Disclosure of Invention
The invention aims to provide a preparation process of a cryopreservation protection solution for umbilical cord mesenchymal stem cells, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation process of a cryopreservation protection solution for umbilical cord mesenchymal stem cells comprises the following steps:
the method comprises the following steps: cleaning treatment: firstly, using sterilized surgical scissors to cut and spread umbilical cord tissues, fixing the spread umbilical cord tissues, then using a disinfectant to clean the surfaces of the umbilical cord tissues, using a cotton ball to dip the disinfectant to repeatedly disinfect and clean the surfaces of the umbilical cord during cleaning, wherein the disinfection and cleaning frequency is 3-5 times, then using a cell pretreatment cleaning buffer solution to clean the umbilical cord tissues, and during cleaning, using the cotton ball to dip the buffer solution to repeatedly clean the umbilical cord tissues, wherein the cleaning frequency is 3-5 times, so that the proportion of red blood cells on the umbilical cord tissues is effectively reduced, and then collecting the umbilical cord tissues for later use;
step two: and (3) digestion treatment: then, carrying out shearing treatment on the umbilical cord tissue obtained in the step one, and controlling the shearing size of the umbilical cord tissue during the shearing treatment so that the area of the upper end surface of a single umbilical cord tissue is controlled to be 0.08cm2-0.2cm2Adding pancreatin and EDTA into the minced umbilical cord tissue for digestion treatment, wherein during the digestion treatment, the pancreatin and EDTA completely immerse the umbilical cord tissue, during the digestion treatment, the umbilical cord tissue is repeatedly turned over, then the umbilical cord tissue is removed after the digestion treatment, then a mesenchymal stem cell culture medium is added into an enzymolysis liquid, so that the digestion is stopped, finally, the cells obtained after the enzymolysis are cleaned, and finally, a cell suspension is obtained;
step three: cell culture: centrifuging the cells subjected to enzymolysis and digestion obtained in the step two, wherein during centrifugation, the cells are centrifuged at a rotating speed of 5000-8000 rpm to ensure the rupture of the cells, and then performing density passage after complete culture medium reselection to continuously grow the cells for 24-72 h;
step four: centrifuging and taking a supernatant: centrifuging the liquid obtained in the third step, wherein the centrifugation is carried out at the rotating speed of 1500-1800 rpm for 10-15 minutes, and then taking the supernatant for later use;
step five: freezing treatment: and D, adding dimethyl sulfoxide into the supernatant obtained in the fourth step, mixing, storing at low temperature overnight after mixing, and finally transferring into liquid nitrogen for long-term storage.
Preferably, 0.02mL/cm is added to the cells in the second step20.2% pancreatin and 0.03% EDTA, and a digestion time of 20 min.
Preferably, 0.01mL/cm is added to the cells in the second step20.3% pancreatin and 0.04% EDTA and digestion time 15 min.
Preferably, 0.03mL/cm is added to the cells in the second step20.1% pancreatin and 0.02% EDTA, and a digestion time of 25 min.
Preferably, the ratio of 5000 cells/cm in step three28000 cells/cm2The passage density of (2) is subjected to passage.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, by the technical process, the use of bovine serum can be effectively avoided, so that heterologous viruses and sensitization sources are introduced as little as possible, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells can be effectively improved.
