CN113249299A - Preparation method of double-layer skin containing microvascular lumen - Google Patents
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Abstract
The double-layer skin containing the microvascular lumen prepared by the invention has two layers of skin structures, namely a epidermis layer and a dermis layer containing the microvascular lumen from top to bottom; the epidermal layer is composed of epidermal cell germinal multiple layers, the dermal layer containing the microvascular lumen is constructed by fibroblasts, umbilical cord mesenchymal stem cells and umbilical vein endothelial cells, the fibroblasts, the umbilical cord mesenchymal stem cells and the umbilical vein endothelial cells are mixed according to a certain proportion, a multi-inoculation method is used, and the extracellular matrix secretion capacity and a paracrine system of the cells are utilized, so that on one hand, the histological structure of the skin dermis is formed, on the other hand, the proliferation, migration, lumen formation and stability of the endothelial cells are promoted. The tissue construction method simulates the assembly of normal human skin and achieves the formation of a dermis layer and the formation of a micro-lumen in the dermis through the interaction and the mutual influence among cells, thereby avoiding the instability of structure and function and the fussy operation caused by artificially adding an exogenous stent and a angiogenesis factor.
Description
The divisional application is based on application number 201510619910.2, application date is 2015, 9, 25 and the name of the invention is 'a double-layer skin containing a microvascular lumen and a preparation method thereof'.
Technical Field
The invention belongs to the technical field of tissue engineering in biomedical engineering, and mainly relates to a preparation method of double-layer skin containing a microvascular lumen.
Background
The requirements of normal skin cells on oxygen and nutrition and the removal of metabolic waste are realized by the blood diffusion and transportation function near the cells, but the current double-layer skin constructed based on fibroblast composite scaffold materials and epidermal cells cannot meet the requirements of long-term survival and functionality due to lack of a vascular structure, so that the dysfunction of the fibroblasts is caused, the migration capability and the proliferation capability of keratinocytes are reduced, and finally the function of skin tissues is limited; the constructed double-layer skin is transplanted to a wound surface, and the acquisition of the nutrient components of the living cells in the double-layer skin and the excretion of waste are mainly completed by seepage of a wound surface substrate, so that the material exchange speed of the cells is low, and the survival of the living cells in the double-layer skin and the realization of the functions of the living cells are seriously influenced. Besides improving the survival time and function of the skin, the double-layer skin with the differentiated structure can be used for basic scientific research of vascular mechanism and screening drugs with inhibiting or promoting effect on angiogenesis.
Therefore, how to construct a double-layered skin containing blood vessels and accelerate vascularization of an artificial dermis substitute have attracted attention of a great number of researchers. Most of the existing skin in the vascularization engineering is constructed by taking extracellular matrix (ECM) substitutes as a scaffold, for example, 5 months in 2004, and related patents (Chinese patent application No. 03134540.9) are applied to Jinshi et al. Although constituting the external microenvironment for cell growth, the extracellular matrix substitutes bring many exogenous substances, such as bovine collagen, allogeneic dermis, artificial polymers, etc., which have certain defects, namely, immunogenicity of different exogenous substances, degradation rate, toxicity of degradation products, host immune response, tissue fibrosis caused by scaffold degradation, and mechanical mismatch with surrounding tissues, which may become key factors affecting long-term bioactivity and major biological functions of the skin structure. And the extracellular matrix substitutes are not secreted by cells, and exogenous addition is not beneficial to the conduction of intercellular signals, so that the structural and functional performance of the constructed skin is influenced.
In addition, the current double-layer skin model containing the microvessels is mainly constructed by adding endothelial cells into a dermis layer mainly containing fibroblasts, and simultaneously adding a pro-angiogenic factor to promote angiogenesis in the skin. Commonly used additional factors are: vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), Platelet Derived Growth Factor (PDGF), etc., which often require repeated implementation or strict control of the release system, such as the concentration of the added factors, the ratio of different added factors, the action time of each factor, etc., all of which inevitably cause complexity and instability of the whole construction process, limiting the application thereof to a certain extent. Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a double-layer skin containing a microvascular lumen and a preparation method thereof. The tissue construction method simulates the assembly of normal human skin and achieves the formation of a dermis layer and the formation of a micro-lumen in the dermis through the interaction and the mutual influence among cells, thereby avoiding the instability of structure and function and the fussy operation caused by artificially adding an exogenous stent and a angiogenesis factor.
The double-layer skin containing the microvascular lumen prepared by the invention has two layers of skin structures, namely a epidermis layer and a dermis layer containing the microvascular lumen from top to bottom; the epidermal layer consists of epidermal cell germinal multiple layers; the dermis containing the microvascular lumen is constructed by fibroblasts, umbilical cord mesenchymal stem cells and umbilical vein endothelial cells.
