CN113322229A - Stem cell culture method capable of differentiating and regenerating and delaying organism aging - Google Patents
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Abstract
The invention discloses a stem cell culture method capable of differentiating and regenerating and delaying organism aging, which comprises the following specific steps: cleaning the obtained tissue with liquid to remove surface blood stains, removing non-culture required tissues such as adhered connective tissues and the like with surgical forceps, digesting and separating fine tissue blocks into cell clusters or dispersed single cells by adopting digestive enzymes to be beneficial to further culture, counting cells of a cell suspension by using a counting plate, adjusting the cell number to the density required by an experiment by using culture solution, subpackaging in a culture bottle, and re-suspending the mononuclear cells in a basal culture medium; washing with PBS 3 times to remove nonadherent cells; the remaining adherent mononuclear cells are continuously cultured, and the beneficial effects are as follows: the stem cell culture medium has the advantages of clear components, simple formula and lower cost, can effectively maintain the self-renewal capacity and multidirectional differentiation capacity of the mesenchymal stem cells after long-term in-vitro passage and amplification, and delays cell aging caused by in-vitro passage.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a stem cell culture method capable of differentiating and regenerating and delaying body senescence.
Background
Many researches show that the stem cells can not only self-renew, but also differentiate into other functional cells under proper conditions, so that the stem cells are expected to become an effective means for treating human intractable diseases. However, stem cells are present in a very small amount in normal adult tissues, and how to rapidly expand and culture stem cells in vitro is an important technique for studying the mechanism of action of stem cells and exploring therapeutic methods for treating human diseases.
Stem cells are present in minor amounts, but are widely distributed in various tissues and organs of mammals, including, but not limited to, bone marrow, umbilical cord, adipose tissue, brain tissue, retina, heart, liver, lung, and skin. Numerous studies have shown that these low stem cells can be further expanded in vitro by suitable methods, one of the most common culture media for stem cells is medium supplemented with fetal bovine serum. However, the stem cells cultured by the culture medium not only grow slowly, but also have greatly reduced potential of differentiating into functional cells after the transfer times exceed 5 generations, and the scale of the bovine serum is greatly influenced by the potential non-repeatability of the bovine serum batches; on the other hand, bovine serum is a heterologous thing with respect to humans, thereby limiting its clinical application. Therefore, it is a very important research content to explore the optimal combination of culture conditions and their medium composition that rapidly expands stem cells without affecting their potency.
An effective solution to the problems in the related art has not been proposed yet.
Disclosure of Invention
Aiming at the problems in the related art, the invention provides a stem cell culture method capable of differentiating and regenerating and delaying the aging of an organism, so as to overcome the technical problems in the prior related art.
The technical scheme of the invention is realized as follows:
a stem cell culture method capable of differentiating and regenerating and delaying body aging comprises the following specific steps:
s1: shearing tissue, cleaning the obtained tissue with liquid to remove blood stain on the surface, and removing non-culture tissue such as connective tissue with forceps;
s2: after cleaning again, cutting the tissue into a plurality of small pieces by using a scalpel, transferring the small pieces into a small penicillin bottle or a small beaker, adding a proper amount of buffer solution, repeatedly shearing the tissue by using an elbow ophthalmic scissors until the tissue is pasty with the size of about 1mn3, standing for a moment, sucking the upper liquid by using a suction pipe, adding the proper amount of buffer solution, and cleaning again;
s3: digesting and separating, namely digesting and separating the fine tissue blocks into cell clusters or dispersed single cells by adopting digestive enzyme so as to be beneficial to further culture;
s4: culturing, counting cells of the cell suspension by using a counting plate, adjusting the cell number to the density required by the experiment by using a culture solution, and subpackaging the cell suspension in a culture bottle, wherein the cell suspension is slightly higher than the bottom of the culture bottle after being covered;
s5: suspending the mononuclear cells in a basal medium, and culturing the mononuclear cells in a 5% CO2 incubator at 37 ℃ for 24 hours; washing with PBS 3 times to remove nonadherent cells; continuously culturing the residual adherent monocytes, and changing the liquid every 3 days;
s6: when the mononuclear cells are proliferated to 75-80% of density, completely absorbing the culture medium in the basic culture medium, carrying out passage, and continuously culturing by using the basic culture medium;
s7: after the monocytes are cultured to the fifth generation, the culture is continued with the anti-aging stem cell culture medium.
