CN105230608B - Preservation method of tissue engineering skin and tissue engineering skin - Google Patents
Preservation method of tissue engineering skin and tissue engineering skin Download PDFInfo
- Publication number
- CN105230608B CN105230608B CN201510697694.3A CN201510697694A CN105230608B CN 105230608 B CN105230608 B CN 105230608B CN 201510697694 A CN201510697694 A CN 201510697694A CN 105230608 B CN105230608 B CN 105230608B
- Authority
- CN
- China
- Prior art keywords
- tissue engineering
- engineering skin
- solution
- skin
- agar
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the field of tissue engineering skin preservation, in particular to a preservation method of tissue engineering skin and the tissue engineering skin. When the tissue engineering skin is stored, the tissue engineering skin is semi-embedded in the mixed solution of the agar solution and the basal culture solution, so that the storage time of the tissue engineering skin can be prolonged. The embodiment of the invention provides a preservation method of tissue engineering skin, which comprises the following steps of: step 1), placing the epidermis layer of the tissue engineering skin upwards in a culture dish; and 2) injecting a mixed solution of the basic culture solution and the agar solution into the culture dish, so that the dermis layer of the tissue engineering skin is wrapped in the mixed solution, and the epidermis layer is exposed.
Description
Technical Field
The invention relates to the field of tissue engineering skin preservation, in particular to a preservation method of tissue engineering skin and the tissue engineering skin.
Background
In recent years, with research and development of tissue engineering skin, tissue engineering skin is currently classified into single-layer skin and double-layer skin, and tissue engineering skin containing living cells and tissue engineering skin containing no living cells are classified according to whether or not living cells are contained in tissue engineering skin. Because living cells can participate in wound repair, when treating skin diseases, tissue engineering skin containing living cells can accelerate wound repair speed and relieve pain of patients, but harsh preservation conditions of tissue engineering skin containing living cells become main factors restricting application of tissue engineering skin containing living cells.
In the prior art, in order to further improve the preservation time of tissue engineering skin containing living cells, various preparation methods for preserving tissue engineering skin have been developed, wherein a low-temperature preservation method of a skin model is disclosed in a patent document with application number 201410418456.X, specifically: step 1) preparing a basal culture solution, wherein the basal culture solution comprises the following components: trehalose, sucrose, glycine, alanine, adenosine, tocopherol, vitamin C, deferoxamine, fructose-1, 6-bisphosphate, etc.; step 2) dissolving agarose with low melting point (gelling at 28-32 ℃ and melting at 62-68 ℃) with deionized water, and sterilizing for later use; step 3) mixing the basic culture solution and agarose solution in a ratio of 1:1, and preparing a solid culture medium after solidification; and 4) embedding the prepared tissue engineering skin and a culture vessel into the solid culture medium, and refrigerating and preserving. The tissue engineering skin obtained by the preparation method is completely encapsulated in the solid medium, so that the epidermis layer is easy to be necrotic due to the inability to contact air, thereby influencing the preservation time of the tissue engineering skin, and is easy to cause damage when the prepared tissue engineering skin is embedded in the solid medium together with a culture vessel.
Disclosure of Invention
The main object of the present invention is to provide a preservation method of tissue engineering skin and tissue engineering skin. When the tissue engineering skin is preserved, the tissue engineering skin is semi-embedded in the mixed solution of the agar solution and the basic culture solution, so that the cell activity of the tissue engineering skin can be maintained, and the preservation time of the tissue engineering skin can be prolonged.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in one aspect, an embodiment of the present invention provides a method for preserving tissue engineering skin having an epidermis layer and a dermis layer, comprising:
step 1), placing the epidermis layer of the tissue engineering skin upwards in a culture dish;
and 2) injecting a mixed solution of the basic culture solution and the agar solution into the culture dish, so that the dermis layer of the tissue engineering skin is wrapped in the mixed solution, and the epidermis layer is exposed.
Preferably, step 1) specifically comprises: placing tissue engineering skin in a container with a through hole at the bottom, and placing the container in the culture dish, wherein a gap is formed between the edge of the container and the side wall of the culture dish.
