CN111110921A - In-vitro construction method of tissue engineering posterior lamella cornea - Google Patents
In-vitro construction method of tissue engineering posterior lamella cornea Download PDFInfo
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Abstract
The invention relates to an in vitro construction method of a tissue engineering posterior lamellar cornea, which comprises a human corneal stromal cell and a human corneal endothelial cell of a corneal seed cell and an acellular porcine corneal stroma with a posterior elastic layer as a tissue engineering cornea carrier bracket. The invention overcomes the international problem that human corneal endothelial cells can not proliferate in vitro, and the corneal endothelial cells form a monolayer endothelial structure with extremely high density and tight connection, and the monolayer endothelial structure is up to 3400-3600 cells/mm2(ii) a Inoculating corneal stromal cells by injection inoculation, i.e.Greatly shortens the in vitro culture time and can keep the integrity of the carrier bracket structure. Therefore, the high-quality requirement can be met, and the mass production can be realized at low cost so as to be popularized and applied.
Description
Technical Field
The invention belongs to a construction technology in the field of regenerative medicine tissue engineering, and particularly relates to an in-vitro construction method of a tissue engineering posterior lamella cornea.
Background
The cornea is located at the foremost end of the eyeball and is the only immune privileged area of the human body, has no blood vessels and is transparent, and provides most of the refractive power for the eyes. The tissue structure of the cornea is composed of a corneal epithelial layer, a front elastic layer, a matrix layer, a back elastic layer and an inner cortical layer from outside to inside in sequence. Wherein the corneal endothelial layer is formed by embedding single-layer, flat and tightly connected hexagonal endothelial-like cells, plays a key role in maintaining the transparency and normal thickness of the cornea and has the function of a cornea-aqueous humor barrier. There are no blood vessels in the cornea and only nutrients/oxygen can be obtained from the aqueous humor through the endothelial layer or for the epithelial portion through the tear fluid. Human corneal endothelial cells lose dividing capacity after adulthood and die at a rate of 0.3% to 0.6% per year. Local cell damage can only be followed by adjacent cell enlargement, expansion and migration to fill the defect area to maintain the monolayer morphology and function of the corneal endothelial layer. Therefore, it is very difficult to culture the human corneal endothelial cells in vitro by continuous expansion, and it is an international problem at present. In addition, surgical injuries such as cataract and the like, long-term ocular hypertension (glaucoma), continuous contact of a formed vitreous body with corneal endothelium, continuous severe iritis, severe anterior iris synechia and the like can cause damage and rapid death of a large number of corneal endothelial cells, and once the density of the corneal endothelial cells is lower than the critical density (600-800 cells/mm) for maintaining the physiological functions of endothelium2) The cornea will have irreversible lesions, which will eventually cause corneal endothelium decompensation; the corneal stroma layer consists of 200-250 layers of regularly arranged collagen lamellae and corneal stroma cells scattered in the collagen lamellae, and plays an important role in maintaining corneal thickness and transparency. When the cornea is deeply wounded and infected, the deep corneal stroma and endothelial layer, namely the posterior lamellar cornea, are often damaged, and light people cause corneal edema and corneal endothelial decompensation, and serious people even cause blindness. The existing method for treating the injury of the posterior lamellar cornea mainly carries out a plurality of times of cornea transplantation operations through a corneal stroma layer and an endothelial layer, thereby increasing the risks of operation trauma and infection and having a plurality of problems in the aspects of cornea healing and scar residue of a transplantation area. Thus, tissue engineered posterior lamellar corneas useful for transplantation are constructed in vitroThe current situation that donor cornea materials are seriously deficient is solved, and a new way or material member is necessary to be developed for treating the diseases.
Disclosure of Invention
The invention aims to provide a method for constructing a tissue engineering posterior lamella cornea, namely constructing the tissue engineering posterior lamella cornea in vitro to make up for the defects of the prior art and materials.
The invention is based on a non-transfection human corneal stromal cell line and a human corneal endothelial cell line which are successfully established in our laboratory, and human corneal stromal cells and human corneal endothelial cells with normal karyotype and normal structural functions are screened out from the non-transfection human corneal stromal cell line and the human corneal endothelial cell line, and the problem of source of sufficient normal seed cells required by lamellar cornea scale construction after tissue engineering is successfully solved. And the tissue engineering cornea carrier bracket (ZL 201410459064.8) which has good transparency, compact structure, strong mechanical property, good biocompatibility with cornea seed cells, easy adhesion and growth of the cornea seed cells and is suitable for batch production is successfully prepared by utilizing animal corneas in Yangtongjun and the like in 2014. Therefore, the establishment of a method for further constructing the tissue engineering posterior lamella cornea in vitro by using the corneal stromal cells and the corneal endothelial cells as seed cells and the carrier scaffold as soon as possible, and the realization of the large-scale production and clinical application of the tissue engineering posterior lamella cornea is the key for benefiting patients with abnormal corneal endothelial layers and stromal layers all over the world. Therefore, on the basis of the prior art, the tissue engineering posterior lamellar cornea is constructed in vitro by utilizing human corneal stroma cells and human corneal endothelial cells or corneal stroma-like cells and corneal endothelial-like cells which are induced and differentiated from limbal stem cells and inoculating the corneal stroma-like cells and the corneal endothelial-like cells to a carrier scaffold.
