CN110894490A

CN110894490A - Umbilical cord hematopoietic stem cell in-vitro amplification culture system and umbilical cord hematopoietic stem cell in-vitro amplification method

Info

Publication number: CN110894490A
Application number: CN201811069944.9A
Authority: CN
Inventors: 李陶
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2020-03-20

Abstract

The invention belongs to the technical field of stem cells, and particularly relates to an in-vitro amplification culture system and an in-vitro amplification method for umbilical cord hematopoietic stem cells. The invention provides an in-vitro amplification culture system for umbilical cord hematopoietic stem cells, which comprises the following components: G1/S-specific cyclin-D1 and bone morphogenetic protein 4. The result shows that the G1/S-specific cyclin-D1 and bone morphogenetic protein 4 are added into the in-vitro expansion culture system of the umbilical cord hematopoietic stem cells, so that the in-vitro expansion efficiency of the umbilical cord hematopoietic stem cells can be obviously improved and the dryness of the umbilical cord hematopoietic stem cells can be maintained.

Description

Umbilical cord hematopoietic stem cell in-vitro amplification culture system and umbilical cord hematopoietic stem cell in-vitro amplification method

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to an in-vitro amplification culture system and an in-vitro amplification method for umbilical cord hematopoietic stem cells.

Background

Hematopoietic Stem Cells (HSCs), one of the blood components, are the most starting cells for the production of various blood cells, also called hematopoietic multipotent stem cells, and are present in bone marrow, embryonic liver, peripheral blood and umbilical cord blood. HSCs have both a high self-renewal capacity and the ability to further differentiate into hematological progenitors. At present, the HSCs are widely applied to hematopoietic stem cell transplantation clinically, and have wide application prospects in gene therapy, immunotherapy, mature blood cell product preparation and the like. Bone marrow has been the major source of HSCs, but the difficulty in finding suitable donors has limited the clinical use of bone marrow HSCs. The use of granulocyte colony stimulating factor (G-CSF) can mobilize the stem/progenitor cells to infiltrate the circulation, making peripheral blood a practical source of HSCs, but G-CSF after use can cause rupture of the spleen and thus donor injury. The HSCs from the cord blood have various advantages, but the content of the HSCs is low, and the actual requirement of clinical application is far from being met, so that the in-vitro amplification of the HSCs from the cord blood becomes more important.

The characteristic that HSCs are easy to differentiate in vitro culture becomes an important bottleneck restricting the theory and practical application of HSCs at present. Aiming at how to amplify HSCs in large quantity in vitro and keep long-term hematopoietic ability, numerous scholars at home and abroad make a great deal of research on HSCs in vitro amplification, and some achievements are obtained at present. Currently, the in vitro amplification methods of HSCs are mainly divided into a co-culture method using stromal cells as feeder layers and a cytokine combination method. As the operation of the co-culture method of the stromal cells as the feeder layer is complicated, and the risk of introducing exogenous pollutants is increased, the method is difficult to be popularized to clinical large-scale amplification production. The CD34 antigen is a common indicator for the detection of hematopoietic stem cells, is expressed on all hematopoietic stem/progenitor cells, and is more positive for CD34 as differentiated primitive cells are obtained. Although cytokine combination methods can be used to expand HSCs, successful transplantation of hematopoietic stem cells requires at least 2.5X 10 cells per kilogram of body weight⁶And CD34+ cells. The content of CD34+ cells in cord blood is about 1% -2%, and the existing in vitro expansion culture system of cord hematopoietic stem cells still has difficulty in meeting the large clinical demand for HSCs.

Disclosure of Invention

In view of the above, the invention provides an in vitro amplification culture system for umbilical cord hematopoietic stem cells and an in vitro amplification method for umbilical cord hematopoietic stem cells, which are used for solving the problem that the existing in vitro amplification culture system for umbilical cord hematopoietic stem cells still cannot meet the large clinical demand on HSCs.