The preparation method of the double-layer skin containing the microvascular lumen provided by the invention comprises the following steps:
step one, extracting fibroblasts and epidermal cells:
soaking the skin tissue block in 75% ethanol solution, and then transferring into PBS solution for soaking and washing until no blood stain exists; adding Dispase digestive juice into the tissue block, and placing the tissue block at the temperature of 4 ℃ for 16-17 h; adding collagenase, incubating at 37 ℃ for about 1-3 h, centrifuging at 1000r/min for 8-10 min after digestion is ended, adding fibroblast culture solution (Gibico) at 37 ℃ for 5%, and culturing under CO2 condition; putting the epidermis stripped by the tissue block into 0.25% pancreatin/EDTA digestive juice (Gibico) preheated at 37 ℃, digesting for 8-10 min at 37 ℃, stopping after the digestion is finished, filtering the obtained epidermis, and centrifuging for 5-6 min at 800 r/min; after the centrifugation is finished, pouring out supernatant, adding PBS buffer solution, and centrifuging for 5-6 min at 800 r/min; centrifuging, pouring out supernatant, adding epidermal cell culture solution (Gibico), and performing inoculation culture;
step two, primarily extracting umbilical cord mesenchymal stem cells and umbilical vein endothelial cells
Mixing 0.8-1.2 cm3Washing the umbilical cord tissues with the size of the umbilical cord tissues by PBS buffer solution, removing blood vessels and connective tissues, placing the umbilical cord tissues in a centrifugal tube at a speed of 300-800 r/min, centrifuging for 5-8 min, removing supernatant, and centrifuging again until the supernatant is red-free; inoculating the tissue blocks at intervals of 0.5cm, standing under aseptic condition for 10-20 min, and adding non-essential amino acid (0.1-0.6 mmol/L), L-glutamine (1-10 mmol/L), and 1 × 10 when the tissue blocks surface is dried5High glucose DMEM medium containing U/L penicillin, streptomycin, beta-mercaptoethanol (0.1-1.0 mmol/L) and 5-10% fetal calf serum at 37 deg.C and 5% CO2Culturing under the condition of (1) changing the culture solution every 3-5 days; removing tissue blocks after 80% of cells are fused, adding 0.25% of pancreatin/EDTA for digestion and subculture, digesting the cells of each generation for 2-3 min after 80% of cells are fused, centrifuging for 5-10 min at 500-1000 r/min, and subculturing;
under the aseptic condition, respectively washing two sections of umbilical cords with lengths of 18-20 cm and regular sections with PBS buffer solution until no blood stain exists, and ligating two ends of an artery of the umbilical cords to prevent pollution; then 0.1-0.5% collagenase II is injected into the umbilical vein of a section of umbilical cord to fill the umbilical vein, and the other section of the umbilical cord is injected with 0.1-0.5% collagenase I and 0.25% pancreatin/EDTA and other ratio mixed digestive juice by the same method to fill the umbilical vein; incubating the umbilical cord at 37 ℃ for 10-20 min; collecting digestive juice in two sections of umbilical veins, injecting RPMI-1640 culture solution containing 10% fetal calf serum to wash the lumen again, collecting the digestive juice and the washing solution together, centrifuging for 5-8 min at 500-1000 r/min, discarding the supernatant, adding RPMI-1640 complete culture solution, blowing and beating uniformly for inoculation, placing the mixture in 5% CO2, culturing for 8-24 h at 37 ℃, then replacing the culture solution, removing cells which are not attached to the wall, and then replacing the culture solution for 1 time at a semi-quantitative interval of 18-24 h until the cells grow and are paved at the bottom of the bottle;
the RPMI-1640 complete culture solution is prepared by adding 5-20% fetal bovine serum, 6-20 ng/mL VEGF, 1-10 mmol/L-glutamine, 100U/mL penicillin and 100 mu g/mL streptomycin into the RPMI-1640 culture solution;
step three, constructing a dermis layer containing a microvascular lumen structure:
when the third generation of fibroblasts, umbilical cord mesenchymal stem cells and endothelial cells are fused to 70% -80%, the three cells are mixed at 1.0 × 105~1.5×105Resuspending the strains in a constructed basic culture medium at the density of each strain/ml and centrifuging the strains at 500-1000 rpm/min for 5-8 min; the basic culture medium is constructed by adding 1-35 ug/ml of insulin, 10-50 ug/ml of adenine, 0.1-4.5 ug/ml of hydrocortisone, 20-80 ug/ml of VC and 5-10% of fetal calf serum into a mixed culture solution of alpha-MEM and RPMI-1640 in a volume ratio of 1: 1;
the culture process is as follows: the first day, three kinds of cells mixed in a ratio of 1:1:1 were seeded in a cell culture chamber (Nunc, hereinafter referred to as chamber) previously coated with 1 to 5% gelatin at a seeding density of 0.15X 106~0.5×106Removing the constructed basic culture medium after 18-24 h, then continuously paving 1-5% gelatin, incubating for 45-60 min at 37 ℃, removing the gelatin, inoculating three kinds of cells mixed according to the proportion of 1:1:1 again, wherein the inoculation density is 0.15 multiplied by 106~0.5×106Culturing in a cell/chamber for 70-72 h, spreading 1-5% gelatin, continuously incubating for 45-60 min, removing gelatin, inoculating cell suspension of fibroblast and umbilical cord mesenchymal stem cell according to the volume ratio of 2:1, wherein the inoculation density is 0.15 multiplied by 106~0.5×106And (5) continuously culturing for 2-3 days in each chamber, and changing the liquid every other day.
This step is a key step in construction. The dermal tissue structure containing the microvascular lumen is established by co-culturing fibroblasts, umbilical cord mesenchymal stem cells and umbilical vein endothelial cells in different proportions in a three-dimensional environment. The traditional mesenchymal stem cells are obtained from bone marrow, and are gradually limited in application due to the risks that the material is invasive, the cell activity and the number of the mesenchymal stem cells are reduced along with the increase of the age of a donor, and the like. Later studies have shown that umbilical cord mesenchymal stem cells have most of the biological characteristics of bone marrow mesenchymal stem cells, such as: cytokine profile, phenotypic characteristics, etc. The umbilical cord is the waste after delivery, has wide source and high yield, and can meet the requirements of later-stage industrialization.
The advantages of introducing umbilical cord mesenchymal stem cells into the dermis layer are as follows: 1) the capability of secreting extracellular matrix is strong, and the extracellular matrix and fibroblasts can synergistically play a role in secreting the framework components of the dermis layer, so that the thickness of the dermis layer can be increased; 2) the proliferation of blood vessels is promoted by paracrine action. The umbilical cord mesenchymal stem cells can secrete angiogenesis promoting active factors such as VEFG, bFGF and the like. VEGF is a highly specific mitogenic factor for endothelial cells, stimulates the proliferation and differentiation of endothelial cells, and induces angiogenesis; bFGF can stimulate the up-regulation of VEGF receptor, and has synergistic effect with VEGF to promote the formation of capillary lumen. In addition, the umbilical cord mesenchymal stem cells can secrete extracellular matrix components required by angiogenesis, such as integrin, cadherin and the like to participate in the generation of blood vessels; 3) the umbilical cord mesenchymal stem cells have differentiation capacity, and when the umbilical cord mesenchymal stem cells are co-cultured with human umbilical vein endothelial cells, the microenvironment formed by the endothelial cells influences the differentiation direction of the umbilical cord mesenchymal stem cells, so that the umbilical cord mesenchymal stem cells are induced and differentiated towards vascular endothelium to further form a microvascular lumen-like structure.
