CN109234230B - Primary isolation method of skin mesenchymal stem cells - Google Patents
Primary isolation method of skin mesenchymal stem cells Download PDFInfo
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- CN109234230B CN109234230B CN201811156966.9A CN201811156966A CN109234230B CN 109234230 B CN109234230 B CN 109234230B CN 201811156966 A CN201811156966 A CN 201811156966A CN 109234230 B CN109234230 B CN 109234230B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to the field of stem cells, and discloses a primary separation method of skin mesenchymal stem cells, which comprises the steps of pretreating skin tissues, adding Dispase II Dispase for digestion, repeatedly blowing tissue blocks after cleaning to separate cells, centrifuging after filtering, resuspending obtained cell precipitates by using a Lonza complete culture medium, and inoculating the cell precipitates in a culture dish for culture for 60-90 min; inoculating the cell suspension without adherence to a fibronectin-coated culture plate for culture, and carrying out passage after the cell confluence reaches 80-90%. The cell membrane of the skin mesenchymal stem cell separated and cultured by the primary separation method is not damaged, the cell culture is stable, mycoplasma pollution is not introduced, a large amount of skin mesenchymal stem cells can be separated, the cell activity is good, and the skin mesenchymal stem cell can be stably cultured subsequently.
Description
Technical Field
The invention belongs to the field of stem cells, and particularly relates to a primary separation method of skin mesenchymal stem cells.
Background
Stem cells are a class of primitive cells that have self-renewal capacity and multisystemic differentiation potential in a suitable microenvironment. According to the different sequences in the development process of individuals, stem cells can be divided into embryonic stem cells, pluripotent stem cells and oriented stem cells in various tissues, such as hematopoietic stem cells, neural stem cells and the like. At present, there have been reported experiments using stem cells for the treatment of myocardial infarction, lupus erythematosus, rheumatoid arthritis, nerve damage, muscular atrophy, and the like. The human stem cell is a super-energy stem cell group with multi-series differentiation potential existing in human tissues, and can be induced to differentiate into various tissue cells under specific environment. Among human stem cells, mesenchymal stem cells are widely present in various tissues throughout the body, particularly, bone marrow, fatty umbilical cord, skin and placenta. While the existing research on mesenchymal stem cells focuses mainly on obtaining mesenchymal stem cells from bone marrow and umbilical cord, the research on skin mesenchymal stem cells is very rare. Because the skin is the largest tissue organ in the human body and the materials are convenient to obtain, the mesenchymal stem cells of the skin are selected as the seed cells for cell therapy, so that the prospect is wide.
Skin stem cells play an important role in maintaining normal tissue architecture and intracellular homeostasis of the skin and mucosa. The skin stem cells play an extremely important role in the gene therapy of hereditary skin diseases and the reconstruction of the function and structure of damaged skin and mucous membranes. The skin stem cells have wide application prospects due to the characteristics thereof, such as: tissue engineering skin construction, application in wound repair, gene research and treatment of hereditary diseases, research on cell lineages and the like.
In the prior art, the method for separating the skin mesenchymal stem cells is to digest skin tissues by trypsin containing EDTA, and inoculate the skin tissues in a specific culture medium after filtration. Trypsin belongs to proteolytic enzymes, and the cell membrane can be seriously damaged by dispersing tissues and separating cells by using the method, is unstable during cell culture, and even introduces mycoplasma pollution.
Disclosure of Invention
In view of the above, the present invention provides an efficient, economical, stable and convenient primary isolation method of skin mesenchymal stem cells, so as to isolate a large amount of skin mesenchymal stem cells, and enable subsequent stable culture.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a primary isolation method of skin mesenchymal stem cells comprises the following steps:
A. pretreating skin tissue, adding Dispase II to digest, repeatedly blowing and beating tissue blocks after cleaning to separate cells, centrifuging after filtering, re-suspending the obtained cell precipitate by using a Lonza complete culture medium, and inoculating the cell precipitate into a culture dish for culture;
B. inoculating the cell suspension without adherence to a fibronectin-coated culture plate for culture, and carrying out passage after the cell confluence reaches 80-90%.
The separation method of the present invention first pre-treats the skin tissue.
Wherein the pretreatment is that skin tissues are cut into a rectangle of 3-5mm, washed by sodium chloride injection and then placed in 75% ethanol for washing, and then the skin tissues are cut into fine tissue blocks after being washed by the sodium chloride injection.
