CN110951686A

CN110951686A - Hematopoietic stem cell in-vitro amplification culture system and method

Info

Publication number: CN110951686A
Application number: CN201911111383.9A
Authority: CN
Inventors: 陈杰; 孔德照; 方松刚
Original assignee: Wuhan Jiyuan High Tech Co ltd
Current assignee: Wuhan Jiyuan High Tech Co ltd
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2020-04-03

Abstract

The invention discloses an in vitro expansion culture system and a method for hematopoietic stem cells, which adopts one or a plurality of combinations of gelatin, collagen, laminin and fibronectin for coating in advance, takes a serum-free culture medium as a basic culture medium, and is added with polyvinyl alcohol, dexamethasone, nicotinamide, epinephrine substances, and the combination of cell factors TPO (thrombopoietin) and SCF (stem cell growth factor). Compared with the traditional culture system and method, the culture system and method can efficiently expand the number of CD34+ cells, can expand the number of cells by 200 to 1000 times in vitro culture for about 4 weeks, and can maintain the active hematopoietic stem cells for a long time. The invention can provide a large amount of clinical hematopoietic stem cells, and effectively solves the problem of insufficient application of the clinical hematopoietic stem cells.

Description

Hematopoietic stem cell in-vitro amplification culture system and method

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a composition for in vitro expansion of stem cells, in particular to a culture system for in vitro expansion of hematopoietic stem cells and a method for efficient expansion culture of hematopoietic stem cells.

Background

Hematopoietic Stem Cells (HSCs) have the ability to self-renew cells to proliferate and differentiate into various blood tissue cells. Hematopoietic stem cells play a crucial role in the development, maintenance and regeneration of the blood system. At present, hematopoietic stem cell transplantation is widely applied clinically to treat diseases such as malignant hematological diseases, partial malignant tumors, partial genetic diseases, immunodeficiency and the like.

The biggest obstacle limiting the clinical application at home and abroad at present is the insufficient transplantation quantity of hematopoietic stem cells. The in vitro amplification technology of the source hematopoietic stem cells is a hot point of research, the most applied method at present is a method of applying a feeder layer and factor combination, but the general effect of cell amplification is poor, the amplification quantity is only about 10 times, the treatment effect is influenced by various blood cells differentiated from other hematopoietic stem cells, the operation is complicated, and the introduction of exogenous substance components increases the risk of clinical application.

Disclosure of Invention

The purpose of the present invention is to provide a hematopoietic stem cell culture system and a hematopoietic stem cell culture method that allow hematopoietic stem cells to be cultured in vitro for about 4 weeks, efficiently expand CD34 cells 200 to 1000 times, and maintain hematopoietic stem cells active for a long period of time. The hematopoietic stem cells of the present invention are hematopoietic stem cells enriched by magnetic beads or other methods, and are derived from tissue cells such as umbilical cord blood, peripheral blood, bone marrow, and hematopoietic stem cells derived from induced pluripotent stem cells. According to the system and the method provided by the invention, because animal serum and feeder layer components are not contained, the purity of the CD34 hematopoietic stem cells is high, other types of blood cells obtained by differentiation of the hematopoietic stem cells are few, the method is simple to operate, a large number of clinical hematopoietic stem cells can be cultured, and the problem of insufficient application of the clinical hematopoietic stem cells is effectively solved.

In particular, the invention relates to the following:

1. a hematopoietic stem cell culture system comprising:

the basic serum-free culture medium is one or a combination of L-DMEM, IMDM, DMEM-F12, F12 or α -MEM and the like;

also contains other additive components, such as polyvinyl alcohol with the final concentration of 10-80 mg/ml, dexamethasone with the final concentration of 0.2-0.3 nM, nicotinamide with the final concentration of 1-4 ug/ml, adrenaline with the final concentration of 1-100 ng/ml; and the final concentration of the cytokine TPO is 10-100 ng/ml, and the final concentration of the SCF factor is 1-10 ng/ml.

2. A method for culturing hematopoietic stem cells, comprising:

one or more combined matrix components of gelatin, collagen, laminin and fibronectin are coated in a culture bottle or a dish in advance, the final concentration of gelatin coating is 1-20 ug/ml, the final concentration of collagen coating is 1-10 ug/ml, the final concentration of laminin coating is 1-5 ug/ml, and the final concentration of fibronectin coating is 1-10 ug/ml.

Drawings

FIG. 1 is a photograph of a microscope observation of hematopoietic stem cell culture on day 5;

FIG. 2 is a photograph of a 14 th day of enlarged culture of hematopoietic stem cells;

FIG. 3 shows the results of flow assay of hematopoietic stem cells cultured for 21 days.

Detailed Description

The following examples are intended to illustrate the invention in further detail, but are not intended to limit the invention.

