CN111631212A - Low-temperature storage method of multifunctional 3D recombinant skin model - Google Patents
Low-temperature storage method of multifunctional 3D recombinant skin model Download PDFInfo
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Abstract
The invention discloses a low-temperature preservation method of a multifunctional 3D recombinant skin model, which is characterized in that cytochalasin B is applied to solid preservation of the skin model for the first time and is treated by a three-step method, the cell activity of the model in the transportation process can be maintained by the treatment method, the hypoxic injury of cells is reduced, the accuracy of 3D heavy skin model test and the cell activity are ensured, and the preservation time is prolonged to the greatest extent.
Description
Technical Field
The invention relates to a low-temperature storage method of a multifunctional 3D recombinant skin model, and belongs to the field of biomedicine.
Background
With the development of in vitro organ culture medium in vitro substitution technology, 3D recombinant skin models constructed based on 3D in vitro cell culture show a special trend in the aspects of safety and effectiveness evaluation of irritation tests. The 3D epidermis model constructed in vitro is similar to a human skin structure, and can be used for in vitro substitution tests of cosmetic detection; the 3D full-layer skin constructed in vitro is tissue engineering skin with living cells, has an epidermal layer and a dermal layer, can participate in the repair of wound surfaces and wounds, can be used for repairing skin defects caused by various reasons, accelerates the healing of skin wounds, and forms functional skin, so that the pain of a patient is relieved, and the life quality of the patient is improved.
However, due to the characteristics of the in vitro 3D recombinant skin model, the model is required to have stability and well maintain the original characteristics in the long-distance transportation process, so that the influence of chemical substances or other factors on the model can be tested and checked, and an accurate result can be obtained. The whole layer of skin is clinically used for overcoming the difficulty of remote transportation so as to ensure that the transported skin has certain activity and further ensure the effect of clinical application. At present, relevant reports on solid culture media suitable for cryopreservation of transportation skin models and tissue engineering skins exist (such as Chinese patent 201410418456.X, 201510697694.3, 201510697887.9), but at present, the solid preservation still has great limitations, and the preservation time is still a limiting factor.
Cytochalasin B has multiple biological functions, especially the influence on the structure of a microfilament, can be used as a cryoprotectant to increase the elasticity of a cytoskeleton, which is very important for low-temperature preservation, and at present, Chinese patent 201410418456.X adds cytochalasin B into a preservation solution, but the utilization of cytochalasin B in reported documents occurs in a liquid environment, and the difference of dosage addition and treatment time has great influence on cells or tissues. Due to the poor mobility of solids compared to liquid storage solutions, many molecules are restricted in their movement, resulting in a reduced mass exchange capacity, which limits the extension of the shelf life of solids to a large extent.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a low-temperature preservation method of a multifunctional 3D recombinant skin model, cytochalasin B is applied to solid preservation of the skin model for the first time, the cell activity of the model in the transportation process can be maintained by the processing method, the hypoxic injury of cells is reduced, the accuracy of 3D heavy skin model test and the cell activity are ensured, and the preservation time is prolonged to the greatest extent.
A cryopreservation method of a multifunctional 3D recombinant skin model comprises the following steps:
1) culturing the 3D recombinant skin model by using a cell culture technology;
2) replacing cell culture solution when the 3D recombinant skin model cultured in the step 1) is at the final stage of culture, adding cytochalasin B into the culture solution after the solution is replaced, wherein the final concentration of the added cytochalasin B in the culture solution is 0.1-5ug/ml, and treating at 37 ℃ for 1-24 h;
3) placing the 3D recombinant skin model treated in the step 2) in an improved liquid preservation solution, and precooling for 0.5-12 h at 4 ℃;
4) preparing an improved solid culture medium, adding the obtained solid culture medium to the outside of the 3D recombinant skin model treated in the step 3), solidifying, and then placing into a sterile packaging bag for sealing.
Further, the cell culture solution in the step 2) is a culture solution with a basic culture medium as a main body and added with growth factors;
the basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12.