In some embodiments, the freshly collected skin tissue is repeatedly washed with saline containing the diabody, then washed with 75% alcohol, and finally stored in glucose solution containing the diabody at 4 ℃ for subsequent pretreatment. Wherein the double antibody is a mixed solution of streptomycin. Penicillin-streptomycin solution (100X) contains 10000U/ml penicillin and 10mg/ml streptomycin. The concentration of the double antibody in the normal saline and the glucose solution is preferably 1 x, namely the working concentration of the penicillin is 100U/ml, and the working concentration of the streptomycin is 0.1 mg/ml.
The separation method of the invention adopts Dispase II Dispase to digest skin tissues. Dispase II Dispase, a neutral protease, is a non-specific metalloprotease that does not harm cell membranes and, because it is derived from bacteria, does not introduce mycoplasma or any animal viruses; the difference of temperature, PH, serum components and the like has little influence on Dispase II Dispase, the activity of the Dispase II Dispase is greatly reduced after the Dispase II Dispase is diluted, and the subsequent cell culture is facilitated.
Preferably, the concentration of Dispase II in the step A is 0.5U/ml-2.5U/ml. In some embodiments, the concentration of Dispase II Dispase is 2U/ml.
Preferably, the digestion in step A is at 37 ℃ for 1 h.
Preferably, the cleaning in the step a is sodium chloride injection cleaning.
The separation method of the invention removes impurities by filtration after separating cells. Preferably, the filtration in step A is a 100 μm disposable cell screen filtration.
Preferably, the centrifugation in step A is 400g for 10 min.
Preferably, the culture in step A is performed at 37 ℃ and CO2Culturing for 60-90 min.
Preferably, the non-adherent cell suspension of step B is seeded at a density of 5X 104/ml。
Preferably, the fibronectin concentration of the coated culture plate in the step B is 10-50 μ g/ml. In some embodiments, the fibronectin concentration of the coated plate is 20 μ g/ml.
Preferably, the coating method in the step B is incubation at 37 ℃ for 30-60 min.
Preferably, the culture in step B is at 37 ℃ and 5% CO2Culturing at the concentration.
Preferably, the first liquid change of the culture in the step B is carried out at intervals of 72 hours, and then every 1 to 3 days.
According to the technical scheme, the invention provides a primary separation method of skin mesenchymal stem cells. The cell membrane of the skin mesenchymal stem cell separated and cultured by the primary separation method is not damaged, the cell culture is stable, and mycoplasma pollution is not introduced. Experiments show that the skin mesenchymal stem cells separated and cultured by the primary separation method can separate a large amount of skin mesenchymal stem cells, have good cell activity and can be stably cultured subsequently.
Detailed Description
The invention discloses a primary separation method of skin mesenchymal stem cells. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, 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.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. Wherein Dispase II Dispase was purchased from Sigma; lonza complete medium was purchased from swiss dragon sand; fibronectin was purchased from Corning.
Example 1 Primary isolation of skin mesenchymal Stem cells
1. Spreading fibronectin FN 2ml of 20 μ g/ml onto 10cm culture plate, and incubating at 37 deg.C for 30-60 min;
2. taking skin tissue (30mm × 5mm), repeatedly washing with 1 × double antibody-containing normal saline for several times, washing with 75% alcohol once, storing in double antibody-containing glucose solution, storing at 4 deg.C, and taking back to laboratory for 24 hr;
3. cutting skin tissues into a rectangle of 3-5mm, cleaning with sodium chloride injection, then placing in 75% ethanol for cleaning, and finally cleaning with sodium chloride injection once;
4. cutting the rectangle into small tissue blocks, and digesting for 1h at 37 ℃ by Dispase II Dispase of 2U/ml;
after 5.1h, taking out the tissue block, and cleaning the tissue block once by using sodium chloride injection;
6. repeatedly blowing and beating the tissue block to separate the cells, and removing impurities by a disposable cell screen with the particle size of 100 mu m;
7. centrifuging the filtered cells at 400g for 10 min;
8. resuspending the obtained cell precipitate with Lonza complete culture medium, inoculating into a 10cm dish, and culturing in a 37 deg.C carbon dioxide incubator for 60-90min to remove fibroblasts;
9. the nonadherent cell suspension was counted at 5X 104Inoculating the culture plate coated with FN at a density of/ml;
after 10.72h, the liquid is changed for the first time, and then the liquid is changed every 2 days, and the cells grow directly until the confluence degree is 80% -90% for passage.