Optionally, step 2) specifically includes: and injecting a preset volume of mixed liquid into the gap along the edge of the culture dish.
Preferably, after the step 2), the method further comprises: standing to solidify the mixed solution, and storing at 2-8deg.C.
Further, the container with the through hole at the bottom is a leather bracket.
Optionally, the temperature of the basic culture solution is 4-8 ℃, and the temperature of the agar solution is 60-68 ℃.
Preferably, the concentration of the agar solution is 1.0-1.5%.
Further, the agar solution is sterilized under the condition of 5.0-10.0 MPa.
Optionally, the basal culture solution comprises: vitamin C with the mass concentration of 10-30mg/L, glutathione with the mass concentration of 0.2-1.0mg/L, L-glutamine with the mass concentration of 0.5-1.0g/L, tryptophan with the mass concentration of 0.4-1.0g/L and alpha-ketoglutarate with the mass concentration of 0.1-0.3 g/L.
Further, the basal culture solution further comprises: DMEM with a mass concentration of 14.4-15.0g/L, F12 with a mass concentration of 4.0-5.6g/L, fetal bovine serum with a volume concentration of 5% -15%, 4-hydroxyethyl piperazine ethane sulfonic acid with a molar concentration of 30-50 mM.
Preferably, the basal culture solution further comprises: sodium bicarbonate with mass concentration of 5.0-7.0g/L, basic fibroblast growth factor bFGF with mass concentration of 0.6-6.0ng/L, and insulin with mass concentration of 4-16 mug/L.
Further, the basal culture solution further comprises: hydrogenated cortisone with a mass concentration of 0.1-0.3mg/L, transferrin with a mass concentration of 6-20mg/L, adenine with a mass concentration of 20-40mg/L and EGF with a mass concentration of 0.1-1.0 ng/L.
In another aspect, an embodiment of the present invention provides a tissue engineering skin, where the tissue engineering skin is obtained by preservation according to the method described above.
The embodiment of the invention provides a preservation method of tissue engineering skin and the tissue engineering skin. The epidermis layer of the tissue engineering skin with the epidermis layer and the dermis layer is upwards placed in the culture dish, and the mixed solution of the agar solution and the basal culture solution is injected into the culture dish, so that the epidermis layer of the tissue engineering skin is exposed outside, the dermis layer is wrapped in the mixed solution, and when the tissue engineering skin is preserved, the epidermis layer of the tissue engineering skin can be directly contacted with air because the tissue engineering skin is semi-embedded in the mixed solution of the agar solution and the basal culture solution, thereby the necrosis of the epidermis layer can be avoided, the growth of the epidermis layer is promoted, and the dermis layer is preserved in the mixed solution of the agar solution and the basal culture solution, so that nutrient substances in the basal culture solution can be absorbed.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flowchart of a method for preserving tissue engineering skin according to an embodiment of the present invention;
FIG. 2 is a flowchart of another method for preserving tissue engineering skin according to an embodiment of the present invention;
FIG. 3 is a flowchart of another method for preserving tissue engineering skin according to an embodiment of the present invention;
fig. 4 is a flowchart of a method for preserving tissue engineering skin according to an embodiment of the present invention.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment. Thus, all other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are intended to be within the scope of the invention.
The materials to which embodiments of the present invention relate are commercially available or available to the applicant.
In one aspect, referring to fig. 1, a flowchart of a method for preserving tissue engineering skin having an epidermis layer and a dermis layer is provided for an embodiment of the present invention, including:
step 1), placing the epidermis layer of the tissue engineering skin upwards in a culture dish;
and 2) injecting a mixed solution of the basic culture solution and the agar solution into the culture dish, so that the dermis layer of the tissue engineering skin is wrapped in the mixed solution, and the epidermis layer is exposed.
