CN115725497A - Method for efficiently separating and extracting rat cartilage cells - Google Patents
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Abstract
The invention discloses a method for efficiently separating and extracting rat cartilage cells, which relates to the technical field of medical treatment and comprises the following steps: the method comprises the following steps: selecting one healthy SPF (specific pathogen free) SD rat of 5 days old, killing the rat by means of cervical dislocation, then soaking the killed rat in 75% alcohol for 30 seconds, and in experiments carried out by the method for efficiently separating and extracting the chondrocytes of the rat, the serum concentration in each step in the extraction process of the chondrocytes has obvious influence on the number of the finally obtained chondrocytes.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a method for efficiently separating and extracting rat cartilage cells.
Background
Chondrocytes are located in cartilage and primarily function to synthesize certain collagenous and non-collagenous macromolecular extracellular matrices, including: type II collagen, proteoglycans, catenin, type IX collagen, and type XI collagen.
Regulation of proliferation and differentiation of chondrocytes, essential for the coordination of skeletal development in vertebrates, produce and react with a large number of peptide growth factors and cytokines, such as: insulin-like growth factor-1 and interleukin-1, the culture of chondrocytes is a very important in vitro model in the research of the regeneration and repair of cartilage, the action of cytokines and growth factors on cartilage, the regulation and control action of specific genes on arthritis and the pathophysiological processes of arthritis.
At present, a great number of researchers have conducted research on the culture and pharmacological properties of mouse/rat chondrocytes, and the existing research mostly adopts 0.25% pancreatin 37 ℃ water bath digestion for 1 hour, and then uses type II collagenase digestion for about 5 hours, which has the disadvantage that most enzymes have violent digestion action under 37 ℃ condition, so that on one hand, the digestion speed can be increased, but on the other hand, the cells are greatly damaged, especially pancreatin. Therefore, the digestion time is not easy to master, the digestion time is usually too long, cell fragments are more, mixed cells are more, cartilage cells are fewer, the obtained cells have poor viability, viable cartilage cells are less and less along with the prolonging of the culture time and the passage, the cells die gradually, the obtained cartilage cells are smaller and cannot be obtained in large quantity, and therefore, the method for efficiently separating and extracting the rat cartilage cells is provided.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for efficiently separating and extracting rat chondrocytes, which solves the problems that the digestion time is not easy to master, the digestion time is usually too long, cell fragments are more, mixed cells are more, the number of chondrocytes is less, the activity of the obtained cells is poor, the viable chondrocytes are less and less along with the prolonging of the culture time and the passage, the cells die gradually, the obtained chondrocytes are smaller, and a large number of chondrocytes cannot be obtained.
In order to achieve the purpose, the invention is realized by the following technical scheme: a method for efficiently separating and extracting rat chondrocytes comprises the following steps:
the method comprises the following steps: selecting one healthy SPF (specific pathogen free) SD rat with the age of 5 days, killing the rat in a cervical dislocation mode, and then soaking the killed rat in 75% alcohol for 30 seconds;
step two: taking out the rat soaked in the treatment in the step one, cutting the epidermis upwards along the femur from the body surface projection position of the tibial plateau at the outer side of the knee joint of the hind limb, and cutting off the muscle attached near the joint cavity by using a micro-shear to expose the cartilage at the two sides of the rat;
step three: shearing the tibia and the femur of the rat treated in the step two along the epiphyseal line, and separating to obtain cartilage tissues;
step four: preliminarily cleaning soft tissues on the surfaces of the cartilage tissues by using micro-scissors;
step five: the cleaned cartilage tissue was transferred to 4 ml of 0.25% EDTA-pancreatin and the remaining soft tissue was digested for 15 minutes using a 37 ℃ constant temperature shaker;
step six: centrifuging the mixture of the tissue and the pancreatin by using a centrifuge, and removing the supernatant to obtain digested cartilage tissue;
step seven: cutting cartilage tissue into fine sand with a scalpel, transferring to 5 ml of 0.22% type II collagenase solution containing Australian fetal bovine serum, and digesting intercellular collagen with a 37 ℃ constant temperature shaker for 2.5 hours;
step eight: filtering the digested solution by a 70-micron filter screen, and centrifuging by using a centrifuge;
step nine: centrifuging by a centrifuge, discarding the centrifuged supernatant, resuspending by an F12-DMEM medium, and centrifuging by a centrifuge;
step ten: the centrifuged supernatant was discarded, and the cells were suspended in F12-DMEM medium containing 10% Australian fetal bovine serum, seeded on a petri dish, placed in a cell plating chamber, plated for 2 days, and then changed.