The specific technical scheme of the invention is as follows:

an in vitro amplification culture system for umbilical cord hematopoietic stem cells, comprising: G1/S-specific cyclin-D1 and bone morphogenetic protein 4.

Preferably, the concentration of G1/S-specific cyclin-D1 is 0.01. mu.g/L-0.2. mu.g/L.

Preferably, the concentration of the bone morphogenetic protein 4 is 0.01-0.2. mu.g/L.

Preferably, the concentration of G1/S-specific cyclin-D1 is 0.1. mu.g/L;

the concentration of the bone morphogenetic protein 4 is 0.1 mug/L.

Preferably, the method further comprises the following steps: serum-free medium, serum substitute, L-glutamine, stem cell growth factor, interleukin 3, interleukin 6 and erythropoietin.

Preferably, the method comprises the following steps: serum-free culture medium, 2-15% of serum substitute, 0.5-5 mM L-glutamine, 0.1-0.5 mu g/L of dry cell growth factor, 0.1-0.5 mu g/L of interleukin 3, 0.1-0.5 mu g/L of interleukin 6, 0.01-0.2 mu g/L of erythropoietin, 0.01-0.2 mu g/L of L G1/S-specific cyclin-D1 and 0.01-0.2 mu g/L of bone morphogenetic protein 4.

Preferably, the method comprises the following steps: serum-free medium, 5% serum substitute by mass, 2mM L-glutamine, 0.25. mu.g/L stem cell growth factor, 0.25. mu.g/L interleukin 3, 0.25. mu.g/L interleukin 6, 0.05. mu.g/L erythropoietin, 0.1. mu.g/L G1/S-specific cyclin-D1 and 0.1. mu.g/L bone morphogenetic protein 4.

Preferably, the serum replacement is plasma.

Preferably, the plasma is cord blood plasma.

The invention also provides an in-vitro amplification method of the cord blood hematopoietic stem cells, which inoculates the cord blood hematopoietic stem cells into the culture system of the technical scheme for culture.

In summary, the present invention provides an in vitro amplification culture system for umbilical cord hematopoietic stem cells, comprising: G1/S-specific cyclin-D1 and bone morphogenetic protein 4. The result shows that the G1/S-specific cyclin-D1 and bone morphogenetic protein 4 are added into the in-vitro expansion culture system of the umbilical cord hematopoietic stem cells, so that the in-vitro expansion efficiency of the umbilical cord hematopoietic stem cells can be obviously improved and the dryness of the umbilical cord hematopoietic stem cells can be maintained.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a flow chart of cord blood mononuclear cells collected in accordance with an embodiment of the present invention for detecting CD34+ cells;

FIG. 2 is a graph showing the fold expansion of cord blood mononuclear cells in a control group and an experimental group according to an embodiment of the present invention;

FIG. 3 is a graph showing the change in the ratio of CD34+ cells in cord blood mononuclear cells expanded in the control group and the experimental group according to the example of the present invention;

FIG. 4 is a diagram of cord blood mononuclear cell colonies 10 days after inoculation of experimental and control groups in an example of the present invention;

FIG. 5 is a graph showing the number of mononuclear cell colonies from cord blood at 6 days and 10 days after the inoculation of the experimental group and the control group in the examples of the present invention.

Detailed Description

The invention provides an in-vitro amplification culture system and method for umbilical cord hematopoietic stem cells, which are used for solving the problem that the existing in-vitro amplification culture system for umbilical cord hematopoietic stem cells still cannot meet the large clinical requirements on HSCs.

