CN114651812A - Fat mesenchymal stem cell cryopreservation protective solution and cryopreservation method - Google Patents
Fat mesenchymal stem cell cryopreservation protective solution and cryopreservation method Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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Abstract
The invention discloses a fat mesenchymal stem cell cryopreservation protective solution, which comprises a basic culture medium and the following components added in the culture medium: glycerol, procaine microcapsule, bergapten, resveratrol, vitamin C, and insulin; the final concentration of each component in the culture medium is: 20-30 mu g/mL of glycerin, 1-5ng/mL of procaine microcapsule, 3-8ng/mL of bergapten, 5-10 mu g/mL of resveratrol, 1-2mg/mL of vitamin C and 0.5-1mg/mL of insulin. The protective solution provided by the invention is added with the procaine microcapsule, the bergapten, the resveratrol and other components, so that the damage of the cells after cryopreservation is reduced, the survival rate of the cells is improved, the cells after cryopreservation still have good proliferation activity, and all the components in the preservation solution are safe and stable, thereby being beneficial to the long-term preservation of the cells. The invention also provides a cryopreservation method of the adipose tissue-derived mesenchymal stem cells, which improves the preservation efficiency of the cells and fully ensures the application of the adipose tissue-derived mesenchymal stem cells in various fields.
Description
Technical Field
The invention relates to a cell cryopreservation protective solution, in particular to a fat mesenchymal stem cell cryopreservation protective solution and a cryopreservation method.
Background
Mesenchymal stem cells are present in various tissues such as bone marrow, umbilical cord tissue, placenta tissue, adipose tissue, and the like. Researches show that the mesenchymal stem cells still have multidirectional differentiation potential after continuous subculture and cryopreservation, can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathological changes, and have remarkable clinical application advantages.
The adipose-derived mesenchymal stem cells are mesenchymal stem cells derived from adipose tissues, have the capacity of self-renewal and multidirectional differentiation, and can be differentiated in the directions of bones, cartilages, fats, muscles, nerves, endothelia and the like. Meanwhile, the adipose-derived mesenchymal stem cells have wide sources, adipose tissues can be easily obtained through liposuction or surgical operation, the trauma to a donor is small, the ethical problem is not involved, the immunogenicity is low, and the adipose-derived mesenchymal stem cells can be greatly amplified under in vitro conditions, so that the adipose-derived mesenchymal stem cells are one of the most popular stem cells in the field of tissue engineering.
Because a long culture period is usually needed to obtain a large amount of adipose-derived mesenchymal stem cells, and the possibility of different places usually exists between a donor and a receptor for cell transplantation, the timely use of the cells is difficult to ensure, and the sufficient amount of adipose-derived mesenchymal stem cells is one of important factors for determining whether the clinical curative effect can be achieved. In order to provide sufficient adipose-derived mesenchymal stem cells for clinical and basic research at any time, the cells need to be frozen in liquid nitrogen. The cell cryopreservation means that the cells are preserved for a long time even if the cells are temporarily separated from the growth state, and the original biological characteristics of the cells are maintained, so that the cells can be normally used after rapid recovery when needed.
In order to reduce damage to cells in the process of cryopreservation, a protective solution is usually added, dimethyl sulfoxide is commonly used at present, and is a toxic reagent, and the dimethyl sulfoxide reacts with hydrophobic groups of proteins to cause protein denaturation, so that certain potential safety hazards exist, and therefore a safe and stable protective solution is needed to be provided for cryopreservation of adipose mesenchymal stem cells.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the adipose-derived mesenchymal stem cell cryopreservation protective solution, which is safe and stable in components and does not contain DMSO.
The second purpose of the invention is to provide a cryopreservation method of adipose tissue-derived mesenchymal stem cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a protective solution for cryopreservation of adipose tissue-derived mesenchymal stem cells comprises a basic culture medium and the following components added in the culture medium: glycerin, procaine microcapsules, bergapten, resveratrol, vitamin C and insulin; the final concentration of each component in the culture medium is: 20-30 mu g/mL of glycerol, 1-5ng/mL of procaine microcapsules, 3-8ng/mL of bergapten, 5-10 mu g/mL of resveratrol, 1-2mg/mL of vitamin C and 0.5-1mg/mL of insulin.
