CN108048396A - A kind of separation method of Sheep Muscle stem cell - Google Patents
A kind of separation method of Sheep Muscle stem cell Download PDFInfo
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- CN108048396A CN108048396A CN201711467657.9A CN201711467657A CN108048396A CN 108048396 A CN108048396 A CN 108048396A CN 201711467657 A CN201711467657 A CN 201711467657A CN 108048396 A CN108048396 A CN 108048396A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses a kind of separation methods of Sheep Muscle stem cell, by the muscle cell for cutting sheep leg, the muscle cell is carried out disinfection, clears up, shreds, digests and centrifuged and etc., muscle stem cell is isolated, and the muscle stem cell is placed in culture dish and is cultivated；The present invention is compared with general muscle method for separating stem cell, and separative efficiency is high, and infected probability is small in separation process, stem cell survival rate after separation is high, and be easy to cultivate, increase the quantity of stem cell, improve stem cell and carry out again in therapeutic process to the speed and efficiency of muscle reparation.
Description
Technical field
The invention belongs to cell separation field, more specifically more particularly to a kind of separation side of Sheep Muscle stem cell
Method.
Background technology
Stem cell is a kind of multipotential cell with the of self-replication capacity.Under certain condition, it can be divided into more
Kind functioning cell.Stage of development according to residing for stem cell is divided into embryonic stem cell and adult stem cell.According to the hair of stem cell
Potential is educated to be divided into three classes：Myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell.Stem cell is a kind of inabundant differentiation, still not
Ripe cell, has the potential function for regenerating various histoorgans and human body, and medical field is known as " general-purpose cell ".Muscle is done carefully
Born of the same parents can Development And Differentiation be sarcoblast, the latter can merge into each other the muscle fibre as multinuclear, form the most basic structure of skeletal muscle.
Muscle stem cell as research how to repair impaired musculature and how to imitate treatment the urinary incontinence it is essential
Object of Development, but muscle stem cell of the prior art separation is more troublesome, the cell survival time is short after separation, influences to study
And treatment.
The content of the invention
It is an object of the invention to provide a kind of separation method of Sheep Muscle stem cell, to solve in above-mentioned background technology
The problem of proposition.
To achieve the above object, the present invention provides following technical solution：
A kind of separation method of Sheep Muscle stem cell, includes the following steps：
S1, the sheep for choosing 2-3 ages, sheep is cleaned up and is led into desinfection chamber, rejects hair at thigh, remaining position is adopted
It is wrapped up, reused at alcohol washes thigh 2-3 times with antiseptic gauze, taken out leg muscle 4-8g, be put into alcohol and impregnate；
S2, alcohol disinfecting 10-20min are removed connective tissue and haemocyte on leg muscle, and are cleaned using physiological saline,
Ensure to clean up sheep leg muscle under the precursor of cell integrity；
S3, the sheep leg muscle after being cleaned in S2 is shredded to 0.8-1.2mm3Fragment, in culture dish holding, first to training
It supports and Type I collagen enzymic digestion 5-8min is added in ware, add type Ⅳ collagen enzymic digestion 4-6min, be eventually adding II Hes of Dispase
7-10min is digested in the mixed enzyme of pancreatin；
All digestive ferments that removal digestion uses after S4, digestion, are screened using 100 mesh screens, and physiology salt is used after screening
Water rinses 2-3 times, and centrifugal treating in centrifuge is put into after flushing, upper liquid is removed after centrifugation, subnatant is put into culture dish,
And complete culture solution 10-12ml is added in into culture dish, then culture dish is put into constant incubator, keep constant temperature incubation box temperature
Degree is at 37-37.5 DEG C.
Preferably, added in S2 after digestive ferment using mechanical agitation, ensure that digestion is complete, and improve the speed of digestion.
Preferably, the speed setting of centrifuge is 2100-2300rpm, time 5-7min in S4.
Preferably, the subnatant after centrifugation is taken out in S4, is seeded in and has been with the addition of in the complete culture solution of 0.3% gelatin, then
10% hyclone and 3% dual anti-is added into culture dish, and the concentration of regulation of carbon dioxide is 5%.