The secretion of extracellular matrix in the dermis needs to be achieved to a certain extent to maintain normal skin structure and provide nutrients for the epidermis. Therefore, three inoculations were used in the present invention during the cultivation of the dermis layer. Due to the nodular growth characteristics of the umbilical cord mesenchymal stem cells and the function of secreting a large amount of extracellular matrix, the model is easy to shrink into balls when the dermis layer is cultured for a long time, so that the dermis layer is failed to be constructed. Therefore, the method uses a pre-coating method before inoculation of 1-5% gelatin and a mode of continuous three times of inoculation of the dermis layer to relieve the shrinkage of the dermis layer, increase the thickness of the dermis layer and shorten the culture period of the dermis layer. At the time of the third dermal layer inoculation, the ratio of fibroblast: the umbilical cord mesenchymal stem cells are cultured and constructed according to the proportion of =2:1, and the aim is to avoid the influence of umbilical cord mesenchymal stem cell paracrine factors on epidermal layer hair growth.
Step four, constructing the double-layer skin containing the capillary lumen structure: resuspending the fourth representative skin cell with the construction culture medium SKc1 and centrifuging the cell with the fusion rate of about 60 percent, wherein the cell is resuspended with 0.1-0.8 multiplied by 106Cell density of individual/chamber was seeded. The culture was performed under liquid on day 1 of SKc1, and gas-liquid surface culture was performed from day 2 of SKc 1. And (3) continuously culturing for 2-3 d under liquid, then, changing the SKc2 culture solution gas-liquid surface for 2-3 d, then, changing the SKc3 culture solution gas-liquid surface for 2-3 d, and finally, changing the SKc4 culture solution gas-liquid surface for 3-4 d, thereby completing the construction process.
The components of the culture solution are as follows: composition of basal medium: adding 1-35 ug/ml of insulin, 10-50 ug/ml of adenine, 0.1-4.5 ug/ml of hydrocortisone and 5-10% of fetal calf serum into a mixed solution of alpha-MEM and F12 in a volume ratio of 1: 1; the culture solution SKc1 is prepared by adding 10-50 mu g/ml of BPE, 0.1-0.5 ng/ml of EGF, 0.01-0.09 nM of ethanolamine phosphate, 2-10 nM of progesterone and 60-90 mu g/ml of commercial heparin into a basic culture medium; the culture solution SKc2 is prepared by adding 10-50 μ g/ml BPE, 0.1-0.5 ng/ml EGF and 0.5-2.5 mM CaCl into basic culture medium220-80 mu g/ml of Vc, 0.01-0.09 nM of ethanolamine phosphate and 2-10 nM of progesterone; the culture solution SKc3 is prepared by adding 10-50 mu g/ml of BPE, 0.1-0.5 ng/ml of EGF, 2.6-3.5 mM of CaCl2, 20-80 mu g/ml of Vc, 0.01-0.09 nM of ethanolamine phosphate and 2-10 nM of progesterone to a basic culture medium; the culture solution SKc4 is prepared by adding 10-50 μ g/ml BPE, 0.1-0.5 ng/ml EGF and 3.6-4.5 mM CaCl into basic culture medium220-80 mu g/ml Vc, 0.01-0.09 nM ethanolamine phosphate, 2-10 nM progesterone and 60-90 mu g/ml commercial heparin; the step simulates the hair growth process of the epidermis layer of the normal human skin, adds various growth factors at different hair growth stages of the epidermis, and leads the epidermis layer to grow normally through the change of a culture modeThe hair is composed of basal layer, spinous layer, granular layer and cuticle. The added factors not only support the growth of the epidermis layer, but also can maintain the growth of the dermis layer and the formation of the lumen structure of the microvasculature.
In the invention, in the construction of a double-layer skin model containing a microvascular lumen, fibroblasts, umbilical cord mesenchymal stem cells and umbilical vein endothelial cells are used as seed cells, a dermal layer multiple inoculation method is used, and nutritional factors are added at different construction stages to ensure the growth of the dermis and the epidermal layer. The introduced umbilical cord mesenchymal stem cells have wide sources, are easy to obtain, have high yield and can meet the requirements of later-stage industrialization. The extracellular matrix secretion capacity and paracrine capacity of the umbilical cord mesenchymal stem cells are utilized to participate in forming a dermal layer skeleton and a blood vessel lumen. Multiple continuous inoculation is adopted, the thickness of the dermis layer is increased, the culture time is shortened, the problem of shrinkage caused by mechanical traction of cells due to extracellular matrix secretion when the dermis layer is cultured for a long time is avoided, and favorable conditions are provided for the inoculation of the epidermis layer in the next step. The whole construction process does not introduce exogenous stents and angiogenesis factors, and the extracellular matrix secretion function of cells and a paracrine system are utilized to assemble a dermal tissue skeleton to form a microvascular lumen structure, so that the normal growth process of human skin is simulated, and the high similarity of histology and the human skin structure is achieved. The invention can construct double-layer skin containing the microvascular lumen in a short time, has simple and convenient operation process and strong repeatability, and can realize industrialization.
Drawings
FIG. 1 is a photograph of normal human umbilical vein endothelial cell morphology;
FIG. 2, 2D co-culture of three cells, human fibroblasts, umbilical cord mesenchymal stem cells and human umbilical vein endothelial cells in 24-well plates, photograph after 6D. As shown in the figure, human umbilical vein endothelial cells can be connected end to form a lumen-like structure by means of the co-culture of three cells without adding exogenous angiogenic factors.
FIG. 3 shows the immunohistochemical staining result of endothelial cell marker CD31 expressed in the lumen-like structure formed after co-culturing three cells, namely human fibroblasts, umbilical cord mesenchymal stem cells and human umbilical vein endothelial cells.
Detailed Description
Fibroblast culture medium, epidermal cell culture medium, and pancreatin/EDTA digest used in the examples were purchased from Gibico, and cell culture chambers used were purchased from Nunc.
