CN107164319B - Method for primary culture of canine umbilical cord-derived mesenchymal stem cells - Google Patents
Method for primary culture of canine umbilical cord-derived mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a method for primary culture of canine umbilical cord-derived mesenchymal stem cells. The mesenchymal stem cells are derived from umbilical cords of newborn dogs, have wide sources and easily obtained materials, and make mass production and clinical application of the cells possible in the later period. The culture method is simple to operate and good in repeatability, and less hybrid cells and pollution-free pure mesenchymal stem cells can be obtained. The collagenase digestion method used in the culture method is mild, has little damage to cells, can obtain a large amount of mesenchymal stem cells with strong proliferation capacity, and can be used for the subsequent establishment of the cells, the characteristics of the stem cells, the treatment of diseases and the like.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for primary culture of canine umbilical cord-derived mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are adult Stem Cells with multidirectional differentiation capability, can be directionally differentiated into various Cells such as muscle, cartilage, blood vessels, endothelium, nerves and the like under specific conditions, are the adult Stem Cells with the most clinical application prospect at present, and have a remarkable effect in the treatment of cardiovascular diseases, blood diseases, joint injuries and other diseases proved by multiple studies. The mesenchymal stem cells from the umbilical cord have the advantages of easily obtained materials and low immunogenicity, avoid ethical and moral disputes in disease treatment, and have wide market prospect. The treatment technology of the diseases of the dogs, which are pets gradually welcomed by people, is rapidly developed, however, no effective treatment medicine for the diseases such as arthritis, nerve injury, kidney injury and the like exists at present, and a new thought is provided for the treatment of the mesenchymal stem cells.
At present, human mesenchymal stem cells are deeply researched, but research on canine umbilical cord mesenchymal stem cells is rarely reported. In order to deeply research the application of the canine umbilical cord mesenchymal stem cells in the treatment of serious diseases, an efficient separation and culture method is particularly important.
Disclosure of Invention
In order to overcome the defects and shortcomings in the process of separating the mesenchymal stem cells by a common method, the invention provides a method for separating and culturing the canine umbilical cord mesenchymal stem cells, and lays a foundation for in vitro culture, establishment and application of the canine umbilical cord mesenchymal stem cells.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a method for primary culture of canine umbilical cord-derived mesenchymal stem cells comprises the following steps:
1) collecting umbilical cords of newborn dogs:
stripping placenta with a sterile instrument when a puppy emerges from a mother body, shearing an umbilical cord, and immediately putting the umbilical cord into PBS (phosphate buffer solution) containing 10% double-antibody solution;
2) isolating mesenchymal stem cell-rich glial tissue from umbilical cord:
cutting off the umbilical cord capsule to expose the middle tubular structure, removing artery and vein, and collecting the remaining hollow milky white tube without blood as the required tubular tissue;
3) washing and cutting tissue pieces:
soaking the separated tubular tissue in PBS buffer solution containing 10%, 5% and 1% double antibody solution for 5 min, transferring into container, and cutting to 1mm with tissue scissors3Size;
4) collagenase digestion:
adding 1% collagenase type I in an amount of about 10 times the volume of the tissue mass into a container containing the tissue mass, and digesting the mixture in an incubator at 37 ℃ for more than 12 hours;
5) amplification culture:
diluting the digested liquid with umbilical cord mesenchymal stem cell culture solution, sieving with 200 mesh sieve, centrifuging at 1200rpm for 5 min, discarding supernatant, adding umbilical cord mesenchymal stem cell culture solution, blowing uniformly, transferring into culture dish, and adding 5% CO2The culture was carried out in an incubator at 37 ℃.
Further, the method also comprises the following steps: 6) and respectively carrying out liquid exchange of the umbilical cord mesenchymal stem cell culture solution after 24h and 72h of culture, and carrying out passage according to the growth condition of the mesenchymal stem cells.
The umbilical cord mesenchymal stem cell culture solution in the step 5 consists of a human umbilical cord mesenchymal stem cell complete culture medium with the volume ratio of 88%, fetal bovine serum with the volume ratio of 10%, a double-antibody solution with the volume ratio of 1% and a glutamine solution with the volume ratio of 1%.
The invention has the beneficial effects that:
(1) the mesenchymal stem cells are derived from umbilical cords of newborn dogs, have wide sources and easily obtained materials, so that mass production and clinical application of the cells at the later stage become possible;
(2) the method is simple to operate and good in repeatability, and less hybrid cells and pollution-free pure mesenchymal stem cells can be obtained;
(3) the collagenase digestion method used in the invention is mild, has little damage to cells, can obtain a large amount of mesenchymal stem cells with strong proliferation capacity, and can be used for the subsequent establishment of the cell lines, the characteristics of the stem cells, the treatment of diseases and the like.