Preferably, the cut dead point in the second step is a femoral head body surface projection.
Preferably, the cleaning method in the fourth step is that the micro scissors are tightly attached to the surface of the cartilage tissue, and macroscopic soft tissue is built in parallel to the tissue surface.
Preferably, the EDTA-pancreatin content of 0.25% in the fifth step is produced by GIBCO, inc., having a product number of 25200056.
Preferably, the digestion conditions of the constant temperature shaking table in the fifth step and the seventh step are as follows: the temperature was 37 ℃ and the rotation speed was 200rpm.
Preferably, the centrifugation conditions in the sixth, eighth and ninth steps are as follows: the temperature of room temperature is 37 ℃, and the rotation speed of the centrifuge is 1000RPM.
Preferably, the method for preparing the type II collagenase solution containing 5 ml of Australian fetal bovine serum in 0.22% in the seventh step comprises:
s1: 11mgII type collagenase (product number 1148090, manufactured by SIGMA corporation) was dissolved in 4.5 ml of PBS solution,
s2: 0.5 ml of Australian fetal bovine serum (manufactured by GIBCO, cat # 10100147) was added thereto, and mixed by inversion.
Preferably, the F12-DMEM medium in the ninth step is produced by GIBCO, and has the product number of 11320033.
Preferably, the preparation method of the culture medium containing 10% Australian fetal bovine serum F12-DMEM in the step ten comprises the following steps: 1 ml of Australian fetal calf serum is dissolved in 9 ml of F12-DMEM medium to prepare 10 ml of mixed medium.
Preferably, the environmental conditions of the cell plating box in the step ten are as follows: the temperature was 37 ℃,5% carbon dioxide concentration, 95% humidity.
The invention provides a method for efficiently separating and extracting rat chondrocytes, which has the following beneficial effects:
1. in experiments carried out by the method for efficiently separating and extracting rat chondrocytes, the serum concentration in each step in the process of extracting the chondrocytes has obvious influence on the number of the finally obtained chondrocytes, and the previous extraction method does not add serum in the digestion of type II collagenase, so that a plurality of chondrocytes are apoptotic in a long digestion time. Serum is added in the digestion process of type II collagenase, which is helpful for providing nutrition and promoting survival of chondrocytes, and meanwhile, a culture medium with high concentration of serum is used in inoculation, so that adherence and proliferation of chondrocytes can be effectively promoted, and a large amount of chondrocytes can be efficiently obtained.
Drawings
FIG. 1 is a schematic representation of the structure under a chondrocyte mirror 4 days after plating 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.
Referring to fig. 1, the present invention provides a technical solution: a method for efficiently separating and extracting rat chondrocytes comprises the following steps:
the method comprises the following steps: selecting one healthy SPF (specific pathogen free) SD rat with the age of 5 days, killing the rat in a cervical dislocation mode, and then soaking the killed rat in 75% alcohol for 30 seconds;
step two: taking out the rat soaked in the treatment in the step one, cutting the epidermis upwards along the femur from the body surface projection position of the tibial plateau at the outer side of the knee joint of the hind limb, and cutting off the muscle attached near the joint cavity by using a micro-shear to expose the cartilage at the two sides of the rat;
step three: cutting off the tibia and the femur of the rat treated in the step two along the epiphyseal line, and separating to obtain cartilage tissues;
step four: preliminarily cleaning soft tissues on the surface of the cartilage tissue by using micro scissors;
step five: the cleaned cartilage tissue was transferred to 4 ml of 0.25% EDTA-pancreatin and the remaining soft tissue was digested for 15 minutes using a 37 ℃ constant temperature shaker;
step six: centrifuging the mixture of the tissue and the pancreatin by using a centrifuge, and removing the supernatant to obtain digested cartilage tissue;
step seven: cutting cartilage tissue into fine sand with a scalpel, transferring to 5 ml of 0.22% type II collagenase solution containing Australian fetal bovine serum, and digesting intercellular collagen with a 37 ℃ constant temperature shaker for 2.5 hours;
step eight: filtering the digested solution by a 70-micron filter screen, and centrifuging by using a centrifuge;
step nine: centrifuging by a centrifuge, discarding the centrifuged supernatant, resuspending by an F12-DMEM medium, and centrifuging by a centrifuge;
step ten: the centrifuged supernatant was discarded, and the cells were suspended in F12-DMEM medium containing 10% Australian fetal bovine serum, seeded on a petri dish, placed in a cell plating chamber, plated for 2 days, and then changed.
