CN114591910B - Method for enriching cord blood hematopoietic stem cells by utilizing mesenchymal stem cells cultured in low-oxygen three-dimensional environment - Google Patents
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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 low-oxygen 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 hypoxia condition; umbilical cord blood mononuclear cells are inoculated to mesenchymal stem cells for co-culture, thereby enriching hematopoietic stem cells. The invention utilizes a specific low-oxygen three-dimensional environment to make the mesenchymal stem cells enrich CD34 positive hematopoietic stem cells in the mononuclear cells of the cord blood, and directly separates the hematopoietic stem cells from the mononuclear cells of the cord blood 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 stem property and activity of hematopoietic stem cells in vitro.
Description
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 low-oxygen three-dimensional environment.
Background
Hematopoietic stem cells (hematopoietic stem cell, HSCs) retain unique self-renewal and multipotent differentiation capabilities 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 to maintaining blood and immune function. Hematopoietic stem cell transplantation has been used as a standard treatment for a variety of hematological disorders such as severe combined immunodeficiency, congenital neutropenia, and malignancy for the past decades.
There are a number of HSCs in bone marrow, mobilized peripheral blood and umbilical cord blood, since umbilical cord blood (umbilical cord blood, UCB) collected from the postpartum placenta and umbilical cord has a higher success rate of matching, and the self-renewal rate, regeneration capacity and activity of umbilical cord blood HSCs are much higher than those of bone marrow and peripheral blood-derived HSCs. UCB is therefore considered the best source of HSC (Smith R A, wagner E J. Alternative haematopoietic stem cell sources for transplantation: place of umbilical cord blood. Br J Haemato. 2009Oct;147 (2): 246-61.). However, the limited number of HSCs in a single donor UCB tends to result in delayed neutrophil and platelet recovery in the patient after transplantation, resulting in still limited success rate of transplantation. On the other hand, although the sequelae caused by UCB transplantation are low, T cells in UCB mononuclear cells still cause a degree of Graft Versus Host Disease (GVHD). Therefore, there is a need to develop efficient methods of HSC in vitro expansion and efficient ways of isolation to obtain HSCs of high purity, thereby improving therapeutic efficacy. In addition, the separated hematopoietic stem cells can be used as drug screening model cells in various aspects of graft anti-tumor treatment, tolerance induction, gene therapy and the like.
The CD34 antigen is the most commonly used surface molecule for identification of hematopoietic stem cells in basic or clinical studies, and CD34 positive HSCs are often sorted from mononuclear cells in UCB using flow sorting or immunomagnetic bead. However, flow sorting is expensive, the amount of single isolated cells is small, the immunomagnetic bead procedure is time consuming, and long treatment results in reduced drying and activity of HSCs. In addition, it has been demonstrated that the presence of small numbers of HSCs negative for CD34 with greater megakaryocyte/erythrocyte differentiation potential in UCB, using flow sorting and magnetic bead sorting, lost this fraction of cells (Sumide K, matsuoka Y, kawamura H, et al A revised road map for the commitment of human cord blood CD-negative hematopoietic stem cells. Nat Commun.2018Jun 6;9 (1): 2202.). There is therefore a great clinical need for a low cost and cell friendly method of isolation of CD34 positive HSCs in cord blood.
Mesenchymal stem cells (Mesenchymal stem Cell, MSC) can interact with HSCs directly Cell-to-Cell via the N-cadherin, integrins and SDF-1/CXCR4 axes, and the niches provided by MSC help to maintain the dryness and activity of the HSCs (Walenda T, bark S, horn P, et al Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells J Cell Mol Med.2010Jan;14 (1-2): 337-50.). Thus, MSC co-culture with cord blood mononuclear cells has become a potential method for enriching HSC. The SDF-1/CXCR4 axis has been shown to be a major participant in the interaction between MSC and HSC. However, when cultured in vitro, MSC had significantly reduced CXCR4 expression levels, affecting HSC migration to the niche (Ponte L A, marais E, gallay N, et al in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activites.stem cells.2007Jul;25 (7): 1737-45.). In addition, the cell volume gradually increases and proliferation rate becomes slow with increasing passage times of MSC during conventional Two-dimensional (2D) culture, which is disadvantageous for HSC enrichment (Mo M, zhou Y, li S, et al, three-Dimensional Culture Reduces Cell Size By Increasing Vesicle, culture. Stem cells.2018Feb;36 (2): 286-292.).