The method comprises the following steps: the tissue engineering cornea carrier support is characterized in that the corneal stroma seed cells are inoculated in the carrier support to construct a stroma layer part of the tissue engineering posterior lamellar cornea, and then the corneal endothelial seed cells are inoculated on the posterior elastic layer of the constructed stroma layer part, so that the tissue engineering posterior lamellar cornea with the tissue structure and the function similar to the normal posterior lamellar cornea is constructed.
The corneal stroma seed cells and the corneal endothelial seed cells can be replaced by corneal stroma-like cells and corneal endothelial-like cells induced by the limbal stem cells.
The stroma layer part is constructed by firstly rehydrating a tissue engineering cornea carrier scaffold with carrier scaffold rehydration solution, then preparing human cornea stromal cells into cornea stromal cell suspension with special construction culture solution A, injecting the cell suspension into the carrier scaffold rehydrated by the carrier scaffold rehydration solution, then adding the special construction culture solution A for in vitro culture, and then replacing the special construction culture solution B for continuous culture to obtain the stroma layer part of the tissue engineering lamellar cornea; and preparing the human corneal endothelial cells into corneal endothelial cell suspension by using the special culture solution C for construction, inoculating the cell suspension on a rear elastic layer of the stroma layer part of the lamellar cornea after the tissue engineering for in vitro culture, replacing the special culture solution C for construction with the special culture solution D for construction for culture, and then replacing the special culture solution D for construction for culture to continue culture, thereby obtaining the lamellar cornea after the tissue engineering with the tissue structure and the function similar to those of the normal lamellar cornea.
The preparation method of the corneal stromal cell suspension comprises the following steps: firstly, taking human corneal stromal cells or corneal stromal-like cells induced and differentiated from limbal stem cells, culturing the cells to a full monolayer by using DMEM/F12 culture solution containing 15-20% calf serum, digesting the cells by using 0.25% trypsin solution, centrifugally collecting cell precipitates, and finally resuspending the cell precipitates by using a special construction culture solution A to obtain homogeneous corneal stromal cell suspension.
The above corneal stromal cell suspension has a cell concentration range of 2X 105-6×105Individual cells/mL.
The specific steps of the in vitro construction of the corneal stroma part are as follows: sucking the corneal stromal cell suspension by a micro-injector, injecting the cell suspension into 6-9 needles in parallel and uniformly along the direction of a corneal stroma plate, wherein each needle is 1-2 mu L, adding 1mL of the special construction culture solution A, placing the special construction culture solution A into a 5% carbon dioxide incubator at 37 ℃ for culture, replacing the special construction culture solution B once every 12-24 hours, and continuously culturing for 48-96 hours to obtain the corneal stroma part.
The preparation method of the corneal endothelial cell suspension comprises the following steps: firstly, taking human corneal endothelial cells or corneal endothelial-like cells induced and differentiated from limbal stem cells, culturing the cells to a full monolayer by using DMEM/F12 culture solution containing 15-20% calf serum, digesting the cells by using 0.25% trypsin solution, centrifugally collecting cell precipitates, and finally resuspending the cell precipitates by using construction special culture solution C to obtain homogeneous corneal endothelial cell suspension.
The above corneal endothelial cell suspension has a cell concentration range of 3X 105-5×105Individual cells/mL.
The inoculation and culture method of the corneal endothelial cells comprises the following steps: spreading the back elastic layer of the corneal stroma part in a built 48-hole culture plate with the back elastic layer facing upwards, inoculating 1mL of corneal endothelial cell suspension suspended in the special construction culture solution C on the back elastic layer, placing the back elastic layer in a 37 ℃ and 5% carbon dioxide incubator for 12 hours, sucking out the special construction culture solution C in a 48-hole plate, adding 1mL of the special construction culture solution D into the 48-hole culture plate, placing the 48-hole culture plate in the 37 ℃ and 5% carbon dioxide incubator for in-vitro culture, replacing the special construction culture solution D once every 12-24 hours, and continuously culturing for 72-120 hours to obtain the tissue engineering posterior lamellar cornea.
The carrier scaffold rehydration solution was DMEM/F12 medium containing 10mg/mL tobramycin.
The formula of the special culture solution A for construction is as follows: DMEM/F12 medium containing 15% fetal bovine serum, 0.2-0.4mg/mL fibronectin and 0.01-0.02mg/mL basic fibroblast growth factor.
The formula of the special culture solution B for construction is as follows: DMEM/F12 medium containing 15% calf serum, 0.3-0.5mg/mL tranexamic acid, 0.1-0.5mg/mL epsilon-aminocaproic acid, 1-5mg/mL ascorbic acid and 1-5. mu.g/mL matrix metalloproteinase inhibitor BB-94.