Example 2 Primary isolation of skin mesenchymal Stem cells
1. Spreading fibronectin FN 2ml 10 μ g/ml into 10cm culture plate, and incubating at 37 deg.C for 30-60 min;
2. taking skin tissue (30mm × 5mm), repeatedly washing with 1 × double antibody-containing normal saline for several times, washing with 75% alcohol once, storing in double antibody-containing glucose solution, storing at 4 deg.C, and taking back to laboratory for 24 hr;
3. cutting skin tissues into a rectangle of 3-5mm, cleaning with sodium chloride injection, then placing in 75% ethanol for cleaning, and finally cleaning with sodium chloride injection once;
4. cutting the rectangle into fine tissue blocks, and digesting for 1h at 37 ℃ by Dispase II Dispase of 0.5U/ml;
after 5.1h, taking out the tissue block, and cleaning the tissue block once by using sodium chloride injection;
6. repeatedly blowing and beating the tissue block to separate the cells, and removing impurities by a disposable cell screen with the particle size of 100 mu m;
7. centrifuging the filtered cells at 400g for 10 min;
8. resuspending the obtained cell precipitate with Lonza complete culture medium, inoculating into a 10cm dish, and culturing in a 37 deg.C carbon dioxide incubator for 60-90min to remove fibroblasts;
9. the nonadherent cell suspension was counted at 5X 104Inoculating the culture plate coated with FN at a density of/ml;
after 10.72h, the liquid is changed for the first time, and then the liquid is changed every 2 days, and the cells grow directly until the confluence degree is 80% -90% for passage.
Example 3 Primary isolation of skin mesenchymal Stem cells
1. Spreading fibronectin FN 2ml 50 μ g/ml into 10cm culture plate, and incubating at 37 deg.C for 30-60 min;
2. taking skin tissue (30mm × 5mm), repeatedly washing with 1 × double antibody-containing normal saline for several times, washing with 75% alcohol once, storing in double antibody-containing glucose solution, storing at 4 deg.C, and taking back to laboratory for 24 hr;
3. cutting skin tissues into a rectangle of 3-5mm, cleaning with sodium chloride injection, then placing in 75% ethanol for cleaning, and finally cleaning with sodium chloride injection once;
4. cutting the rectangle into small tissue blocks, and digesting for 1h at 37 ℃ by Dispase II Dispase of 2.5U/ml;
after 5.1h, taking out the tissue block, and cleaning the tissue block once by using sodium chloride injection;
6. repeatedly blowing and beating the tissue block to separate the cells, and removing impurities by a disposable cell screen with the particle size of 100 mu m;
7. centrifuging the filtered cells at 400g for 10 min;
8. resuspending the obtained cell precipitate with Lonza complete culture medium, inoculating into a 10cm dish, and culturing in a 37 deg.C carbon dioxide incubator for 60-90min to remove fibroblasts;
9. the nonadherent cell suspension was counted at 5X 104Inoculating the culture plate coated with FN at a density of/ml;
after 10.72h, the liquid is changed for the first time, and then the liquid is changed every 2 days, and the cells grow directly until the confluence degree is 80% -90% for passage. Comparative example 1 Primary isolation of skin mesenchymal Stem cells
1. Taking skin tissue (30mm × 5mm), repeatedly washing with 1 × double antibody-containing normal saline for several times, washing with 75% alcohol once, storing in double antibody-containing glucose solution, storing at 4 deg.C, and taking back to laboratory for 24 hr;
2. cutting skin tissues into a rectangle of 3-5mm, cleaning with sodium chloride injection, then placing in 75% ethanol for cleaning, and finally cleaning with sodium chloride injection once;
3. cutting the rectangle into small tissue blocks, and digesting for 1h at 37 ℃ by 0.1% trypsin;
taking out the tissue block after 4.1h, and cleaning once by using sodium chloride injection;
5. repeatedly blowing and beating the tissue block to separate the cells, and removing impurities by a disposable cell screen with the particle size of 100 mu m;
6. centrifuging the filtered cells at 400g for 10 min;
7. the resulting cell pellet was cultured in Lonza complete medium at 5X 104Inoculating the strain in a 10cm dish at a density of one ml, and culturing in a carbon dioxide incubator at 37 ℃;
after 8.72h, the liquid is changed for the first time, and then the liquid is changed every 2 days, and the cells grow directly until the confluence degree is 80% -90% for passage. Comparative example 2 Primary isolation of skin mesenchymal Stem cells