We found that the serum concentration in each step during the extraction of chondrocytes had a significant effect on the number of chondrocytes obtained, and that previous extraction methods did not add serum during collagenase II digestion, resulting in apoptosis of many chondrocytes over a prolonged digestion period. Serum is added in the digestion process of type II collagenase, which is helpful for providing nutrition and promoting survival of chondrocytes, and meanwhile, a culture medium with high concentration of serum is used in inoculation, so that adherence and proliferation of chondrocytes can be effectively promoted, and a large amount of chondrocytes can be efficiently obtained.
And the cut stopping point in the second step is the femoral head body surface projection.
The cleaning method in the fourth step is that the micro scissors are tightly adhered to the surface of the cartilage tissue and parallel to the tissue surface to build macroscopic soft tissue.
0.25% in step five of EDTA-pancreatin manufactured by GIBCO, cat # 25200056.
The digestion conditions of the constant temperature shaking table in the fifth step and the seventh step are as follows: the temperature was 37 ℃ and the rotation speed was 200rpm.
The centrifugation conditions in the sixth step, the eighth step and the ninth step are as follows: the temperature of room temperature is 37 ℃, and the rotation speed of the centrifuge is 1000RPM.
The method for preparing 5 ml of type II collagenase solution containing 0.22% Australian fetal bovine serum in the seventh step comprises:
s1: 11mg of collagenase type II (manufactured by SIGMA, inc., cat. 1148090) was dissolved in 4.5 ml of PBS solution,
s2: 0.5 ml of Australian fetal bovine serum (manufactured by GIBCO, inc., cat No. 10100147) was added thereto, and the mixture was mixed by inversion.
The F12-DMEM medium in the ninth step is produced by GIBCO company, and has the product number of 11320033.
The preparation method of the culture medium containing 10% Australia fetal bovine serum F12-DMEM in the step ten comprises the following steps: 1 ml of Australian fetal calf serum is dissolved in 9 ml of F12-DMEM medium to prepare 10 ml of mixed medium.
The environmental conditions of the cell applying box in the step ten are as follows: the temperature was 37 ℃,5% carbon dioxide concentration, 95% humidity.
In summary, the method for efficiently separating and extracting rat chondrocytes includes the steps of selecting a healthy 5-day-old SPF-grade SD rat, killing the rat by dislocation of cervical vertebrae, immersing the killed rat in 75% alcohol for 30 seconds, taking out the immersed rat, cutting the skin upward along the femur from the surface projection of the tibial plateau outside the knee joint of the hind limb, dissecting the muscles attached to the vicinity of the joint cavity with a microscope, exposing bilateral cartilage of the rat, cutting the tibia and the femur along the epiphysical line, separating to obtain cartilage tissue, primarily cleaning the soft tissue surface with the microscope, transferring the cleaned cartilage tissue to 4 ml of 0.25-g EDTA-pancreatin, digesting the remaining soft tissue with a 37 ℃ constant temperature shaker for 15 minutes, centrifuging the mixture of the tissue and pancreatin a centrifuge, discarding the supernatant to obtain digested cartilage tissue, cutting the cartilage tissue with a scalpel to form fine sand, transferring 5 ml of a solution of 0.22-g II-containing bovine serum with a constant temperature shaker at 37 ℃ to remove collagen for 2.5 hours, centrifuging the digested cartilage tissue to remove collagen in a culture medium, centrifuging the supernatant containing 12-g bovine serum, centrifuging the supernatant containing 12-g bovine serum, and then centrifuging the cultured cells, and centrifuging the cultured bovine serum in a centrifuge to remove 10-g serum containing filtration medium, and then centrifuging the supernatant to obtain a 10-F culture medium.