The growth factor comprises hydrocortisone, adenine, ITT, L-glutamine, EOP (ethanolamine and catalytic phosphatidylethanolamine), human epidermal growth factor (hEGF), Bovine Pituitary Extract (BPE), selenium and fetal bovine serum.
Further, the final concentration of hydrocortisone in a basal medium is 0.1-2 ug/ml, the final concentration of adenine in the basal medium is 10-30 mM, the final concentration of ITT in the basal medium is 5.0-10.0 ug/ml, the concentration of L-glutamine is 30-60 mM, the final concentration of EOP in the basal medium is 1-10 mM, and human epidermal growth factor (hEGF) in the basal mediumThe final concentration of the selenium is 0.1 to 1ng/ml, and the final concentration of the selenium in a basic culture medium is 10-3And (3) about 0.1nM, the final concentration of the Bovine Pituitary Extract (BPE) in the basal medium is about 0.1-2 ug/ml, and the volume fraction of fetal calf serum in the basal medium is about 2-10%.
Further, the method for preparing the improved liquid storage solution in the step 3) includes the steps of:
s1 preparing a cell culture solution;
s2 adding cytochalasin B, trehalose, sucrose, glycine, alanine, adenosine, vitamin C, vitamin E, hyaluronic acid, chondroitin sulfate, deferoxamine, and fructose-1, 6-diphosphate to the cell culture solution obtained in step S1 to obtain an improved liquid storage solution.
Further, in the step S2, the final concentration of cytochalasin B in the liquid preservation solution is 1-2ug/ml, the final concentration of trehalose in the liquid preservation solution is 1-20 mM, the final concentration of sucrose in the liquid preservation solution is 50-100 mM, the final concentration of glycine in the liquid preservation solution is 1-20 mM, the final concentration of alanine in the liquid preservation solution is 1-10 mM, the final concentration of adenosine in the liquid preservation solution is 1-10 mM, the final concentration of vitamin E in the liquid preservation solution is 10-30 mM, the final concentration of vitamin C in the liquid preservation solution is 10-40 mM, the final concentration of deferoxamine in the liquid preservation solution is 1-10 mM, the final concentration of 1, 6-diphosphofructose in the liquid preservation solution is 1-15 mM, and the volume fraction of hyaluronic acid in the liquid preservation solution is 0.01-1 mM, The final concentration of the chondroitin sulfate in the liquid preservation solution is 0.5-2 g/L.
Further, the improved solid culture medium in the step 4) is formed by uniformly mixing an agarose solution and an improved liquid preservation solution according to the volume ratio of 1:1 and then solidifying the mixture.
Furthermore, the concentration of the agarose solution is 0.25% -5%, the agarose is low-melting-point agarose, the melting point is 60-70 ℃, and the solidification temperature is 28-32 ℃.
Has the advantages that:
(1) the method of the invention can reduce the damage of the cell skeleton caused by the adverse factors such as low temperature, high permeability and the like encountered by the recombinant model in the transportation process to the maximum extent, maintain the cell activity of the recombinant skin, and ensure the accuracy of the test result of the model and the good effect of clinical application.
(2) The method prolongs the storage time to more than 120h, greatly prolongs the storage time to ensure long-distance low-temperature transportation, and is more beneficial to the realization of tissue engineering application industrialization.
(2) The cytochalasin B can slow down the influence of temperature change on the deformation of cytoskeleton from proper low concentration to high concentration; the second step of the three-step method is a treatment, the model is preserved in liquid at 4 ℃ in advance, and the liquid preservation has stronger fluidity than the solid preservation, so that the capability of exchanging substances between various protective components in the preservation liquid and the skin model can be improved, the internal and external osmotic pressure of the skin when the temperature is reduced is maintained to the maximum degree, and the subsequent solid preservation is carried out.
(3) The method can be suitable for low-temperature transportation of more than 2 3D recombinant skin models, and can be used for storage of various 3D recombinant skin models (3D epidermis model, 3D full-thickness skin model and 3D dermis model).