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a method for enriching umbilical blood hematopoietic stem cells by utilizing mesenchymal stem cells cultured in a low-oxygen three-dimensional environment, which solves the problems of high cost and reduced activity of 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 low-oxygen 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 hypoxia condition;
s2: umbilical cord blood mononuclear cells are inoculated to mesenchymal stem cells for co-culture, thereby enriching hematopoietic stem cells.
Further, the micro-topology comprises a plurality of spaced apart micro-islands.
The beneficial effects are that: the micro island structure is more favorable for the adhesion, proliferation, cytoskeletal development and expression of extracellular matrix protein genes of the mesenchymal stem cells, thereby being more favorable for promoting the growth of the mesenchymal stem cells. The arrangement of the micro islands includes regular arrangement, semi-random arrangement and completely random arrangement.
Further, the diameter of the micro-island is 5-100 μm, and the height is 20-200 μm.
The beneficial effects are 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 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 of the micro islands on the cell culture substrate is 90% or more.
Further, the material of the cell culture substrate is polystyrene.
In step S1, the concentration of oxygen under the condition of low oxygen is 1% -5%, and the time of culture is 12-48 hours. Preferably, the concentration of oxygen is 1% and the incubation time is 24 hours.
Further, in step S2, the cord blood mononuclear cells are inoculatedThe density of (2) is 1 to 5X 10 6 The co-culture time is 2-6 hours per mL.
Preferably, the cord blood mononuclear cell seeding density is 1×10 6 And (3) per mL, wherein the co-culture time is 2 hours.
Further, the method further comprises the step S3: performing digestion treatment on the co-cultured cord blood mononuclear cells and mesenchymal stem cells, wherein the used digestion solution is pancreatin containing 0.025% -0.1% EDTA; preferably, the digestive juice is pancreatin with 0.1% edta.
In addition, in the invention, mesenchymal stem cells are extracted by a conventional tissue mass method, umbilical cord blood mononuclear cells are prepared by a conventional method, and the preparation method of the micro-topology structure is the conventional method.
In the invention, the establishment method of the low-oxygen environment is a conventional method, and specifically comprises the following steps:
(1) Simultaneously introducing two gases of nitrogen and carbon dioxide into an incubator;
(2) The concentration of oxygen in the incubator is measured by an oxygen meter, and the concentration of oxygen in the incubator is controlled by adjusting a pressure reducing valve of nitrogen, while the concentration of carbon dioxide is maintained.
In the present invention, the term "3D" means cultivation under three-dimensional conditions; "2D" means cultivation under two-dimensional conditions; "micron islands" means blocks of three dimensions, all micron-scale, the shape of which includes square, cylindrical, etc.
It is known in the art that reference to a concentration of a gas, such as oxygen, and carbon dioxide, both refer to a volume percentage, such as 1% oxygen, and refer to the volume of oxygen as a percentage of the volume of air. The concentration unit for EDTA is g/ml.
