except that: the concentration of oxygen was 5% and the incubation time was 12 h.
Example 4 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, except that: the concentration of oxygen was 3% and the incubation time was 48 h.
Example 5 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, except that: the density of the mononuclear cell inoculation of the cord blood is 5 multiplied by 106One cell/mL, and the culture time is 6 h.
Example 6 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, except that: adherent cells were digested with pancreatin containing 0.025% EDTA.
Example 7 detection of mesenchymal Stem cells and hematopoietic Stem cells
2.1 detection of mesenchymal Stem cell morphology
Setting the mesenchymal stem cells cultured in 3D under the low oxygen condition in example 1 as a low oxygen 3D group; and setting the mesenchymal stem cells cultured in 2D under the normoxic condition into a 2D group according to the same method, and observing the form of the mesenchymal stem cells. The results are shown in FIG. 4.
As can be seen from fig. 4: the mesenchymal stem cells in the 2D group are similar to the fibroblasts in shape, while the mesenchymal stem cells in the hypoxia 3D group are obviously reduced in cell volume and slightly raised, and the cells grow in an ordered arrangement.
2.2 detection of the adsorption Capacity of mesenchymal Stem cells
Setting a mixture of mesenchymal stem cells and cord blood mononuclear cells cultured in 3D under hypoxic conditions in example 1 as a hypoxic 3D group; according to the same method, a mixture of mesenchymal stem cells cultured under the condition of normal oxygen and cord blood mononuclear cells is set as a 2D group, and the number of mononuclear cells adsorbed by the mesenchymal stem cells is observed under a light microscope. The results are shown in FIG. 5.
As can be seen from fig. 5: the mesenchymal stem cells between the two groups adsorb a certain amount of mononuclear cells. Under a 100 Xlight microscope, the number of mononuclear cells adsorbed by the hypoxia 3D group is more than that of the mononuclear cells adsorbed by the 2D group, which indicates that the hypoxia 3D environment is favorable for the adsorption of the mesenchymal stem cells to the mononuclear cells.
2.3 detection of proliferative Capacity of mesenchymal Stem cells
Setting the mesenchymal stem cells cultured in 3D under the low oxygen condition in example 1 as a low oxygen 3D group; and according to the same method, setting the 2D-cultured mesenchymal stem cells under the normoxic condition as a 2D group and the 3D-cultured mesenchymal stem cells under the normoxic condition as a 3D group, and detecting the proliferation condition of the mesenchymal stem cells by CFSE. The results are shown in FIG. 6.
As can be seen from fig. 6, after 48 hours, the mesenchymal stem cells in the three groups all split into the second generation, but the number of the second generation cells in the 2D group is significantly less than that of the second generation cells in the 3D group and the hypoxic 3D group, which indicates that the proliferation capacity of the mesenchymal stem cells is improved in the three-dimensional environment, and the proliferation capacity of the mesenchymal stem cells is further enhanced under the hypoxic condition.
2.4 detection of the Collection Rate of CD34 Positive cells
(1) The mixture of the mesenchymal stem cells cultured in 3D under hypoxic condition and the cord blood mononuclear cells in example 1 was set as a hypoxic 3D group, and according to the same method, the mixture of the mesenchymal stem cells cultured in 2D under normoxic condition and the cord blood mononuclear cells was set as a 2D group, the mixture of the mesenchymal stem cells cultured in 3D under normoxic condition and the cord blood mononuclear cells was set as a 3D group, and the mesenchymal stem cells cultured in 2D under normoxic condition was set as a control group, and CD34 positive cells were detected by flow cytometry. The results are shown in FIG. 7.
As can be seen from fig. 7, since the mesenchymal stem cells do not express CD34, no event occurred in the control group. Whereas the 2D, 3D and hypoxic 3D groups all exhibited lymphocyte populations and all exhibited CD34 positive cells. By comparing the proportion of CD34 positive cells of the three groups of cells, the proportion of CD34 positive cells of the 2D group and the 3D group has no significant difference, but the proportion of CD34 positive cells of the hypoxia 3D group is significantly higher than that of the former two groups (p <0.001), which indicates that the hypoxia environment can improve the expression of mesenchymal stem cell adhesion factors so as to improve the adsorption capacity to CD34 positive cells.
(2) To quantitatively count the collection rate of CD34 positive cells, the collection rate of CD34 positive cells was calculated and compared between the 2D group and the hypoxic 3D group. The results are shown in fig. 8 and table 1.