Further, the final concentration of each component in the culture medium is: 25 mu g/mL of glycerin, 3ng/mL of procaine microcapsule, 5ng/mL of bergapten, 8 mu g/mL of resveratrol, 1.5mg/mL of vitamin C and 0.7mg/mL of insulin.
Further, the basic medium is DMEM/F12.
Further, the preparation method of the procaine microcapsule comprises the following steps: dissolving wall materials in absolute ethyl alcohol, wherein the mass concentration of the wall materials is 1-2%, adding core material procaine powder, uniformly mixing, and performing spray drying to obtain the procaine microcapsule.
Further, the wall material is sodium carboxymethyl cellulose, and the mass ratio of the sodium carboxymethyl cellulose to the procaine powder is 1: 0.2-0.3.
The second purpose of the invention is realized by adopting the following technical scheme:
a cryopreservation method of adipose tissue-derived mesenchymal stem cells adopts the protection solution for cryopreservation.
Further, the cryopreservation method of the adipose tissue-derived mesenchymal stem cells comprises the following steps: and (3) resuspending the adipose-derived mesenchymal stem cells to be preserved by adopting a protective solution, carrying out programmed cooling to-80 ℃, preserving for 4-6h at-80 ℃, and then transferring to liquid nitrogen for freezing.
Further, the density of adipose-derived mesenchymal stem cells in the protective solution is 106-107one/mL.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a protective solution for cryopreservation of adipose-derived mesenchymal stem cells, which is characterized in that procaine microcapsules, bergapten, resveratrol and other components are added into the protective solution, so that damage of the cells after cryopreservation is reduced, the survival rate of the cells is improved, the adipose-derived mesenchymal stem cells after cryopreservation still have good proliferation activity, and each component in the preservation solution is safe and stable, so that the long-term preservation of the adipose-derived mesenchymal stem cells is facilitated.
The invention also provides a cryopreservation method of the adipose tissue-derived mesenchymal stem cells, which improves the preservation efficiency of the adipose tissue-derived mesenchymal stem cells and fully ensures the application of the adipose tissue-derived mesenchymal stem cells in various fields.
Drawings
Fig. 1 is a proliferation curve of adipose-derived mesenchymal stem cells cryopreserved for 3 months in example 1 of the present invention, comparative example 1 to comparative example 4.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A protective solution for cryopreservation of adipose-derived mesenchymal stem cells comprises a DMEM/F12 culture medium and the following components added in the culture medium: glycerin, procaine microcapsules, bergapten, resveratrol, vitamin C and insulin; the final concentration of each component in the culture medium is: 25 mu g/mL of glycerol, 3ng/mL of procaine microcapsules, 5ng/mL of bergapten, 8 mu g/mL of resveratrol, 1.5mg/mL of vitamin C and 0.7mg/mL of insulin.
The preparation method of the procaine microcapsule comprises the following steps: dissolving wall material sodium carboxymethyl cellulose in absolute ethyl alcohol, wherein the mass concentration of the wall material is 1%, adding core material procaine powder, and the mass ratio of the sodium carboxymethyl cellulose to the procaine powder is 1:0.2, uniformly mixing, and performing spray drying to obtain the procaine microcapsule.
Example 2
A protective solution for cryopreservation of adipose-derived mesenchymal stem cells comprises a DMEM/F12 culture medium and the following components added in the culture medium: glycerin, procaine microcapsules, bergapten, resveratrol, vitamin C and insulin; the final concentration of each component in the culture medium is: 20 mu g/mL of glycerin, 1ng/mL of procaine microcapsule, 3ng/mL of bergapten, 5 mu g/mL of resveratrol, 1mg/mL of vitamin C and 0.5mg/mL of insulin.
The preparation method of the procaine microcapsule comprises the following steps: dissolving wall material sodium carboxymethyl cellulose in absolute ethyl alcohol, wherein the mass concentration of the wall material is 2%, adding core material procaine powder, and the mass ratio of the sodium carboxymethyl cellulose to the procaine powder is 1:0.3, uniformly mixing, and performing spray drying to obtain the procaine microcapsule.