Example 1: preparation method of double-layer skin containing microvascular lumen
Step one, extracting fibroblasts and epidermal cells:
soaking skin tissue in 75% ethanol solution, and soaking in PBS solution until there is no blood stain; adding Dispase digestive juice into the tissue block, and placing for 16 h at the temperature of 4 ℃; adding collagenase, incubating at 37 deg.C for 1 hr, digesting at 1000rpm/min, centrifuging for 8min, adding fibroblast culture solution, culturing, placing the peeled epidermis in 8 ml of 37 deg.C preheated 0.25% pancreatin/EDTA digestive solution, transferring into 37 deg.C incubator, digesting for 8min, terminating after digestion, filtering to obtain epidermis, and centrifuging at 800r/min for 5 min; adding PBS buffer solution after centrifugation, and centrifuging for 5min at 800 r/min; pouring out the supernatant, adding epidermal cell culture solution, and performing inoculation culture;
step two, primary extraction of umbilical cord mesenchymal stem cells and umbilical vein endothelial cells
Mixing 0.8-1.2 cm3Washing umbilical cord tissues with the size of the umbilical cord tissues with PBS buffer solution, removing blood vessels and connective tissues, placing the umbilical cord tissues in a 50ml centrifugal tube at 300rpm/min, centrifuging for 5min, removing supernatant, and centrifuging again until the supernatant is red-free; inoculating the tissue blocks into a 10 cm culture dish at an interval of 0.5cm, placing in a super clean bench for 10min, adding 4ml of high-sugar DMEM medium containing non-essential amino acid (0.1mmol/L), L-glutamine (2mmol/L), 1 × 10U/L penicillin, streptomycin, beta-mercaptoethanol (0.2mmol/L) and 10% fetal calf serum when the surfaces of the tissue blocks are dried, and culturing in an incubator at 37 ℃ and 5% CO2, wherein the solution is changed for 1 time every 3 d; after 80% of cells are fused, removing tissue blocks, adding 0.25% of pancreatin/EDTA for digestion and subculture. Then, after 80% of cells of each generation are fused, 0.25% of pancreatin/EDTA is digested for 3min, and the cells are centrifuged at 1000r/min for 5min and then are passaged.
On a clean bench, the umbilical cord is placed in a PBS culture dishIn the method, blood in the umbilical cord is squeezed out, two sections are trimmed by scissors and divided into 20cm sections, the umbilical vein is found out and inserted into the umbilical vein from one end by using an injector with a flat needle, vascular clamps are fixed, then preheated PBS is used for flushing for more than 3 times, blood stains are completely flushed, in order to prevent residual blood in the umbilical artery from mixing, the artery can be slightly separated and ligated by using a disinfection line, and the other end is clamped by using hemostatic clamps. Injecting 0.1% collagenase II into the washed needle to fill the needle, and injecting mixed digestive juice of 0.1% collagenase I and 0.25% pancreatin in the same way; taking out the needle, clamping the injection end with hemostatic clamp, transferring into sterile beaker, and incubating in 37 deg.C incubator for 15 min; taking out, loosening vascular forceps at the injection end, collecting digestive juice in umbilical vein, injecting RPMI-1640 culture solution containing 10% fetal calf serum into the vessel, washing the vessel, collecting digestive juice and washing solution in a centrifuge tube at 1000r/min, centrifuging for 8min, discarding supernatant, adding RPMI-1640 complete culture solution, blowing and beating with Pasteur pipette, inoculating into culture bottle, placing in 5% CO2Culturing in an incubator at 37 ℃, changing the culture solution after 24h to remove the cells which are not attached to the wall, and then changing the culture solution for 1 time at intervals of 24h in a semi-quantitative manner until the cells grow and are paved on the bottom of the bottle.
The RPMI-1640 complete culture solution is prepared by adding 5-20% fetal bovine serum, 6-20 ng/mL VEGF, 1-10 mmol/L-glutamine, 100U/mL penicillin and 100 mu g/mL streptomycin into the RPMI-1640 culture solution.
Step three, constructing a dermis layer containing a microvascular lumen structure: transferring the cells to a third generation, fusing 70-80% umbilical cord mesenchymal stem cells and endothelial cells as fibroblasts, respectively resuspending the three cells in a constructed basal culture medium at a density of 1.5 multiplied by 10/ml, and centrifuging the three cells at 800rpm/min for 5 min; wherein the composition of the constructed basic culture medium is as follows: alpha-MEM, RPMI-1640 (volume ratio alpha-MEM: RPMI-1640=1: 1), insulin 15ug/ml, adenine 25ug/ml, hydrocortisone 3ug/ml, Vc50ug/ml,10% fetal calf serum.
The culture process is as follows: inoculating three kinds of cells mixed in a ratio of 1:1:1 in a cell culture chamber coated with 1% gelatin in advance on the first day, wherein the inoculation density is 0.2 multiplied by 10 cells per chamber, discarding cell culture solution after 18h, continuously paving 1% gelatin, incubating in an incubator for 45min, abandoning gelatin, inoculating three kinds of cells mixed in a ratio of 1:1:1, the inoculation density is 0.2 multiplied by 10 cells per chamber, paving 1% gelatin after 72h, incubating in the incubator for 45min, abandoning gelatin, inoculating fibroblasts: umbilical cord mesenchymal stem cells =2:1 cell suspension, the seeding density is 0.2 × 10/chamber, the culture is continued for 3d, and the liquid is changed every other day.
Step four, constructing the double-layer skin containing the capillary lumen structure: the epidermal cells were passaged to the fourth passage, and when the cells were cultured until the fusion rate reached about 60%, the cells were resuspended in the construction medium SKc1 and centrifuged, and seeded into the cell culture chamber at a cell density of 0.5 × 10/chamber. The culture was performed under liquid on day 1 of SKc1, and gas-liquid surface culture was performed from day 2 of SKc 1. And after continuous submerged culture for 2d, changing the SKc2 culture solution gas-liquid level for culturing for 2d, then changing the SKc3 culture solution gas-liquid level for culturing for 2d, and finally changing the SKc4 culture solution gas-liquid level for culturing for 3d, thereby completing the construction process.
