CN113481153A - Isolated culture method of adipose-derived mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a separation culture method of adipose tissue-derived stem cells, which comprises the following steps: (1) taking out isolated adipose tissues under aseptic condition, washing with normal saline, and cutting into pieces for later use; (2) adding digestive juice into the fat tissue fragments in the step (1) for digestion treatment; (3) adding a culture medium added with sodium valproate and iron (II) phthalocyanine into the digested adipose tissues in the step (2) for resuspension, replacing the culture medium after culturing for 1-2 days, and adding the following components into the culture medium: norcantharidin and sanggenon C, N-nicotinoyl glycine are cultured until the cell fusion degree reaches 80-90%, and the fat mesenchymal stem cells for subculture are obtained after digestion. The invention provides a separation culture method of adipose-derived mesenchymal stem cells, the fusion degree of the cells can reach 80-90% in a period of time after continuous culture, the proliferation activity of the cells in the primary culture process is improved, and the clinical application of the adipose-derived mesenchymal stem cells is fully guaranteed.
Description
Technical Field
The invention relates to the field of stem cells, in particular to a separation culture method of adipose tissue-derived mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a group of pluripotent cells that can be isolated from a variety of tissues including fat, bone marrow, and umbilical cord tissue. Because of their plasticity, these cells are of great clinical and fundamental research interest.
Adipose-derived stem cells (ADSCs) are stem cells with multi-directional differentiation potential separated from adipose tissues and can be differentiated into cartilage, osteogenic, adipose, neural tissues and other tissues or organs under the action of certain induction factors. The adipose-derived mesenchymal stem cells are widely used in clinical research at present, have application values mainly expressed in tissue repair and reconstruction, are suitable for repairing various tissues lacking in regeneration capacity, such as cartilage, muscle, cardiac muscle, nerves and the like, have sufficient sources, can be extracted in large quantities by a simpler method, and have wide research and application prospects.
The in vitro isolation and culture process of the adipose-derived mesenchymal stem cells relates to the safety of clinical application of the stem cells. The in vitro culture process comprises isolation culture and subculture, wherein the isolation culture processes adipose tissues after being processed to obtain adipose-derived mesenchymal stem cells for subculture, so that the isolation culture process influences the quality of the cells for subculture, and the existing adipose-derived mesenchymal stem cell isolation culture process has the following problems: the culture medium added with the fetal calf serum is used in the culture process, the cost of the fetal calf serum is high, and the fetal calf serum is used as an exogenous substance derived from animals, can generate potential risks to the safety of clinical use of stem cells, and cannot meet the clinical requirements. Therefore, it is necessary to provide a method for isolated culture of adipose-derived mesenchymal stem cells, which can improve the efficiency of in vitro proliferation of adipose-derived mesenchymal stem cells.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the method for separating and culturing the adipose-derived mesenchymal stem cells, fetal calf serum is not added, the activity of the primary adipose-derived mesenchymal stem cells obtained by culturing is good, the number of the primary adipose-derived mesenchymal stem cells is large, and conditions are provided for subculturing the cells.
The purpose of the invention is realized by adopting the following technical scheme:
an isolated culture method of adipose-derived mesenchymal stem cells comprises the following steps:
(1) taking out isolated adipose tissues under aseptic condition, washing with normal saline, and cutting into pieces for later use;
(2) adding a digestive juice into the fat tissue fragments obtained in the step (1) for digestion treatment, adding a D-hanks solution for dilution after digestion is finished, and removing a supernatant after centrifugation;
(3) adding a culture medium added with sodium valproate and iron (II) phthalocyanine into the precipitate after centrifugation in the step (2) for resuspension, inoculating the mixture into a culture dish, and performing 5% CO treatment at 37 DEG C2The culture medium is replaced after 1 to 2 days of culture in the incubator, and the following components are added into the culture medium: norcantharidin and sanggenon C, N-nicotinoyl glycine, replacing the culture medium added with the components every 2-3 days, culturing until the cell fusion degree reaches 80-90%, and digesting to obtain the adipose mesenchymal stem cell for subculture.
Further, the culture medium in the step (3) is DMEM/F12 culture medium.
Further, the concentrations of the sodium valproate and the iron (II) phthalocyanine in the culture medium are as follows: sodium valproate 8-13ng/mL and iron (II) phthalocyanine 1-4.5 ng/mL.