The formula of the special culture solution C for construction is as follows: DMEM/F12 medium containing 10% fetal bovine serum, 0.2-0.4mg/mL fibronectin, 0.01-0.02mg/mL basic fibroblast growth factor and 0.01-0.02mg/mL epidermal growth factor.
The formula of the special culture solution D for construction is as follows: DMEM/F12 culture medium containing 20% calf serum, 0.2-0.4mg/mL glucosamine hydrochloride, 0.1-0.5mg/mL epsilon-aminocaproic acid, 30-50ng/mL human bone morphogenetic protein 7, 1-5mg/mL ascorbic acid and 1-5. mu.g/mL matrix metalloproteinase inhibitor BB-94.
The seed cells in the invention are from non-transfected human corneal stromal cells and human corneal endothelial cells, or corneal stromal-like cells and corneal endothelial-like cells induced and differentiated from limbal stem cells, technically ensure that the cells have good growth state and no any tumorigenic risk, and can provide sufficient normal seed cells for large-scale in-vitro construction of tissue engineering lamellar corneas through continuous passage; the carrier support is obtained by the preparation method in the patent granted by the inventor, no toxic chemical reagent or medicine is used, the safety is high, the antigenicity is small, the carrier support after rehydration has the characteristics of good transparency, compact and complete structure, strong mechanical property, good biocompatibility with corneal seed cells, easiness in seed cell attachment, migration and growth, low production cost and the like, and can meet the requirement of sufficient carrier support materials required by lamellar cornea batch construction after tissue engineering.
The invention adopts the special culture solution for construction, which can simulate the physiological environment of the human corneal endothelial cells to the maximum extent, thereby overcoming the international problem that the human corneal endothelial cells can not be proliferated in vitro; the injection inoculation method is adopted for inoculating the corneal stromal cells, so that the time of in vitro culture is greatly shortened, and the integrity of the carrier scaffold structure can be better maintained compared with the direct inoculation method. In the finally constructed tissue engineering posterior lamellar cornea, the distribution of corneal stromal cells in the carrier bracket is quite uniform, and corneal endothelial cells form a monolayer endothelial structure with extremely high density and tight connection (as high as 3400-3600 cells/mm)2Equivalent to children 0-3 years old). Therefore, the high-quality requirement and the low cost of the tissue engineering posterior lamella cornea can be met, and the mass production is realized so as to be popularized and applied.
Obviously, the construction process of the tissue engineering posterior lamellar cornea is scientific and reasonable, the constructed tissue engineering posterior lamellar cornea is similar to the normal human posterior lamellar cornea in morphological structure and transparency and has the biological function of the normal posterior lamellar cornea, and the discovery of the transplantation of the posterior lamellar cornea of New Zealand rabbits and domestic cats can respectively keep the transparency of the transplanted cornea for more than 2 months, which indicates that the tissue engineering posterior lamellar cornea constructed by the invention can be completely used as a substitute of donated cornea for repairing the abnormity of the corneal endothelial layer and deep basal layer.
Detailed Description
An in vitro construction method of tissue engineering posterior lamellar cornea comprises human corneal stromal cells and human corneal endothelial cells which are taken as corneal seed cells, and acellular porcine corneal stroma with a posterior elastic layer which is taken as a tissue engineering corneal carrier bracket, and is characterized in that corneal stromal cells are inoculated in the carrier bracket to construct a stromal layer part of the tissue engineering posterior lamellar cornea, and then corneal endothelial seed cells are inoculated on the posterior elastic layer of the constructed stromal layer part to construct the tissue engineering posterior lamellar cornea with the tissue structure and the function similar to those of the normal posterior lamellar cornea.
The corneal stroma seed cells and the corneal endothelial seed cells can be replaced by corneal stroma-like cells and corneal endothelial-like cells induced by the limbal stem cells. That is, the corneal stroma-like cells and corneal endothelium-like cells induced to differentiate from the limbal stem cells can be used as the corneal stroma seed cells and corneal endothelium seed cells. Since both have the characteristics of corneal seed cells, basically the same method and culture solution can be used.
The tissue engineering posterior lamellar cornea comprises a corneal stroma layer part and a corneal endothelium layer part, so that human corneal stromal cells and human corneal endothelium cells or corneal stromal-like cells and corneal endothelium-like cells induced and differentiated from limbal stem cells are used as seed cells and are sequentially inoculated on a carrier bracket retaining a posterior elastic layer, and the tissue engineering posterior lamellar cornea with high quality can be successfully constructed in vitro.
The preparation method of various solutions comprises the following steps:
1. preparing carrier bracket rehydration solution: taking 80mL of conventionally prepared DMEM/F12 culture solution, adding 1g of tobramycin, filtering and sterilizing by using a 0.22 mu m microporous filter membrane after complete dissolution, and supplementing the DMEM/F12 culture solution to 100mL to obtain the carrier scaffold rehydration solution.