The points to be finally explained are: first, in the description of the present application, it should be noted that, unless otherwise specified and limited, the terms "mounted," "connected," and "connected" should be understood broadly, and may be a mechanical connection or an electrical connection, or a communication between two elements, and may be a direct connection, and "upper," "lower," "left," and "right" are only used to indicate a relative positional relationship, and when the absolute position of the object to be described is changed, the relative positional relationship may be changed;
secondly, the method comprises the following steps: in the drawings of the disclosed embodiments of the invention, only the structures related to the disclosed embodiments are referred to, other structures can refer to common designs, and the same embodiment and different embodiments of the invention can be combined with each other without conflict;
and finally: 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 are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. A method for efficiently separating and extracting rat chondrocytes is characterized in that: the method comprises the following steps:
the method comprises the following steps: selecting one healthy SPF (specific pathogen free) SD rat with the age of 5 days, killing the rat in a cervical dislocation mode, and then soaking the killed rat in 75% alcohol for 30 seconds;
step two: taking out the rat soaked in the treatment in the step one, cutting the epidermis upwards along the femur from the body surface projection position of the tibial plateau at the outer side of the knee joint of the hind limb, and cutting off the muscle attached near the joint cavity by using a micro-shear to expose the cartilage at the two sides of the rat;
step three: cutting off the tibia and the femur of the rat treated in the step two along the epiphyseal line, and separating to obtain cartilage tissues;
step four: preliminarily cleaning soft tissues on the surface of the cartilage tissue by using micro scissors;
step five: transferring the cleared cartilage tissue to 4 ml of 0.25% EDTA-pancreatin, and digesting the remaining soft tissue using a 37 ℃ constant temperature shaker for 15 minutes;
step six: centrifuging the mixture of the tissue and the pancreatin by using a centrifuge, and removing the supernatant to obtain digested cartilage tissue;
step seven: cutting cartilage tissue into fine sand with a scalpel, transferring to 5 ml of 0.22% type II collagenase solution containing Australian fetal bovine serum, and digesting intercellular collagen with a 37 ℃ constant temperature shaker for 2.5 hours;
step eight: filtering the digested solution by a 70-micron filter screen, and centrifuging by using a centrifuge;
step nine: centrifuging by a centrifuge, discarding the centrifuged supernatant, resuspending by an F12-DMEM culture medium, and centrifuging by the centrifuge;
step ten: the supernatant after centrifugation was discarded, and the cells were suspended in F12-DMEM medium containing 10% Australian fetal bovine serum, seeded on a petri dish, placed in a cell plating chamber, plated for 2 days, and then changed.
2. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: and the cut stopping point in the second step is the femoral head body surface projection position.
3. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the cleaning method in the fourth step is that the micro scissors are tightly attached to the surface of the cartilage tissue and the macroscopic soft tissue is built in parallel to the surface of the tissue.
4. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: 0.25% in the fifth step EDTA-pancreatin was produced by GIBCO, having a product number of 25200056.
5. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the digestion conditions of the constant temperature shaking table in the fifth step and the seventh step are as follows: the temperature was 37 ℃ and the rotation speed was 200rpm.
6. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the centrifugation conditions in the sixth step, the eighth step and the ninth step are as follows: the temperature was 37 ℃ and the centrifuge speed was 1000RPM.
7. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the method for preparing the type II collagenase solution containing 0.22 percent of Australia fetal bovine serum in 5 milliliters in the step seven comprises the following steps:
s1: taking 11mgII collagenase, dissolving in 4.5 ml PBS solution,
s2: 0.5 ml of Australian fetal bovine serum was added, and the mixture was inverted and mixed.
8. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the F12-DMEM medium in the ninth step is produced by GIBCO company, and the product number is 11320033.
9. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the preparation method of the culture medium containing 10% Australia fetal bovine serum F12-DMEM in the step ten comprises the following steps: 1 ml of Australian fetal calf serum is dissolved in 9 ml of F12-DMEM medium to prepare 10 ml of mixed medium.
10. The method for efficiently separating and extracting rat chondrocytes according to claim 1, wherein: the environmental conditions of the cell dressing box in the step ten are as follows: the temperature was 37 ℃,5% carbon dioxide concentration, 95% humidity.
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| CN114058585A (en) * | 2021-12-03 | 2022-02-18 | 无锡市第二人民医院 | Culture method of mouse bone marrow-derived dendritic cells |
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| CN114058585A (en) * | 2021-12-03 | 2022-02-18 | 无锡市第二人民医院 | Culture method of mouse bone marrow-derived dendritic cells |
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