Drawings
FIG. 1 is a graph of the growth obtained in example 4;
FIG. 2 is a graph showing the results of the identification obtained in example 5;
FIG. 3 is a graph showing the differentiation results obtained in example 6.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: (collagenase method)
1) Collecting umbilical cords of newborn dogs:
stripping placenta with a sterile instrument when a puppy emerges from a mother body, shearing an umbilical cord, and immediately putting the umbilical cord into PBS (phosphate buffer solution) containing 10% double-antibody solution;
2) isolating mesenchymal stem cell-rich glial tissue from umbilical cord:
cutting off the umbilical cord capsule to expose the middle tubular structure, removing artery and vein, and collecting the remaining hollow milky white tube without blood as the required tubular tissue;
3) washing and cutting tissue pieces:
(3-1) soaking the separated tubular tissue in a PBS (phosphate buffer solution) containing 10% double antibody for 5 minutes, and taking out the tubular tissue by using forceps;
(3-2) soaking the tubular tissue taken out in the step (3-1) in a PBS (phosphate buffer solution) containing 5% double antibody for 5 minutes, and taking out the tubular tissue by using forceps;
(3-3) soaking the tubular tissue taken out in the step (3-2) in a PBS (phosphate buffer solution) containing 1% double antibody for 5 minutes, and taking out the tubular tissue by using forceps;
(3-4) transferring the tubular tissue taken out in the step (3-2) into a sterile 100mL small beaker, and shearing the tubular tissue to 1mm by using tissue scissors3Size;
4) collagenase digestion:
adding 1% collagenase type I in an amount of about 10 times the volume of the mixture, and placing the mixture in an incubator at 37 ℃ for digesting for more than 12 hours until no tissue mass is visible to naked eyes;
5) amplification culture:
diluting with umbilical cord mesenchymal stem cell culture solution, sieving with 200 mesh sieve, centrifuging at 1200rpm for 5 min, discarding supernatant, adding umbilical cord mesenchymal stem cell culture solution, blowing uniformly, transferring into culture dish, and adding 5% CO2The culture was carried out in an incubator at 37 ℃.
The reagents used in this example include:
the preparation formula of the PBS buffer solution is as follows:
the complete culture medium of human umbilical cord mesenchymal stem cells is purchased from Cyagen company, and the commercial numbers are as follows: HUXUC-03011-440, which is prepared into umbilical mesenchymal stem cell culture solution according to the use instruction, has the following formula:
the double antibody solution and the serum are purchased from Hyclone company
Collagenase type i was purchased from SIGMA.
Example 2: (Trypsin method)
This example is one of the comparative examples, and the culture method is different from that of example 1 in that:
and 4) adding 0.25% trypsin with the volume about 5 times that of the mixture, digesting the mixture in a water bath kettle at 37 ℃ for 30min, and shaking the mixture from time to enable the digestive juice to fully react with the tissue blocks.
The trypsin was purchased from Hyclone.
Example 3: (tissue block method)
This example was used as one of the comparative examples, and the culture method was different from that of example 1 in steps 4) and 5):
4-1) uniformly spreading the cut umbilical cord tissue blocks on the bottom of a T25 culture flask with the cut umbilical cord tissue blocks downward, and then placing the culture flask with the bottom surface upward in 5% CO2Culturing in 37 deg.C incubator for 30 min;
4-2) taking out the culture bottle, adding 1ml of umbilical cord mesenchymal stem cell culture solution (just submerging the bottom of the bottle to avoid suspending tissue blocks), placing the culture bottle with the bottom face downward in 5% CO2Culturing in an incubator at 37 ℃ for 2 h;
4-3) taking out the culture bottle, adding 2ml of umbilical cord mesenchymal stem cell culture solution, completely immersing the tissue block, placing the tissue block on 5% CO with the bottom surface facing downwards2And cultured in an incubator at 37 ℃.