The components of the culture solution are as follows: constructing a basic culture medium: alpha-MEM, F12 (volume ratio alpha-MEM: F12=1: 1), insulin 1ug/ml, adenine 22ug/ml, hydrocortisone 0.22ug/ml, 10% fetal calf serum. The culture solution SKc1 is prepared by adding BPE 15 μ g/ml, EGF 0.1ng/ml, ethanolamine phosphate 0.03nM, progesterone 3nM, and commercial heparin 60 μ g/ml to the constructed basic culture medium; culture solution SKc1 is prepared by adding BPE 15 μ g/ml, EGF 0.5ng/ml, ethanolamine phosphate 0.04nM, progesterone 5nM, and commercial heparin 75 μ g/ml to the constructed basic culture medium; the culture solution SKc2 is prepared by adding 15 μ g/ml BPE, 0.1ng/ml EGF, 0.03nM ethanolamine phosphate, 3nM progesterone, 2.5 mM CaCl2, and 50 μ g/ml Vc to a constructed basic culture medium; the culture solution SKc3 is prepared by adding BPE 15 μ g/ml, EGF 0.1ng/ml, ethanolamine phosphate 0.03nM, progesterone 3nM, CaCl2 3.5 mM, and Vc50 μ g/ml to a constructed basic culture medium; culture solution SKc4 was prepared by adding 15. mu.g/ml BPE, 0.1ng/ml EGF, 0.03nM ethanolamine phosphate, 4.5mM CaCl2, 3nM progesterone, 50. mu.g/ml Vc, and 60. mu.g/ml commercial heparin to the constructed basal medium.
Example 2: preparation method of double-layer skin containing microvascular lumen
Step one, extracting fibroblasts and epidermal cells:
soaking skin tissue in 75% ethanol solution to remove blood stain, and soaking in PBS solution until there is no blood stain; adding Dispase digestive juice into the tissue block, and placing for 17 h at the temperature of 4 ℃; adding collagenase, incubating at 37 deg.C for about 2 hr, terminating digestion, centrifuging at 1000rpm/min for 10min, and adding fibroblast culture solution for culturing; placing the peeled epidermis into preheated 0.25% pancreatin/EDTA digestive juice at 37 deg.C, transferring into 37 deg.C incubator for digestion for 10min, filtering to obtain epidermis, and centrifuging at 800rpm/min for 5 min;
after the centrifugation is finished, the supernatant is poured out, 10 mL of PBS buffer solution is added, and the mixture is centrifuged for 5min at 800 rpm/min; centrifuging, pouring out supernatant, adding epidermal cell culture solution, and performing inoculation culture;
step two, primary extraction of umbilical cord mesenchymal stem cells and umbilical vein endothelial cells
Mixing 0.8-1.2 cm3Washing umbilical cord tissues with the size of the umbilical cord tissues with PBS buffer solution, removing blood vessels and connective tissues, placing the umbilical cord tissues in a 50ml centrifugal tube at 300rpm/min, centrifuging for 5min, removing supernatant, and centrifuging again until the supernatant is red-free; inoculating the tissue blocks into a culture dish at an interval of 0.5cm, placing in an ultra-clean bench for 10min, carefully adding a high-sugar DMEM culture medium containing non-essential amino acid (0.1mmol/L), L-glutamine (2mmol/L), 1 × 10U/L penicillin, streptomycin, beta-mercaptoethanol (0.2mmol/L) and 10% fetal calf serum when the surfaces of the tissue blocks are dried, culturing in an incubator at 37 ℃ and 5% CO2, and changing the culture solution for 1 time every 3-5 days; after 80% of cells are fused, removing tissue blocks, adding 0.25% of pancreatin/EDTA for digestion and subculture; then digesting the cells of each generation by 0.25% pancreatin/EDTA for 2min after 80% fusion, centrifuging for 5min at 1000r/min, and carrying out 1: passage 3.
Placing the umbilical cord into a culture dish containing preheated PBS in advance under aseptic condition, cutting off the part with clamp marks and hematoma, squeezing out blood in the umbilical cord, trimming two sections with scissors, and dividing into sections of 20 cm. Inserting an injector with a flat needle into the umbilical vein from one end, fixing the umbilical vein by using vascular clamps, and flushing the umbilical vein by using preheated PBS until no blood mark exists; injecting 0.1% collagenase II into the washed needle to fill the needle, and injecting mixed digestive juice of 0.1% collagenase I and 0.25% pancreatin in the same way; taking out the needle, and clamping with hemostatic clampThe injection end was transferred to a sterile beaker and incubated in an incubator at 37 ℃ for 15 min. Taking out, loosening vascular forceps at injection end, collecting digestive juice in umbilical vein, injecting RPMI-1640 culture solution containing 10% fetal calf serum into the vessel, washing the vessel, collecting digestive juice and washing solution in centrifuge tube at 1000r/min, centrifuging for 8min, discarding supernatant, adding RPMI-1640 complete culture solution, inoculating into culture bottle, placing in 5% CO2Culturing in an incubator at 37 ℃, changing the culture solution after 24h to remove the cells which are not attached to the wall, and then changing the culture solution for 1 time at intervals of 24h in a semi-quantitative manner until the cells grow and are paved on the bottom of the bottle.
The RPMI-1640 complete culture solution is prepared by adding 5-20% fetal bovine serum, 6-20 ng/mL VEGF, 1-10 mmol/L-glutamine, 100U/mL penicillin and 100 mu g/mL streptomycin into the RPMI-1640 culture solution.
Step three, constructing a dermis layer containing a microvascular lumen structure: the cells are transmitted to the third generation, as the fiber cells, the umbilical vein mesenchymal stem cells and the endothelial cells are fused to 70-80%, and the three cells are respectively resuspended in a constructed basic culture medium at the density of 1.0 multiplied by 10/ml and centrifuged at 800r/min for 5 min.
Wherein the composition of the constructed basic culture medium is as follows: alpha-MEM, RPMI-1640 (volume ratio alpha-MEM: RPMI-1640=1: 1), insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml, Vc50ug/ml,10% fetal calf serum.
