WO2018153049A1

WO2018153049A1 - Preparation method for human breast milk stem cells and application thereof

Info

Publication number: WO2018153049A1
Application number: PCT/CN2017/100137
Authority: WO
Inventors: 姜舒; 张芸; 纪惜銮; 杨顺; 罗朝霞
Original assignee: 深圳市茵冠生物科技有限公司
Priority date: 2017-02-21
Filing date: 2017-09-01
Publication date: 2018-08-30
Also published as: CN106701663B; CN106701663A

Abstract

A preparation method for human breast milk stem cells and an application thereof. The preparation method comprises the following steps: collecting breast milk; separating human breast milk cells to obtained purified breast milk cell suspension; transferring the purified breast milk cell suspension into a culture plate coated with gelatin having a volume percentage of 0.01-0.05%, putting the culture plate into a CO2 incubator for culturing, changing liquid once every day, separating individual cells on the sixth day for cloning and inoculating the cells into a new ultralow-adherency culture plate to promote clone formation; when the cells are fused to 80% or above, adding trypsin, performing digestion at 37°C for 5 min, ending the digestion, collecting cell sediments, washing and centrifugating the cells, resuspending the cells with a fresh cell culture liquid, and perform passage according to the ratio of 1:3. The technical solution is easy to operate, needs no co-culture of feeder cells, and can better maintain the undifferentiated state of the human breast milk stem cells and obtain a larger number of human breast milk stem cells.

Description

Description: A method for preparing a human mother ¥1 stem cell and an application thereof

[0001] The present invention belongs to the technical field of cell biology, and in particular relates to a method for preparing human breast milk stem cells and an application thereof.

Background technique

[0002] Stem cells are those cells that have the ability to self-replicate and multi-differentiate, which are constantly self-renewing and differentiate under certain conditions into one or more cells that make up a body's tissues or organs. The use of stem cells to transplant damaged tissue restores tissue regeneration and repairs its function. Stem cell replacement therapy offers great promise for the treatment of various diseases and tissue damage. Stem cells exist in embryos, mature tissues, and blood. In recent years, studies have found that human breast milk also contains stem cell components. Breast milk stem cells exhibit multipotential differentiation potential similar to human embryonic stem cells, and can express various pluripotency genes, such as OCT4. , SOX 2, NANOG, etc. These genes play an important role in maintaining the undifferentiated state and pluripotency of cells. Human breast milk stem cells have many natural advantages: they can be obtained by non-invasive methods, and can be provided by lactating women, with abundant sources. Therefore, human breast milk stem cells can be a stable and abundant source of cell replacement therapy.

[0003] There are currently few literatures on methods for preparing human breast milk stem cells, and there is no known standard preparation method for obtaining approval. According to the current literature reports, human breast milk stem cells are cultured in DMEM or RPMI medium supplemented with 10%-20 <3⁄4 fetal bovine serum. Based on this, some researchers have added insulin, epidermal growth factor and other substances. Scholars added feeder cells. However, substances such as insulin and epidermal growth factor do not have a very good effect on maintaining the undifferentiated state of human breast milk stem cells. Feeder cell culture can maintain the undifferentiated state of human breast milk stem cells slightly better than the former, but the experimental procedure It is more cumbersome and requires more time. Therefore, there is an urgent need to find a preparation method which is simple in operation and can better maintain the undifferentiated state of human breast milk stem cells.

technical problem

[0004] Type a paragraph describing the technical problem here.

Problem solution Technical solution

[0005] In view of the above technical problems, the present invention discloses a method for preparing human breast milk stem cells and an application thereof, which are simple in operation and do not require co-culture of feeder cells.

[0006] In this regard, the technical solution adopted by the present invention is:

[0007] A method for preparing human breast milk stem cells, comprising the steps of:

[0008] Step S1: collecting breast milk;

[0009] Step S2: separating human breast milk cells to obtain a purified breast milk cell suspension;

[0010] Step S3: breast cell suspension was transferred to purified coated plates volume percent to 0.01 to 0.05% of gelatin and placed in a CO ₂ incubator culture, medium was changed once a day, 6 days Isolate a single cell clone and inoculate it into a new culture plate to promote colony formation; preferably, the culture plate is a commercially available ultra-low adhesion culture plate;

[0011] Step S4: When the cells are fused to 80% (percent of the number of cell fusions) or more, trypsin is added, trypsin covers the surface of the cells, digest at 37 ° C for 5 min, and contain 10% in 2 volumes (volume percentage) The DMEM/F12 medium of fetal bovine serum was digested and centrifuged at 100 rpm for 10 min. The cell pellet was collected, washed once with PBS buffer, centrifuged at 1000 rpm for 10 min, and resuspended in fresh cell culture medium, passaged 1:3.

