Cow milk-derived stem cell and isolation and culture method therefor

Abstract

Provided are a cow milk-derived stem cell, and an isolation and culture method therefor. The method comprises the following steps: (1) taking fresh cow milk, and isolating cells therefrom; (2) coating a culture dish with a matrix gel in advance, discarding the coating solution, then washing the culture dish with a washing solution, and discarding the washing solution; (3) inoculating the cells treated in the step (1) on the coated culture dish, and culturing the cells with a stem cell culture medium; and (4) when the cell confluence reaches 80% or more, digesting the cells with a digestion enzyme, terminating the digestion, isolating the cells, resuspending the cells using a CTFR culture solution, then inoculating the cells on a feeder layer for culture, promoting colony formation, and performing subculture to obtain cow milk-derived stem cells. The cow milk-derived stem cell has OCT4, SOX2, NANOG and SSEA4 markers.

Description

Bovine milk-derived stem cells and a separation and culture method thereof

[0001] CROSS-REFERENCE

[0002] The present application claims priority to the Chinese patent application No. 202410643505.3, filed on May 23, 2024, with the title of "Bovine milk-derived stem cells and a separation and culture method thereof", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the technical field of stem cell culture, and particularly relates to bovine milk-derived stem cells and a separation and culture method thereof. BACKGROUND

[0004] Stem cells are a class of primitive undifferentiated cells with multilineage differentiation potential and self-replication capacity, and are the original cells for forming various tissues and organs of mammals. Stem cells have common characteristics in morphology, usually being round or oval, with a relatively large nucleus, and the cell nucleus is mostly euchromatin with high telomerase activity. Stem cells can be divided into embryonic stem cells and adult stem cells.

[0005] Embryonic stem cells are totipotent stem cells with the potential to differentiate into any type of cell. Adult stem cells can only differentiate into one or more types of cells, with limited differentiation capacity. However, in the field of stem cell therapy, adult stem cells are often differentiated into target cell types because embryonic stem cells are almost impossible to collect for individual therapy, and the conditions for embryonic stem cells to differentiate into single type cells are more complex. Therefore, the separation of stem cells from adult tissues has become a key research direction.

[0006] Bovine stem cells are usually obtained from bovine umbilical cord, bovine bone marrow or bovine embryos, and mesenchymal stem cells or totipotent embryonic stem cells are obtained. In recent years, it has been found that human breast milk also contains stem cell components (reference Breastmilk is a novel source of stem cells with multilineage differentiation potential. Stem Cells.), and breast milk stem cells exhibit multilineage differentiation potential similar to human embryonic stem cells, and can express various pluripotency genes such as OCT4, SOX2, NANOG, etc. These genes play a very important role in maintaining the undifferentiated state and pluripotency of cells. However, there are few literature materials on the preparation method of bovine milk-derived stem cells at present, and there is no recognized standardized preparation method. There is also no related literature report on the separation and culture of bovine milk-derived stem cells.

[0007] The inventors found in the research that the isolation and culture of stem cells from cow milk are difficult, and the culture system is not perfect, which leads to the difficulty in culture and isolation. SUMMARY

[0008] OBJECTIVE

[0009] The present application aims to provide a stem cell from cow milk and a method for isolating and culturing the same. The stem cell is isolated and cultured from cow milk for the first time, and the obtained stem cell has OCT4, SOX2, NANOG and SSEA4 markers. A new source of cow stem cells is provided.

[0010] SOLUTION

[0011] In a first aspect, the present application provides a stem cell from cow milk, which is obtained by isolation and culture from cow milk, and the expression markers of the stem cell are OCT4, SOX2, NANOG and SSEA4.