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

This example performed the preparation of cord blood mononuclear cells, comprising the following steps:

heparin sodium (20mg/L) antibody was added to a 100ml glass bottleAnd (3) coagulating, and collecting fresh umbilical blood of full-term pregnancy, wherein infectious diseases and various family genetic diseases are eliminated by collecting the umbilical blood, and strict aseptic operation is performed during umbilical blood collection. The average volume of the collected cord blood is 50ml to 100ml, and the cord blood is separated within 4 hours after collection. During separation, the cord blood is subpackaged into a plurality of sterile centrifuge tubes and centrifuged at 1500rpm for 10 min. After centrifugation, transferring the upper plasma layer to a new centrifuge tube, inactivating at 56 deg.C for 30min, standing at-20 deg.C for 10min, centrifuging at 2000rpm for 10min, and collecting the upper plasma layer for use. Centrifuging at 1500rpm for 10min to obtain blood cells in the lower layer, adding physiological saline to dilute the blood cells in the lower layer to the original volume, taking several sterile centrifuge tubes, adding into the centrifuge tubes with specific gravity of 1.077g/cm³15ml of lymphocyte separation liquid, slowly adding diluted blood cells into each tube according to 30ml to form an obvious interface, centrifuging at 2000rpm/min for 20min, carefully sucking the middle leucocyte layer, washing with Dulbecco's phosphate buffer solution (D-PBS) for 2 times, and completely discarding the supernatant to obtain the cord blood mononuclear cells.

Example 2

This example performed the assay of CD34+ cells in cord blood mononuclear cells prepared in example 1. The detection result by flow cytometry is shown in figure 2, and the result shows that the content of CD34+ cells in cord blood mononuclear cells is 1.25%.

Example 3

In this example, the cord blood mononuclear cells prepared in example 1 were divided into eight groups, and cultured in 24-well plates using different culture systems, each having a volume of 0.5ml per well and a cell seeding density of 2X 10, for the following different experimental groups⁶one/mL, half the liquid change was performed once every two days. The plasma in the culture system was the plasma prepared in example 1. The cell growth density was observed under an inverted microscope at

weeks

1, 2, 3 and 4, and the suspension cells were counted, as shown in FIG. 2, the proliferation rate of cord blood mononuclear cells was faster in experimental group 1 to experimental group 7 than in control group 1, indicating that G1/S-specific cyclin-D1 (cyclins D1) and bone morphogenetic protein 4 (BMP 4) can promote cord blood concentration within the range of 0.01 μ G/L to 0.2 μ G/LThe proliferation speed of the mononuclear cells, and the promoting effect of the mononuclear cells tends to be flat along with the increase of the concentration; the proliferation rates were slower in experimental group 6 and experimental group 7 compared to experimental group 3. The embodiment shows that cyclins D1 and BMP4 can obviously improve the efficiency of in vitro expansion of cord blood stem cells, and cyclins D1 and BMP4 have synergistic effect on the improvement of the efficiency of in vitro expansion of cord blood stem cells.

TABLE 1 culture systems of different groups

Example 4

In this example, the cord blood mononuclear cells cultured in example 3 were collected at

weeks

1, 2, 3, and 4, respectively, and subjected to flow assay. The specific method comprises the following steps: respectively collecting cell suspensions of different groups, subpackaging into flow cell tubes, dividing cells of each culture system into a control group and a CD34 positive group, wherein the cell number of each tube is 1x10⁶Adding PBS to wash the cells twice, adding 100 mu l PBS to each tube to resuspend the cells, adding 5 mu l CD34 antibody to the CD34 positive group, and mixing uniformly; the control group did not contain any antibody. Incubating for 30min at room temperature in a dark place, adding 500 μ l PBS to each tube after incubation, centrifuging for 5min at 1500rpm, discarding the supernatant, repeatedly washing once, adding 200 μ l PBS to resuspend the cells, and detecting on a machine.

FIG. 3 is a graph showing the ratio of CD34+ cells in cord blood mononuclear cells expanded in the control group and the experimental group according to the example of the present invention. The results showed that the proportion of CD34+ cells among the cord blood mononuclear cells increased faster in experimental group 1 to experimental group 5 compared to control group 1. In addition, the proportion of CD34+ cells in cord blood mononuclear cells of the experimental groups 1 to 5 is rapidly increased in 1 to 3 weeks and is increased in the fourth week and falls back, wherein the high concentrations of cyclins D1 and BMP4 in the culture system are most obvious. The proportion of CD34+ reached a maximum of 23.4% in test group 4 at week four. This example shows that cyclins D1 and BMP4 can maintain the dryness of cord blood stem cells.