Detailed Description
Example 1: the invention provides a technical scheme that:
a preparation process of a cryopreservation protection solution for umbilical cord mesenchymal stem cells comprises the following steps:
the method comprises the following steps: cleaning treatment: firstly, using sterilized surgical scissors to cut and spread umbilical cord tissues, fixing the spread umbilical cord tissues, then using a disinfectant to clean the surfaces of the umbilical cord tissues, using a cotton ball to dip the disinfectant to repeatedly disinfect and clean the surfaces of the umbilical cord during cleaning, wherein the disinfection and cleaning frequency is 3-5 times, then using a cell pretreatment cleaning buffer solution to clean the umbilical cord tissues, and during cleaning, using the cotton ball to dip the buffer solution to repeatedly clean the umbilical cord tissues, wherein the cleaning frequency is 3-5 times, so that the proportion of red blood cells on the umbilical cord tissues is effectively reduced, and then collecting the umbilical cord tissues for later use;
step two: and (3) digestion treatment: then, carrying out shearing treatment on the umbilical cord tissue obtained in the step one, and controlling the shearing size of the umbilical cord tissue during the shearing treatment so that the area of the upper end surface of a single umbilical cord tissue is controlled to be 0.08cm2-0.2cm2Then adding pancreatin and EDTA into the minced umbilical cord tissue for digestion treatment, wherein the pancreatin and EDTA completely immerse the umbilical cord tissue during the digestion treatment, repeatedly turning over the umbilical cord tissue during the digestion treatment, removing the umbilical cord tissue after the digestion treatment, then adding a mesenchymal stem cell culture medium into an enzymolysis solution,thereby terminating digestion, and finally cleaning cells obtained after enzymolysis to finally obtain cell suspension;
step three: cell culture: centrifuging the cells subjected to enzymolysis and digestion obtained in the step two, wherein during centrifugation, the cells are centrifuged at a rotating speed of 5000-8000 rpm to ensure the rupture of the cells, and then performing density passage after complete culture medium reselection to continuously grow the cells for 24-72 h;
step four: centrifuging and taking a supernatant: centrifuging the liquid obtained in the third step, wherein the centrifugation is carried out at the rotating speed of 1500-1800 rpm for 10-15 minutes, and then taking the supernatant for later use;
step five: freezing treatment: and D, adding dimethyl sulfoxide into the supernatant obtained in the fourth step, mixing, storing at low temperature overnight after mixing, and finally transferring into liquid nitrogen for long-term storage.
In the second step, 0.03mL/cm is added to the cells20.1% pancreatin and 0.02% EDTA, and a digestion time of 25min, in steps three according to 5000 cells/cm28000 cells/cm2The passage density of the umbilical cord mesenchymal stem cells is increased, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells is effectively guaranteed.
According to the invention, by the technical process, the use of bovine serum can be effectively avoided, so that heterologous viruses and sensitization sources are introduced as little as possible, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells can be effectively improved.
Example 2: the invention provides a technical scheme that:
a preparation process of a cryopreservation protection solution for umbilical cord mesenchymal stem cells comprises the following steps:
the method comprises the following steps: cleaning treatment: firstly, using sterilized surgical scissors to cut and spread umbilical cord tissues, fixing the spread umbilical cord tissues, then using a disinfectant to clean the surfaces of the umbilical cord tissues, using a cotton ball to dip the disinfectant to repeatedly disinfect and clean the surfaces of the umbilical cord during cleaning, wherein the disinfection and cleaning frequency is 3-5 times, then using a cell pretreatment cleaning buffer solution to clean the umbilical cord tissues, and during cleaning, using the cotton ball to dip the buffer solution to repeatedly clean the umbilical cord tissues, wherein the cleaning frequency is 3-5 times, so that the proportion of red blood cells on the umbilical cord tissues is effectively reduced, and then collecting the umbilical cord tissues for later use;
step two: and (3) digestion treatment: then, carrying out shearing treatment on the umbilical cord tissue obtained in the step one, and controlling the shearing size of the umbilical cord tissue during the shearing treatment so that the area of the upper end surface of a single umbilical cord tissue is controlled to be 0.08cm2-0.2cm2Adding pancreatin and EDTA into the minced umbilical cord tissue for digestion treatment, wherein during the digestion treatment, the pancreatin and EDTA completely immerse the umbilical cord tissue, during the digestion treatment, the umbilical cord tissue is repeatedly turned over, then the umbilical cord tissue is removed after the digestion treatment, then a mesenchymal stem cell culture medium is added into an enzymolysis liquid, so that the digestion is stopped, finally, the cells obtained after the enzymolysis are cleaned, and finally, a cell suspension is obtained;
step three: cell culture: centrifuging the cells subjected to enzymolysis and digestion obtained in the step two, wherein during centrifugation, the cells are centrifuged at a rotating speed of 5000-8000 rpm to ensure the rupture of the cells, and then performing density passage after complete culture medium reselection to continuously grow the cells for 24-72 h;
step four: centrifuging and taking a supernatant: centrifuging the liquid obtained in the third step, wherein the centrifugation is carried out at the rotating speed of 1500-1800 rpm for 10-15 minutes, and then taking the supernatant for later use;
step five: freezing treatment: and D, adding dimethyl sulfoxide into the supernatant obtained in the fourth step, mixing, storing at low temperature overnight after mixing, and finally transferring into liquid nitrogen for long-term storage.