The culture process is as follows: three cells mixed in a ratio of 1:1:16 were seeded on day one in a cell culture chamber precoated with 2% gelatin at a seeding density of 0.2X106And (4) removing the cell culture solution after 24 hours, continuously paving with 2% gelatin, incubating in an incubator for 45min, removing the gelatin, inoculating three kinds of cells mixed according to a ratio of 1:1:1, and inoculating at a density of 0.2x106Chamber, 72h later, discard gelatin and inoculate fibroblasts: umbilical cord mesenchymal stem cell =2:1 cell suspension, seeding density of 0.15 x106And (4) continuously culturing for 3d every chamber, and changing the liquid every other day.
Step four, constructing the double-layer skin containing the capillary lumen structure: subculturing the epidermal cells to the fourth generation, and when the cells are cultured until the fusion rate reaches about 60%, re-suspending the cells by using a construction culture solution SKc1, centrifuging, and performing cell culture at 0.5 × 106Cell density per chamber it was seeded into cell culture chambers. The culture was performed under liquid on day 1 of SKc1, and gas-liquid surface culture was performed from day 2 of SKc 1. And after continuous submerged culture for 2d, changing the SKc2 culture solution gas-liquid level for culturing for 2d, then changing the SKc3 culture solution gas-liquid level for culturing for 2d, and finally changing the SKc4 culture solution gas-liquid level for culturing for 4d, thereby completing the construction process.
The components of the culture solution are as follows: constructing a basic culture medium: alpha-MEM, F12 (volume ratio alpha-MEM: F12=1: 1), insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml,10% fetal calf serum.
Culture solution SKc1 is prepared by adding BPE 15 μ g/ml, EGF 0.5ng/ml, ethanolamine phosphate 0.04nM, progesterone 5nM, and commercial heparin 75 μ g/ml to the constructed basic culture medium; culture solution SKc2 was prepared by adding 15. mu.g/ml BPE, 0.5ng/ml EGF, 0.04nM ethanolamine phosphate, 5nM progesterone, 2.0 mM CaCl to a constructed basal medium2Vc 35. mu.g/ml; culture solution SKc3 was prepared by adding 15. mu.g/ml BPE, 0.5ng/ml EGF, 0.04nM ethanolamine phosphate, 5nM progesterone, 3.0 mM CaCl to a constructed basal medium2Vc 35. mu.g/ml; culture solution SKc4 was prepared by adding 15. mu.g/ml BPE, 0.5ng/ml EGF, 0.04nM ethanolamine phosphate, 5nM progesterone, 4.0 mM CaCl to a constructed basal medium235. mu.g/ml Vc, commercial heparin 75. mu.g/ml.
Example 3: preparation method of double-layer skin containing microvascular lumen
Step one, extracting fibroblasts and epidermal cells:
soaking skin tissue in 75% ethanol solution, removing blood stain, and soaking in PBS solution; adding Dispase digestive juice into the tissue block, and placing for 16 h at the temperature of 4 ℃; adding collagenase, incubating at 37 deg.C for about 2 hr, terminating digestion, centrifuging at 1000rpm/min for 10min, and adding fibroblast culture solution for culturing; placing the peeled epidermis into preheated 0.25% pancreatin/EDTA digestive juice at 37 deg.C, transferring into 37 deg.C incubator for digestion for 10min, filtering to obtain epidermis, and centrifuging at 800rpm/min for 5 min; after the centrifugation is finished, the supernatant is poured out, 10 mL of PBS buffer solution is added, and the mixture is centrifuged for 5min at 800 rpm/min; centrifuging, pouring out supernatant, adding epidermal cell culture solution, and performing inoculation culture;
step two, primary extraction of umbilical cord mesenchymal stem cells and umbilical vein endothelial cells
Washing umbilical cord tissue with PBS buffer solution, removing blood vessel and connective tissue, placing in 50ml centrifugal tube at 300rpm/min, centrifuging for 5min, discarding supernatant, and centrifuging again until supernatant is red-free; inoculating the tissue blocks into a culture dish at an interval of 0.5cm, placing in a super clean bench for 10min, carefully adding high sugar DMEM medium containing non-essential amino acids (0.1mmol/L), L-glutamine (2mmol/L), 1 × 10U/L penicillin, streptomycin, beta-mercaptoethanol (0.2mmol/L) and 10% fetal calf serum when the tissue blocks surface is dry, and culturing at 37 deg.C and 5% CO2Culturing in an incubator, and replacing the culture solution for 1 time every 3-5 days; after 80% of cells are fused, removing tissue blocks, adding 0.25% of pancreatin/EDTA for digestion and subculture; then digesting the cells of each generation by 0.25% pancreatin/EDTA for 2min after 80% fusion, centrifuging for 5min at 1000r/min, and carrying out 1: passage 3.
Placing the umbilical cord into a culture dish containing preheated PBS in advance under aseptic condition, cutting off the part with clamp marks and hematoma, squeezing out blood in the umbilical cord, trimming two sections with scissors, and dividing into sections of 20 cm. Inserting an injector with a flat needle into the umbilical vein from one end, fixing the umbilical vein by using vascular clamps, and flushing the umbilical vein by using preheated PBS until no blood mark exists; injecting 0.1% collagenase II into the washed needle to fill the needle, and injecting mixed digestive juice of 0.1% collagenase I and 0.25% pancreatin in the same way; the needle was removed, the injection end was clamped with a hemostat, transferred to a sterile beaker and incubated in an incubator at 37 ℃ for 15 min. Taking out, loosening vascular forceps at injection end, collecting digestive juice in umbilical vein, injecting RPMI-1640 culture solution containing 10% fetal calf serum into the vessel, washing the vessel, collecting digestive juice and washing solution in centrifuge tube at 1000r/min, centrifuging for 8min, discarding supernatant, adding RPMI-1640 complete culture solution, inoculating into culture bottle, placing in 5% CO2Culturing in an incubator at 37 ℃, changing the culture solution after 24h to remove the cells which are not attached to the wall, and then changing the culture solution for 1 time at intervals of 24h in a semi-quantitative manner until the cells grow and are paved on the bottom of the bottle.