Further, the liquid cleaning adopts D-Hanks or Hanks liquid for cleaning.
Further, trypsin or collagenase is used as the digestive enzyme.
Further, the basic culture medium is prepared by adding 100ml of fetal calf serum, 10ml of penicillin and 10ml of streptomycin into 880ml of alpha-MEM culture medium in sequence, mixing uniformly and storing at 4 ℃.
Further, the stem cell culture medium comprises amino acids, vitamins, salts, lipids, cytokines and protein polypeptides.
Further, the lipids include dexamethasone, oleic acid, cholesterol, ethanolamine, linoleic acid, lipoic acid, and lipids.
The invention provides a stem cell culture method capable of differentiating and regenerating and delaying organism aging, which has the following beneficial effects:
(1) the obtained tissue is washed by liquid to remove blood stain on the surface, adhered connective tissue and other tissues which are not needed to be cultured are removed by surgical forceps, the tissue is cut into a plurality of small blocks by a surgical knife after being washed again, the small blocks are moved into a penicillin bottle or a small beaker, a proper amount of buffer solution is added, the tissue is repeatedly cut by an elbow ophthalmic scissors until the tissue is pasty and has the size of about 1mn3, the upper layer liquid is absorbed by a suction pipe after the tissue is kept still for a moment, the buffer solution is added for washing again, the tiny tissue blocks are digested and separated into cell clusters or dispersed single cells by digestive enzyme, so as to be beneficial to further culture, cell counting is carried out on cell suspension by a counting plate, the cell count is adjusted to the density needed by experiment by culture solution, the cell clusters are subpackaged in a culture bottle, the amount of the cells is better than the bottom of the culture bottle after being covered, and the mononuclear cells are resuspended in a basic culture medium, culturing at 37 deg.C in 5% CO2 incubator for 24 hr; washing with PBS 3 times to remove nonadherent cells; the rest adherent mononuclear cells are continuously cultured, liquid is changed every 3 days, when the mononuclear cells are proliferated to 75% -80% of density, the culture medium in the basic culture medium is completely sucked, passage is carried out, the mononuclear cells are continuously cultured by the basic culture medium, when the mononuclear cells are proliferated to 75% -80% of density, the culture medium in the basic culture medium is completely sucked, passage is carried out, and the mononuclear cells are continuously cultured by the basic culture medium.
(2) And D-Hanks or Hanks liquid is adopted for cleaning.
(3) Trypsin or collagenase is used as the digestive enzyme.
(4) The basic culture medium is prepared by adding fetal calf serum 100ml, penicillin 10ml and streptomycin 10ml into 880ml alpha-MEM culture medium in sequence, mixing, and storing at 4 deg.C.
(5) The stem cell culture medium comprises amino acids, vitamins, salts, lipids, cytokines and protein polypeptides.
(6) Lipids include dexamethasone, oleic acid, cholesterol, ethanolamine, linoleic acid, lipoic acid and lipids.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a stem cell culture method capable of differentiation regeneration and delaying senescence in an organism according to an embodiment of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in 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 of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
The invention is further described with reference to the following drawings and detailed description:
the first embodiment is as follows:
referring to fig. 1, a stem cell culture method capable of differentiation and regeneration for delaying aging according to an embodiment of the present invention includes the following steps:
s1: shearing tissue, cleaning the obtained tissue with liquid to remove blood stain on the surface, and removing non-culture tissue such as connective tissue with forceps;
s2: after cleaning again, cutting the tissue into a plurality of small pieces by using a scalpel, transferring the small pieces into a small penicillin bottle or a small beaker, adding a proper amount of buffer solution, repeatedly shearing the tissue by using an elbow ophthalmic scissors until the tissue is pasty with the size of about 1mn3, standing for a moment, sucking the upper liquid by using a suction pipe, adding the proper amount of buffer solution, and cleaning again;
s3: digesting and separating, namely digesting and separating the fine tissue blocks into cell clusters or dispersed single cells by adopting digestive enzyme so as to be beneficial to further culture;
s4: culturing, counting cells of the cell suspension by using a counting plate, adjusting the cell number to the density required by the experiment by using a culture solution, and subpackaging the cell suspension in a culture bottle, wherein the cell suspension is slightly higher than the bottom of the culture bottle after being covered;
s5: suspending the mononuclear cells in a basal medium, and culturing the mononuclear cells in a 5% CO2 incubator at 37 ℃ for 24 hours; washing with PBS 3 times to remove nonadherent cells; continuously culturing the residual adherent monocytes, and changing the liquid every 3 days;
s6: when the mononuclear cells are proliferated to 75-80% of density, completely absorbing the culture medium in the basic culture medium, carrying out passage, and continuously culturing by using the basic culture medium;
s7: after the monocytes are cultured to the fifth generation, the culture is continued with the anti-aging stem cell culture medium.