[0012] In a second aspect, the present application provides a method for isolating and culturing the stem cell from cow milk according to the first aspect, which is performed according to the following steps:

[0013] (1) Fresh cow milk is taken, and cells in the cow milk are isolated;

[0014] (2) The culture dish is coated with a stem cell adherent culture matrix gel in advance, and then the liquid is discarded, and then the culture dish is washed with a cleaning solution and the liquid is discarded;

[0015] (3) The cells treated in step (1) are inoculated on the coated culture dish, and cultured with a stem cell culture medium;

[0016] (4) When the cells are fused to more than 80%, the cells are digested with a digestive enzyme, the digestion is terminated, the cells are isolated, resuspended with a stem cell culture solution, inoculated on feeder cells for culture, and cloned to obtain cow milk stem cells. Further, in step (1), the cell isolation method comprises: centrifuging the cow milk to separate layers, removing the upper cream and turbid liquid, retaining the bottom precipitate and transparent cell layer; resuspending the cell layer, separating the cells from the bottom precipitate, and isolating and culturing the obtained cells.

[0017] Further, the centrifugation conditions of the cow milk are 2500-3500 rpm for 15-25 min.

[0018] Further, the cell separation method in the cell solution is centrifugation, and the cells are obtained by centrifuging the supernatant.

[0019] Further, the resuspension solution is PBS solution; alternatively, the amount of PBS solution added is 1-3 times the volume of the remaining bottom precipitate.

[0020] Further, in step (1), the fresh cow milk is obtained by disinfecting the udder surface of the milking cow, and the disinfection is performed by wiping with alcohol, and the number of wiping is 4-8 times, and the concentration of alcohol is 75%.

[0021] Further, in step (2), the coating time is 1-2 hours, and the cell adhesion substrate is Vitronectin XF, and the culture temperature is 25-35℃.

[0022] Further, the cleaning solution is CellAdhere Dilution Buffer.

[0023] Further, in step (3), the stem cell culture medium is mTeSR Plus, which is beneficial for obtaining bovine milk stem cells.

[0024] Further, in step (3), the culture condition in the incubator is 37℃ and 5% CO2.

[0025] Further, in step (4), the stem cell culture medium is CTFR culture medium.

[0026] Further, in step (4), the stem cell culture medium is changed every day.

[0027] Further, in step (4), the feeder layer cells are mouse fetal fibroblasts.

[0028] Further, in step (4), the digestion enzyme is Tryple, and the digestion time is 2-4 minutes.

[0029] Further, in step (4), the digestion is terminated by using 2 times the amount of Wash of the digestion enzyme.

[0030] Further, in step (4), the method for separating the cells is centrifugation, and the centrifugation condition is 400-600 rpm for 4-6 minutes.

[0031] In a third aspect, the application provides a use of the bovine milk-derived stem cells of the first aspect or the bovine milk stem cells separated by the separation and culture method of the second aspect in the preparation of a stem cell replacement therapy or a product for promoting tissue repair or promoting stem cell differentiation.

[0032] A possible specific method for separating stem cells from bovine milk can be as follows:

[0033] (1) use the medical gauze soaked with alcohol to wipe the dairy cow's udder, take the milk and put it in the centrifuge tube, centrifuge, the four layers from top to bottom are yellow opaque cream layer, turbid liquid layer, transparent cell layer and white precipitate layer, discard the yellow opaque cream layer and the liquid of turbid liquid layer;

[0034] (2) dilute the transparent cell layer and white precipitate layer of step (1) with PBS solution, centrifuge, discard the upper liquid, add equal volume of PBS solution to blow the cell mass, do not blow the lower white precipitate; transfer the supernatant to another centrifuge tube, add equal volume of PBS solution, centrifuge, discard the upper liquid with a pipette, and retain the precipitate;

[0035] (3) coat the 6-well plate in advance (usually one hour in advance), discard the liquid, then wash with the washing solution, discard the liquid;

[0036] (4) resuspend the precipitate prepared in step (2) with stem cell culture medium, inoculate in the 6-well plate, and place in the incubator for culture;

[0037] (5) when the cells grow to 75-85%, digest them with digestive enzyme, then terminate with termination solution, centrifuge, discard the supernatant, resuspend with stem cell culture solution, and inoculate on the feeder layer cells for culture, pass 6-10 generations to obtain stem cells. Beneficial effects:

[0038] The application first separates and feeds stem cells from cow's milk, and the stem cells obtained by specific disinfection and specific culture method have OCT4, SOX2, NANOG, and SSEA4 markers. A new type of stem cell is provided for inducing differentiation of bovine cells. BRIEF DESCRIPTION OF DRAWINGS

[0039] One or more embodiments are illustrated by way of example in the figures that constitute a part of this patent document. The illustrative examples set forth in the figures are not to be construed as limiting the embodiments. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0040] Figure 1 is an optical microscope image of P0 cells (primary isolated cells) of Example 1.