Example 3
A protective solution for cryopreservation of adipose-derived mesenchymal stem cells comprises a DMEM/F12 culture medium and the following components added in the culture medium: glycerin, procaine microcapsules, bergapten, resveratrol, vitamin C and insulin; the final concentration of each component in the culture medium is: 30 mu g/mL of glycerin, 5ng/mL of procaine microcapsule, 8ng/mL of bergapten, 10 mu g/mL of resveratrol, 2mg/mL of vitamin C and 1mg/mL of insulin.
The preparation method of the procaine microcapsule comprises the following steps: dissolving wall material sodium carboxymethyl cellulose in absolute ethyl alcohol, wherein the mass concentration of the wall material is 1.5%, adding core material procaine powder, and the mass ratio of the sodium carboxymethyl cellulose to the procaine powder is 1:0.25, uniformly mixing, and performing spray drying to obtain the procaine microcapsule.
Comparative example 1
Comparative example 1 provides a protective solution for cryopreservation of adipose-derived mesenchymal stem cells, which is different from example 1 in that: the procaine microcapsules were omitted and the procedure was as in example 1.
Comparative example 2
Comparative example 2 provides a cryopreservation protective solution for adipose mesenchymal stem cells, which is different from that in example 1 in that: the procaine microcapsules were prepared as procaine powder, and the rest was the same as in example 1.
Comparative example 3
Comparative example 3 provides a cryopreservation protective solution for adipose-derived mesenchymal stem cells, which is different from example 1 in that: the bergapten was omitted and the rest was the same as in example 1.
Comparative example 4
Comparative example 4 provides a cryopreservation protective solution for adipose-derived mesenchymal stem cells, which is different from example 1 in that: the procaine microcapsules and bergapten were omitted and the rest was the same as in example 1.
Washing fat sample with PBS twice, centrifuging, collecting fat tissue, packaging in 50mL centrifuge tube, adding 1% type II collagenase with equal volume, shaking for digestion for 30min, centrifuging at 1200rpm for 10min, collecting cell precipitate at bottom layer, adding into culture mediumNutrient medium (DMEM/F12+ 10% FBS) was resuspended and cell density adjusted to 5X 104Culturing in T75 culture flask at 37 deg.C and 5% CO2The culture box is used for subculturing after the cell fusion degree reaches 80%, and the P3 generation cells in the logarithmic phase are taken for freezing experiments.
The P3 generation adipose-derived mesenchymal stem cells obtained in the culture experiment are respectively resuspended by the protective solution of example 1 and comparative examples 1 to 4, and the cell density is 106Subpackaging the seeds/mL in a 2mL freezing tube, and carrying out programmed cooling to-80 ℃, wherein the programmed cooling process comprises the following steps: cooling to-80 deg.C at 4 deg.C/min, storing at-80 deg.C for 4h, and transferring to liquid nitrogen for freezing.
The P3 generation adipose-derived mesenchymal stem cells obtained from the above culture experiment were resuspended in the protective solution of example 2 at a cell density of 5X 106Subpackaging the seeds/mL in a 2mL freezing tube, and carrying out programmed cooling to-80 ℃, wherein the programmed cooling process comprises the following steps: cooling to-80 deg.C at 4 deg.C/min, storing at-80 deg.C for 5h, and transferring to liquid nitrogen for freezing.
The P3 generation adipose-derived mesenchymal stem cells obtained from the culture experiment are taken to be re-suspended by the protective solution in the example 3, and the cell density is 107Subpackaging the seeds/mL in a 2mL freezing tube, and carrying out programmed cooling to-80 ℃, wherein the programmed cooling process comprises the following steps: cooling to-80 deg.C at 4 deg.C/min, storing at-80 deg.C for 6h, and transferring to liquid nitrogen for freezing.