(4) Before using the model, the model is put into a recovery culture medium to be cultured for 21 +/-3 h, so that the model can be used for detection, the use is convenient and quick, and the function of the recovered original skin model has no adverse effect.
Drawings
Figure 1 different application effects of solid and liquid preservation on skin models.
FIG. 2 the effect of different concentrations of cytochalasin B on the activity of dermal fibroblasts and epidermal cells of the skin.
Figure 33D epidermal model and 3D full-thickness skin model cryo-preserved resuscitation results are compared.
FIG. 4 shows the results of H & E staining of epidermal model under different conditions.
FIG. 5 full-thickness skin model grafting results under normal conditions.
FIG. 6 the full thickness skin model preserved the mice transplantation results after 120h resuscitation by the method of the present invention.
FIG. 7 preservation of the results of transplantation of mice after 120h resuscitation using the solid method of cytochalasin B directly.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.
Example 1 different applications of solid and liquid preservation to skin models
Firstly, experimental steps
1. Preparing liquid preserving liquid
(1) The basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12.
(2) The added factors are added into the basic culture solution according to the concentration, and the factor concentration is as follows: hydrocortisone at a concentration of 1ug/ml, adenine at 20nM, ITT at 10.0ug/ml, L-glutamine at 40mM, EOP (ethanolamine and catalytic phosphatidylethanolamine) at 10nM, human epidermal growth factor (hEGF) at 0.5ng/ml, Bovine Pituitary Extract (BPE) at 1ug/ml, selenium at 10ug/ml-2nM, fetal bovine serum volume fraction of 5%. After the preparation, a filter membrane of 0.22um is used for filtration and sterilization to obtain the sterilized culture solution.
(3) And (3) adding a protective agent into the degerming culture solution obtained in the step (2) according to the concentration and the sequence of the additives, and filtering and sterilizing after the protective agent is fully dissolved. The protectant concentrations were as follows: the concentration of cytochalasin was 2ug/ml, the concentration of trehalose was 10mM, the concentration of sucrose was 100mM, the concentration of glycine was 10mM, the concentration of alanine was 5mM, the concentration of adenosine was 5mM, the concentration of vitamin E was 20mM, the concentration of vitamin C was 30mM, the concentration of Deferoxamine (DFO) was 10mM, the concentration of fructose-1, 6-diphosphate (FDP) was 10mM, the volume fraction of hyaluronic acid was 0.01%, and the concentration of chondroitin sulfate was 1g/L, to obtain a liquid protective solution.
(4) And (4) putting the 3D full-thickness skin model into the liquid preservation solution obtained in the step (3), and preserving in a refrigerator at 4 ℃. Such as a (full skin fluid preservation) in fig. 1.
2. Preparing solid preserving liquid
The liquid storage solution was obtained as described in (1) to (4) in the above 1.
(5) And (4) adding 2.5% of agarose solution into the liquid storage culture solution obtained in the step (4) according to the volume ratio of 1:1, uniformly mixing, cooling at room temperature until the culture medium is solidified, and then carrying out solid storage on the skin model. As in B (epidermal model solid preservation) of fig. 1.
Second, experimental results
In FIG. 1, A is a graph showing the effect after storage in a liquid, and B is a graph showing the effect after storage in a solid. As shown in fig. 1B, solid storage is significantly more beneficial to transportation of the in vitro epidermis model than liquid storage, so that any other reagent on the surface of the epidermis model is prevented from contacting, and the accuracy of the detection result is ensured to the maximum extent. The skin model is not contacted with any liquid when used for reagent detection, otherwise adverse effects are caused, such as inaccurate detection results, if the preservation effect of A in FIG. 1 is used, the liquid is contacted with the surface skin (the skin model grows along the surface of the culture dish), and the problem can be effectively avoided by using solid preservation.