2. Preparation of a special culture solution A: taking 85mL of conventionally prepared sterile DMEM/F12 culture solution, and adding 15mL of fetal calf serum to obtain a special construction culture solution A;
in order to improve the adhesion effect of human corneal stromal cells in a carrier scaffold, 20-40mg of fibronectin is added to the formula of the special culture solution A for construction; in order to maintain the activity of human corneal stromal cells, 1-2mg of basic fibroblast growth factor is added into the formula of the special culture solution A for construction;
3. preparing a special culture solution B: taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 30-50mg tranexamic acid, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of calf serum, and supplementing the DMEM/F12 culture solution to 100mL, thus obtaining a special construction culture solution B;
in order to improve the adhesion and migration effects of human corneal stromal cells in a carrier scaffold, 10-50mg of epsilon-aminocaproic acid is added into the formula of the special culture solution B for construction; in order to promote the human corneal stromal cells to secrete more extracellular matrix components, 0.1-0.5g of ascorbic acid is added into the formula of the special culture solution B; in order to inhibit the slight degradation of the carrier scaffold, 0.1-0.5mg of matrix metalloproteinase inhibitor BB-94 is added to the formula of the special culture solution B.
4. Preparing a special culture solution C: taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 20-40mg of fibronectin, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 10mL of fetal calf serum, and supplementing the DMEM/F12 culture solution to 100mL, thus obtaining the special culture solution C.
In order to maintain the activity of human corneal stromal cells and human corneal endothelial cells, 1-2mg of basic fibroblast growth factor and 1-2mg of epidermal growth factor are added into the formula of the special culture solution C for construction;
5. preparation of a special culture solution D: taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 10-50mg of epsilon-aminocaproic acid, 0.1-0.5g of ascorbic acid and 0.1-0.5mg of matrix metalloproteinase inhibitor BB-94, filtering and sterilizing by using a 0.22 mu m microporous filter membrane after complete dissolution, adding 20mL of calf serum, and supplementing the DMEM/F12 culture solution to 100mL, thus obtaining the special culture solution D.
In order to keep the human corneal endothelial cells attached to the posterior elastic layer in an ideal growth state, 20-40mg of glucosamine hydrochloride is added into the formula of the special culture solution D for construction; in order to inhibit mesenchymal transformation of the human corneal endothelial cells cultured in vitro, 3-5 mu g of human bone morphogenetic protein 7 is added into the formula of the special culture solution D for construction.
The method comprises the following specific implementation steps:
1. preparation of the carrier scaffold: soaking the carrier bracket in carrier bracket rehydration solution for 1-2 hours for rehydration treatment;
2. the preparation method of the corneal stroma seed cell comprises the following steps: taking human corneal stromal cells or corneal stromal-like cells induced and differentiated from limbal stem cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 15-20% calf serum, sucking out the culture solution after the cells grow into a monolayer, adding 0.25% trypsin solution for digestion for 1-2 minutes, adding the old culture solution sucked out before the digestion is stopped, and suspending cell precipitates by using 4mL of special construction culture solution A to prepare homogeneous corneal stromal cell suspension; after cell counting, the cell concentration was adjusted to 2X 10 with the above-mentioned special culture solution A5-6×105Individual cells/mL;
3. in vitro construction of the corneal stromal part: sucking the corneal stromal cell suspension by a micro-syringe, and uniformly injecting 6-9 needles of 1-2 mu L per needle on each carrier bracket; placing the elastic layer surface of the carrier bracket after cell suspension injection into a 48-hole culture plate, adding 1mL of the special culture solution A for construction, placing the culture solution A in a 5% carbon dioxide incubator at 37 ℃ for culture, then replacing the special culture solution B for construction once every 12-24 hours, and continuously culturing for 48-96 hours to obtain the corneal stroma layer part.
4. Preparation of corneal endothelial seed cells: taking human corneal endothelial cells or corneal endothelial-like cells induced and differentiated from limbal stem cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 15-20% calf serum, sucking out the culture solution after the cells grow into a monolayer, adding 0.25% trypsin solution for digestion for 1-2 minutes, adding the old culture solution sucked out before to stop digestion, and suspending cell precipitates uniformly by using 4mL of special construction culture solution C to prepare homogeneous corneal endothelial cell suspension, wherein the old culture solution is used for stopping digestion, and the 1000-1500 rpm/separation center is used for 10-15 minutes; after cell counting, the cell concentration was adjusted to 3X 10 by using the above-mentioned culture medium C specially for construction5-5×105Individual cells/mL;
5. in vitro construction of tissue engineered lamellar corneas: inoculating 1mL of corneal endothelial cell suspension on the rear elastic layer, placing in a 37 ℃ and 5% carbon dioxide incubator for 12 hours, sucking out the special culture solution C for construction, adding 1mL of special culture solution D for construction in a 48-hole culture plate, placing in a 37 ℃ and 5% carbon dioxide incubator for culture, replacing the special culture solution D for construction once every 12-24 hours, and continuously culturing for 72-120 hours to obtain the tissue engineering posterior lamella cornea.
The invention is further illustrated in connection with the following examples.
Example 1
Injecting human corneal stromal cells into a carrier bracket with a rear elastic layer, adding a special culture solution A for construction for culture, and then replacing a special culture solution B for construction for continuous culture to prepare a corneal stromal layer part; then inoculating the human corneal endothelial cells on the obtained posterior elastic layer of the corneal stroma layer part, and then replacing and constructing the special culture solution D for continuous culture to obtain the tissue engineering posterior lamellar cornea with the tissue structure and function similar to those of the normal posterior lamellar cornea.
Taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 1g of tobramycin, completely dissolving, filtering and sterilizing by using a 0.22-micron microporous filter membrane, and supplementing the DMEM/F12 culture solution to 100mL to obtain carrier scaffold rehydration solution;
soaking the carrier support in the carrier support rehydration solution for 2 hours for rehydration treatment, and taking the carrier support as a tissue engineering posterior lamella cornea carrier support for later use;
taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 40mg of fibronectin and 1mg of basic fibroblast growth factor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of fetal calf serum, and supplementing the DMEM/F12 culture solution to 100mL to prepare a special construction culture solution A; taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 30mg of tranexamic acid, 50mg of epsilon-aminocaproic acid, 0.1g of ascorbic acid and BB-940.1 mg of matrix metalloproteinase inhibitor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of calf serum, and supplementing the DMEM/F12 culture solution prepared conventionally to 100mL to prepare a special construction culture solution B;
then taking the existing human corneal stroma cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 15% calf serum, sucking out the culture solution after the cells grow into a monolayer, adding 0.25% trypsin solution for digestion for 2 minutes, adding the old culture solution sucked out before the digestion, stopping the digestion, centrifuging at 1000 rpm for 15 minutes to collect cell precipitates, and suspending the cell precipitates into uniform corneal stroma cell suspension by using 4mL of the special culture solution A for construction; after cell counting, the cell concentration was adjusted to 6.0X 10 by using the above-mentioned culture solution A specially used for construction5Individual cells/mL;
sucking the corneal stromal cell suspension by a micro-syringe, and uniformly injecting 6 needles of 1 mu L per needle on the carrier bracket; then placing the carrier bracket injected with the cell suspension into a 48-hole culture plate, adding 1mL of the special culture solution A for construction, placing the culture solution A in a 5% carbon dioxide incubator at 37 ℃ for culture, then replacing the special culture solution B for construction once every 12 hours, and continuously culturing for 48 hours to obtain a corneal stroma layer part constructed in vitro;
then taking 80mL of conventionally prepared DMEM/F12 culture solution, adding 20mg of fibronectin, 1mg of basic fibroblast growth factor and 1mg of epidermal growth factor, completely dissolving, filtering and sterilizing by using a 0.22-micrometer microporous filter membrane, adding 10mL of fetal calf serum, and supplementing the DMEM/F12 culture solution to 100mL to prepare a special construction culture solution C;
taking the existing human corneal endothelial cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 15% calf serum, sucking out the culture solution after the cells grow into a monolayer, adding 0.25% trypsin solution for digestion for 2 minutes, adding the sucked out old culture solution to stop digestion, centrifuging at 1500 rpm/10 minutes to collect cell precipitates, and suspending the cell precipitates uniformly into corneal endothelial cell suspension by using 4mL of the special culture solution C for construction; after cell counting, the cell concentration was adjusted to 5X 10 by using the above-mentioned culture medium C specially for construction5Individual cells/mL;
then inoculating 1mL corneal endothelial cell suspension on the back elastic layer surface of the corneal stroma part, and placing the corneal stroma part in a 5% carbon dioxide incubator at 37 ℃ for 12 hours;
then taking 80mL of DMEM/F12 culture solution prepared conventionally, and adding 20mg of glucosamine hydrochloride, 10mg of epsilon-aminocaproic acid, 73 mu g of human bone morphogenetic protein, 0.1g of ascorbic acid and BB-940.1 mg of matrix metalloproteinase inhibitor; filtering with 0.22 μm microporous membrane for sterilization after complete dissolution, adding 20mL calf serum, and supplementing the DMEM/F12 culture solution to 100mL to obtain a special constructed culture solution D; and (3) after the culture medium is placed in a 37 ℃ and 5% carbon dioxide incubator for 12 hours, sucking out the special culture medium C for construction, adding 1mL of the special culture medium D for construction into a 48-hole culture plate, placing the culture medium D in the 37 ℃ and 5% carbon dioxide incubator for culture, replacing the special culture medium D for construction once every 12 hours, and continuously culturing for 72 hours to obtain the tissue engineering posterior lamellar cornea.
Example 2
Injecting human corneal stromal cells into a carrier bracket with a rear elastic layer, adding a special culture solution A for construction for culture, and then replacing a special culture solution B for construction for continuous culture to prepare a corneal stromal layer part; then inoculating the human corneal endothelial cells on the obtained posterior elastic layer of the corneal stroma layer part, and then replacing and constructing the special culture solution D for continuous culture to obtain the tissue engineering posterior lamellar cornea with the tissue structure and function similar to those of the normal posterior lamellar cornea.
Taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 1g of tobramycin, completely dissolving, filtering and sterilizing by using a 0.22-micron microporous filter membrane, and supplementing the DMEM/F12 culture solution to 100mL to obtain carrier scaffold rehydration solution;
soaking the carrier support in the carrier support rehydration solution for 2 hours for rehydration treatment, and taking the carrier support as a tissue engineering posterior lamella cornea carrier support for later use;
taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 20mg of fibronectin and 1.5mg of basic fibroblast growth factor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of fetal calf serum, and supplementing the DMEM/F12 culture solution to 100mL to prepare a special construction culture solution A; taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 40mg of tranexamic acid, 50mg of epsilon-aminocaproic acid, 0.3g of ascorbic acid and BB-940.3 mg of matrix metalloproteinase inhibitor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of calf serum, and supplementing the DMEM/F12 culture solution prepared conventionally to 100mL to prepare a special construction culture solution B;
then taking existing human corneal stroma cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 20% calf serum, sucking out the culture solution after the cells grow into a monolayer, adding 0.25% trypsin solution for digestion for 1.5 minutes, adding the old culture solution sucked out before the digestion is stopped, centrifuging at 1500 rpm/10 minutes to collect cell precipitates, and suspending the cell precipitates into corneal stroma cell suspension uniformly by using 4mL of the special culture solution A for construction; after cell counting, the cell concentration was adjusted to 4.0X 10 by using the above-mentioned culture solution A specially used for construction5Individual cells/mL;
sucking the corneal stromal cell suspension by a micro-syringe, and uniformly injecting 6 needles of 2 mu L per needle on the carrier bracket; then placing the carrier bracket injected with the cell suspension into a 48-hole culture plate, adding 1mL of the special culture solution A for construction, placing the culture solution A in a 5% carbon dioxide incubator at 37 ℃ for cultivation, then replacing the special culture solution B for construction once every 12 hours, and continuously culturing for 72 hours to obtain a corneal stroma layer part constructed in vitro;
then taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 30mg of fibronectin, 1.5mg of basic fibroblast growth factor and 1.5mg of epidermal growth factor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 10mL of fetal bovine serum, and supplementing the DMEM/F12 culture solution to 100mL to prepare a special construction culture solution C;
taking the existing human corneal endothelial cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 20% calf serum, sucking out the culture solution after the cells grow into a monolayer, adding 0.25% trypsin solution for digestion for 1.5 minutes, adding the old culture solution sucked out before the digestion is stopped, centrifuging at 1000 rpm for 10 minutes to collect cell precipitates, and suspending the cell precipitates uniformly into corneal endothelial cell suspension by using 4mL of the special culture solution C for construction; after cell counting, the cell concentration was adjusted to 4X 10 by using the above-mentioned culture medium C specially for construction5Individual cells/mL;
then inoculating 1mL corneal endothelial cell suspension on the back elastic layer surface of the corneal stroma part, and placing the corneal stroma part in a 5% carbon dioxide incubator at 37 ℃ for 12 hours;
then taking 80mL of DMEM/F12 culture solution prepared conventionally, 30mg of glucosamine hydrochloride, 30mg of epsilon-aminocaproic acid, 74 mu g of human bone morphogenetic protein, 0.3g of ascorbic acid and BB-940.3 mg of matrix metalloproteinase inhibitor; filtering with 0.22 μm microporous membrane for sterilization after complete dissolution, adding 20mL calf serum, and supplementing the DMEM/F12 culture solution to 100mL to obtain a special constructed culture solution D; and (3) after the culture medium is placed in a 37 ℃ and 5% carbon dioxide incubator for 12 hours, sucking out the special culture medium C for construction, adding 1mL of special culture medium D for construction into a 48-hole culture plate, placing the culture medium D in the 37 ℃ and 5% carbon dioxide incubator for culture, replacing the special culture medium D for construction once every 18 hours, and continuously culturing for 96 hours to obtain the tissue engineering posterior lamella cornea.
Example 3
Injecting corneal stroma-like cells obtained by inducing and differentiating limbal stem cells into a carrier bracket with a rear elastic layer, adding a special culture solution A for construction for culture, and then replacing a special culture solution B for construction for continuous culture to obtain a corneal stroma layer part; then, the corneal endothelial-like cells induced and differentiated from the limbal stem cells are inoculated on the posterior elastic layer of the obtained corneal stroma layer part, and then the special culture solution D for construction is replaced to continue culturing, thus obtaining the tissue engineering posterior lamellar cornea with the tissue structure and function similar to the normal posterior lamellar cornea.
Taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 1g of tobramycin, completely dissolving, filtering and sterilizing by using a 0.22-micron microporous filter membrane, and supplementing the DMEM/F12 culture solution to 100mL to obtain carrier scaffold rehydration solution;
soaking the carrier support in the carrier support rehydration solution for 2 hours for rehydration treatment, and taking the carrier support as a tissue engineering posterior lamella cornea carrier support for later use;
taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 25mg of fibronectin and 2mg of basic fibroblast growth factor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of fetal calf serum, and supplementing the DMEM/F12 culture solution to 100mL to prepare a special construction culture solution A; taking 80mL of DMEM/F12 culture solution prepared conventionally, adding 50mg of tranexamic acid, 30mg of epsilon-aminocaproic acid, 0.5g of ascorbic acid and BB-940.5 mg of matrix metalloproteinase inhibitor, filtering and sterilizing by using a 0.22-micron microporous filter membrane after complete dissolution, adding 15mL of calf serum, and supplementing the DMEM/F12 culture solution prepared conventionally to 100mL to prepare a special construction culture solution B;
then taking corneal stroma-like cells obtained by inducing differentiation of limbal stem cells, carrying out amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 20% calf serum, sucking out the culture solution after the cells grow to be full of a monolayer, adding 0.25% trypsin solution for digestion for 1 minute, adding the old culture solution sucked out before the digestion is stopped, and suspending the cell sediment uniformly into corneal stroma-like cell suspension by using 4mL of the special culture solution A for construction after the cell sediment is collected at 1200 rpm/separation for 15 minutes; after cell counting, the cell concentration was adjusted to 2.0X 10 by using the above-mentioned culture solution A specially used for construction5Individual cells/mL;
sucking the corneal stroma-like cell suspension by using a micro-syringe, and uniformly injecting 9 needles of 1.5 mu L per needle on the carrier bracket; then placing the carrier bracket injected with the cell suspension into a 48-hole culture plate, adding 1mL of the special culture solution A for construction, placing the culture solution A in a 5% carbon dioxide incubator at 37 ℃ for cultivation, then replacing the special culture solution B for construction once every 24 hours, and continuously culturing for 96 hours to obtain a corneal stroma layer part constructed in vitro;
then taking 80mL of conventionally prepared DMEM/F12 culture solution, adding 40mg of fibronectin, 2mg of basic fibroblast growth factor and 2mg of epidermal growth factor, completely dissolving, filtering and sterilizing by using a 0.22-micrometer microporous filter membrane, adding 10mL of fetal calf serum, and supplementing the DMEM/F12 culture solution to 100mL to prepare a special construction culture solution C;
taking corneal endothelial-like cells obtained by inducing and differentiating corneal limbal stem cells, performing amplification culture at 37 ℃ by using DMEM/F12 culture solution containing 20% calf serum, sucking out the culture solution after the cells grow to be full of a monolayer, adding 0.25% trypsin solution for digestion for 1 minute, adding the old culture solution sucked out before the digestion is stopped, performing 1200 rpm/separation for 15 minutes to collect cell precipitates, and suspending the cell precipitates uniformly into corneal endothelial-like cell suspension by using 4mL of the special culture solution C for construction; after cell counting, the cell concentration was adjusted to 3X 10 by using the above-mentioned culture medium C specially for construction5Individual cells/mL;
then inoculating 1mL of corneal endothelium-like cell suspension on the rear elastic layer surface of the corneal stroma part, and placing the corneal endothelium-like cell suspension in a 5% carbon dioxide incubator at 37 ℃ for 12 hours;
then taking 80mL of DMEM/F12 culture solution prepared conventionally, and adding 30mg of glucosamine hydrochloride, 50mg of epsilon-aminocaproic acid, 75 mu g of human bone morphogenetic protein, 0.5g of ascorbic acid and BB-940.5 mg of matrix metalloproteinase inhibitor; filtering with 0.22 μm microporous membrane for sterilization after complete dissolution, adding 20mL calf serum, and supplementing the DMEM/F12 culture solution to 100mL to obtain a special constructed culture solution D; and (3) after the culture medium is placed in a 37 ℃ and 5% carbon dioxide incubator for 12 hours, sucking out the special culture medium C for construction, adding 1mL of the special culture medium D for construction into a 48-hole culture plate, placing the culture medium D in the 37 ℃ and 5% carbon dioxide incubator for culture, replacing the special culture medium D for construction once every 24 hours, and continuously culturing for 120 hours to obtain the tissue engineering posterior lamellar cornea.
Therefore, the tissue engineering posterior lamellar cornea constructed in vitro by the method can be used as a substitute for donated cornea for repairing corneal diseases with abnormal corneal endothelial layer and stroma layer, and is easy to realize large-scale production.
Claims (8)
1. An in vitro construction method of tissue engineering posterior lamellar cornea comprises human corneal stromal cells and human corneal endothelial cells as corneal seed cells, and acellular porcine corneal stroma with a posterior elastic layer as a tissue engineering corneal carrier scaffold, and is characterized in that corneal stromal cells are inoculated in the carrier scaffold to construct a stromal layer part of the tissue engineering posterior lamellar cornea, and then corneal endothelial seed cells are inoculated on the posterior elastic layer of the constructed stromal layer part to construct the tissue engineering posterior lamellar cornea with a tissue structure and a function similar to that of a normal posterior lamellar cornea.
2. The method of in vitro constructing tissue-engineered posterior lamellar cornea of claim 1, wherein said stromal corneal seed cells and corneal endothelial seed cells are replaced by stromal corneal-like cells and corneal endothelial-like cells induced by limbal stem cells.