The embodiment of the invention provides a preservation method of tissue engineering skin. The epidermis layer of the tissue engineering skin with the epidermis layer and the dermis layer is upwards placed in the culture dish, and the mixed solution of the agar solution and the basal culture solution is injected into the culture dish, so that the epidermis layer of the tissue engineering skin is exposed outside, the dermis layer is wrapped in the mixed solution, and when the tissue engineering skin is preserved, the epidermis layer of the tissue engineering skin can be directly contacted with air because the tissue engineering skin is semi-embedded in the mixed solution of the agar solution and the basal culture solution, thereby the necrosis of the epidermis layer can be avoided, the growth of the epidermis layer is promoted, and the dermis layer is preserved in the mixed solution of the agar solution and the basal culture solution, so that nutrient substances in the basal culture solution can be absorbed.
The basic culture solution is not limited to be obtained, and the basic culture solution can be obtained through commercial paths or self-made.
In addition, when the basal culture solution is prepared by self, in order to avoid insufficient dissolution and ensure the quality of the final product, the basal culture solution needs to be filtered to obtain a clear solution, and in order to obtain sterile tissue engineering skin during operation, the preparation method is convenient for later use, and preferably, the prepared basal culture solution is subjected to sterile filtration in step 1).
The agar solution is not limited, and agar is melted in water after being heated to 95 ℃ and solidified after being cooled to 30 ℃, so that the agar solution can be obtained by heating agar in water to 95 ℃ by utilizing the characteristic of agar.
Wherein the concentration of the agar solution is not limited, and preferably the concentration of the agar solution is 1.0 to 1.5%.
In order to increase the preservation time of the tissue engineering skin, the tissue engineering skin needs to be strictly sterilized when preserved, and the agar solution may be sterilized when the agar solution is used for preparing a solid medium. Wherein the sterilization conditions of the agar solution are not limited, and preferably, the agar solution is sterilized under the conditions of 5.0 to 10.0 MPa.
In one embodiment of the present invention, the temperature of the basal culture solution is 4-8deg.C, and the temperature of the agar solution is 60-68deg.C. Since the basic culture solution contains a plurality of active ingredients, when the basic culture solution is mixed with the agar solution, the active ingredients in the basic culture solution are deactivated if the temperature of the agar solution is too high, and when the agar solution is mixed with the basic culture solution, the agar solution is coagulated due to the fact that the temperature is lower than 30 ℃, so that the subsequent operation is not facilitated.
The specific method of the semi-embedding of the tissue engineering skin is not limited, and the dermis layer of the tissue engineering skin may be enclosed after the mixed solution of the basal culture solution and the agar solution is injected into the culture dish, and the epidermis layer may be exposed.
In a preferred embodiment of the present invention, step 1) specifically comprises: placing tissue engineering skin in a container with a through hole at the bottom, and placing the container in the culture dish, wherein a gap is formed between the edge of the container and the side wall of the culture dish. The process is simple and convenient, and simultaneously, when the tissue engineering skin is placed in the culture dish, the tissue engineering skin can be prevented from being damaged by clamping by forceps, and the tissue engineering skin is polluted by other modes (for example, the tissue engineering skin is manually clamped and placed), further, the bottom of the container is provided with a through hole, after the mixed solution is injected into the culture dish, the mixed solution wraps the dermis layer of the tissue engineering skin through the through hole at the bottom of the container, and the dermis layer of the tissue engineering skin can be fully contacted with the mixed solution, so that the preservation time of the tissue engineering skin is further prolonged.
In order to avoid that the mixed solution is injected onto the epidermis layer of the tissue engineering skin during the operation when the mixed solution is injected into the culture dish, it is preferable that the step 2) specifically comprises: and injecting a preset volume of mixed liquid into the gap along the edge of the culture dish.
The preset volume is not limited, and can be flexibly determined according to the volume of the culture dish and the volume of the container for placing the tissue engineering skin.
Specifically, the preset volume of the mixed solution is determined according to the size of the gap.
The preset volume of the mixed solution may be determined by visually inspecting the size of the gap when the mixed solution is injected into the gap, or may be determined by calculating the size of the gap in advance, and is not limited herein.
The container with the through hole at the bottom is not limited, and preferably the container with the through hole at the bottom is a leather bracket. When the tissue engineering skin is prepared, the skin support is used as a mould, so that the tissue engineering skin is more similar to natural skin, and after the tissue engineering skin is prepared, the prepared tissue engineering skin is not required to be transferred, and the skin support and the tissue engineering skin are directly placed into the culture dish together, so that pollution and even damage to the tissue engineering skin can be avoided.