1. Taking skin tissue (30mm × 5mm), repeatedly washing with 1 × double antibody-containing normal saline for several times, washing with 75% alcohol once, storing in double antibody-containing glucose solution, storing at 4 deg.C, and taking back to laboratory for 24 hr;
2. cutting skin tissues into a rectangle of 3-5mm, cleaning with sodium chloride injection, then placing in 75% ethanol for cleaning, and finally cleaning with sodium chloride injection once;
3. cutting the rectangle into small tissue blocks, and digesting for 1h at 37 ℃ by 0.1% trypsin;
taking out the tissue block after 4.1h, and cleaning once by using sodium chloride injection;
5. repeatedly blowing and beating the tissue block to separate the cells, and removing impurities by a disposable cell screen with the particle size of 100 mu m;
6. centrifuging the filtered cells at 400g for 10 min;
7. resuspending the obtained cell precipitate with Lonza complete culture medium, inoculating into a 10cm dish, and culturing in a 37 deg.C carbon dioxide incubator for 60-90min to remove fibroblasts;
8. the nonadherent cell suspension was counted at 5X 104Inoculating the strain in a 10cm culture dish at a density of one ml;
after 9.72h, the liquid is changed for the first time, and then the liquid is changed every 2 days, and the cells grow directly until the confluence degree is 80% -90% for passage. Test examples, comparison of cell growth and cell survival Rate
The cell proliferation and cell viability after 7 days of culture by the methods of examples 1 to 3 and comparative examples 1 to 2, respectively, were compared, and the results are shown in Table 1.
TABLE 1 cell proliferation and cell viability rates after 7 days of culture
As can be seen from the above table, the primary isolation method described in examples 1-3 can obtain more cells, and the mesenchymal stem cells of skin obtained by the method have higher cell viability. In contrast, in comparative example 1, the mesenchymal stem cells are attached to the wall in a differential speed for 60-90min after the cells are not digested, and the number of the obtained mesenchymal stem cells is zero. The differential adherence is necessary for 60-90min after cell digestion, and the rest of the hybrid cells can be effectively removed by the step based on different cell characteristics (different adherence time). And the cell is greatly damaged by other enzymes such as trypsin, so that the cell viability is poor and the cell cannot be proliferated better.
Claims (7)
1. A primary isolation method of skin mesenchymal stem cells is characterized by comprising the following steps:
A. pretreating skin tissue, adding 0.5-2.5U/ml dispase II at 37 deg.C, digesting for 1 hr, cleaning, repeatedly blowing tissue block to separate cells, filtering, centrifuging to obtain cell precipitate, re-suspending with Lonza complete culture medium, inoculating into culture dish, culturing at 37 deg.C with 5% CO2Carrying out differential adherence for 60-90 min;
B. inoculating the cell suspension without adherence to a fibronectin-coated culture plate for culture, and carrying out passage after the cell confluence reaches 80-90%.
2. The primary isolation method of claim 1, wherein the pretreatment in step a is that the skin tissue is stored in glucose solution containing double antibody at 4 ℃ during transportation, the skin tissue is cut into 3-5mm rectangles, washed with sodium chloride injection, then washed with 75% ethanol, washed with sodium chloride injection, and then cut into fine tissue blocks.
3. The primary isolation method of claim 1, wherein the washing in step a is sodium chloride injection washing.
4. The primary isolation method of claim 1, wherein the filtration of step a is a 100 μ ι η disposable cell screen filtration.
5. The primary separation method of claim 1, wherein the centrifugation of step a is 400g for 10 min.
6. Primary isolation according to claim 1The method is characterized in that the seeding density of the nonadherent cell suspension in the step B is 5 multiplied by 104/ml。
7. The primary isolation method of claim 1, wherein the fibronectin concentration of the coated plate of step B is 10-50 μ g/ml; the coating method comprises incubation at 37 deg.C for 30-60 min; the culture is carried out at 37 ℃ and 5% CO2Culturing under the concentration; the first liquid change of the culture is carried out at intervals of 72 hours, and then the liquid is changed every 1-3 days.
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