The technical principle of the invention is as follows:
when the mesenchymal stem cells are separated from the organism to the surface of the monolayer cell culture substrate for culture, the original signal transmission path, mechanism and the like are destroyed, thereby affecting the growth behavior of the mesenchymal stem cells. The inventor finds through repeated research and screening that when the mesenchymal stem cells are cultured on a cell culture substrate with a micro-topological structure under the condition of hypoxia, the growth of the mesenchymal stem cells and the expression of cytokines can be effectively promoted, thereby being beneficial to the direct contact of the mesenchymal cells and the hematopoietic stem cells. The inventor further found 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. Furthermore, when the mesenchymal stem cells are cultured in a low-oxygen three-dimensional environment, the effect of proliferation and enrichment of the hematopoietic stem cells is obviously better than that in a normoxic three-dimensional environment, and the effect in the normoxic three-dimensional environment is obviously better than that in a normoxic 2D environment, which indicates that the proliferation and enrichment of the hematopoietic stem cells are synergistically promoted by the low-oxygen and three-dimensional environment. This is probably due to the fact that the hypoxic three-dimensional environment mimics the local microenvironment of stem cell survival, and the regulation of the morphology and function of mesenchymal stem cells is achieved through the interaction of cells and extracellular matrix, secretion of cytokines including N-cadherin, integrins, CXCR4 and the like, and activation of signal pathways.
Compared with the prior art, the invention has the following beneficial effects:
(1) The mesenchymal stem cells are treated by hypoxia to improve the expression of cell adhesion molecules, so that the adsorption efficiency of the mesenchymal stem cells on 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 the MSC is further improved in a low-oxygen three-dimensional culture environment, so that the collection rate of HSC is improved.
(2) The hypoxia and three-dimensional environment has synergistic effect on the culture of mesenchymal stem cells, promotes the proliferation of mesenchymal stem cells, the secretion of cytokines and the proliferation of hematopoietic stem cells, maintains the stem property and activity of hematopoietic stem cells, thereby improving the success rate of stem cell transplantation and the cure rate of diseases.
(3) According to the invention, the specific low-oxygen three-dimensional environment is utilized to enable the mesenchymal stem cells to enrich the CD34 positive hematopoietic stem cells in the umbilical blood mononuclear cells, and the hematopoietic stem cells in the umbilical 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) The micro-topology of the cell culture substrate is prepared in conjunction with 3D printing techniques, and the chemical, mechanical or optical properties of the cell culture substrate do not occur, thereby eliminating uncontrollable variables.
(6) The cell mixture of the mesenchymal stem cells and the hematopoietic stem cells obtained by the invention does not influence the immune rejection after 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 structure of 2D-group culture dishes and 3D-group culture dishes according to example 1 of the present invention.
FIG. 2 is a flow chart depicting the identification of umbilical cord mesenchymal stem cells in example 1 of the present invention.
FIG. 3 is a graph showing the ratio of CD 34-positive cells of cord blood mononuclear cells in example 1 of the present invention.
FIG. 4 is a morphology diagram of mesenchymal stem cells of the 2D group and the hypoxia 3D group of example 7 of the present invention after culturing for 24 hours.
FIG. 5 is a photomicrograph of the 2D and hypoxic 3D groups of example 7 of the invention after 2 hours of cord blood mononuclear cell culture.
FIG. 6 is a graph showing CFSE cell proliferation after 48 hours of culture of mesenchymal stem cells of 2D group, 3D group and hypoxia 3D group in example 7 of the present invention.
FIG. 7 is a plot of CD34 flow scattergrams for the control, 2D, 3D, and hypoxic 3D groups of example 7, in accordance with the invention.
FIG. 8 is a statistical chart of the collection rate of CD34 positive cells in the 2D group and the hypoxia 3D group in example 7 of the present invention.
FIG. 9 is a graph of CFU-GM colonies formed on 14 days for the 2D and hypoxic 3D groups of cells according to example 7 of the invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples. In the following examples, umbilical cord mesenchymal stem cells are described as an example, and other tissue-derived mesenchymal stem cells can also be used by the same principle.