Example 1:

in the hematopoietic stem cell culture system of this embodiment, a hematopoietic stem cell serum-free medium is prepared by using F12 medium as a basal medium, which is specifically as follows: the following components and their contents (final concentrations) were added to the prepared F12 medium: 80mg/ml of polyvinyl alcohol, 0.2nM of dexamethasone, 4ug/ml of nicotinamide and 50ng/ml of epinephrine; and cytokine TPO 100ng/ml, SCF factor 50 ng/ml.

Example 2:

peripheral blood hematopoietic stem cells were cultured in a serum-free medium for hematopoietic stem cells obtained in example 1 in the following manner.

Coating: aseptically preparing laminin coating solution with final concentration of 5ug/ml, sucking 12ml of coating solution with pipette, and adding into T75cm²And (3) slowly and horizontally shaking the bottom of the cell culture bottle to uniformly coat the coating liquid, sealing the coating bottle by using a sealing film, and slowly and horizontally putting the cell culture bottle into a refrigerator at 4 ℃ overnight in a dark place or a constant-temperature constant-humidity incubator at 37 ℃ in a dark place for 0.5-1 hour.

Separation and culture: collecting 50ml of peripheral blood of healthy volunteers, enriching the peripheral blood hematopoietic stem cells by magnetic beads or other methods, washing the cells by normal saline, centrifuging for 10 min at 400 g, adding 20ml of the hematopoietic stem cell complete culture medium prepared in the embodiment 1, blowing and uniformly mixing, and counting and adjusting the cell density to 0.5-1 × 106/ml. The coating solution was aspirated off, the cell suspension was slowly added to the flask, taking care not to blow and touch the flask coating substrate, and the cell growth factors TPO were added rapidly at 100ng/ml, SCF factor 50ng/ml, and the cells were immediately cultured in a humidified incubator or bioreactor at 37 deg.C, 5% CO 2.

Liquid changing: when the cells grew to the third day, the medium was centrifuged (340 g for 10 min) to replace 100ng/ml of hematopoietic stem cell complete medium and 50ng/ml of SCF factor TPO prepared in example 1, the flask was not replaced, the cells were cultured in a humidified incubator or bioreactor at 37 ℃ and 5% CO2, the cell state was observed every day, and a photograph was taken by microscopic observation on the 5 th day. FIG. 1 shows the growth state of hematopoietic stem cells on day 5 of culture.

And (3) amplification culture: when the volume of the culture solution is more than 150 ml, the culture solution needs to be transferred into a culture bag for culture (bubbles are emptied as much as possible so as to avoid influencing cell growth), and the number of the cells is more than 1.5-2.5 multiplied by 10⁶In some cases, a complete hematopoietic stem cell culture medium prepared in example 1 was added in time. At day 21 of culture, samples were counted and phenotyped with CD34+ CD45 RA-hematopoietic stem cells. FIG. 2 shows the growth state of hematopoietic stem cells on day 14 of expansion culture; FIG. 3 shows the results of flow assay of hematopoietic stem cells cultured for 21 days.

Claims

1. An in vitro expansion culture system and a method for hematopoietic stem cells are characterized in that the culture system is a basic serum-free culture medium, and polyvinyl alcohol, dexamethasone, nicotinamide, epinephrine substances, cytokine TPO and SCF are added. During the culture process, one or more of gelatin, collagen, laminin and fibronectin is adopted to coat the culture bottle or dish in advance.

2. The hematopoietic stem cell of claim 1 which is a hematopoietic stem cell positive for expression of the CD34 molecule and not a hematopoietic progenitor cell.

3. The cell according to claim 1, which is derived from tissue cells such as umbilical cord blood, peripheral blood, bone marrow, and hematopoietic stem cells derived from induced pluripotent stem cells.

4. The culture system of claim 1, wherein the basic serum-free medium is one or more selected from the group consisting of L-DMEM, IMDM, DMEM-F12, F12, and α -MEM.

5. The culture system according to claim 1, wherein: the final concentration of polyvinyl alcohol is 10 mg-80 mg/ml, the final concentration of dexamethasone is 0.2-0.3 nM, the final concentration of nicotinamide is 1-4 ug/ml, and the final concentration of epinephrine is 1-100 ng/ml.

6. The culture system according to claim 1, wherein the cytokine TPO is contained at a final concentration of 10 to 100ng/ml and the SCF factor is contained at a final concentration of 1 to 10 ng/ml.

7. The culture method according to claim 1, wherein: the final concentration of gelatin coating is 1-20 ug/ml, the final concentration of collagen coating is 1-10 ug/ml, the final concentration of laminin coating is 1-5 ug/ml, and the final concentration of fibronectin coating is 1-10 ug/ml.