Respectively sampling and detecting the survival rate of the cells before and after the cells are frozen for 3 months, wherein the detection method comprises the following steps: incubating and thawing the frozen tube in water bath at 37 deg.C, adding DMEM/F12 medium with the same volume, diluting, centrifuging, discarding supernatant, adding DMEM/F12 medium, resuspending, and adjusting cell density to 1 × 104one/mL. Then, 10. mu.L of the mixture was mixed with 10. mu.L of trypan blue, and the cell count was performed using a full-automatic cell counter for counting the cell survival rate. Cell viability is the number of viable cells/total number of cells x 100%. The results are shown in Table 1.
TABLE 1
As can be seen from table 1, the adipose-derived mesenchymal stem cells of examples 1 to 3 had high cell survival rate after being frozen for 3 months. The ingredients of the protective solution were adjusted in comparative examples 1 to 4, and the survival rate of adipose-derived mesenchymal stem cells was decreased to a different degree from that of example 1. The preservation solution can effectively improve the survival rate of the cells after cryopreservation and reduce the cell damage caused by cryopreservation.
Washing the frozen P3 generation cells for 3 months with PBS, centrifuging, adding culture medium (DMEM/F12+ 10% FBS), blowing to obtain cell suspension, and adjusting cell density to 1 × 104each/mL, inoculated in 12-well plates, incubated at 37 ℃ with 5% CO 23 wells per day were cultured in the incubator of (1), and the cells were counted by trypan blue staining, averaged, and the cell growth curve was plotted, as shown in FIG. 1.
It can be seen from FIG. 1 that the proliferation rate of the cells recovered in example 1 was high, the number of the cells collected after 7 days of culture was the largest, the proliferation rate of the cells in comparative examples 1 to 4 and the proliferation rate of the cells in example 1 were decreased to different degrees from each other, and the composition of the protective solution was adjusted in comparative examples 1 to 4. The proliferation activity of the adipose tissue-derived mesenchymal stem cells after the frozen storage by the protective solution is better.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.
Claims (8)
1. The cryopreservation protective solution for the adipose tissue-derived stem cells is characterized by comprising a basic culture medium and the following components added in the culture medium: glycerol, procaine microcapsule, bergapten, resveratrol, vitamin C, and insulin; the final concentration of each component in the culture medium is: 20-30 mu g/mL of glycerin, 1-5ng/mL of procaine microcapsule, 3-8ng/mL of bergapten, 5-10 mu g/mL of resveratrol, 1-2mg/mL of vitamin C and 0.5-1mg/mL of insulin.
2. The cryopreservation protection solution for the adipose-derived mesenchymal stem cells according to claim 1, wherein the final concentration of each component in the culture medium is as follows: 25 mu g/mL of glycerol, 3ng/mL of procaine microcapsules, 5ng/mL of bergapten, 8 mu g/mL of resveratrol, 1.5mg/mL of vitamin C and 0.7mg/mL of insulin.
3. The cryopreservation protection solution for adipose-derived mesenchymal stem cells according to claim 1, wherein the basic culture medium is DMEM/F12.
4. The cryopreservation protection solution for the adipose-derived mesenchymal stem cells according to claim 1 or 2, wherein the preparation method of the procaine microcapsule comprises the following steps: dissolving wall materials in absolute ethyl alcohol, wherein the mass concentration of the wall materials is 1-2%, adding core material procaine powder, uniformly mixing, and performing spray drying to obtain the procaine microcapsule.
5. The cryopreservation protection solution for the adipose-derived mesenchymal stem cells according to claim 4, wherein the wall material is sodium carboxymethyl cellulose, and the mass ratio of the sodium carboxymethyl cellulose to the procaine powder is 1: 0.2-0.3.
6. A cryopreservation method of adipose-derived mesenchymal stem cells, which is characterized in that the protective solution of any one of claims 1 to 5 is adopted for cryopreservation.
7. The cryopreservation method of adipose mesenchymal stem cells according to claim 6, comprising the following processes: and (3) resuspending the adipose-derived mesenchymal stem cells to be preserved by adopting a protective solution, carrying out programmed cooling to-80 ℃, preserving for 4-6h at-80 ℃, and then transferring to liquid nitrogen for freezing.
8. The cryopreservation method of adipose-derived mesenchymal stem cells according to claim 7, wherein the density of adipose-derived mesenchymal stem cells in the protective solution is 106-107one/mL.
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