EXAMPLE 1 enrichment of cord blood hematopoietic Stem cells
1. Preparation of three-dimensional cell culture substrates
(1) The SolidWorks software is used for designing a 3D model with a diameter of 5-100 mu m and a height of 20-200 mu m and a cylindrical micro island structure, then 3D data of the model are converted into 3D printing codes, and then the 3D printing codes are input into a 3D printer. The designed micro-island structure was printed layer by layer on the substrate using negative photoresist IP-S as a starting material using a 3D lithography system (Photonic Professional GT, nanoscales GmbH) equipped with a 780nm femtosecond laser and an objective lens (25×,0.8NA, zeiss, plan Apochromat). In the printing process, controlling the movement of a laser focus through a current scanner and controlling the movement of a substrate through a high-resolution xyz platform, so that the positions of the micro island structures are arranged randomly and cover more than 90% of a cell culture substrate; the diameter and height of each micro island structure are controlled by changing laser power, voxel distance and scanning line gap, so that the diameter and height of each micro island structure are not identical. After laser direct writing, the substrate was developed with 25mL propylene glycol methyl ether acetate for 10 minutes and then rinsed with 25mL isopropyl alcohol for 5 minutes.
(2) The printed substrate and polystyrene film were placed in an embosser. After removing the air from the chamber with a vacuum pump, the chamber was heated to 145 ℃ and the substrate was pressed into the polystyrene film by normal downward force. After the substrate and the polystyrene film are separated, the polystyrene film is cut according to the size of a cell culture dish, and finally fixed on the surface of the cell culture dish, and then dried overnight in a drying oven at 56 ℃, sterilized by 75% alcohol and sterilized by an ultraviolet lamp, thus obtaining the three-dimensional cell culture dish. The microstructure of the three-dimensional cell culture dish was observed under a light microscope, and the result was shown in fig. 1 using a conventional 2D cell culture dish as a control.
From the results, the 2D cell culture dish surface was smooth, while the three-dimensional cell culture dish surface was rugged, and microscopically formed a large number of peak shapes with different diameters and heights.
2. Extraction of mesenchymal stem cells: sterile umbilical cord is obtained, and cut into 1mm after the adventitia and arteriovenous are stripped 3 Tissue pieces, placed in complete medium (purchased from pramoxine), placed at 37 ℃,5% co 2 Culturing in cell incubator, wherein adherent cells can climb out from tissue edge after 3 days, can be spread on bottle bottom after about 7 days, and can be digested with trypsin solution at a ratio of 3×10 4 /cm 2 And (3) taking third-generation cells for flow identification and subsequent experiments. The results of the stream authentication are shown in fig. 2.
The results show that the positive surface markers CD44, CD73, CD90 and CD105 of the obtained mesenchymal stem cells are higher than 99%, the endothelial cell marker CD34, the leucocyte marker CD45 and the lymphocyte marker HLA-DR are lower than 0.05%, and the standard of international requirements is met.
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 at 20℃for 30 minutes. The lower blood cells were collected and transferred to a Ficoll tube after being diluted with an equal volume of physiological saline. After centrifugation at 800g for 20 min at 20℃the intermediate buffy coat was carefully aspirated and washed with physiological saline and centrifuged at 2000rpm for 10 min at 20℃for CD34 flow assay of cells. The results of the flow detection are shown in fig. 3.
From the results, the CD34 positive cells of the cord blood mononuclear cells account for 1.01% of the cord blood mononuclear cells.
4. Enrichment of cord blood hematopoietic stem cells: inoculation 10 6 And (3) introducing the mesenchymal stem cells into the three-dimensional cell culture dish prepared in the step (1). After cell attachment, the three-dimensional cell culture dish was placed at 37℃with 5% CO 2 And 1%O 2 Is cultured in an incubator for 24 hours. When the cell confluency rate reaches about 80%, the cell confluency rate is 1×10 into the mesenchymal stem cells 6 The cord blood mononuclear cells were seeded at a density of individual/mL. The cells were placed in 5% CO at 37 ℃ 2 After co-cultivation in the incubator for 2 hours, the medium was discarded and washed once with PBS, and finally the 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, the difference is that: the micro islands have a diameter of 50 μm and a height of 100 μm.
EXAMPLE 3 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, the difference is that: the concentration of oxygen was 5%, and the incubation time was 12 hours.
EXAMPLE 4 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, the difference is that: the concentration of oxygen was 3%, and the incubation time was 48 hours.