Further, the concentrations of the sodium valproate and the iron (II) phthalocyanine in the culture medium are as follows: sodium valproate 10ng/mL, iron (II) phthalocyanine 3 ng/mL.
Further, the concentration of norcantharidin and sanggenon C, N-nicotinoyl glycine in the culture medium is as follows: norcantharidin 1-5ng/mL, sanggenon C10-20 μ g/mL, and N-nicotinoyl glycine 15-25 μ g/mL.
Further, the concentration of norcantharidin and sanggenon C, N-nicotinoyl glycine in the culture medium is as follows: norcantharidin 3.5ng/mL, sanggenon C15 μ g/mL, and N-nicotinoyl glycine 20 μ g/mL.
Further, in the step (3), the density of the cells in the culture medium added with norcantharidin and sanggenon C, N-nicotinoyl glycine is 1-4 x 105one/mL.
Further, in the step (2), collagenase I with the concentration of 0.2-0.3% is added into the fat tissue fragments for digestion treatment, and the volume of the collagenase I is 3-5 times of that of the fat tissue fragments.
Further, in step (3), the cells were digested with 0.25% of pancreatin.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a separation culture method of adipose-derived mesenchymal stem cells, which is characterized in that a culture medium added with sodium valproate and iron (II) phthalocyanine is adopted to pre-culture digested adipose tissues for 1-2 days in the culture process, so that the adherent growth of the adipose-derived mesenchymal stem cells is facilitated. After the culture medium is replaced, norcantharidin and sanggenon C, N-nicotinoyl glycine are added into the culture medium to improve the proliferation activity of the adipose-derived mesenchymal stem cells, and the fusion degree of the cells can reach 80-90% in a short time after continuous culture. The proliferation activity of the cells in the primary culture process is improved in the culture process, the number of the cells obtained after the culture is large, the activity is good, the number of the cells for subculture is increased, and a sufficient guarantee is provided for the clinical application of the adipose mesenchymal stem cells.
Detailed Description
The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.
Example 1
An isolated culture method of adipose-derived mesenchymal stem cells comprises the following steps:
(1) washing isolated adipose tissue with normal saline under aseptic condition, and cutting into 1-2mm pieces3Breaking the blocks for later use;
(2) adding 0.2% collagenase I into the fat tissue fragments obtained in the step (1) for digestion for 1 hour, adding the collagenase I with the volume 5 times that of the fat tissue, adding a D-hanks solution for dilution after the digestion is finished, centrifuging at 1500rpm for 30min, and removing a supernatant after the centrifugation;
(3) adding DMEM/F12 medium added with sodium valproate and iron (II) phthalocyanine into the sediment after centrifugation in the step (2) for resuspension, wherein the concentration of the sodium valproate and the iron (II) phthalocyanine in the medium is as follows: sodium valproate 10ng/mL and iron (II) phthalocyanine 3ng/mL, inoculating in 10cm culture dish at 37 deg.C and 5% CO2The medium was changed after 1 day of culture in the incubator, and the density of the cells in the medium was 2X 105The culture medium is characterized in that the following components are added into DMEM/F12 culture medium: norcantharidin 3.5ng/mL, sanggenon C15 μ g/mL, and N-nicotinoyl glycine 20 μ g/mL, replacing culture medium with the above components every 2 days, culturing until cell fusion degree reaches 80%,and digesting with 0.25% pancreatin to obtain the adipose-derived mesenchymal stem cells for subculture.
Example 2
An isolated culture method of adipose-derived mesenchymal stem cells comprises the following steps:
(1) washing isolated adipose tissue with normal saline under aseptic condition, and cutting into 1-2mm pieces3Breaking the blocks for later use;
(2) adding 0.25% collagenase I into the fat tissue fragments obtained in the step (1) for digestion for 40min, wherein the volume of the collagenase I is 4 times that of the fat tissue, adding a D-hanks solution for dilution after the digestion is finished, centrifuging at 1500rpm for 30min, and removing a supernatant after the centrifugation;
(3) adding DMEM/F12 medium added with sodium valproate and iron (II) phthalocyanine into the sediment after centrifugation in the step (2) for resuspension, wherein the concentration of the sodium valproate and the iron (II) phthalocyanine in the medium is as follows: valproate sodium 8ng/mL, iron (II) phthalocyanine 1ng/mL, inoculated in 10cm petri dish at 37 deg.C and 5% CO2The medium was changed after 2 days of culture in the incubator, and the density of the cells in the medium was 1X 105The culture medium is characterized in that the following components are added into DMEM/F12 culture medium: 1ng/mL of norcantharidin, 10 mu g/mL of sanggenon C, and 15 mu g/mL of N-nicotinoyl glycine, replacing the culture medium added with the components once every 2 days, culturing until the cell fusion degree reaches 85%, and digesting with 0.25% pancreatin to obtain the adipose mesenchymal stem cell for subculture.