Compared with prior art, the beneficial effects of the invention are as follows：The present invention compared with general muscle method for separating stem cell,
Separative efficiency is high, and infected probability is small in separation process, and the stem cell survival rate after separation is high, and is easy to cultivate, and increases dry
The quantity of cell improves stem cell and carries out again in therapeutic process to the speed and efficiency of muscle reparation.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
A kind of separation method of Sheep Muscle stem cell, includes the following steps：
S1, the sheep for choosing 2-3 ages, sheep is cleaned up and is led into desinfection chamber, rejects hair at thigh, remaining position is adopted
It is wrapped up, reused at alcohol washes thigh 2-3 times with antiseptic gauze, taken out leg muscle 4-8g, be put into alcohol and impregnate；
S2, alcohol disinfecting 10-20min are removed connective tissue and haemocyte on leg muscle, and are cleaned using physiological saline,
Ensure to clean up sheep leg muscle under the precursor of cell integrity；
S3, the sheep leg muscle after being cleaned in S2 is shredded to 0.8-1.2mm3Fragment, in culture dish holding, first to training
It supports and Type I collagen enzymic digestion 5-8min is added in ware, add type Ⅳ collagen enzymic digestion 4-6min, be eventually adding II Hes of Dispase
7-10min is digested in the mixed enzyme of pancreatin；
All digestive ferments that removal digestion uses after S4, digestion, are screened using 100 mesh screens, and physiology salt is used after screening
Water rinses 2-3 times, and centrifugal treating in centrifuge is put into after flushing, upper liquid is removed after centrifugation, subnatant is put into culture dish,
And complete culture solution 10-12ml is added in into culture dish, then culture dish is put into constant incubator, keep constant temperature incubation box temperature
Degree is at 37-37.5 DEG C.
Specifically, ensure that digestion is complete using mechanical agitation after digestive ferment is added in S2, and improve the speed of digestion.
Specifically, the speed setting of centrifuge is 2100-2300rpm, time 5-7min in S4.
Specifically, taking out the subnatant after centrifugation in S4, it is seeded in and has been with the addition of in the complete culture solution of 0.3% gelatin, then
10% hyclone and 3% dual anti-is added into culture dish, and the concentration of regulation of carbon dioxide is 5%.
The present invention is compared with general muscle method for separating stem cell, by cutting the muscle cell of sheep leg, to the flesh
Meat cell is carried out disinfection, clears up, shreds, digests and centrifuged, and isolates muscle stem cell, and the muscle is done carefully
Born of the same parents are placed in culture dish and cultivate；Separative efficiency is high, and infected probability is small in separation process, and the stem cell survival rate after separation is high,
And be easy to cultivate, increase the quantity of stem cell, improve stem cell and carry out again in therapeutic process to the speed and effect of muscle reparation
Rate.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (4)
1. a kind of separation method of Sheep Muscle stem cell, which is characterized in that include the following steps：
S1, the sheep for choosing 2-3 ages, sheep is cleaned up and is led into desinfection chamber, rejects hair at thigh, remaining position is adopted
It is wrapped up, reused at alcohol washes thigh 2-3 times with antiseptic gauze, taken out leg muscle 4-8g, be put into alcohol and impregnate；
S2, alcohol disinfecting 10-20min are removed connective tissue and haemocyte on leg muscle, and are cleaned using physiological saline,
Ensure to clean up sheep leg muscle under the precursor of cell integrity；
S3, the sheep leg muscle after being cleaned in S2 is shredded to 0.8-1.2mm3Fragment, in culture dish holding, first to training
It supports and Type I collagen enzymic digestion 5-8min is added in ware, add type Ⅳ collagen enzymic digestion 4-6min, be eventually adding II Hes of Dispase
7-10min is digested in the mixed enzyme of pancreatin；
All digestive ferments that removal digestion uses after S4, digestion, are screened using 100 mesh screens, and physiology salt is used after screening
Water rinses 2-3 times, and centrifugal treating in centrifuge is put into after flushing, upper liquid is removed after centrifugation, subnatant is put into culture dish,
And complete culture solution 10-12ml is added in into culture dish, then culture dish is put into constant incubator, keep constant temperature incubation box temperature
Degree is at 37-37.5 DEG C.
2. a kind of separation method of Sheep Muscle stem cell according to claim 1, it is characterised in that：Digestion is added in S2
Using mechanical agitation after enzyme, ensure that digestion is complete, and improve the speed of digestion.
3. a kind of separation method of Sheep Muscle stem cell according to claim 1, it is characterised in that：Centrifuge in S4
Speed setting is 2100-2300rpm, time 5-7min.
4. a kind of separation method of Sheep Muscle stem cell according to claim 1, it is characterised in that：Centrifugation is taken out in S4
Subnatant afterwards is seeded in and has been with the addition of in the complete culture solution of 0.3% gelatin, then add into culture dish 10% hyclone and
3% is dual anti-, and the concentration of regulation of carbon dioxide is 5%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112294993A (en) * | 2020-04-29 | 2021-02-02 | 广州市天河诺亚生物工程有限公司 | Umbilical cord deep disinfection method for effectively reducing pollution rate of umbilical cord blood collection and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112294993A (en) * | 2020-04-29 | 2021-02-02 | 广州市天河诺亚生物工程有限公司 | Umbilical cord deep disinfection method for effectively reducing pollution rate of umbilical cord blood collection and application thereof |
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