As shown in fig. 1B, since the in vitro epidermis model is suitable for detecting various influences of certain chemical substances on the skin, such as whether certain detection substances in cosmetic detection cause irritation and corrosion to the skin, and scientifically evaluating the degree of the irritation and corrosion to the skin in order to obtain accurate results, the model is required to maintain its original characteristics and have good stability during long-distance transportation. The liquid preservation has great limitation on the application of the model, and the detection result has great error, so the laboratory invents the solid preservation culture medium on the basis of the liquid preservation and adopts a three-step method for preservation.
Example 2 Effect of cytochalasin B concentration on skin cell Activity
Firstly, experimental steps
Step 1 cytochalasin B, at a concentration of 20mg/ml, was diluted to the following assay concentrations: 1mg/ml, 10ug/ml, 5ug/ml, 1ug/ml, 0.1 ug/ml.
And 2, digesting, centrifuging and counting dermal fibroblasts and epidermal cells in a good logarithmic phase state, and then respectively resuspending the dermal fibroblasts and the epidermal cells by using a dermal culture medium and an epidermal culture medium, wherein 60 ten thousand dermal cells and 80 ten thousand epidermal cells are inoculated into a 96-hole cell culture plate, and each hole volume is 100 ul.
Step 3 after the cells adhere to the wall, adding the cytochalasin B with different concentrations in the step 1, the volume is 100ul, each concentration is set to be three times, the dermis and the epidermis contrast groups are respectively added with the dermis culture medium and the epidermis culture medium with the volume of 100ul, and the mixture is placed at the temperature of 37 ℃ and 5 percent CO2Culturing in an incubator.
Step 4 cell viability was measured using the MTT method after 24h of culture.
Second, experimental results
As shown in FIG. 2, due to the side effects of cytochalasin B in different concentrations and different cells to different degrees, it was verified that the cells used in the present invention still maintain better activity at 0.1-5ug/ml when cultured for 24h, and dermal fibroblasts are more active than epidermal cells.
Example 33D epidermal model cryopreservation
First, experiment operation
1. Setting up an experimental group
The method comprises the steps of culturing a 3D epidermis model by using a cell culture technology, dividing the epidermis model after the culture is finished into a control group, an experiment group 1 and an experiment group 2 when the culture of the 3D epidermis model is finished, wherein the control group treatment method is that MTT detection is directly carried out without preservation treatment after the culture is finished, the experiment groups 1 and 2 are respectively directly carried out traditional solid preservation and solid preservation by adopting the three-step method of the invention, then, the epidermis model is revived, and the cell viability before and after the reviving is compared.
2. Treatment of Experimental group 1
(1) The basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12.
(2) The added factors are added into the basic culture solution according to the concentration, and the factor concentration is as follows: hydrocortisone at a concentration of 1ug/ml, adenine at 20nM, ITT at 10.0ug/ml, L-glutamine at 40mM, EOP (ethanolamine and catalytic phosphatidylethanolamine) at 10nM, human epidermal growth factor (hEGF) at 0.5ng/ml, Bovine Pituitary Extract (BPE) at 1ug/ml, selenium at 10ug/ml-2nM, fetal bovine serum volume fraction of 5%. Filtering with 0.22um filter membrane for sterilization after preparation to obtain sterilized culture solution, and storing for use.
(3) And (3) adding a protective agent into the degerming culture solution obtained in the step (2) according to the concentration and the sequence of the additives, and filtering and sterilizing after the protective agent is fully dissolved. The protectant concentrations were as follows: the liquid protective solution was obtained at a cytochalasin concentration of 2ug/ml, a trehalose concentration of 10mM, a sucrose concentration of 100mM, a glycine concentration of 10mM, an alanine concentration of 5mM, an adenosine concentration of 5mM, a vitamin E concentration of 20mM, a vitamin C concentration of 30mM, a Desferrioxamine (DFO) concentration of 10mM, a fructose-1, 6-diphosphate (FDP) concentration of 10mM, a hyaluronic acid volume fraction of 0.01%, and a chondroitin sulfate concentration of 1 g/L.