Example 3
An isolated culture method of adipose-derived mesenchymal stem cells comprises the following steps:
(1) washing isolated adipose tissue with normal saline under aseptic condition, and cutting into 1-2mm pieces3Breaking the blocks for later use;
(2) adding 0.3% collagenase I into the fat tissue fragments obtained in the step (1) for digestion for 30min, wherein the volume of the collagenase I is 3 times that of the fat tissue, adding a D-hanks solution for dilution after the digestion is finished, centrifuging at 1500rpm for 30min, and removing a supernatant after the centrifugation;
(3) adding sodium valproate and iron (II) phthalein into the precipitate obtained after centrifugation in the step (2)The cyanine DMEM/F12 culture medium is used for resuspension, and the concentrations of sodium valproate and iron (II) phthalocyanine in the culture medium are as follows: sodium valproate 13ng/mL, iron (II) phthalocyanine 4.5 ng/mL, inoculated in 10cm petri dish at 37 deg.C and 5% CO2The medium was changed after 2 days of culture in the incubator, and the density of the cells in the medium was 4X 105The culture medium is characterized in that the following components are added into DMEM/F12 culture medium: 5ng/mL of norcantharidin, 20 mu g/mL of sanggenon C and 25 mu g/mL of N-nicotinoyl glycine, replacing the culture medium added with the components once every 3 days, culturing until the cell fusion degree reaches 90%, and digesting with 0.25% of pancreatin to obtain the adipose mesenchymal stem cell for subculture.
Comparative example 1
Comparative example 1 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: the iron (II) phthalocyanine was omitted and the rest was the same as in example 1.
Comparative example 2
Comparative example 2 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: the iron (II) phthalocyanine was omitted, and the amount of sodium valproate was adjusted to 13ng/mL, and the procedure was repeated as in example 1.
Comparative example 3
Comparative example 3 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: sodium valproate was omitted and the procedure was as in example 1.
Comparative example 4
Comparative example 4 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: norcantharidin was omitted and the rest was the same as in example 1.
Comparative example 5
Comparative example 5 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: sanggenon C was omitted and the procedure was as in example 1.
Comparative example 6
Comparative example 6 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: sanggenon C was replaced with sanggenon G, and the rest was the same as in example 1.
Comparative example 7
Comparative example 7 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: n-nicotinoylglycine was omitted and the procedure was as in example 1.
Comparative example 8
Comparative example 8 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: the procedure of example 1 was repeated except that N-nicotinoylglycine was omitted and the amount of sanggenon C was adjusted to 35. mu.g/mL.
Comparative example 9
Comparative example 9 provides a method for isolated culture of adipose-derived mesenchymal stem cells, which is different from example 1 in that: norcantharidin, sanggenon C, N-nicotinoylglycine, sodium valproate, iron (II) phthalocyanine were added to the medium together, and the rest was the same as in example 1.
The statistics of the time required for the cell fusion degree of the adipose-derived mesenchymal stem cells in example 1 and comparative examples 1 to 9 to reach 80% in the isolation culture process are shown in table 1.
TABLE 1
| Group of | Time (h) |
| Example 1 | 112 |
| Comparative example 1 | 167 |
| Comparative example 2 | 152 |
| Comparative example 3 | 159 |
| Comparative example 4 | 178 |
| Comparative example 5 | 172 |
| Comparative example 6 | 147 |
| Comparative example 7 | 151 |
| Comparative example 8 | 145 |
| Comparative example 9 | 134 |
As can be seen from Table 1, the time taken for example 1 was the least and the time taken for comparative examples 1 to 9 was extended to various degrees when the adipose-derived mesenchymal stem cells reached 80% confluency. In comparative examples 1 to 9, the components added to the medium during the culture and the timing of addition of the components were adjusted, respectively, and the above adjustments all had a certain effect on the proliferation activity of the cells.
The numbers of adipose-derived mesenchymal stem cells in examples 1 to 3 and comparative examples 1 to 9 were counted by trypan blue staining method after the isolation culture was completed, respectively, and the results are shown in table 2.