[0041] Figure 2 is an optical microscope image of P8 cells (cells cultured for 8 passages) of Example 1.

[0042] Figure 3 is a PCR product electrophoresis map of Example 1.

[0043] Figure 4 is the result of the immunofluorescence experiment of Example 1. DETAILED DESCRIPTION

[0044] For the purposes of promoting an understanding of the principles of the application, the application will now be described more fully hereinafter. However, it should be understood that the application can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0045] Because the isolation and culture of stem cells from bovine milk are difficult, such as the sampling process is easily contaminated, the culture system is not perfect, leading to difficulty in isolation and obtaining, the inventors have tried continuously, and the selected isolation and culture method is more conducive to the growth of stem cells, so that bovine milk stem cells are obtained.

[0046] The isolation and culture method of the stem cells from bovine milk is carried out according to the following steps:

[0047] (1) A medical gauze soaked with 75% alcohol by volume is used to wipe the udder of a dairy cow, and the wiping is performed 5 times. The bovine milk is placed in a centrifuge tube, and centrifuged at 3000 rpm for 20 min. The centrifugation is divided into four layers from top to bottom, which are a yellow opaque cream layer, a turbid liquid layer, a transparent cell layer, and a white precipitate layer. The yellow opaque cream layer and the turbid liquid layer are discarded;

[0048] (2) The transparent cell layer and the white precipitate layer prepared in step (1) are diluted with PBS liquid at a volume ratio of 1:1, and then centrifuged. The supernatant liquid is discarded, and an equal volume of PBS liquid is added to blow the cell mass. The lower white precipitate is not blown. The supernatant liquid is transferred to another centrifuge tube, and an equal volume of PBS liquid is added. The mixture is centrifuged at 3000 rpm for 20 min. The supernatant liquid is discarded with a pipette, and the precipitate is retained;

[0049] (3) A 6-well plate is coated with coating liquid Vitronectin XF (purchased from STEM CELL, 07108) for 1 h, and then washed with cleaning liquid CellAdhere Dilution Buffer (purchased from STEM CELL, 07183). The liquid is discarded;

[0050] (4) The precipitate prepared in step (2) is resuspended with culture liquid mTeSR Plus (purchased from STEM CELL, 100-0276) and inoculated into a 6-well plate. The plate is placed in a 37℃, 5% CO2 incubator for culture. The liquid is changed after 1 day of inoculation;

[0051] (5) When the cells grow to 80%, they are digested with Tryple (purchased from GIBCO, 12604021) for 3 min, then terminated with Wash (DMEM / F-12 (purchased from GIBCO, 21041025) supplemented with BSA (purchased from GIBCO, 1891702)), centrifuged at 500 rpm for 5 min, the supernatant is discarded, resuspended with CTFR culture solution (mTeSR Plus supplemented with FGF2 and IWR1), and then inoculated on feeder cells (mouse fetal fibroblasts) for culture, the medium is changed every day, and the cells are subcultured every 3 days.

[0052] Experimental Example 1, Cell Morphology Observation

[0053] The isolated and cultured method of bovine milk-derived stem cells of Example 1 and the stem cells prepared thereby were used as the detection object, and the P0 generation of cells (P0 generation cultured with mTeSR Plus and Matrigel) and the P8 generation of cells (P8 generation cultured with CTFR and feeder cells) were observed by electron microscopy, as shown in FIG. 1 and FIG. 2, and the results showed that the morphology of the isolated stem cells was good. TM Plus and Matrigel) and the P8 generation of cells (P8 generation cultured with CTFR and feeder cells) were observed by electron microscopy, as shown in FIG. 1 and FIG. 2, and the results showed that the morphology of the isolated stem cells was good.