In an embodiment of the present invention, after the step 2), the method further includes: standing, solidifying the mixed solution, and preserving at 2-8deg.C. Because the agar solution and the basic culture solution are mixed and then the agar solution starts to solidify when the temperature is lower than 30 ℃, a solid culture medium is obtained after the agar solution and the basic culture solution are solidified, the dermis layer of the tissue engineering skin is semi-embedded in the solid culture medium, and when the tissue engineering skin is preserved at the temperature of 2-8 ℃, compared with a normal-temperature preservation method, the metabolism rate of living cells can be relatively slowed down, the preservation time can be prolonged, compared with a freezing preservation method, the cost is relatively low, the transportation is convenient, and the cell viability in the tissue engineering skin can be maintained.
Wherein, the components of the basic culture solution are not limited, and in a preferred embodiment of the present invention, the basic culture solution comprises: vitamin C with the mass concentration of 10-30mg/L, glutathione with the mass concentration of 0.2-1.0mg/L, L-glutamine with the mass concentration of 0.5-1.0g/L, tryptophan with the mass concentration of 0.4-1.0g/L and alpha-ketoglutarate with the mass concentration of 0.1-0.3 g/L. The vitamin C has the functions of resisting oxidization and eliminating free radicals, can protect tissue engineering skin from being damaged at low temperature when being used for preserving tissue engineering skin, eliminates free radicals generated by the tissue engineering skin in the preservation process, the glutathione has the function of protecting the tissue engineering skin from being damaged at low temperature, the degradation products of the glutamine provide nutrient substances for the tissue engineering skin, the tryptophan can prevent the tissue engineering skin from being damaged due to hypoxia at low temperature, and the alpha-ketoglutarate can protect the tissue engineering skin from being damaged at low temperature.
Further, the basal culture solution further comprises: DMEM with a mass concentration of 14.4-15.0g/L, F12 with a mass concentration of 4.0-5.6g/L, fetal bovine serum with a volume concentration of 5% -15%, 4-hydroxyethyl piperazine ethane sulfonic acid with a molar concentration of 30-50 mM. Wherein, the DMEM and F12 in the basic culture solution can be obtained commercially, the DMEM is a culture medium containing various amino acids and glucose, and F12 contains rich components, and the two culture media can provide various nutritional components for the growth of fibroblasts and epidermal cells; the 4-hydroxyethyl piperazine ethane sulfonic acid is a biological buffer agent, has relatively strong buffer capacity, and can keep the pH value of the basic culture solution in a constant range for a long time.
The forms of the DMEM and the F12 are not limited, the DMEM and the F12 may be powder or solution, and when the DMEM and the F12 are liquid, the addition amount of the DMEM and the F12 may be calculated according to the concentrations of the DMEM and the F12; when the DMEM and F12 are powders, the formulation may be performed according to the concentration of the DMEM and F12.
Further preferably, the basal culture solution further comprises: sodium bicarbonate with mass concentration of 5.0-7.0g/L, basic fibroblast growth factor bFGF with mass concentration of 0.6-6.0ng/L, and insulin with mass concentration of 4-16 mug/L. The sodium bicarbonate has a buffer effect, can maintain the constant pH value of the tissue engineering skin in the preservation process, the alkaline fibroblast growth factor has the effect of promoting the growth of dermal cells in the tissue engineering skin, and the insulin can accelerate the glucose utilization rate of the tissue engineering skin.
Still further, the basal culture solution further comprises: hydrogenated cortisone with a mass concentration of 0.1-0.3mg/L, transferrin with a mass concentration of 6-20mg/L, adenine with a mass concentration of 20-40mg/L and EGF with a mass concentration of 0.1-1.0 ng/L. The hydrogenated cortisone has the function of keeping the osmotic pressure of tissue engineering skin within a normal range, so that solute damage of the tissue engineering skin caused by high osmotic pressure in cells is avoided, and the transferrin has the function of repairing the cells and reduces the necrosis proportion of the cells; the adenine is a DNA composition and has the function of providing energy for tissue engineering skin, and the epidermal growth factor has the function of promoting the growth of epidermal cells.