EXAMPLE 5 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, the difference is that: cord blood mononuclear cell inoculation density of 5×10 6 The incubation time was 6h per mL.
EXAMPLE 6 enrichment of cord blood hematopoietic Stem cells
Similar to example 1, the difference is 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 3D-cultured under the hypoxia condition in example 1 as a hypoxia 3D group; and according to the same method, setting up the mesenchymal stem cells 2D cultured under normal oxygen condition as a 2D group, and observing the morphology of the mesenchymal stem cells. The results are shown in FIG. 4.
As can be seen from fig. 4: the morphology of the mesenchymal stem cells of the 2D group is similar to that of the fibroblasts, and the cell volume of the mesenchymal stem cells of the hypoxia 3D group is obviously reduced and slightly raised, and the cells are orderly arranged and grown.
2.2 detection of adsorption Capacity of mesenchymal Stem cells
Setting the mixture of the mesenchymal stem cells and the cord blood mononuclear cells 3D-cultured under the hypoxia condition in the example 1 as a hypoxia 3D group; and according to the same method, setting a mixture of the mesenchymal stem cells and the umbilical cord blood mononuclear cells which are 2D cultured under normal oxygen condition as a 2D group, and observing the quantity of the mesenchymal stem cells adsorbed to the mononuclear cells under a light microscope. The results are shown in FIG. 5.
As can be seen from fig. 5: both groups of mesenchymal stem cells adsorbed a certain number of mononuclear cells. The number of mononuclear cells adsorbed by the hypoxia 3D group is more than that of the 2D group under 100 x light, 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 proliferation Capacity of mesenchymal Stem cells
Setting the mesenchymal stem cells 3D-cultured under the hypoxia condition in example 1 as a hypoxia 3D group; and according to the same method, setting a 2D group of mesenchymal stem cells cultured under normoxic conditions and a 3D group of mesenchymal stem cells cultured under normoxic conditions, and detecting proliferation 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 of three groups all divide into the second generation, but the number of the second generation cells of the 2D group is significantly smaller than that of the second generation cells of the 3D group and the hypoxia 3D group, which indicates that the proliferation capacity of the mesenchymal stem cells is improved in a three-dimensional environment, and the proliferation capacity of the mesenchymal stem cells is further enhanced in a hypoxia condition.
Detection of 2.4CD34 Positive cell Collection Rate
(1) The mixture of the mesenchymal stem cells and the umbilical blood mononuclear cells 3D-cultured under the hypoxia condition in example 1 was set as a hypoxia 3D group, and the mixture of the mesenchymal stem cells and the umbilical blood mononuclear cells 2D-cultured under the normoxic condition was set as a 2D group, the mixture of the mesenchymal stem cells and the umbilical blood mononuclear cells 3D-cultured under the normoxic condition was set as a 3D group, and the mesenchymal stem cells 2D-cultured under the normoxic condition was set as a control group, and CD34 positive cells were detected by flow cytometry according to the same method. The results are shown in FIG. 7.
As can be seen from fig. 7, since the mesenchymal stem cells did not express CD34, no event occurred in the control group. Whereas the 2D, 3D and hypoxic 3D groups all present lymphocyte populations and all present CD34 positive cells. By comparing the CD34 positive cell ratios of the three groups of cells, there is no significant difference in the CD34 positive cell ratios of the 2D group and the 3D group, but the CD34 positive cell ratio of the hypoxic 3D group is significantly higher than that of the first two groups (p < 0.001), indicating that the hypoxic environment can increase the expression of mesenchymal stem cell adhesion factors and thus increase the adsorption capacity to CD34 positive cells.
(2) To quantitatively calculate the collection rate of CD34 positive cells, the collection rate of CD34 positive cells in the 2D group was calculated and compared with that in the hypoxic 3D group. The results are shown in FIG. 8 and Table 1.