[0054] Experimental Example 2, RT-PCR Determination of the Expression Amount of OCT4, SOX2, NANOG, and KLF4 mRNA in the Obtained Stem Cells

[0055] The total RNA in the stem cells obtained in Example 1 was extracted by using Qiagen RNA-easy Isolation Reagent R701 kit, 1 pg to 1 ug of mRNA was taken as a template, and the mRNA was reversely transcribed into cDNA by using Qiagen HiScript II Q Select RT SuperMix for qPCR(+gDNA wiper) R233-01 kit and operation method. Then, the cDNA was used as a template for PCR amplification to obtain the target gene or detect the gene expression. The primer was entrusted to be synthesized, and the primer sequence is shown in Table 1 (selected primer reference: Hassiotou F, Beltran A, Chetwynd E, Stuebe AM, Twigger AJ, Metzger P, Trengove N, Lai CT, Filgueira L, Blancafort P, Hartmann PE. Breast milk is a novel source of stem cells with multilineage differentiation potential. Stem Cells. 2012 Oct; 30(10): 2164-74. doi: 10.1002 / stem.1188. PMID: 22865647; PMCID: PMC3468727.).

[0056] Table 1, primer list:

[0057] The PCR product was taken for electrophoresis, and the results are shown in Figure 3. The results show that the mRNA of OCT4, SOX2, NANOG and KLF4 has strong expression in the prepared stem cells.

[0058] Experimental Example 3, determination of the expression amount of OCT4, SOX2, NANOG and SSEA4 protein in the obtained stem cells by immunofluorescence

[0059] I. Cell preparation:

[0060] When subcultured, the cells were inoculated into a 35 mm culture dish in which a cover glass was previously placed, and the cover glass was taken out after the cells grew.

[0061] II. Fixation and permeation

[0062] 1. The cover glass with cells climbed in the culture dish was washed with 1xPBS for 3 times, 3 min each time.

[0063] 2. The climbed cover glass was fixed with 2 ml of 4% paraformaldehyde for 15 min.

[0064] 3. 0.4% Triton X-100 (1x PBS) to permeabilize cells for 30 min at room temperature.

[0065] III. Blocking

[0066] 4. Discard the solution, add 500 uL of 3% BSA to the slide and block for 1 h at room temperature.

[0067] IV. Antibody Incubation

[0068] 5. Absorb the blocking solution with a paper towel, add 300 uL of 0.3% BSA diluted primary antibody (OCT4 (purchased from Cell Signaling Technology, 2840T) and SOX2 (purchased from Cell Signaling Technology, 4900), NANOG (purchased from Cell Signaling Technology, 4903T) and SSEA4 (purchased from Cell Signaling Technology, 4755Tb)) to each slide and place in a humidified box, incubate overnight at 4°C.

[0069] 6. Add fluorescent secondary antibody: 0.3% BSA to wash the slide 3 times for 10 min each time, absorb the excess liquid on the slide with a paper towel, add 300 uL of 0.3% BSA diluted fluorescent secondary antibody (Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) Cell Signaling Technology 4412S, Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor 647 Conjugate) Cell Signaling Technology 4410S) to the slide, incubate in a humidified box at 37°C for 1 h, 0.3% BSA to wash the slide 3 times for 10 min each time.

[0070] V. Fixation and Photography

[0071] 7. Counterstain the nucleus: add DAPI and incubate in the dark for 10 min to stain the nucleus of the specimen, 0.3% BSA to wash away the excess DAPI 3 times for 5 min each time.

[0072] 8. Absorb the liquid on the slide with a paper towel, mount the slide with an anti-fluorescence quencher containing mounting medium, and observe and collect images under a laser confocal microscope.

[0073] The results of the immunofluorescence are shown in FIG. 4, which shows that the cells obtained from the bovine milk express stem cell marker proteins OCT4, SOX2, NANOG, and SSEA4.