In another aspect, an embodiment of the present invention provides a tissue engineering skin, where the tissue engineering skin is obtained by preservation according to the method described above.
The embodiment of the invention provides tissue engineering skin. The epidermis layer of the tissue engineering skin is exposed outside, the dermis layer is wrapped in the mixed solution, and the epidermis layer of the tissue engineering skin can be in direct contact with air, so that the necrosis of the epidermis layer can be avoided, the growth of the epidermis layer is promoted, and the dermis layer is stored in the mixed solution of the agar solution and the basal culture solution, so that nutrient substances in the basal culture solution can be absorbed, therefore, the tissue engineering skin stored by the method can keep the cell activities of the dermis layer and the epidermis layer in the tissue engineering skin, and the storage time of the tissue engineering skin is prolonged.
Detailed Description
The present invention will be described in detail below with reference to examples, comparative examples and test examples of the present invention. These examples are given solely for the purpose of illustrating the invention, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these examples, comparative examples and test examples.
Control example:
for convenience, the tissue engineering skin obtained in the control example was designated as G.
In the control example, the tissue engineering skin G was stored in cold storage at 2 ℃ for 72 hours.
Example 1
For convenience, the tissue engineering skin obtained in example 1 was designated as a, and the basal culture solution used in example 1 was designated as a formulation.
The formula A is prepared:
the method comprises the following steps: adding sterile ultrapure water into DMEM powder, adding a factor combination, dissolving to a certain volume, fully dissolving to obtain 500ml of basic culture solution A, filtering with a 0.22-micrometer filter membrane, and packaging to obtain the formula A. Wherein, the formula A is shown in the table 1.
TABLE 1
Referring to fig. 2, the preparation of a is specifically:
step 1), placing the prepared formula A in a refrigerator at 4 ℃ for heat preservation;
step 2) preparing 100ml of agar solution with the concentration of 1.0%, sterilizing under the condition of 5.0MPa, and preserving heat in a water bath kettle at 60 ℃;
step 3) placing the tissue engineering skin together with the pitot into a culture dish with the height of 7 cm;
step 4) evenly mixing 50ml of the formula A prepared in the step 1) with 50ml of the agar solution prepared in the step 2), pumping 30ml of the mixed solution along the edge of the culture dish, injecting the mixed solution into the culture dish in the step 3), standing, solidifying and refrigerating at 2 ℃.
Example 2
For convenience, the tissue engineering skin obtained in example 2 was designated as B, and the basal culture solution used in example 2 was designated as B formulation.
And (3) preparing a formula B:
the method comprises the following steps: adding sterile ultrapure water into DMEM powder, adding factor combination, dissolving to a certain volume, fully dissolving to obtain 500ml of basic culture solution B, filtering with a 0.22-micrometer filter membrane, and packaging to obtain the formula B. Wherein, the formula B is shown in Table 2.
TABLE 2
| Name of the name | Content of |
| DMEM | 15.0g/L |
| F12 | 5.6g/L |
| Fetal bovine serum | 15％ |
| 4-hydroxyethyl piperazine ethanesulfonic acid | 50mM |
| Vitamin C | 30mg/L |
| Glutathione | 1.0mg/L |
| L-glutamine | 1.0g/L |
| Tryptophan | 1.0g/L |
| Alpha-ketoglutaric acid | 0.3g/L |
Referring to fig. 3, the preparation of b is specifically:
step 1), placing the prepared formula B in a refrigerator at the temperature of 6 ℃ for heat preservation;
step 2) preparing 100ml of agar solution with the concentration of 1.5%, sterilizing under the condition of 10.0MPa, and preserving heat in a water bath kettle at 65 ℃;
step 3) placing the tissue engineering skin together with the pitot into a culture dish with the height of 7 cm;
step 4) evenly mixing 50ml of the formula B prepared in the step 1) with 75ml of the agar solution prepared in the step 2), pumping 30ml of the mixed solution along the edge of the culture dish, injecting the mixed solution into the culture dish in the step 3), standing, solidifying and refrigerating at 5 ℃.