[0012] As a further improvement of the present invention, in the step S4, the composition of the cell culture solution is: containing 10% (volume percentage, the same below) KOSR (knockout serum replacement), 10-20 ng/mL alkali Human fibroblast growth factor, 10-20 ng/mL epidermal growth factor, 100-300 U/mL leukemia inhibitory factor, DMEM/F12 medium containing 0.1-0.5 mol/mL retinol.

[0013] As a further improvement of the present invention, the composition of the cell culture solution is: containing 10% KOSR, 12-15 ng/mL basic fibroblast growth factor, 12-18 ng/mL epidermal growth factor, 150- 250 U/mL leukemia inhibitory factor, DMEM/F12 medium containing 0.2-0.4 mol/mL retinol.

[0014] As a further improvement of the present invention, the composition of the cell culture fluid is: 10% KOSR, 10 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor, 100 U/mL leukemia inhibition Factor, DMEM/F12 medium containing 0.5 mol/mL retinol.

[0015] As a further improvement of the present invention, in step S1, before the collection of breast milk, the donor ABO/Rh blood type, HLA typing and syphilis, HIV, HBV, HCV are detected, and then the donor's breast milk is collected under aseptic conditions. 5- 200mL.

[0016] As a further improvement of the present invention, in step S2, the separating human breast milk cells comprises: mixing breast milk with an equal volume of PBS buffer, performing centrifugation at 1500-2000 rpm at 4 ° C, aspirating the fat layer and Skim milkmilk ingredients.

[0017] As a further improvement of the present invention, the remaining cell pellet was washed three times with PBS buffer, centrifuged at 1000-2 OOO rpm for 5-15 min, and the cell pellet was resuspended in fresh culture to obtain a purified breast milk cell suspension. . Wherein, the fresh culture liquid is a freshly arranged culture liquid, and may be a culture liquid commonly used in the art. The fresh medium may be DMEM medium containing 10% by volume of fetal calf serum.

[0018] As a further improvement of the present invention, the mother milk and an equal volume of PBS buffer are mixed, and centrifuged at 20 rpm for 20 minutes at 4 ° C to absorb the fat layer and the skim milk component.

[0019] As a further improvement of the present invention, the remaining cell pellet was washed three times with PBS buffer, centrifuged at 1500 rpm for 10 min each time, and the cell pellet was resuspended in fresh culture to obtain a purified breast milk cell suspension. Wherein, the fresh culture liquid is a freshly arranged culture liquid, and may be a culture liquid commonly used in the art. The fresh culture solution may be DMEM medium containing 10% by volume of fetal calf serum.

[0020] As a further improvement of the present invention, the purified breast milk cell suspension is transferred to a 0.01-0.05% gelatin culture plate before coating, the gelatin culture plate is coated for 1 hour in advance, and washed with PBS at least 2 times before cell culture.

[0021] As a further improvement of the present invention, the gelatin culture plate has a gelatin percentage of 0.01% by volume.

[0022] The present invention also discloses the use of a human breast milk stem cell prepared by the method for preparing human breast milk stem cells according to any one of the above, which is applied to cell replacement therapy to promote human tissue repair. in.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] By adopting the technical scheme of the invention, the operation is simple, the feeder cell co-culture is not needed, and the undifferentiated state of the human breast milk stem cells can be better maintained, and the number of human breast milk stem cells obtained is more.

Advantageous effects of the invention

Beneficial effect

[0025] Type a paragraph of beneficial effects description here. Brief description of the drawing

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a view showing the results of flow cytometry identification of human breast milk stem cells on day 20 of the culture of Example 1 of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0027] The paragraphs describing the best mode of the invention are entered here.

Embodiments of the invention

[0028] The preferred embodiments of the present invention are further described in detail below with reference to the accompanying drawings. It is understood that the specific embodiments described below are merely illustrative of the invention and are not intended to limit the invention.

Embodiment 1

[0030] A method for preparing human breast milk stem cells, comprising the steps of:

[0031] 1. Collecting breast milk

[0032] Human breast milk collection: Before collecting breast milk, the donor ABO/Rh blood type, HLA typing and syphilis, HI V, HBV, HCV are detected, and then the donor's breast milk is collected under sterile conditions, 5-200 mL, and immediately sent Preparation of human breast milk stem cells was performed in the laboratory.

[0033] 2. Isolation of human breast cells

[0034] The breast milk was mixed with an equal volume of PBS buffer, centrifuged at 2000 rpm for 20 min at 4 ° C, the fat layer and the skim milk component were aspirated, and the remaining cell pellet was washed three times with PBS buffer, and centrifuged at 1500 rpm for 10 min. The fresh culture medium resuspend the cell pellet and harvest the purified breast milk cell suspension.