TABLE 1 statistical table of CD34 positive cell collection rates
Figure BDA0003567931810000071
Figure BDA0003567931810000081
As can be seen from table 1 and fig. 8, the average ratio of CD34 positive cells in the 2D group is 4.31% ± 0.10%, the average ratio of CD34 positive cells in the hypoxic 3D group is 6.40% ± 0.03%, and the average ratio of CD34 positive cells in the two groups is significantly different (p <0.001), and meanwhile, it can be found by calculating the total number of cells and the average collection rate of CD34 cells in the two groups that the average collection rate of CD34 positive cells in the 2D group is 53.06% ± 1.92%, and the average collection rate of CD34 positive cells in the hypoxic 3D group is 91.48% ± 2.42%, and thus the collection rate of CD34 positive cells in the hypoxic 3D group is significantly higher than that in the 2D group (p <0.001), indicating that mesenchymal stem cells cultured under the hypoxic three-dimensional condition can efficiently enrich CD34 positive cells.
(3) The comparison of CFU-GM colony forming ability between the 2D group and the hypoxic 3D group using the methylcellulose method is shown in FIG. 9 and Table 2.
TABLE 2 statistical Table of CFU-GM colony counts for 14 days in methylcellulose cultured 2D and hypoxic 3D groups
Figure BDA0003567931810000082
Hypoxia 3D group compared to 2D group: p < 0.001.
As can be seen from fig. 9: both groups formed CFU-GM colonies at day 14. In the same visual field range, CFU-GM colonies of the hypoxia 3D group are obviously more than those of the 2D group, which indicates that the CD34 positive cells adsorbed by the mesenchymal stem cells after the hypoxia three-dimensional condition treatment have stronger proliferation capacity. From table 2, it can be seen that 35 ± 3.61 CFU-GM colonies were formed after 14 days of culture in the 2D group, 83.33 ± 7.57 CFU-GM colonies were formed in the hypoxic 3D group, and the number of CFU-GM colonies in the hypoxic 3D group was significantly greater than that in the 2D group (p <0.001), and thus it can be seen that the mesenchymal stem cells cultured under the hypoxic three-dimensional condition could maintain the proliferation ability of CD34 positive cells in vitro.
The results of the tests of examples 2-6 were similar to those of example 1.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims (10)

1. A method for enriching hematopoietic stem cells by using mesenchymal stem cells cultured in a hypoxic three-dimensional environment is characterized by comprising the following steps:
s1: inoculating mesenchymal stem cells on a cell culture substrate with a micro-topological structure on the surface, and culturing under a low-oxygen condition;
s2: inoculating the cord blood mononuclear cells into mesenchymal stem cells for co-culture, thereby enriching hematopoietic stem cells.
2. The method for enriching hematopoietic stem cells using mesenchymal stem cells cultured in a hypoxic three-dimensional environment, according to claim 1, wherein the micro-topology structure comprises several micro-islands arranged at intervals.
3. The method for enriching hematopoietic stem cells by using mesenchymal stem cells cultured in a hypoxic three-dimensional environment, according to claim 1, wherein the micro-island has a diameter of 5 to 100 μm and a height of 20 to 200 μm.
4. The method for enriching hematopoietic stem cells using mesenchymal stem cells cultured in a hypoxic three-dimensional environment, according to claim 3, wherein the micro-island has a diameter of 50 μm and a height of 100 μm.
5. The method for enriching hematopoietic stem cells using mesenchymal stem cells cultured in a hypoxic three-dimensional environment according to claim 1, wherein the coverage of the micro-islands on the cell culture substrate is 90% or more.
6. The method for enriching hematopoietic stem cells using mesenchymal stem cells cultured in a hypoxic three-dimensional environment according to claim 1, wherein the material of the cell culture substrate is polystyrene.
7. The method for enriching hematopoietic stem cells by using mesenchymal stem cells cultured in a hypoxic three-dimensional environment according to claim 1, wherein in step S1, the concentration of oxygen under hypoxic conditions is 1% to 5%, and the culturing time is 12 to 48 hours.
8. The method for enriching hematopoietic stem cells by using mesenchymal stem cells cultured in the hypoxic three-dimensional environment according to claim 1, wherein in step S2, the density of the inoculation of cord blood mononuclear cells is 1-5 x 106And (2) one cell per mL, wherein the co-culture time is 2-6 hours.
9. The method for enriching hematopoietic stem cells by using mesenchymal stem cells cultured in a hypoxic three-dimensional environment according to claim 8, wherein the density of the umbilical cord blood mononuclear cell seeding is 1 x 106one/mL, the co-cultivation time is 2 hours.
10. The method for enriching hematopoietic stem cells using mesenchymal stem cells cultured in a hypoxic three-dimensional environment, according to claim 1, further comprising step S3: the cord blood mononuclear cells and the mesenchymal stem cells which are co-cultured are digested, and the used digestive juice is pancreatin containing 0.025-0.1% of EDTA.
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