(4) And (3) adding 2.5% agarose solution into the liquid preservation culture solution obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing, cooling to room temperature until the culture medium is solidified, and performing solid preservation on the 3D epidermis model to obtain an experimental group 1.
3. Treatment of Experimental group 2
(1) The basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12.
(2) The added factors are added into the basic culture solution according to the concentration, and the factor concentration is as follows: hydrocortisone at a concentration of 1ug/ml, adenine at 20nM, ITT at 10.0ug/ml, L-glutamine at 40mM, EOP (ethanolamine and catalytic phosphatidylethanolamine) at 10nM, and human epidermal growth factor (hEGF) at a concentration of 10nMThe content is 0.5ng/ml, the concentration of Bovine Pituitary Extract (BPE) is 1ug/ml, and the concentration of selenium is 10-2nM, fetal bovine serum volume fraction of 5%. After the preparation, a filter membrane of 0.22um is used for filtration and sterilization to obtain the sterilized culture solution.
(3) And (3) adding a protective agent into the degerming culture solution obtained in the step (2) according to the concentration and the sequence of the additives, and filtering and sterilizing after the protective agent is fully dissolved. The protectant concentrations were as follows: the liquid protective solution was obtained at a cytochalasin concentration of 2ug/ml, a trehalose concentration of 10mM, a sucrose concentration of 100mM, a glycine concentration of 10mM, an alanine concentration of 5mM, an adenosine concentration of 5mM, a vitamin E concentration of 20mM, a vitamin C concentration of 30mM, a Desferrioxamine (DFO) concentration of 10mM, a fructose-1, 6-diphosphate (FDP) concentration of 10mM, a hyaluronic acid volume fraction of 0.01%, and a chondroitin sulfate concentration of 1 g/L.
(4) And (3) replacing the cell culture solution when the 3D epidermal model of the experimental group 2 is at the end stage of culture, adding cytochalasin B into the culture solution after the solution is replaced, wherein the final concentration of the added cytochalasin B in the culture solution is 0.1-5ug/ml, and treating for 2h at 37 ℃.
(5) And (4) placing the 3D epidermis model treated in the step (4) in the liquid protective solution obtained in the step (4), and precooling for 1h at 4 ℃.
(6) Preparing an improved solid culture medium (obtaining a liquid protective solution according to the steps (1) - (3), adding 2.5% of agarose solution into the liquid protective solution according to the volume ratio of 1:1 to obtain the improved solid culture medium), adding the obtained solid culture medium to the outside of the 3D epidermis model treated in the step (5), solidifying, and then placing into a sterile packaging bag for sealing.
4. Resuscitation detection
The 3D epidermal models of experimental group 1 and experimental group 2 were stored for 120 hours at refrigerated storage (4 ℃) and then examined for epidermal cell viability and histological structure in the epidermal models using the MTT method, respectively. MTT detection was performed directly without preservation treatment after the control group culture was completed.
Second, experimental results
A in figure 3 is a 3D epidermis model low-temperature preservation recovery result, group 1 in the figure is an experimental group 1, group 2 in the figure is an experimental group 2 of model cell viability pretreated by a three-step method by using cytochalasin B, and the result shows that the epidermis model pretreated by the three-step method has better effect than that of directly preserving solid after preservation and recovery steps, and particularly, the cell or cell rate after recovery is improved by at least 20 percent than that of the direct method.
In fig. 4, a is a staining result of an epidermis model H & E under a normal condition, in fig. 4, B is a staining result of the epidermis model H & E after an experimental group 2 is pretreated and stored for 120 hours by using the three-step method of the invention, and C is a staining result of the epidermis model H & E after the skin model of the experimental group 1 is recovered, and the results show that after the epidermis structure of the model is complete and a thicker cuticle is obviously visible after the epidermis model is pretreated and stored for 120 hours by using the three-step method, but the epidermis structure is loose and incomplete under a direct storage method.