Through the scheme of the invention, the obtained tissue is washed by liquid to remove blood stain on the surface, the surgical forceps is used to remove the tissue which is not needed by culture, such as the adhered connective tissue, and the like, the tissue is cut into a plurality of small blocks by a surgical knife after being washed again, the small blocks are moved into a penicillin bottle or a small beaker, a proper amount of buffer solution is added, the tissue is repeatedly cut by an elbow ophthalmic scissors until the tissue is pasty with the size of about 1mn3, the tissue is kept still for a moment, the upper layer liquid is sucked by a suction pipe, the proper buffer solution is added for washing again, the tiny tissue blocks are digested and separated into cell clusters or dispersed single cells by digestive enzyme, the cell suspension is favorable for further culture, the cell counting is carried out by a counting plate, the cell count is adjusted to the density needed by the culture solution, the cell suspension is subpackaged into a culture bottle, and the amount of the cell suspension is proper to be slightly higher than the bottom of the culture bottle after being covered, the mononuclear cells are resuspended in a basic culture medium and cultured for 24 hours in a 5% CO2 incubator at 37 ℃; washing with PBS 3 times to remove nonadherent cells; the rest adherent mononuclear cells are continuously cultured, liquid is changed every 3 days, when the mononuclear cells are proliferated to 75% -80% of density, the culture medium in the basic culture medium is completely sucked, passage is carried out, the mononuclear cells are continuously cultured by the basic culture medium, when the mononuclear cells are proliferated to 75% -80% of density, the culture medium in the basic culture medium is completely sucked, passage is carried out, and the mononuclear cells are continuously cultured by the basic culture medium.
Example two:
as shown in FIG. 1, the liquid cleaning adopts D-Hanks or Hanks liquid cleaning.
Example three:
as shown in FIG. 1, trypsin or collagenase is used as the digestive enzyme.
Example four:
as shown in FIG. 1, the basic medium was prepared by adding fetal bovine serum 100ml, penicillin 10ml and streptomycin 10ml to 880ml of α -MEM medium in this order, mixing them well, and storing at 4 ℃.
Example five:
as shown in fig. 1, the stem cell culture medium includes amino acids, vitamins, salts, lipids, cytokines, and protein polypeptides.
Example six:
as shown in FIG. 1, the lipids include dexamethasone, oleic acid, cholesterol, ethanolamine, linoleic acid, lipoic acid and lipids.
For the convenience of understanding the technical solutions of the present invention, the following detailed description will be made on the working principle or the operation mode of the present invention in the practical process.