In the second step, 0.02mL/cm is added to the cells20.2% of pancreatin and 0.03% of EDTA, and a digestion time of 20min, in steps three according to 5000 cells/cm28000 cells/cm2The passage density of the umbilical cord mesenchymal stem cells is increased, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells is effectively guaranteed.
According to the invention, by the technical process, the use of bovine serum can be effectively avoided, so that heterologous viruses and sensitization sources are introduced as little as possible, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells can be effectively improved.
Example 3: the invention provides a technical scheme that:
a preparation process of a cryopreservation protection solution for umbilical cord mesenchymal stem cells comprises the following steps:
the method comprises the following steps: cleaning treatment: firstly, using sterilized surgical scissors to cut and spread umbilical cord tissues, fixing the spread umbilical cord tissues, then using a disinfectant to clean the surfaces of the umbilical cord tissues, using a cotton ball to dip the disinfectant to repeatedly disinfect and clean the surfaces of the umbilical cord during cleaning, wherein the disinfection and cleaning frequency is 3-5 times, then using a cell pretreatment cleaning buffer solution to clean the umbilical cord tissues, and during cleaning, using the cotton ball to dip the buffer solution to repeatedly clean the umbilical cord tissues, wherein the cleaning frequency is 3-5 times, so that the proportion of red blood cells on the umbilical cord tissues is effectively reduced, and then collecting the umbilical cord tissues for later use;
step two: and (3) digestion treatment: then, carrying out shearing treatment on the umbilical cord tissue obtained in the step one, and controlling the shearing size of the umbilical cord tissue during the shearing treatment so that the area of the upper end surface of a single umbilical cord tissue is controlled to be 0.08cm2-0.2cm2Adding pancreatin and EDTA into the minced umbilical cord tissue for digestion treatment, wherein during the digestion treatment, the pancreatin and EDTA completely immerse the umbilical cord tissue, during the digestion treatment, the umbilical cord tissue is repeatedly turned over, then the umbilical cord tissue is removed after the digestion treatment, then a mesenchymal stem cell culture medium is added into an enzymolysis liquid, so that the digestion is stopped, finally, the cells obtained after the enzymolysis are cleaned, and finally, a cell suspension is obtained;
step three: cell culture: centrifuging the cells subjected to enzymolysis and digestion obtained in the step two, wherein during centrifugation, the cells are centrifuged at a rotating speed of 5000-8000 rpm to ensure the rupture of the cells, and then performing density passage after complete culture medium reselection to continuously grow the cells for 24-72 h;
step four: centrifuging and taking a supernatant: centrifuging the liquid obtained in the third step, wherein the centrifugation is carried out at the rotating speed of 1500-1800 rpm for 10-15 minutes, and then taking the supernatant for later use;
step five: freezing treatment: and D, adding dimethyl sulfoxide into the supernatant obtained in the fourth step, mixing, storing at low temperature overnight after mixing, and finally transferring into liquid nitrogen for long-term storage.
In the second step, 0.01mL/cm is added to the cells20.3% of pancreatin and 0.04% of EDTA, and the digestion time is 15min, in step three according to 5000 cells/cm28000 cells/cm2The passage density of the umbilical cord mesenchymal stem cells is increased, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells is effectively guaranteed.
According to the invention, by the technical process, the use of bovine serum can be effectively avoided, so that heterologous viruses and sensitization sources are introduced as little as possible, and the quality of the cryopreservation protection solution for the umbilical cord mesenchymal stem cells can be effectively improved.
The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts of the present invention. The foregoing is only a preferred embodiment of the present invention, and it should be noted that there are objectively infinite specific structures due to the limited character expressions, and it will be apparent to those skilled in the art that a plurality of modifications, decorations or changes may be made without departing from the principle of the present invention, and the technical features described above may be combined in a suitable manner; such modifications, variations, combinations, or adaptations of the invention using its spirit and scope, as defined by the claims, may be directed to other uses and embodiments.