[0035] 3. Culture human breast milk stem cells

[0036] Transfer the purified breast milk cell suspension to a culture plate coated with 0.01% gelatin, the coating coated with 0.01% gelatin was pre-coated for 1 h, and washed twice with PBS before cell culture; The cells were cultured in a CO ₂ incubator, and the cells were changed once a day. On the sixth day, individual cell clones were isolated and inoculated into new ultra-low adhesion culture plates to promote colony formation. When the cells are fused to more than 80% sputum, add 0.25% trypsin, trypsin to cover the cell surface, digest at 37 ° C for 5 min, and digest with 2 volumes of DMEM/F12 medium containing 10% fetal bovine serum, lOOOOrpm After centrifugation for 10 min, the cell pellet was collected and washed once with PBS buffer, 100 After centrifugation at Orpm for 10 min, the cells were resuspended in fresh cell culture medium and passaged 1:3.

[0037] the cell culture fluid is 10% KOSR, 10 ng / mL basic fibroblast growth factor (bFGF), 20 ng / mL epidermal growth factor (EGF), 100 U / mL leukemia inhibitory factor (LIF), DMEM/F12 medium of 0.5 mol/mL retinol.

[0038] Human breast milk stem cells cultured on day 20 were identified by flow cytometry, as shown in FIG. 1, the results showed that S0X2

NANOG and 0CT4 were highly expressed, and the positive proportions were 72<3⁄4, 70%, and 53%, respectively. It is indicated that the human breast milk stem cells cultured by the method of the present invention have high purity.

Example 2

[0040] On the basis of Example 1, the steps of culturing human breast milk stem cells are different, as follows: [0041] Transfer the purified breast milk cell suspension to a culture plate coated with 0.01% gelatin, The bag was coated with 0.01% gelatin in advance for 1 h, washed twice with PBS before cell culture, then placed in a CO ₂ incubator, and changed once a day. On day 6, individual cell clones were isolated and inoculated into new ones. In ultra-low adhesion culture plates, promote colony formation. When the cells are fused to more than 80% sputum, add 0.25% trypsin, trypsin to cover the cell surface, digest at 37 ° C for 5 min, and digest with 2 volumes of DMEM/F12 medium containing 10% fetal bovine serum, lOOOOrpm After centrifugation for 10 min, the cell pellet was collected, washed once with PBS buffer, centrifuged at 100 °C for 10 min, and resuspended in fresh cell culture medium, passaged 1:3.

[0042] The cell culture medium is 10% KOSR, 20 ng/mL basic fibroblast growth factor (bFGF), 10 ng/mL epidermal growth factor (EGF), 300

U/mL leukemia inhibitory factor (LIF), Ο.ΙιηοΙ/mL retinol DMEM/F12 medium.

[0043] The human breast milk stem cells cultured on the 20th day were identified by flow cytometry. The results showed that S0X2, NANOG, and O CT4 were highly expressed, and the positive proportions were 65%, 66%, and 42%, respectively. It is indicated that human breast milk stem cells cultured by the method of the present invention have high purity.

Comparative Example 1

[0045] This example differs from the embodiment in that the steps of culturing human breast milk stem cells are different, and a conventional method is employed, as follows:

[0046] The purified breast cell suspension transferred to a cell density of 5x10 ⁴ / cm ² is covered with the mouse embryonic fibroblasts (MEFs, seeding density of 5x10 4 / cm 2) of the dish, placed in a CO ₂ incubator Cultivate in the box, change the liquid once every other day, change the liquid once a day after 1 week, add the appropriate amount of 0.25% trypsin when the cells are fused to more than 80% Enzyme, trypsin covers the surface of the cells, digest at 37 ° C for 5 min, terminate digestion with 2 volumes of DMEM/F12 medium containing 10% fetal bovine serum, centrifuge at 1000 rpm for 10 min, collect the cell pellet, and wash once with PBS buffer. After centrifugation at 1000 rpm for 10 min, the cells were resuspended in fresh cell culture medium and passaged 1:3.

[0047] The cell culture fluid is 10% autologous plasma or fetal bovine serum, 20 ng / mL basic fibroblast growth factor (bFGF), 10 ng / mL epidermal growth factor (EGF), 5 g / mL insulin RPMI1640 Medium.

[0048] The human breast milk stem cells cultured on the 20th day were identified by flow cytometry, and the results showed that the positive proportions of SOX2, NANOG, and OCT4 were 55%, 62%, and 40%, respectively. Comparing with Example 1 and Example 2, it can be seen that the human breast milk stem cell human breast milk stem cells obtained by the methods of Example 1 and Example 2 have higher purity, and can better maintain the undifferentiated state of human breast milk stem cells.