EXAMPLE 43D full-thickness skin model cryopreservation
First, experiment operation
1. Setting up an experimental group
The method comprises the steps of culturing a 3D full-thickness skin model by using a cell culture technology, dividing the cultured 3D full-thickness skin model into a control group, an experiment group 1 and an experiment group 2 when the 3D full-thickness skin model is cultured, carrying out MTT detection directly by using a control group treatment method, namely, carrying out preservation treatment after the 3D full-thickness skin model is cultured, carrying out traditional solid preservation directly and carrying out solid preservation by using the three-step method of the invention respectively for the experiment group 1 and the experiment group 2, then recovering the skin model, and comparing the cell viability before and after recovery.
2. Treatment of Experimental group 1
(1) The basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12.
(2) The added factors are added into the basic culture solution according to the concentration, and the factor concentration is as follows: hydrocortisone at a concentration of 1ug/ml, adenine at 20nM, ITT at 10.0ug/ml, L-glutamine at 40mM, EOP (ethanolamine and catalytic phosphatidylethanolamine) at 10nM, human epidermal growth factor (hEGF) at 0.5ng/ml, Bovine Pituitary Extract (BPE) at 1ug/ml, selenium at 10ug/ml-2nM, volume fraction of fetal bovine serumThe number was 5%. Filtering with 0.22um filter membrane for sterilization after preparation to obtain sterilized culture solution, and storing for use.
(3) And (3) adding a protective agent into the degerming culture solution obtained in the step (2) according to the concentration and the sequence of the additives, and filtering and sterilizing after the protective agent is fully dissolved. The protectant concentrations were as follows: the concentration of cytochalasin was 2ug/ml, the concentration of trehalose was 10mM, the concentration of sucrose was 100mM, the concentration of glycine was 10mM, the concentration of alanine was 5mM, the concentration of adenosine was 5mM, the concentration of vitamin E was 20mM, the concentration of vitamin C was 30mM, the concentration of Deferoxamine (DFO) was 10mM, the concentration of fructose-1, 6-diphosphate (FDP) was 10mM, the volume fraction of hyaluronic acid was 0.01%, and the concentration of chondroitin sulfate was 1g/L, to obtain a liquid protective solution.
(4) And (3) adding 2.5% agarose solution into the liquid preservation culture solution obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing, cooling to room temperature until the culture medium is solidified, and performing solid preservation on the 3D full-thickness skin model of the experimental group 1. This was set as test group 1.
3. Treatment of Experimental group 2
(1) The basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12.
(2) The added factors are added into the basic culture solution according to the concentration, and the factor concentration is as follows: hydrocortisone at a concentration of 1ug/ml, adenine at 20nM, ITT at 10.0ug/ml, L-glutamine at 40mM, EOP (ethanolamine and catalytic phosphatidylethanolamine) at 10nM, human epidermal growth factor (hEGF) at 0.5ng/ml, Bovine Pituitary Extract (BPE) at 1ug/ml, selenium at 10ug/ml-2nM, fetal bovine serum volume fraction of 5%. After the preparation, a filter membrane of 0.22um is used for filtration and sterilization to obtain the sterilized culture solution.
(3) And (3) adding a protective agent into the degerming culture solution obtained in the step (2) according to the concentration and the sequence of the additives, and filtering and sterilizing after the protective agent is fully dissolved. The protectant concentrations were as follows: the concentration of cytochalasin was 2ug/ml, the concentration of trehalose was 10mM, the concentration of sucrose was 100mM, the concentration of glycine was 10mM, the concentration of alanine was 5mM, the concentration of adenosine was 5mM, the concentration of vitamin E was 20mM, the concentration of vitamin C was 30mM, the concentration of Deferoxamine (DFO) was 10mM, the concentration of fructose-1, 6-diphosphate (FDP) was 10mM, the volume fraction of hyaluronic acid was 0.01%, and the concentration of chondroitin sulfate was 1g/L, to obtain a liquid protective solution.