Example 5

This example was carried out by culturing a colony of granulocyte colony forming unit (CFU-GM) from cord blood mononuclear cells of the experimental group 4 and the control group 1 cultured in example 3 at week 4. And (3) detecting the CFU-GM of the cord blood mononuclear cells after the cord blood mononuclear cells are amplified by adopting a methylcellulose semisolid culture medium H4534. The assay was performed in 6-well plates, the density of CFU-GM of CD34+ expanded cells was 5000 per well, and after 6 and 10 days of culture, microscopic observation was performed, and the number of colonies was counted. The culture conditions of the control group 1 were identical to those of the experimental group 4. The results showed that small clusters of cell masses appeared gradually from 3 to 4 days after inoculation, and the cell masses became larger with the increase of the culture time. At 10 days after inoculation, a needle-tip-sized clump of villus-like cells was visible to the naked eye. FIG. 4 shows the mononuclear cell colony of cord blood 10 days after the inoculation of the experimental group and the control group; referring to FIG. 5, the numbers of mononuclear cell colonies from cord blood 6 days and 10 days after the inoculation of the experimental group and the control group are shown. The results showed that experiment group 4 in methylcellulose medium H4534 had a large number of CFU-GM growth, counted as (284. + -. 29) colonies, which was significantly higher than (197. + -.21) colonies of control group 1. This example shows cyclins D1 and BMP4 maintain the dryness of cord blood stem cells.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. An in vitro amplification culture system for umbilical cord hematopoietic stem cells, which is characterized by comprising: G1/S-specific cyclin-D1 and bone morphogenetic protein 4.

2. The culture system according to claim 1, wherein the concentration of G1/S-specific cyclin-D1 is 0.01 μ G/L to 0.2 μ G/L.

3. The culture system of claim 2, wherein the concentration of bone morphogenic protein 4 is between 0.01 μ g/L and 0.2 μ g/L.

4. The culture system of claim 3, wherein the concentration of G1/S-specific cyclin-D1 is 0.1 μ G/L;

the concentration of the bone morphogenetic protein 4 is 0.1 mug/L.

5. The culture system of claim 1, further comprising: serum-free medium, serum substitute, L-glutamine, stem cell growth factor, interleukin 3, interleukin 6 and erythropoietin.

6. The culture system of claim 5, comprising: serum-free culture medium, 2-15% of serum substitute, 0.5-5 mM L-glutamine, 0.1-0.5 mu g/L of dry cell growth factor, 0.1-0.5 mu g/L of interleukin 3, 0.1-0.5 mu g/L of interleukin 6, 0.01-0.2 mu g/L of erythropoietin, 0.01-0.2 mu g/L of L G1/S-specific cyclin-D1 and 0.01-0.2 mu g/L of bone morphogenetic protein 4.

7. The culture system of claim 6, comprising: serum-free medium, 5% serum substitute by mass, 2mM L-glutamine, 0.25. mu.g/L stem cell growth factor, 0.25. mu.g/L interleukin 3, 0.25. mu.g/L interleukin 6, 0.05. mu.g/L erythropoietin, 0.1. mu.g/L G1/S-specific cyclin-D1 and 0.1. mu.g/L bone morphogenetic protein 4.

8. The culture system of claim 6, wherein the serum replacement is plasma.

9. The culture system of claim 8, wherein the plasma is cord blood plasma.

10. An in vitro method for expanding umbilical cord hematopoietic stem cells, comprising culturing umbilical cord hematopoietic stem cells inoculated into the culture system according to any one of claims 1 to 9.