TABLE 1 statistics of CD34 Positive cell collection rate
As can be seen from table 1 and fig. 8, the ratio of CD34 positive cells in the 2D group was 4.31% ± 0.10% on average, the ratio of CD34 positive cells in the hypoxic 3D group was 6.40% ± 0.03% on average, and the ratio of CD34 positive cells in the two groups showed a significant difference (p < 0.001), while the average collection rate of CD34 positive cells in the 2D group was 53.06% ± 1.92% and the average collection rate of CD34 positive cells in the hypoxic 3D group was 91.48% ± 2.42%, thus the collection rate of CD34 positive cells in the hypoxic 3D group was significantly higher than that in the 2D group (p < 0.001), indicating that the mesenchymal stem cells cultured under the hypoxic three-dimensional condition can efficiently enrich CD34 positive cells.
(3) CFU-GM colony forming ability comparisons were performed on the 2D group and the hypoxic 3D group using the methylcellulose method, and the results are shown in fig. 9 and table 2.
TABLE 2 statistical table of CFU-GM colony count for 14 days in methylcellulose culture of 2D group and hypoxia 3D group
Hypoxia 3D group compared to 2D group: * P <0.001.
As can be seen from fig. 9: both groups formed CFU-GM colonies at 14 days. In the same visual field range, CFU-GM colony of the hypoxia 3D group is obviously more than that of the 2D group, which indicates that the CD34 positive cells obtained by the adsorption of the mesenchymal stem cells after the hypoxia three-dimensional condition treatment have stronger proliferation capacity. From Table 2, it can be seen that the 2D group formed 35.+ -. 3.61 CFU-GM colonies after 14 days of culture, the hypoxic 3D group formed 83.33.+ -. 7.57 CFU-GM colonies, and the number of CFU-GM colonies in the hypoxic 3D group was significantly larger than that in the 2D group (p < 0.001), so that the mesenchymal stem cells cultured under the hypoxic three-dimensional condition could maintain the proliferation capacity of CD 34-positive cells in vitro.
The results of the tests of examples 2-6 were similar to those of example 1.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (6)
1. A method for enriching hematopoietic stem cells by utilizing mesenchymal stem cells cultured in a low-oxygen three-dimensional environment, which 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 hypoxia condition;
s2: inoculating umbilical blood mononuclear cells to mesenchymal stem cells for co-culture, thereby enriching hematopoietic stem cells;
the micro-topology structure comprises a plurality of cylindrical micro-islands which are arranged at intervals;
the diameter of the micron island is 50 μm, and the height is 100 μm;
the cell culture substrate is made of polystyrene;
the mesenchymal stem cells are derived from umbilical cord;
the concentration of oxygen under the condition of low oxygen is 1% -5%.
2. The method for enriching hematopoietic stem cells by using a mesenchymal stem cell cultured in a low-oxygen three-dimensional environment according to claim 1, wherein the coverage of the micro-islands on the cell culture substrate is 90% or more.
3. The method for enriching hematopoietic stem cells by culturing mesenchymal stem cells in a three-dimensional environment with hypoxia according to claim 1, wherein the culturing time is 12-48 hours in step S1.
4. The method for enriching hematopoietic stem cells by using mesenchymal stem cells cultured in a low-oxygen three-dimensional environment according to claim 1, wherein in step S2, the density of the cord blood mononuclear cell inoculation is 1-5×10 6 And (3) per mL, wherein the co-culture time is 2-6 hours.
5. The method for enriching hematopoietic stem cells using a low-oxygen three-dimensional environment according to claim 4, wherein the cord blood mononuclear cell seeding density is 1 x 10 6 And (3) per mL, wherein the co-culture time is 2 hours.
6. The method for enriching hematopoietic stem cells by using the mesenchymal stem cells cultured in a low-oxygen three-dimensional environment according to claim 1, further comprising step S3: and (3) performing digestion treatment on the co-cultured cord blood mononuclear cells and the mesenchymal stem cells, wherein the used digestion liquid is pancreatin containing 0.025% -0.1% EDTA.
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