Example 3

[0050] The human breast milk stem cell proliferation activity test was carried out on Example 1 and Comparative Example, and the specific process was as follows:

The human breast milk stem cells of Example 1 and Comparative Example 1 were cultured on the 20th day, 6 fields of view (200x) were taken under an inverted microscope, and the number of undifferentiated cells was counted, and the human breast milk stem cells prepared by the method of Example 1 were prepared. The number of human breast milk stem cells prepared by the method of Comparative Example 1 was 4.17 + 0.75. Therefore, the number of human breast milk stem cells prepared in Example 1 was significantly higher than that of the conventional method.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and it is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.

Industrial applicability

[0053] Enter the paragraph of industrial applicability description here.

Sequence table free content

[0054] Type the sequence table free content description paragraph here.

Claims

Claim

[Claim 1] A method for preparing a human breast milk stem cell, comprising the steps of:

Step S1: collecting breast milk;

Step S2: separating human breast milk cells to obtain a purified breast milk cell suspension;

Step S3: Transfer the purified breast milk cell suspension to a culture plate coated with gelatin in a volume percentage of 0.01-0.05 <3⁄4, culture in a CO ₂ incubator, change the solution once a day, and separate the individual on the 6th day. The cells are cloned and inoculated into an ultra-low-adhesion culture plate to promote colony formation; Step S4: When the cells are fused to more than 80% sputum, trypsin is added, trypsin covers the surface of the cells, and digested at 37 ° C for 5 min, twice The DMEM/F1 2 medium containing 10% fetal bovine serum was centrifuged at 1000 rpm for 10 min. The cell pellet was collected, washed once with PBS buffer, centrifuged at 1000 rpm for 10 min, and resuspended in fresh cell culture medium. 3 pass down.

[Claim 2] The method for preparing human breast milk stem cells according to claim 1, wherein: step S

In 4, the composition of the cell culture solution is: 10% KOSR, 10-20 ng/mL basic fibroblast growth factor, 10-20 ng/mL epidermal growth factor, 100-300 U/mL leukemia inhibitory factor , DMEM/F12 medium containing 0. l-0.5 mol/mL retinol.

[Claim 3] The method for preparing human breast milk stem cells according to claim 2, wherein the composition of the cell culture fluid is: 10% KOSR, 12-15 ng/mL basic fibroblast growth factor, 12-18 ng/mL epidermal growth factor, 150-250 U/mL leukemia inhibitory factor, DMEM/F12 medium containing 0.2-0.4 mol/mL retinol.

[Claim 4] The method for preparing human breast milk stem cells according to claim 2, wherein the composition of the cell culture fluid is: 10% KOSR, 10 ng/mL basic fibroblast growth factor, 20 ng /mL epidermal growth factor, 100 U/mL leukemia inhibitory factor, DMEM/F12 medium containing 0.5 mol/mL retinol.

[Claim 5] The method for preparing human breast milk stem cells according to claim 1, wherein: step S

In 1, before the collection of breast milk, the donor ABO/Rh blood type, HLA typing and syphilis, H IV, HBV, HCV were detected, and then the donor's breast milk 5-200 mL was collected under aseptic conditions.

[Claim 6] The method for preparing human breast milk stem cells according to claim 1, wherein: step S In 2, the separating human breast milk cells comprises: mixing breast milk with an equal volume of PBS buffer, centrifuging at 1500-2000 rpm at 4 ° C, aspirating the fat layer and the skim milk component; the remaining cell pellet is buffered with PBS The solution was washed 3 times, centrifuged at 1000-2000 rpm for 5-15 min each time, and the cell pellet was resuspended in fresh culture to obtain a purified breast milk cell suspension. The method for preparing human breast milk stem cells according to claim 6, wherein: the mother milk is mixed with an equal volume of PBS buffer, centrifuged at 2000 rpm for 20 min at 4 ° C, and the fat layer and the skim milk are absorbed. Ingredients; The remaining cell pellet was washed 3 times with PBS buffer, centrifuged at 1500 rpm for 10 min each time, and the cell pellet was resuspended in fresh culture to obtain a purified breast milk cell suspension.

The method for preparing human breast milk stem cells according to claim 6, wherein: the purified breast milk cell suspension is transferred to a 0.01-0.05% gelatin culture plate, and the gelatin culture plate is coated for 1 hour in advance, and the cells are used. Wash with PBS at least 2 times before culture.

The method for producing human breast milk stem cells according to claim 6, wherein the culture plate has a coated gelatin having a volume percentage of 0.01%.

The invention relates to a human breast milk stem cell, which is characterized in that: the human breast milk stem cell is prepared by the method for preparing human breast milk stem cells according to any one of claims 19 to be used in cell replacement therapy to promote human tissue repair. .