Example 3
For convenience, the tissue engineering skin obtained in example 3 was designated as C, and the basal culture solution used in example 3 was designated as C formulation.
And C, preparing a formula:
the method comprises the following steps: adding sterile ultrapure water into DMEM powder, adding factor combination, dissolving to a certain volume, fully dissolving to obtain 500ml of basic culture solution C, filtering with a 0.22-micrometer filter membrane, and packaging to obtain the formula C. Wherein, the formula C is shown in Table 3.
TABLE 3 Table 3
Referring to fig. 4, the preparation of c is specifically:
step 1), placing the prepared formula C in a refrigerator at 8 ℃ for heat preservation;
step 2) preparing 100ml of agar solution with the concentration of 1.4%, sterilizing under the condition of 8.0MPa, and preserving heat in a water bath kettle at 68 ℃;
step 3) placing the tissue engineering skin together with the pitot into a culture dish with the height of 7 cm;
step 4) evenly mixing 50ml of the formula C prepared in the step 1) with 60ml of the agar solution prepared in the step 2), pumping 30ml of the mixed solution along the edge of the culture dish, injecting the mixed solution into the culture dish in the step 3), standing, solidifying and refrigerating at 8 ℃.
Example 4
For convenience, the tissue engineering skin obtained in example 4 was designated as D, and the basal medium used in example 1 was designated as D formulation.
And D, preparing a formula:
the method comprises the following steps: adding sterile ultrapure water into DMEM powder, adding factor combination, dissolving to a certain volume, fully dissolving to obtain 500ml of basic culture solution D, filtering with a 0.22-micrometer filter membrane, and packaging to obtain the formula D. Wherein the formula D is shown in Table 4.
TABLE 4 Table 4
| Name of the name | Content of |
| DMEM | 14.4g/L |
| F12 | 4.0g/L |
| Fetal bovine serum | 5％ |
| 4-hydroxyethyl piperazine ethanesulfonic acid | 30mM |
| Vitamin C | 25mg/L |
| Glutathione | 0.4mg/L |
| Hydrogenated cortisone | 0.2mg/L |
| Transferrin | 10mg/L |
| Adenine (A) | 30mg/L |
| EpidermisGrowth factor EGF | 0.5ng/L |
| L-glutamine | 0.6g/L |
| Tryptophan | 0.5g/L |
| Alpha-ketoglutaric acid | 0.2g/L |
Preparation of D:
the preparation method of D is basically the same as that of A, except that the basic culture solution adopted is the formula D, and the details are not repeated here.
Example 5
For convenience, the tissue engineering skin obtained in example 5 was designated as E, and the basal medium used in example 5 was designated as E formulation.
And E, preparing a formula:
the method comprises the following steps: adding sterile ultrapure water into DMEM powder, adding factor combination, dissolving to a certain volume, fully dissolving to obtain 500ml of basic culture solution E, filtering with a 0.22-micrometer filter membrane, and packaging to obtain the formula E. Wherein, E formula is shown in Table 5.
TABLE 5
E, preparation:
the preparation method of E is basically the same as that of B, except that the basic culture solution adopted is the formula of E, and the details are not repeated here.
Example 6
For convenience, the tissue engineering skin obtained in example 6 was designated as F, and the basal culture solution used in example 5 was designated as F formulation.
And F, preparing a formula:
the method comprises the following steps: adding sterile ultrapure water into DMEM powder, adding a factor combination, dissolving to a certain volume, fully dissolving to obtain 500ml of basic culture solution F, filtering with a 0.22-micrometer filter membrane, and packaging to obtain the formula F. Wherein, the formula F is shown in Table 6.
TABLE 6
F, preparation:
the preparation method of F is basically the same as that of C, except that the basic culture solution adopted is the formula F, and the details are not repeated here.
Experimental example:
in order to objectively evaluate the cryopreservation effect of the tissue engineering skins A to G, the tissue engineering skins A to G were stored for a certain period of time, respectively, and then the cell viability in the tissue engineering skins was examined by MTT method.
Among them, the MTT method is also called MTT colorimetric method, and is a method for detecting cell survival and growth.