In practical application, the obtained tissue is washed by liquid to remove blood stain on the surface, the surgical forceps are used for removing non-culture required tissues such as adhered connective tissues and the like, the tissue is cut into a plurality of small blocks by a surgical knife after being washed again, the small blocks are moved into a penicillin bottle or a small beaker, a proper amount of buffer solution is added, an elbow ophthalmic scissors are used for repeatedly cutting the tissue until the tissue is pasty with the size of about 1mn3, the tissue is kept still for a moment, a pipette is used for sucking the upper liquid, the proper buffer solution is added for washing again, the tiny tissue blocks are digested and separated into cell clusters or dispersed single cells by digestive enzyme to be beneficial to further culture, cell counting is carried out on cell suspension by a counting plate, the cell count is adjusted to the density required by the culture solution and is distributed in a culture bottle, the amount of the cell suspension is proper to be slightly higher than the bottom of the culture bottle after being covered, and the mononuclear cells are resuspended in a basic culture medium, culturing at 37 deg.C in 5% CO2 incubator for 24 hr; washing with PBS 3 times to remove nonadherent cells; the rest adherent mononuclear cells are continuously cultured, liquid is changed every 3 days, when the mononuclear cells are proliferated to 75% -80% of density, the culture medium in the basic culture medium is completely sucked, passage is carried out, the mononuclear cells are continuously cultured by the basic culture medium, when the mononuclear cells are proliferated to 75% -80% of density, the culture medium in the basic culture medium is completely sucked, passage is carried out, and the mononuclear cells are continuously cultured by the basic culture medium.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. A stem cell culture method capable of differentiating and regenerating and delaying body aging is characterized by comprising the following specific steps:
s1: shearing tissue, cleaning the obtained tissue with liquid to remove blood stain on the surface, and removing non-culture tissue such as connective tissue with forceps;
s2: after cleaning again, cutting the tissue into a plurality of small pieces by using a scalpel, transferring the small pieces into a small penicillin bottle or a small beaker, adding a proper amount of buffer solution, repeatedly shearing the tissue by using an elbow ophthalmic scissors until the tissue is pasty with the size of about 1mn3, standing for a moment, sucking the upper liquid by using a suction pipe, adding the proper amount of buffer solution, and cleaning again;
s3: digesting and separating, namely digesting and separating the fine tissue blocks into cell clusters or dispersed single cells by adopting digestive enzyme so as to be beneficial to further culture;
s4: culturing, counting cells of the cell suspension by using a counting plate, adjusting the cell number to the density required by the experiment by using a culture solution, and subpackaging the cell suspension in a culture bottle, wherein the cell suspension is slightly higher than the bottom of the culture bottle after being covered;
s5: suspending the mononuclear cells in a basal medium, and culturing the mononuclear cells in a 5% CO2 incubator at 37 ℃ for 24 hours; washing with PBS 3 times to remove nonadherent cells; continuously culturing the residual adherent monocytes, and changing the liquid every 3 days;
s6: when the mononuclear cells are proliferated to 75-80% of density, completely absorbing the culture medium in the basic culture medium, carrying out passage, and continuously culturing by using the basic culture medium;
s7: after the monocytes are cultured to the fifth generation, the culture is continued with the anti-aging stem cell culture medium.
2. The method for culturing stem cells capable of differentiation regeneration and delaying senescence of organisms according to claim 1, wherein the cleaning solution is D-Hanks or Hanks solution.
3. The method for culturing stem cells capable of differentiating regeneration and delaying senescence of an organism according to claim 1, wherein the digestive enzyme is trypsin or collagenase.
4. The method for culturing stem cells capable of differentiated regeneration and delaying senescence according to claim 1, wherein the basic culture medium is prepared by sequentially adding 100ml of fetal bovine serum, 10ml of penicillin and 10ml of streptomycin into 880ml of alpha-MEM culture medium, mixing uniformly, and storing at 4 ℃.
5. The method as claimed in claim 1, wherein the stem cell culture medium comprises amino acids, vitamins, salts, lipids, cytokines and protein polypeptides.
6. The method for culturing stem cells capable of differentiating regeneration and delaying senescence of claim 5, wherein the lipid comprises dexamethasone, oleic acid, cholesterol, ethanolamine, linoleic acid, lipoic acid and lipid.
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| CN108611320A (en) * | 2018-05-17 | 2018-10-02 | 广东芙金干细胞再生医学有限公司 | A kind of stem cell media and mescenchymal stem cell cultural method of anti-aging |
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Patent Citations (6)
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| CN102559590A (en) * | 2012-01-16 | 2012-07-11 | 遵义医学院附属医院 | Method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media |
| CN102660501A (en) * | 2012-05-21 | 2012-09-12 | 博雅干细胞科技有限公司 | Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord |
| CN102876630A (en) * | 2012-11-05 | 2013-01-16 | 东南大学 | Method for efficiently separating and expanding mesenchymal stem cells in human umbilical cord blood |
| CN107041894A (en) * | 2016-12-26 | 2017-08-15 | 湖南新起源医疗技术有限公司 | A kind of stem cell liquid for skin delication preparation method for anti-aging |
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| CN108676773A (en) * | 2018-05-17 | 2018-10-19 | 广东芙金干细胞再生医学有限公司 | A kind of culture solution and preparation method delaying mescenchymal stem cell aging |
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Application publication date: 20210831 |
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