The RPMI-1640 complete culture solution is prepared by adding 5-20% fetal bovine serum, 6-20 ng/mLVEGF, 1-10 mmol/L-glutamine, 100U/mL penicillin and 100 mug/mL streptomycin into the RPMI-1640 culture solution.
Step three, constructing a dermis layer containing a microvascular lumen structure: the cells are transmitted to the third generation, as fibroblasts, the umbilical vein mesenchymal stem cells and the endothelial cells are fused to 70-80%, and the three cells are respectively resuspended in a constructed basic culture medium at the density of 1.0 multiplied by 10/ml and centrifuged at 800rpm/min for 5 min.
Wherein the composition of the constructed basic culture medium is as follows: alpha-MEM, RPMI-1640 (volume ratio alpha-MEM: RPMI-1640=1: 1), insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml, Vc50ug/ml,10% fetal calf serum.
The culture process is as follows: three cells mixed in a ratio of 1:1:16 were seeded on day one in a cell culture chamber precoated with 2% gelatin at a seeding density of 0.2X106And (4) removing the cell culture solution after 24 hours, continuously paving with 2% gelatin, incubating in an incubator for 45min, removing the gelatin, inoculating three kinds of cells mixed according to a ratio of 1:1:1, and inoculating at a density of 0.2x106Chamber, 72h later, discard gelatin and inoculate fibroblasts: umbilical cord mesenchymal stem cell =2:1 cell suspension, seeding density of 0.15 x106And (4) continuously culturing for 3d every chamber, and changing the liquid every other day.
Step four, constructing the double-layer skin containing the capillary lumen structure: subculturing the epidermal cells to the fourth generation, and when the cells are cultured until the fusion rate reaches about 60%, re-suspending the cells by using a construction culture solution SKc1, centrifuging, and performing cell culture at 0.5 × 106Cell density per chamber it was seeded into cell culture chambers. The culture was performed under liquid on day 1 of SKc1, and gas-liquid surface culture was performed from day 2 of SKc 1. And after continuous submerged culture for 2d, changing the SKc2 culture solution gas-liquid level for culturing for 2d, then changing the SKc3 culture solution gas-liquid level for culturing for 2d, and finally changing the SKc4 culture solution gas-liquid level for culturing for 4d, thereby completing the construction process.
The components of the culture solution are as follows: constructing a basic culture medium: alpha-MEM, F12 (volume ratio alpha-MEM: F12=1: 1), insulin 10ug/ml, adenine 25ug/ml, hydrocortisone 1ug/ml,10% fetal calf serum.
The culture solution SKc1 is prepared by adding BPE 15 μ g/ml, EGF 0.5ng/ml, ethanolaminePhosphate 0.04nM, progesterone 5nM, commercial heparin 75 μ g/ml; culture solution SKc2 was prepared by adding BPE 15. mu.g/ml, EGF 0.5ng/ml, ethanolamine phosphate 0.04nM, progesterone 5nM, and CaCl 0.5 mM to the constructed basal medium2Vc 35. mu.g/ml; culture solution SKc3 was prepared by adding 15. mu.g/ml BPE, 0.5ng/ml EGF, 0.04nM ethanolamine phosphate, 5nM progesterone, 2.6 mM CaCl to a constructed basal medium2Vc 35. mu.g/ml; culture solution SKc4 was prepared by adding 15. mu.g/ml BPE, 0.5ng/ml EGF, 0.04nM ethanolamine phosphate, 5nM progesterone, 3.6 mM CaCl to a constructed basal medium235. mu.g/ml Vc, commercial heparin 75. mu.g/ml. FIG. 1 is a normal human umbilical vein endothelial cell morphology, showing that the endothelial cells are scattered and evenly distributed; FIG. 2 shows that in the present example, 2D co-culture of three cells, namely human fibroblasts, human umbilical cord mesenchymal stem cells and human umbilical vein endothelial cells, was performed in a 24-well plate, and after 6 days, a significant luminal-like structure was observed; FIG. 3 shows the immunohistochemical staining results of endothelial cell marker CD31 expressed in the lumen-like structure formed after co-culturing three cells, namely human fibroblasts, human umbilical cord mesenchymal stem cells and human umbilical vein endothelial cells.
Claims (9)
1. A preparation method of double-layer skin containing microvascular lumens is characterized by comprising the following steps:
resuspending the fourth representative skin cells with the constructed culture solution SKc1 and centrifuging the cells at the fusion rate of about 60%, wherein the cell suspension is 0.1-0.8 multiplied by 106(ii) cell density of individual/chamber it is seeded into a cell culture chamber containing the dermal layer of the microvascular luminal structure; the 1 st day of the culture of SKc1 was an under-liquid culture mode, and the gas-liquid surface culture was performed from the 2 nd day of the under-liquid culture of SKc 1;
after continuous submerged culture is carried out for 2-3 d, the SKc2 culture solution is changed for gas-liquid surface culture for 2-3 d, then the SKc3 culture solution is changed for gas-liquid surface culture for 2-3 d, and finally the SKc4 culture solution is changed for gas-liquid surface culture for 3-4 d, so that the construction process is completed;
the components of the culture solution are as follows:
the culture solution SKc1 is prepared by adding 10-50 mu g/ml of BPE, 0.1-0.5 ng/ml of EGF, 0.01-0.09 nM of ethanolamine phosphate, 2-10 nM of progesterone and 60-90 mu g/ml of commercial heparin into a basic culture medium;
the culture solution SKc2 is prepared by adding 10-50 μ g/ml BPE, 0.1-0.5 ng/ml EGF and 0.5-2.5 mM CaCl into basic culture medium220-80 mu g/ml of Vc, 0.01-0.09 nM of ethanolamine phosphate and 2-10 nM of progesterone;
the culture solution SKc3 is prepared by adding 10-50 μ g/ml BPE, 0.1-0.5 ng/ml EGF and 2.6-3.5 mM CaCl into basic culture medium220-80 mu g/ml of Vc, 0.01-0.09 nM of ethanolamine phosphate and 2-10 nM of progesterone;
the culture solution SKc4 is prepared by adding 10-50 μ g/ml BPE, 0.1-0.5 ng/ml EGF and 3.6-4.5 mM CaCl into basic culture medium220-80 mu g/ml Vc, 0.01-0.09 nM ethanolamine phosphate, 2-10 nM progesterone and 60-90 mu g/ml commercial heparin.