(4) The 3D full-thickness skin model of the experimental group 2 was changed at the end of the culture, cytochalasin B was added to the culture after the change, the final concentration of the cytochalasin B in the culture was 0.1-5ug/ml, and the treatment was carried out at 37 ℃ for 2 h.
(5) And (4) placing the 3D full-thickness skin model processed in the step (4) in the liquid protective solution obtained in the step (4), and precooling for 1h at 4 ℃.
(6) Preparing an improved solid culture medium (obtaining a liquid protective solution according to the steps (1) - (3), adding 2.5% of agarose solution into the liquid protective solution according to the volume ratio of 1:1 to obtain the improved solid culture medium), adding the obtained solid culture medium to the outside of the 3D full-thickness skin model treated in the step (5), solidifying, and placing into a sterile packaging bag for sealing.
4. Resuscitation detection
The 3D full-thickness skin models of experimental group 1 and experimental group 2 were stored for 120 hours at refrigerated storage (4 ℃) and then examined for epidermal cell viability and histological structure in the epidermal models using the MTT method, respectively. MTT detection was performed directly without preservation treatment after the control group culture was completed.
Second, experimental results
In the figure 3, B is a low-temperature preservation recovery result of the 3D full-thickness skin model, group 1 is a model cell survival rate obtained by adding cytochalasin B with the same concentration in the second step and directly performing solid preservation, and group 2 is a model cell survival rate obtained by using cytochalasin B to perform three-step pretreatment, and after comparison, the cell survival rate obtained after the three-step pretreatment model is recovered is 94.38% and the cell survival rate obtained after the three-step pretreatment model is recovered is 75.34% after the full-thickness skin model is preserved and recovered, which shows that the preservation survival rate of the model can be obviously improved by the three-step pretreatment of cytochalasin B.
EXAMPLE 53D Resuscitation transplantation after cryopreservation of full-thickness skin models
The 3D full-thickness skin model was stored according to the method 3 in example 3, and the 3D full-thickness skin model stored at low temperature (4 ℃) was refrigerated for 120 hours and then revived for 24 + -3 hours before the nude mouse transplantation experiment. The experiment used 3D full-thickness skin under normal conditions as a control. After 2 months after the 3D full-thickness skin was transplanted, the skin at the regeneration site was stained with paraffin sections H & E, and the histological structure of the formed skin was observed.
As shown in fig. 5 to 7, fig. 5 is a control group, i.e., a full-thickness skin model transplantation result under normal conditions, a in fig. 5 is an appearance of regenerated skin formed 2 months after transplantation, B in fig. 5 is a H & E staining result of skin formed 2 months after transplantation, scar of a mouse is well restored 2 months after normal transplantation, and HE staining shows that skin has a dermal layer, an epidermal layer and a subcutaneous tissue, has a differentiated stratum corneum, and has a compact and intact structure. Fig. 6 shows the results of transplantation of mice after 120H resuscitation with cytochalasin B pre-treatment, in which a in fig. 6 is the appearance of regenerated skin formed 2 months after transplantation, and B in fig. 6 is the result of H & E staining of skin formed 2 months after transplantation. Fig. 7 shows the results of mice transplantation after 120H resuscitation by a solid method directly added with cytochalasin B, A in fig. 7 is the appearance image of regenerated skin formed 2 months after transplantation, B in fig. 7 is the result of H & E staining of skin formed 2 months after transplantation, scar healing results show that the wound surface recovery of the transplanted skin surface is poorer by a direct solid preservation method compared with the wound surface recovery of the transplanted skin surface by a control and three-step pretreatment preservation method, HE staining shows that the skin structure is complete but the cuticle layer in the epidermis layer is thinner and is separated, and the dermis layer and the subcutaneous structure are scattered.