Example 4: plotting cell growth curves
(1) Taking well-grown third-generation canine mesenchymal stem cells from examples 1-3, digesting with 0.25% trypsin to prepare cell suspension;
(2) will be provided withCells were diluted to 3X 104Perml/mL into 24-well plates, 0.5mL per well, placed in a 5% CO2Culturing in an incubator at 37 ℃;
(3) randomly extracting 3-hole cells at the same time every day, digesting the cells with trypsin to prepare suspension, counting the cells, and counting the cells in each hole for three times to obtain an average value;
(4) eight days were counted continuously, and based on the counting results, the cell density was plotted as ordinate (counts/mL) and the time as abscissa, to obtain a growth curve as shown in FIG. 1.
Example 5: and (3) immunofluorescence identification:
(1) respectively selecting well-grown third-generation mesenchymal stem cells from examples 1-3, inoculating the well-grown third-generation mesenchymal stem cells on a culture plate coated by gelatin, allowing the cells to adhere to the wall for 12 hours, removing a culture medium, and washing the cells for 2 times by using PBS containing 5% FBS;
(2) adding 4% paraformaldehyde, fixing at room temperature for 30min, removing the fixing solution, and washing with PBS containing 5% FBS for 5 min for 3 times;
(3) adding a permeabilizing agent (0.2% Triton. times.100), incubating on ice for 20 minutes, discarding and washing 3 times with 5% FBS-containing PBS for 5 minutes each;
(4) adding blocking solution (PBS containing 10% FBS), blocking at 37 deg.C for 1 hr, removing blocking solution, and washing with PBS containing 5% FBS for 5 min for 3 times;
(5) adding primary antibodies of three surface marker proteins of CD44, CD34 and CD90 respectively, incubating overnight at 4 ℃ in the dark, discarding the liquid, and washing 3 times with PBS containing 5% FBS for 5 minutes each time;
(6) adding corresponding FITC labeled secondary antibodies respectively, incubating for 30 minutes at 4 ℃ in a dark place, discarding the liquid, and washing for 5 minutes each time for 3 times with PBS containing 5% FBS;
(7) adding 1 mu g/mL DAPI, counterstaining for 5 minutes in a dark place, discarding the liquid, and washing 3 times with PBS containing 5% FBS for 5 minutes each time;
(8) an anti-fluorescence quencher was added, and the staining result was observed under an inverted fluorescence microscope and photographed to obtain an identification result chart as shown in FIG. 2.
Example 6: selecting cells with good growth state for adipogenesis and osteogenesis induced differentiation
The reagents required for this example include:
the formula of the dog umbilical cord mesenchymal stem cell osteogenesis induced differentiation culture medium comprises the following components:
the formula of the medium for inducing differentiation of canine mesenchymal stem cells by adipogenesis comprises the following steps:
the step of inducing adipogenic differentiation:
(1) taking the canine umbilical cord mesenchymal stem cells prepared according to the method in the embodiment 1, and digesting the canine umbilical cord mesenchymal stem cells by using trypsin to prepare cell suspension when the canine umbilical cord mesenchymal stem cells are cultured until 80% of the canine umbilical cord mesenchymal stem cells are fused;
(2) the cell density was adjusted to 2X 104/cm2Adding the mixture into a six-hole plate, and adding 2ml of cell suspension into each hole;
(3) placing at 37 ℃ and 5% CO2Culturing the cells under the condition, and changing the liquid every 3 days;
(4) when the cells reach 100% confluence, removing the culture medium in the holes, washing the cells for 2 times by PBS, and adding 2ml of solution A into each hole;
(5) after three days of culture, the stock culture was discarded, washed 2 times with PBS, and 2ml of solution B was added to each well
(6) After 24 hours of incubation, the stock culture was discarded, washed 2 times with PBS, and 2ml of solution A was added to each well
(7) Repeating the steps (5) and (6) for 3-5 times, and finally culturing the cells in the solution B for 7 days, and changing the solution every 3 days;
(8) oil red O staining analysis: discarding culture solution in the hole, washing with PBS for 2 times, and adding 2ml of 4% formaldehyde solution to fix the cell for 30 minutes; the liquid was then discarded, washed 2 times with PBS, stained with 1ml of oil red O working solution for 30 minutes, washed 2 times with PBS, observed under a microscope and photographed.
(II) inducing osteogenic differentiation:
(1) when the UC-MSC of the dog is cultured to 80 percent fusion, the UC-MSC is digested by trypsin to prepare cell suspension;
(2) adjusting the cell density to 3 × 104/cm2, adding into six-well plate, adding 2ml cell suspension per well;
(3) placing at 37 ℃ and 5% CO2Culturing for 24h under the condition, discarding the original culture solution, washing with PBS for 2 times, and adding 2ml osteogenic differentiation culture solution;
(4) changing the solution every 3 days until alizarin red staining analysis is carried out after 2-3 weeks;
(5) alizarin red staining analysis: removing the original culture solution, washing with PBS for 2 times, and adding 2ml of 4% formaldehyde solution into each hole for fixing for 30 minutes; then, the liquid was discarded and washed with PBS 2 times, 1ml of alizarin red working solution was added to react for five minutes, and then washed with PBS 2 times, observed under an optical microscope and photographed to obtain a differentiation result graph as shown in FIG. 3.