2. The method of claim 1, wherein the basal medium has a composition that: 1-35 ug/ml of insulin, 10-50 ug/ml of adenine, 0.1-4.5 ug/ml of hydrocortisone and 5-10% of fetal calf serum are added into a mixed solution of alpha-MEM and F12 in a volume ratio of 1: 1.
3. The method for preparing a dermal layer of claim 1, wherein the constructing of the dermal layer containing the microvascular luminal structure comprises the steps of:
when the third generation of fibroblasts, umbilical cord mesenchymal stem cells and endothelial cells are fused to 70% -80%, the three cells are mixed at 1.0 × 105~1.5×105Resuspending the strains in a constructed basic culture medium at the density of each strain/ml and centrifuging the strains at 500-1000 r/min for 5-8 min;
three cells mixed in a ratio of 1:1:1 were inoculated in a cell culture chamber previously coated with 1-5% gelatin at an inoculation density of 0.15X 106~0.5 ×106Removing the constructed basic culture medium after 18-24 h, then continuously paving 1-5% gelatin, incubating for 45-60 min at 37 ℃, removing the gelatin, inoculating three kinds of cells mixed according to the proportion of 1:1:1 again, wherein the inoculation density is 0.15 multiplied by 106~0.5 ×106Culturing in a small chamber for 70-72 h, and spreading 1-5% MingGlue, continuously incubating for 45-60 min, removing the gelatin, inoculating cell suspension of fibroblasts and umbilical cord mesenchymal stem cells according to the volume ratio of 2:1, wherein the inoculation density is 0.15 multiplied by 106~0.5 ×106And (5) continuously culturing for 2-3 days in each chamber, and changing the liquid every other day.
4. The preparation method according to claim 3, wherein the basic culture medium is prepared by adding 1-35 ug/ml of insulin, 10-50 ug/ml of adenine, 0.1-4.5 ug/ml of hydrocortisone, 20-80 ug/ml of VC, and 5-10% of fetal calf serum to a mixed culture solution of alpha-MEM and RPMI-1640 at a volume ratio of 1: 1.
5. The method of claim 1, wherein the fibroblasts are extracted by the following steps: soaking the skin tissue block in 75% ethanol solution, and then transferring into PBS solution for soaking and washing until no blood stain exists; adding Dispase digestive juice into the tissue block, and placing the tissue block at the temperature of 4 ℃ for 16-17 h; adding collagenase for incubation for 1-3 h at 37 ℃, centrifuging for 8-10 min at 1000r/min after digestion is ended, adding fibroblast culture solution at 37 ℃ and 5% CO2And (5) performing conditioned culture.
6. The method according to claim 1, wherein the epidermal cells are extracted by the following process: putting the epidermis stripped by the tissue blocks into 0.25% pancreatin/EDTA digestive juice preheated at 37 ℃, digesting for 8-10 min at 37 ℃, stopping after the digestion is finished, filtering to obtain the epidermis, and centrifuging for 5-6 min at 800 r/min; after the centrifugation is finished, pouring out supernatant, adding PBS buffer solution, and centrifuging for 5-6 min at 800 r/min; centrifuging, pouring out supernatant, adding epidermal cell culture solution, and inoculating.
7. The preparation method according to claim 3, wherein the umbilical cord mesenchymal stem cells are extracted as follows: mixing 0.8-1.2 cm3Washing the umbilical cord tissues with the size of the umbilical cord tissues by PBS buffer solution, removing blood vessels and connective tissues, placing the umbilical cord tissues in a centrifugal tube at a speed of 300-800 r/min, centrifuging for 5-8 min, removing supernatant, and centrifuging again until the supernatant is red-free; tissue blocks are arranged according toInoculating at an interval of 0.5cm, standing under aseptic condition for 10-20 min, and adding non-essential amino acids 0.1-0.6 mmol/L, L-glutamine 1-10 mmol/L, and 1 × 105Culturing the high-glucose DMEM culture medium containing U/L penicillin, streptomycin, beta-mercaptoethanol 0.1-1.0 mmol/L and 5-10% fetal calf serum at 37 ℃ under the condition of 5% CO2, and changing the culture medium for 1 time every 3-5 days; and after 80% of cells are fused, removing tissue blocks, adding 0.25% of pancreatin/EDTA for digestion and subculture, then digesting the cells of each generation for 2-3 min by using 0.25% of pancreatin/EDTA after 80% of cells are fused, and centrifuging for 5-10 min at 500-1000 r/min for subculture.
8. The method of claim 3, wherein the umbilical vein endothelial cells are extracted by the following steps: under the aseptic condition, respectively washing two sections of umbilical cords with lengths of 18-20 cm and regular sections with PBS buffer solution until no blood stain exists, and ligating two ends of an artery of the umbilical cords to prevent pollution; injecting 0.1-0.5% collagenase II into umbilical vein of one segment of umbilical cord to fill the umbilical vein, injecting 0.1-0.5% collagenase I and 0.25% pancreatin/EDTA mixed digestive juice in the same method to fill the umbilical vein, and incubating at 37 ℃ for 10-20 min; collecting digestive juice in two umbilical vein segments, injecting RPMI-1640 culture solution containing 10% fetal calf serum to wash the lumen again, collecting the digestive juice and the washing solution together, centrifuging at 500-1000 r/min for 5-8 min, discarding the supernatant, adding RPMI-1640 complete culture solution, blowing and beating uniformly for inoculation, placing in 5% CO2And after culturing for 8-24 h at 37 ℃, changing the culture solution, removing the cells which are not attached to the wall, and then changing the solution for 1 time in a semi-quantitative mode every 18-24 h until the cells grow and cover the bottom of the bottle.
9. The method according to claim 8, wherein the RPMI-1640 complete culture solution is prepared by adding 5-20% fetal bovine serum, 6-20 ng/mL VEGF, 1-10 mmoL/L of L-glutamine, 100U/mL of penicillin and 100 μ g/mL of streptomycin to the RPMI-1640 culture solution.
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