3. The method for in vitro construction of tissue engineered lamellar cornea of claim 1, wherein said stromal layer fraction is constructed by first rehydrating a tissue engineered corneal carrier scaffold with a carrier scaffold rehydration solution, then preparing a suspension of corneal stromal cells from human corneal stromal cells with a construct specific culture solution A, then injecting the suspension of cells into the carrier scaffold rehydrated with the carrier scaffold rehydration solution, then adding the construct specific culture solution A and then culturing in vitro, and then replacing the construct specific culture solution B and continuing culturing to obtain the stromal layer fraction of the tissue engineered lamellar cornea; preparing human corneal endothelial cells into corneal endothelial cell suspension by using a special construction culture solution C, inoculating the cell suspension on a rear elastic layer of a matrix layer part of the tissue-engineered lamellar cornea for in vitro culture, replacing the special construction culture solution C with a special construction culture solution D for culture, and then replacing the special construction culture solution D for culture to continue culture, thereby obtaining the tissue-engineered lamellar cornea with a tissue structure and a function similar to those of a normal posterior lamellar cornea;
the formula of the special culture solution A for construction is DMEM/F12 culture solution containing 15% fetal calf serum, 0.2-0.4mg/mL fibronectin and 0.01-0.02mg/mL basic fibroblast growth factor;
the formula of the special culture solution B for construction is DMEM/F12 culture solution containing 15% calf serum, 0.3-0.5mg/mL tranexamic acid, 0.1-0.5mg/mL epsilon-aminocaproic acid, 1-5mg/mL ascorbic acid and 1-5 mu g/mL matrix metalloproteinase inhibitor BB-94;
the formula of the special culture solution C for construction is DMEM/F12 culture solution containing 10% fetal calf serum, 0.2-0.4mg/mL fibronectin, 0.01-0.02mg/mL basic fibroblast growth factor and 0.01-0.02mg/mL epidermal growth factor;
the formula of the special culture solution D for construction is a DMEM/F12 culture solution containing 20% calf serum, 0.2-0.4mg/mL glucosamine hydrochloride, 0.1-0.5mg/mL epsilon-aminocaproic acid, 30-50ng/mL human bone morphogenetic protein 7, 1-5mg/mL ascorbic acid and 1-5 mu g/mL matrix metalloproteinase inhibitor BB-94.
4. The method for in vitro constructing a tissue-engineered posterior lamellar cornea of claim 3, wherein said preparation of said corneal stromal cell suspension comprises: taking human corneal stromal cells, culturing with DMEM/F12 culture solution containing 15% -20% calf serum to confluent monolayer, digesting with 0.25% trypsin solution, centrifuging to collect cell precipitate, re-suspending the cell precipitate with the special culture solution A, adjusting cell concentration to 2 × 105-6×105And (4) obtaining a homogeneous corneal stromal cell suspension by each cell/ml.
5. The method for in vitro constructing a tissue-engineered posterior lamellar cornea of claim 3, wherein said method for preparing said corneal endothelial cell suspension comprises: firstly, taking human corneal endothelial cells, and adding 15% -20% calf bloodCulturing with clear DMEM/F12 culture solution to confluent monolayer, digesting with 0.25% trypsin solution, centrifuging to collect cell precipitate, re-suspending the cell precipitate with the special culture solution C, and adjusting cell concentration to 3 × 105-5×105And (4) obtaining a homogeneous corneal endothelial cell suspension after each cell per milliliter.
6. The method for in vitro construction of a lamellar cornea after tissue engineering according to claim 3, wherein said corneal stromal cell suspension is injected into a carrier scaffold rehydrated with a carrier scaffold rehydration solution, and then cultured in vitro after adding the special culture solution A for construction, and then cultured continuously by replacing the special culture solution B for construction: sucking the corneal stromal cell suspension by a micro-injector, uniformly injecting 6-9 needles on each carrier bracket, wherein each needle is 1-2 microliters, the back elastic layer faces upwards and is placed in a 48-pore plate, then adding the special construction culture solution A, placing in a 37 ℃ and 5% carbon dioxide incubator, replacing the special construction culture solution B once every 12-24 hours, and continuously culturing for 48-96 hours to obtain the stromal layer part of the tissue engineering posterior lamellar cornea.
7. The method for in vitro constructing a tissue engineered posterior lamellar cornea as in claim 3, wherein said corneal endothelial cell suspension is inoculated onto the posterior elastic layer of the stromal part of said tissue engineered posterior lamellar cornea for in vitro culture, and then said culture solution C for construction is replaced with culture solution D for construction, and then said culture solution D for construction is replaced with new culture solution D for construction, and the culture is continued: 1mL of the corneal endothelial cell suspension is sucked and inoculated on the rear elastic layer surface of the stroma layer part of the tissue engineering posterior lamellar cornea, after 12 hours, the special culture solution C for construction is sucked out and replaced by special culture solution D for construction, the special culture solution D for construction is placed in a 5% carbon dioxide incubator at 37 ℃ and once every 12 to 24 hours, and after continuous culture is carried out for 72 to 120 hours, the tissue engineering posterior lamellar cornea is obtained.
8. The tissue engineered posterior lamellar cornea constructed in accordance with claim 1, characterized in that it is used as a replacement for a donor posterior lamellar cornea for the repair of abnormalities of the corneal endothelial and stromal layers.
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