TABLE 2
| Group of | Number of cells (. times.10)5One) |
| Example 1 | 23.67 |
| Example 2 | 20.79 |
| Example 3 | 21.45 |
| Comparative example 1 | 13.26 |
| Comparative example 2 | 14.07 |
| Comparative example 3 | 13.84 |
| Comparative example 4 | 8.69 |
| Comparative example 5 | 11.32 |
| Comparative example 6 | 15.84 |
| Comparative example 7 | 14.51 |
| Comparative example 8 | 15.17 |
| Comparative example 9 | 17.23 |
As can be seen from table 2, the adipose-derived mesenchymal cells obtained by isolated culture in examples 1 to 3 were increased in number, and decreased in the comparative examples 1 to 9 to different extents.
In comparative examples 1 to 3, when iron (II) phthalocyanine or sodium valproate was omitted, or the amount of sodium valproate was adjusted after the iron (II) phthalocyanine was omitted, the number of adipose-derived mesenchymal stem cells was still decreased to some extent, because the addition of sodium valproate and iron (II) phthalocyanine to the culture medium pre-cultured adipose tissues after digestion for 1 to 2 days, which facilitated the adherent growth of adipose-derived mesenchymal stem cells, maintained the activity, and prepared for the proliferation of cells.
In comparative examples 4 to 8, one of norcantharidin and sanggenon C, N-nicotinoyl glycine is omitted, or the amount of the rest of the components is adjusted after one of the components is omitted, and sanggenon C is replaced by sanggenon G, so that the number of cells is not as large as that in example 1, because the norcantharidin and the sanggenon C, N-nicotinoyl glycine added in the invention can effectively improve the proliferation activity of adipose-derived mesenchymal stem cells in the separation culture process, promote the proliferation of the cells, and can harvest more active adipose-derived mesenchymal stem cells after the separation culture is finished.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (9)
1. A separation culture method of adipose-derived mesenchymal stem cells is characterized by comprising the following steps:
(1) taking out isolated adipose tissues under aseptic condition, washing with normal saline, and cutting into pieces for later use;
(2) adding a digestive juice into the fat tissue fragments obtained in the step (1) for digestion treatment, adding a D-hanks solution for dilution after digestion is finished, and removing a supernatant after centrifugation;
(3) adding into the precipitate after centrifugation in step (2)Adding sodium valproate and iron (II) phthalocyanine into the culture medium, re-suspending, inoculating in a culture dish at 37 deg.C and 5% CO2The culture medium is replaced after 1 to 2 days of culture in the incubator, and the following components are added into the culture medium: norcantharidin and sanggenon C, N-nicotinoyl glycine, replacing the culture medium added with the components every 2-3 days, culturing until the cell fusion degree reaches 80-90%, and digesting to obtain the adipose mesenchymal stem cell for subculture.
2. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 1, wherein the medium in the step (3) is DMEM/F12 medium.
3. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 1, wherein the concentrations of the sodium valproate and the iron (II) phthalocyanine in the culture medium are as follows: sodium valproate 8-13ng/mL and iron (II) phthalocyanine 1-4.5 ng/mL.
4. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 3, wherein the concentrations of the sodium valproate and the iron (II) phthalocyanine in the culture medium are as follows: sodium valproate 10ng/mL, iron (II) phthalocyanine 3 ng/mL.
5. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 1, wherein the concentration of norcantharidin and sanggenon C, N-nicotinoyl glycine in the culture medium is: norcantharidin 1-5ng/mL, sanggenon C10-20 μ g/mL, and N-nicotinoyl glycine 15-25 μ g/mL.
6. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 5, wherein the concentration of norcantharidin and sanggenon C, N-nicotinoyl glycine in the culture medium is as follows: norcantharidin 3.5ng/mL, sanggenon C15 μ g/mL, and N-nicotinoyl glycine 20 μ g/mL.
7. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 1The method is characterized in that the density of the cells in the step (3) in the culture medium added with norcantharidin and sanggenon C, N-nicotinoyl glycine is 1-4 multiplied by 105one/mL.
8. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 1, wherein the adipose tissue fragment is digested by adding collagenase I having a concentration of 0.2-0.3% to the adipose tissue fragment in the step (2), wherein the volume of collagenase I added is 3-5 times that of the adipose tissue fragment.
9. The isolated culture method of adipose-derived mesenchymal stem cells according to claim 1, wherein the cells are digested with 0.25% of pancreatin in step (3).
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