Claims (7)
1. A low-temperature preservation method of a multifunctional 3D recombinant skin model is characterized by comprising the following steps:
1) culturing the 3D recombinant skin model by using a cell culture technology;
2) replacing cell culture solution when the 3D recombinant skin model cultured in the step 1) is at the final stage of culture, adding cytochalasin B into the culture solution after the solution is replaced, wherein the final concentration of the added cytochalasin B in the culture solution is 0.1-5ug/ml, and treating at 37 ℃ for 1-24 h;
3) placing the 3D recombinant skin model treated in the step 2) in an improved liquid preservation solution, and precooling for 0.5-12 h at 4 ℃;
4) preparing an improved solid culture medium, adding the obtained solid culture medium to the outside of the 3D recombinant skin model treated in the step 3), solidifying, and then placing into a sterile packaging bag for sealing.
2. The preservation method according to claim 1, wherein the cell culture solution in step 2) is a culture solution prepared by adding growth factors to a basal medium;
the basal medium is DMEM, F12 or a mixed solution compounded by DMEM and F12, and the volume ratio of DMEM to F12 in the mixed solution compounded by DMEM and F12 is 4: 1-12;
the growth factor comprises hydrocortisone, adenine, ITT, L-glutamine, EOP, human epidermal growth factor, bovine pituitary extract, selenium and fetal calf serum.
3. The preservation method according to claim 2, wherein the final concentration of hydrocortisone in the basal medium is 0.1-2 ug/ml, the final concentration of adenine in the basal medium is 10-30 mM, the final concentration of ITT in the basal medium is 5.0-10.0 ug/ml, the concentration of L-glutamine is 30-60 mM, the final concentration of EOP in the basal medium is 1-10 mM, the final concentration of human epidermal growth factor in the basal medium is 0.1-1 ng/ml, and the final concentration of selenium in the basal medium is 10 ng/ml-3And the concentration of the bovine pituitary extract in a basal culture medium is 0.1-2 ug/ml, and the volume fraction of the fetal bovine serum in the basal culture medium is 2-10%.
4. The preservation method according to claim 1, wherein the preparation method of the improved liquid preservation solution according to step 3) comprises the steps of:
s1 preparing a cell culture solution;
s2 adding cytochalasin B, trehalose, sucrose, glycine, alanine, adenosine, vitamin C, vitamin E, hyaluronic acid, chondroitin sulfate, deferoxamine, and fructose-1, 6-diphosphate to the cell culture solution obtained in step S1 to obtain an improved liquid storage solution.
5. The preservation method according to claim 4, wherein in step S2, the cytochalasin B is present in the liquid preservation solution at a final concentration of 1-2ug/ml, the trehalose is present in the liquid preservation solution at a final concentration of 1-20 mM, the sucrose is present in the liquid preservation solution at a final concentration of 50-100 mM, the glycine is present in the liquid preservation solution at a final concentration of 1-20 mM, the alanine is present in the liquid preservation solution at a final concentration of 1-10 mM, the adenosine is present in the liquid preservation solution at a final concentration of 1-10 mM, the vitamin E is present in the liquid preservation solution at a final concentration of 10-30 mM, the vitamin C is present in the liquid preservation solution at a final concentration of 10-40 mM, the deferoxamine is present in the liquid preservation solution at a final concentration of 1-10 mM, the 1, 6-diphosphate is present in the liquid preservation solution at a final concentration of 1-15 mM, the fructose is present in the liquid preservation solution, The volume fraction of the hyaluronic acid in the liquid preservation solution is 0.01-1%, and the final concentration of the chondroitin sulfate in the liquid preservation solution is 0.5-2 g/L.
6. The preservation method according to claim 1, wherein the modified solid medium in step 4) is prepared by uniformly mixing the agarose solution and the modified liquid preservation solution in a volume ratio of 1:1 and then solidifying the mixture.
7. The preservation method according to claim 6, wherein the concentration of the agarose solution is 0.25 to 5%, the agarose is low-melting agarose, the melting point is 60 to 70 ℃, and the solidification temperature is 28 to 32 ℃.
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