[0074] The above described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it cannot be understood as the limitation of the patent scope of the present application. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Industrial applicability

[0075] The present application relates to a bovine milk-derived stem cell and a separation and culture method thereof. The separation and culture method comprises the following steps: (1) taking fresh bovine milk to separate and obtain cells in the bovine milk; (2) coating a culture dish with a substrate glue in advance, discarding the liquid, then washing with a cleaning liquid, and discarding the liquid; (3) inoculating the cells treated in step (1) on the coated culture dish, and culturing with a stem cell culture medium; (4) when the cells are fused to more than 80%, digesting with a digestive enzyme, stopping digestion, separating the cells, resuspending with a CTFR culture solution, and inoculating on feeder layer cells for culture to promote clone formation, and subculturing to obtain bovine milk stem cells. The present application first separates and cultures stem cells from bovine milk, can better maintain the undifferentiated state of bovine milk stem cells, and the obtained stem cells have OCT4, SOX2, NANOG, and SSEA4 markers. A new type of stem cell is provided for bovine cell induction and differentiation.

Claims

1. A bovine milk-derived stem cell, characterized in that, The stem cells are isolated from cow milk and subcultured, and the expression markers of the stem cells are OCT4, SOX2, NANOG and SSEA4.

2. A method for isolating and culturing bovine milk stem cells as described in claim 1, characterized in that, The following steps are taken: (1) Fresh cow milk is taken, and cells in the cow milk are isolated; (2) The culture dish is coated with a stem cell adherent culture matrix gel in advance, and then the liquid is discarded, and then the culture dish is washed with a washing liquid and the liquid is discarded; (3) The cells treated in step (1) are inoculated on the coated culture dish, and cultured with a stem cell culture medium; (4) When the cells are fused to more than 80%, the cells are digested with a digestive enzyme, the digestion is terminated, the cells are isolated, and then the cells are resuspended with a stem cell culture solution and inoculated on feeder cells for culture, so as to promote the formation of clones and obtain cow milk stem cells through subculture.

3. The separation culture method according to claim 2, characterized by, In step (1), the cell isolation method comprises: centrifuging the cow milk to separate layers, removing the upper cream and turbid liquid, retaining the bottom precipitate and transparent cell layer; resuspending the cell layer, separating the cells from the bottom precipitate, and isolating the obtained cells for culture.

4. The separation culture method according to claim 2, characterized by, In step (1), the surface of the udder of the milk cow is disinfected when fresh cow milk is taken; Or, in step (1), the surface of the udder of the milk cow is disinfected by alcohol wiping when fresh cow milk is taken, and the wiping frequency is 4-8 times.

5. The separation and culture method according to claim 2, wherein In step (2), the coating time is 1-2 hours, And / or, the stem cell adherent culture matrix gel is Vitronectin XF; And / or, the washing liquid is CellAdhere Dilution Buffer.

6. The separation and culture method according to claim 2, wherein In step (3), the stem cell culture medium is mTeSR Plus; And / or, in step (3), the culture conditions in the incubator are 37℃ and 5% CO2.

7. The separation culture method according to any one of claims 2 to 6, characterized by, In step (4), the stem cell culture solution is CTFR culture solution; And / or, in step (4), the stem cell culture solution is changed every day.

8. The separation culture method according to any one of claims 2 to 6, characterized by, In step (4), the feeder cells are mouse fetal fibroblasts.

9. The separation culture method according to any one of claims 2 to 6, characterized by, In step (4), the digestive enzyme is Tryple; And / or, in step (4), the digestion is terminated by using 2 times the amount of Wash of the digestive enzyme; And / or, in step (4), the method for isolating the cells is centrifugation, and the centrifugation conditions are 400-600 rpm for 4-6 min.

10. Use of the stem cells from cow milk of claim 1 or the cow milk stem cells isolated by the isolation and culture method of any one of claims 2 to 9 in the preparation of a stem cell replacement therapy or a product for promoting tissue repair or promoting stem cell differentiation.

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