Claims (2)
1. A method for primary culture of canine umbilical cord-derived mesenchymal stem cells is characterized by comprising the following steps:
1) collecting umbilical cords of newborn dogs:
stripping placenta with a sterile instrument when a puppy emerges from a mother body, shearing an umbilical cord, and immediately putting the umbilical cord into PBS (phosphate buffer solution) containing 10% double-antibody solution;
2) isolating mesenchymal stem cell-rich glial tissue from umbilical cord:
cutting off the umbilical cord capsule to expose the middle tubular structure, removing artery and vein, and collecting the remaining hollow milky white tube without blood as the required tubular tissue;
3) washing and cutting tissue pieces:
sequentially and respectively soaking the separated tubular tissues in PBS buffer solution containing 10%, 5% and 1% double-antibody solution for 5 minutes, then transferring the tubular tissues into a beaker, and shearing the tubular tissues to the size of 1mm3 by using tissue scissors;
4) collagenase digestion:
adding 1% type I collagenase with the volume 10 times that of the mixture, and placing the mixture in an incubator at 37 ℃ for digestion for more than 12 hours;
5) amplification culture:
diluting with an umbilical cord mesenchymal stem cell culture solution, sieving with a 200-mesh screen, centrifuging at 1200rpm for 5 minutes, discarding the supernatant, adding the umbilical cord mesenchymal stem cell culture solution, blowing and beating uniformly, transferring into a culture dish, and culturing in an incubator at the concentration of 5% CO2 and the temperature of 37 ℃, wherein the umbilical cord mesenchymal stem cell culture solution consists of 88% of a human umbilical cord mesenchymal stem cell complete culture medium, 10% of fetal calf serum, 1% of double antibody solution and 1% of glutamine solution in volume ratio.
2. The method of primary culturing canine umbilical cord-derived mesenchymal stem cells according to claim 1, further comprising, after step 5, the steps of: 6) and respectively carrying out liquid exchange of the umbilical cord mesenchymal stem cell culture solution after 24h and 72h of culture, and carrying out passage according to the growth condition of the mesenchymal stem cells.
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| CN107746829B (en) * | 2017-10-19 | 2022-03-22 | 佛山科学技术学院 | Method for separating and primary culturing dog placenta-derived mesenchymal stem cells |
| CN108456657B (en) * | 2018-03-31 | 2020-08-18 | 天津博雅秀岩生物技术有限公司 | Canine umbilical cord mesenchymal stem cells and preparation method and cryopreservation method thereof |
| CN108486050B (en) * | 2018-03-31 | 2020-09-29 | 天津博雅秀岩生物技术有限公司 | Method for preparing mesenchymal stem cells from umbilical cord of dog |
| CN108795853B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Method for preparing canine fetal membrane mesenchymal stem cells and canine fetal membrane mesenchymal stem cells |
| CN108728408B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Canine fetal membrane mesenchymal stem cell, preparation method and culture medium used by same |
| CN109370986A (en) * | 2018-12-11 | 2019-02-22 | 南京农业大学 | The extracting method and its preparation of a kind of dog fat stem cell and application |
| CN112080464B (en) * | 2020-09-16 | 2022-05-13 | 中国科学院昆明动物研究所 | Canine umbilical cord-derived mesenchymal stem cell culture medium and culture method |
| CN114525245B (en) * | 2022-02-15 | 2023-12-12 | 江苏银丰生物工程有限公司 | CMM culture medium and application thereof |
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| CN106282104A (en) * | 2016-07-29 | 2017-01-04 | 中卫华医（北京）生物科技有限公司 | The method of effective acquisition mescenchymal stem cell from umbilical cord tissue |
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| "Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells";Min-Soo Seo 等;《Journal of Veterinary Science》;20091231;第10卷(第3期);第181-187页 * |
| "Maintenance and characterization of multipotent mesenchymal stem cells isolated from canine umbilical cord matrix by collagenase digestion";Lee 等;《Research in Veterinary Science》;20131231;第94卷(第1期);摘要，第144-145页 * |
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