1. Test sample
The tissue engineered skins A-F in examples 1-6 and the tissue engineered skin G in the control.
2. The test analysis method comprises the following steps:
the tissue engineering skin A and the tissue engineering skin D are refrigerated for 15 days, the tissue engineering skin B and the tissue engineering skin E are refrigerated for 20 days, the tissue engineering skin C and the tissue engineering skin F are refrigerated for 25 days, and the MTT method is respectively carried out after the tissue engineering skin is refrigerated for 72 hours in the control example to detect the cell viability in the tissue engineering skin.
The detection principle is as follows: succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water insoluble blue-violet crystalline Formazan (Formazan) and deposits in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value can be measured at 490 or 560nm wavelength by an enzyme-linked immunosorbent assay, so that the number of living cells can be indirectly reflected. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers.
The method comprises the following steps: the prepared tissue engineering skin is measured to have the light absorption value of 1.0676 at the wavelength of 490nm by an MTT method before cold storage and preservation, the tissue engineering skin A-C is resuscitated, a part of the tissue engineering skin A-G is cut and taken as an experimental sample, a certain amount of MTT solution is added, blue-purple skin slices are dissolved by DMSO, the light absorption value of the tissue engineering skin is measured at the wavelength of 490nm by an ELISA detector, the light absorption values of A-F are respectively obtained and are respectively 0.9381, 0.7253, 0.6294, 1.0520, 0.8634 and 0.4753, the cell vitality of the tissue engineering skin A-F is respectively 87.8%,67.9%,58.9%,98.5%,80.8% and 44.5%, and the tissue engineering skin G is measured to have almost no living cells.
3. Experimental results:
the cell activities of the tissue engineering skin A and the tissue engineering skin D after being preserved for 15 days are 87.8% and 98.5%, the cell activities of the tissue engineering skin B and the tissue engineering skin E after being preserved for 20 days are 67.9% and 80.8%, and the cell activities of the tissue engineering skin C and the tissue engineering skin F after being preserved for 25 days are 58.9% and 44.5%, respectively, therefore, the average cell activity of the tissue engineering skin obtained by the preservation method after being preserved for 15 days is 87.8-98.5%, the average cell activity after being preserved for 20 days is about 67.9-80.8%, and the average cell activity after being preserved for 25 days is about 44.5-58.9%, and the tissue engineering skin G in the control example can be preserved for 72 hours without living cells.
From the above, the epidermis layer of the tissue engineering skin is exposed, the dermis layer is wrapped in the mixed solution, and the epidermis layer of the tissue engineering skin can be in direct contact with air, so that necrosis of the epidermis layer can be avoided, growth of the epidermis layer is promoted, and the dermis layer is stored in the mixed solution of the agar solution and the basal culture solution, so that nutrient substances in the basal culture solution can be absorbed, and therefore, the tissue engineering skin stored by the method can keep cell viability of the dermis layer and the epidermis layer in the tissue engineering skin. When the tissue engineering skin is refrigerated and stored, compared with the prior art, the preservation time of the tissue engineering skin can be greatly prolonged.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. A method of preserving tissue engineering skin having an epidermis layer and a dermis layer, comprising:
step 1) placing an epidermis layer of tissue engineering skin upwards in a container with a through hole at the bottom, and placing the container in a culture dish, wherein a gap is formed between the edge of the container and the side wall of the culture dish;
step 2) injecting a mixed solution of a basic culture solution and an agar solution into the culture dish, so that the dermis layer of the tissue engineering skin is wrapped in the mixed solution, and the epidermis layer is exposed; standing to solidify the mixed solution, and storing at 2-8deg.C.
2. The preservation method according to claim 1, wherein step 2) specifically comprises: and injecting a preset volume of mixed liquid into the gap along the edge of the culture dish.
3. The preservation method of claim 1 wherein the container having a through-hole in the bottom is a receptacle.
4. The method according to claim 1, wherein the temperature of the basal culture solution is 4-8deg.C and the temperature of the agar solution is 60-68deg.C.
5. The method of claim 1, wherein the concentration of the agar solution is 1.0-1.5%.
6. The preservation method according to claim 1, wherein the agar solution is sterilized under the condition of 5.0 to 10.0 MPa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510697694.3A CN105230608B (en) | 2015-10-23 | 2015-10-23 | Preservation method of tissue engineering skin and tissue engineering skin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510697694.3A CN105230608B (en) | 2015-10-23 | 2015-10-23 | Preservation method of tissue engineering skin and tissue engineering skin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105230608A CN105230608A (en) | 2016-01-13 |
| CN105230608B true CN105230608B (en) | 2023-05-09 |
Family
ID=55028678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510697694.3A Active CN105230608B (en) | 2015-10-23 | 2015-10-23 | Preservation method of tissue engineering skin and tissue engineering skin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105230608B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201708729D0 (en) * | 2017-06-01 | 2017-07-19 | Ucl Business Plc | Cryopreservation |
| CN113545337B (en) * | 2021-07-21 | 2022-08-26 | 益诺思生物技术南通有限公司 | Method for sizing a mammalian heart valve |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104222068A (en) * | 2014-08-22 | 2014-12-24 | 陕西博鸿生物科技有限公司 | Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103721294B (en) * | 2013-12-27 | 2015-05-13 | 太原理工大学 | Quick construction preparation method of human epidermal tissues |
-
2015
- 2015-10-23 CN CN201510697694.3A patent/CN105230608B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104222068A (en) * | 2014-08-22 | 2014-12-24 | 陕西博鸿生物科技有限公司 | Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium |
Non-Patent Citations (1)
| Title |
|---|
| "体外皮肤模拟物的研究进展";陆毅等;《国外医学皮肤性病学分册》;20031231;第29卷(第1期);第30-33页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105230608A (en) | 2016-01-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104145943B (en) | A kind of frozen protection liquid of people's umbilical cord China Tong Shi glue tissue and preparation and application thereof | |
| CN101720753B (en) | Cryopreservation solution of tissue engineering products and application method thereof | |
| CN104222068B (en) | Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium | |
| AR080221A1 (en) | IMMUNOCOMPATIBLE AMNIOTIC MEMBRANE PRODUCTS | |
| CN105230608B (en) | Preservation method of tissue engineering skin and tissue engineering skin | |
| CN111011363B (en) | Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and recovery method | |
| KR102328832B1 (en) | Composition for cryopreservation of bovine reproductive cells and cryopreservation method therefor | |
| CN103548814A (en) | Non-programmable-controlled cooling cryopreservation method and protection agent for hematopoietic stem cells at minus 80DEG C | |
| CN109673623A (en) | A kind of glass freezing reagent and vitrifying defrosting reagent and its application and application method | |
| CN103478118A (en) | Cryopreservation method for tissue block used for cell culture | |
| JP2001247401A (en) | Cooling preservation liquid for tissue | |
| CN107027741B (en) | Human sperm freezing protection solution without yolk and preservation method | |
| JP5156747B2 (en) | Fish processing method | |
| CN105219700A (en) | A kind of gel and its preparation method and application | |
| CN101536707B (en) | Method for prolonging cooled meat shelf period by combination of chilling and high concentration carbon dioxide modified atmospheric packaging | |
| CN105532641A (en) | Placenta preserving fluid and use method thereof | |
| CN115152743A (en) | Preparation method of human umbilical cord mesenchymal stem cell serum-free frozen stock solution | |
| US20150080300A1 (en) | Growth factor concentrate and the use thereof | |
| CN102246744A (en) | Giant panda semen freezing and preserving fluid and preparation method thereof | |
| CN109122666A (en) | A kind of recovery instant red blood cell cryoprotector and application method | |
| CN107164306A (en) | Cornea model Cord blood solid medium and preparation method and application method | |
| CN111972397A (en) | Skin cold storage preservation solution and application thereof | |
| CN110896946A (en) | Hematopoietic stem cell cryopreservation liquid and hematopoietic stem cell cryopreservation method | |
| EP1922923A2 (en) | Methods and compositions for cryopreserving oocytes | |
| KR20160001454A (en) | Composition for cryopreservating sperm of ankole cow and uses thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |