CN115067321B

CN115067321B - Nutritional capsule for medium-long-term three-dimensional preservation of cornea tissue and preparation method thereof

Info

Publication number: CN115067321B
Application number: CN202210772669.7A
Authority: CN
Inventors: 崔文国; 赵婧; 蔡正伟; 周行涛; 张哲�
Original assignee: SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS; Eye and ENT Hospital of Fudan University
Current assignee: SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS; Eye and ENT Hospital of Fudan University
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2023-08-08
Anticipated expiration: 2042-06-30
Also published as: CN115067321A

Abstract

The invention provides a nutritional capsule for long-term three-dimensional preservation in cornea tissue and a preparation method thereof, belonging to the technical field of cornea in-vitro tissue preservation. According to the invention, the nutrient preservation solution containing sodium alginate and calcium ions are rapidly diffused and coordinated to form the three-dimensional buffer capsule, and the capsule ball structure sequentially comprises a lipid simulation layer, a water liquid simulation layer and a lubricating layer from outside to inside, so that the nutrient capsule capable of maintaining the stable state of human tears is constructed. The nutrition capsule structure is highly similar to the composition of human tear structure, and can be used for preserving cornea stroma lens, thereby playing good roles of three-dimensional preservation, nutrition supply and stable buffering. The light transmittance, cell activity and collagen fiber density of the cornea stroma lens after long-term preservation in the nutrition capsule are all superior to those of the existing cornea preservation solution, and are equivalent to those of a fresh cornea stroma lens, so that the characteristics of fresh tissues can be well maintained, and the medium-term preservation of the cornea tissues can be realized.

Description

Nutritional capsule for medium-long-term three-dimensional preservation of cornea tissue and preparation method thereof

Technical Field

The invention belongs to the technical field of cornea in-vitro tissue preservation, and particularly relates to a nutritional capsule for long-term three-dimensional preservation in cornea tissue and a preparation method thereof.

Background

Keratopathy is the second blinding disease in China, and most of patients with vision disorder are disabled due to cornea blindness, and the second is caused by cornea dysfunction. Cornea transplantation is currently the primary means of restoring vision because of the irreversibility of corneal damage. However, since the medical cornea material has limited sources, the preservation of the cornea tissue activity is limited by the type of preservation solution and the preservation time, and the demand of patients who need cornea transplantation is large, so that the cornea donor material is seriously deficient. Meanwhile, a short plate exists in cornea transplantation, so that the number and quality of cornea donors are limited, the development of surgical operation is hindered, and in addition, complications such as infection, transplantation failure, immune response and the like can occur.

Intensive studies on the structure of cornea tissue have been carried out, and the studies show that the cornea tissue structure has distinct layers and can be divided into five layers from outside to inside: an epithelial layer, a front elastic layer, a matrix layer, a rear elastic layer and an endothelial layer. The cornea preservation technology for cornea transplantation at present mainly focuses on how to maintain the activity and integrity of the endothelium, prolong the activity preservation time of endothelial cells, and has less preservation research on cornea stroma.

The review document "current state of cornea preservation method and progress" in the international journal of ophthalmology refers to a conventional method of cornea preservation, which classifies cornea preservation into short-term preservation, medium-term preservation, long-term preservation and ultra-long-term preservation. (1) For short term preservation, endothelial cell activity will decrease by 50% after 2d in wet room preservation; (2) For mid-term preservation, the cornea preservation solution can maintain the activity of endothelial cells for 4-14 d, and the activity of the cornea endothelial cells is gradually improved along with the improvement of preservation technology and the continuous change of the components of the cornea preservation solution; (3) For long-term preservation, although the organ culture method can better preserve the activity of endothelial cells and greatly reduce rejection rate after transplantation, the organ culture method has higher requirements on equipment and detection, complex operation, easy infection occurrence, higher cost and difficult popularization in the environment of the current stage in China; (4) For ultra-long preservation, the glycerol preservation method belongs to an inactive cornea preservation method, and the preserved cornea is generally only used for emergency keratoprosthesis which can not obtain fresh cornea material in a short time when lamellar transplantation or perforation and fracture injury of corneal ulcer are carried out, and is less used for penetrating keratoplasty; the deep low temperature preservation method generally reduces the temperature to-80 ℃ under the protection of a refrigerant, stores the cornea in liquid nitrogen at-196 ℃ to enable the corneal endothelial cells to be in a dormant state, and carries out proper rewarming treatment to restore the cornea activity when in use, but the method has the advantages of expensive preservation equipment, complex process, higher technical requirement and easy damage to the corneal endothelial cells during rewarming. It can be seen that the existing cornea preservation methods, no matter how long, focus on maintaining the activity and integrity of endothelial cells, and are less involved in preserving the cornea stroma.

The corneal stroma consists of corneal stromal cells (HCSCs), which are mesenchymal cells derived from the neural crest, which are sandwiched between laminae, maintaining the stromal component of the lamina connective tissue, highly organized collagen laminae providing the mechanical support and biophysical properties required for corneal transparency. Maintenance of the fibrous structure and transparency of the corneal stroma, as well as maintenance of the activity of the corneal stromal cells over time, are therefore key factors in determining the quality of preservation thereof.

Femtosecond laser small incision corneal stroma lens extraction (Small Incision Lenticule Extraction, SMILE) is a typical representative of the rapid development of the field of corneal refractive surgery in recent years, and has gained worldwide acceptance as a safe, effective, minimally invasive myopia correction laser surgery technique. The lens tissue taken out of the cornea stroma layer in the SMILE operation is called as a cornea stroma lens, the SMILE cornea stroma lens has very abundant sources, more than 600 tens of thousands of cases/eyes of SMILE operation have been carried out in more than 80 countries worldwide, tens of thousands of human cornea stroma lenses can be produced, and more than one million SMILE operation has been carried out in China each year. However, the portion of the "stromal corneal lens" removed during SMILE surgery is often discarded as surgical waste and is not effectively utilized.

The corneal stroma lens is a part of cornea tissue, is composed of orderly arranged collagen, has no blood vessel and lymph tissue, has extremely low probability of immunological rejection reaction during transplantation, can be reused as a good cornea donor material, can be used for correcting ametropia such as hyperopia, myopia, presbyopia and the like, and can be used for treating keratoconus, corneal dystrophy, corneal ulcer, corneal perforation, limbic corneal degeneration (peripheral cornea immunity, non-infectious diseases) and corneal stroma thinning and other corneal diseases, and uses the cornea stroma lens to construct a carrier of tissue engineering biological cornea and the like.

In recent years, some students have carried out research on changing the waste cornea matrix lens into valuables, and clinical preliminary results show that the SMILE lens has excellent biocompatibility and safety after implantation and has very good application prospect. However, the SMILE lenses used in these studies were only those obtained from fresh corneal tissue from a donor or from cornea that had been cryopreserved, and did not have to be transplanted after long term preservation of the corneal stroma lens. For the reuse of such a large amount of surgical wastes, whether the fiber structure and transparency of the stromal corneal lens can be effectively maintained, and how to maintain the activity of stromal corneal cells for a long time are the primary problems solved in the long-term preservation process in the stromal corneal lens.

However, the prior studies on the preservation method of the corneal stroma lens only involve the ultralow-temperature long-term preservation of the corneal lens, but the activity for the medium-term preservation with preservation solution at 4 ℃ is not good and the preservation time is short.

Chinese patent document CN 111066777A discloses a method for preserving the cornea lens at ultralow temperature for a long time, which comprises the steps of aseptically packaging the cornea lens preservation solution into an asepsis freezing tube, transferring the cornea lens into the freezing tube, then placing the cornea lens into a deep low temperature refrigerator at-60 ℃ to-80 ℃ for program cooling and overnight, and then placing the cornea lens into liquid nitrogen at-196 ℃ for long-term preservation. The method can be used for long-term preservation of the cornea lens collected by SMILE operation, but is complex, the adopted equipment is expensive, popularization and application cannot be performed, and meanwhile, the activity of the cornea stromal cells cannot be maintained for a long time, and the cornea stromal cells are in a dormant state for long-term preservation.

The experimental study of tissue engineering stromal corneal scaffolds constructed by corneal stromal lenses derived from Smile, which is a document of phyllanthus urinaria, describes that human stromal corneal cells are isolated and cultured from the corneal stromal lenses derived from Smile, the toxic effect of human fibrin adhesive (FS) on cells is detected by MTT method, and the double-layer lens scaffolds constructed by adhering FS to double-layer stromal corneal lenses are respectively stored in different media (anhydrous glycerin, sodium hyaluronate, fetal bovine serum, simulated wet room environment) and different temperatures (normal temperature, 4 ℃ and-20 ℃), and the study result is that the invention: after the cornea stroma lens bracket is stored for 14d at the temperature of 4 ℃, a certain degree of cracking phenomenon occurs, the transparency of the cornea stroma lens bracket is influenced, and besides cracking, serious shrinkage and edema phenomena occur in the cornea stroma lens bracket in a wet room environment, so that the cornea stroma lens bracket is finally obtained to be stored in the absolute glycerol at the temperature of-20 ℃ with good effect, and the stability and the transparency of the lens bracket can be maintained. This study result shows that it is difficult to achieve mid-term preservation of the stromal corneal lens under preservation conditions of 4 ℃ with conventional cornea preservation solutions.

The report shows that the cornea preservation method (such as the common preservation liquid in mid-term preservation: optisonl liquid) which is commonly used at present has a good effect on the maintenance of the activity of endothelial cells, but has a great disadvantage on the preservation of cornea stroma lenses, particularly on the maintenance of the activity of cornea stroma cells. The following disadvantages are mainly present: (1) The conventional preservation solution is only suitable for mid-stage low-temperature preservation of cornea, the preservation time is only 14 days at maximum, and the longer the preservation time is, the abnormal morphology and activity of cells can be caused, so that the long-term transportation of corneal lens tissues is not facilitated; (2) The cornea thickness becomes thin after preservation, and the maintenance of the collagen fiber structure and transparency of the lens is lacking; (3) The medium-term preservation solution takes the cell culture medium as the base solution to provide the cornea tissue with needed nutrient substances, but can not meet all the nutrient substances needed by the physiological metabolism of cornea stromal cells; these drawbacks limit the reuse and popularization of the stromal corneal lens.

Therefore, how to achieve the middle-long term maintenance of the collagen fiber structure and transparency of the corneal stroma lens and how to achieve the middle-long term maintenance of the activity of the corneal stroma cells provides a preservation method of the corneal stroma or lens tissue, which provides possibility for the middle-long term preservation and long-distance transportation of the corneal stroma lens, and becomes a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to solve the technical problems, and provides a nutritional capsule for long-term three-dimensional preservation in cornea tissues and a preparation method thereof. The technical purpose of the invention is that: on one hand, the cornea preservation solution solves the problems that the existing cornea preservation solution is mainly only suitable for medium-term preservation of endothelial cells, has shorter preservation time for cornea stroma lenses and can not realize medium-term preservation and long-distance transportation of cornea stroma lenses; on the other hand, the problem that the existing cornea preservation solution is difficult to maintain the collagen fiber structure and transparency of the cornea stroma lens and can not maintain the activity of cornea stroma cells for a long time is solved.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

the invention provides a nutritional capsule for long-term three-dimensional preservation in cornea tissue, which is of a capsule ball structure containing nutritional liquid inside, wherein the capsule ball structure sequentially comprises a lipid simulation layer, a water liquid simulation layer and a lubricating layer from outside to inside, the lipid simulation layer comprises a shell layer formed by complexing sodium alginate solution and calcium ions through diffusion coordination, and the water liquid simulation layer comprises a cornea basic culture medium and nutritional substances; the lubricating layer includes chondroitin sulfate.

The nutrition capsule provided by the invention can play a good physical barrier and buffer role on the cornea matrix lens stored in the capsule shell when being used for storing the cornea matrix lens, can realize the maintenance of the structure and transparency of the collagen fiber in the middle and long periods, can well maintain the activity of the cornea matrix cells, has an activity maintaining effect superior to that of the Optiosol preservation solution and the glycerol, and has an activity maintaining time of at least 28 days. In addition, in the long-distance transportation process, when the nutrition capsule is used for preserving cornea tissues, the damage of the cornea tissues caused by external force can be avoided, and the preservation effect is obviously better than that of the existing cornea preservation solution. The nutrition capsule is not only suitable for preserving the cornea stroma lens, but also suitable for preserving other cornea tissues.

The human bionics research shows that the natural body fluid microenvironment of the human organ is the best preservation solution for the tissue, can continuously provide fresh culture solution for tissue cells, keeps the cells to maintain sufficient and stable nutrition, simultaneously continuously eliminates metabolic wastes to enable the cells to grow in a relatively stable environment, and keeps higher cell activity and tissue complete structure. Therefore, an ideal cornea tissue preservation solution needs to have the following characteristics: 1) Can simulate the external body fluid of natural cornea tissue, meet the nutrient substances required by the physiological metabolism of cells and prevent the cells from being physiologically damaged; 2) Can maintain the tissue morphology and physiological structure of the corneal organ; 3) Can provide three-dimensional buffer for long-distance transport of cornea tissues and avoid structural damage of transplanted organs or tissues caused by external force.

The inventor creatively obtains a three-dimensional buffer capsule ball shell when sodium alginate and calcium ions are subjected to rapid diffusion coordination, and the capsule ball shell has a good three-dimensional structure and can be automatically formed. Based on the above, the inventor further carries out a great deal of research on the structure of the nutrition capsule, and after repeated fumbling, finally designs a three-layer structure of the nutrition capsule, which sequentially comprises a lipid simulation layer, a water liquid simulation layer and a lubrication layer from outside to inside, and experimental results show that the three-layer structure is highly similar to the structural composition of human tear, can well simulate the function of human tear, and realizes the medium-long-term high-efficiency preservation of cornea tissue.

The nutrition capsule structure designed by the invention is a spherical structure with a solid gel shell outside and a nutrient solution filled in an internal hollow structure, wherein the solid gel shell formed by complexing sodium alginate and calcium ions through diffusion coordination is provided with a gap; the substances such as sodium chondroitin sulfate, sodium hyaluronate and dextran are added into the capsule ball as nutrient preservation solution, and the water solution simulation layer can perform nutrient supply, substance metabolism and pathogen blocking through gaps of the shell of the capsule ball; the innermost layer is composed of chondroitin sulfate, and can generate electrostatic action with the surface of the cornea matrix lens stored in the nutrient solution (namely the water solution simulation layer) to form a lubricating layer, so that a good lubricating function is achieved. The invention constructs a novel nutrition capsule with a structure similar to that of human tears, which can be well used for medium-long-term three-dimensional preservation of cornea tissues and has multiple functions of excellent physical barrier, nutrition supply, surface lubrication and the like. After stereoscopic preservation for at least 28 days, the collagen fiber structure and transparency of the stromal lens of the cornea can be well maintained, and the activity of stromal cells of the cornea can be also highly maintained.

When the present inventors prepared the nutrition capsule to be solid, as shown in comparative example 1, it was found that the preservation effect of the nutrition capsule on the corneal stromal lens was significantly deteriorated, and the activity of the corneal stromal cells was significantly reduced, which would be disadvantageous for the medium-long-term preservation of the corneal stromal lens.

Compared with the existing cornea preservation solution, the nutrition capsule provided by the invention has the advantages that the preservation period can be prolonged to at least 28 days, the aims of long-term preservation and long-distance transportation of cornea tissues are well realized, meanwhile, the three-dimensional capsule shell structure can have good physical blocking and buffering effects on cornea tissues preserved inside, the cornea tissues are not damaged due to external force even in the long-distance transportation process, and the preservation effect is obviously better than that of the existing cornea preservation solution.

After the nutrition capsule is used for long-term preservation of the cornea tissue lens (preservation is carried out for 28 days), the preserved cornea tissue lens is used for cornea lens transplanting operation, and good clinical effects are obtained for treating diseases such as cornea malnutrition, keratoconus and the like, and the safety and the effectiveness are good. It can be seen that the present invention provides a simple, safe, controllable optimization strategy for preservation, transportation and reuse of the stromal corneal lens. Meanwhile, the nutrition capsule can be used for medium-long term preservation of other cornea isolated tissues or organs, and the effect of the nutrition capsule is superior to that of the existing cornea preservation solution.

Further, the spherical diameter of the cornea tissue three-dimensional preservation nutrition capsule is 1-20mm, and the thickness of the shell of the nutrition capsule is 10-500 mu m. The size range can well meet the preservation requirement of cornea tissues required by clinic, and meanwhile, the size of the nutrition capsule and the thickness of the shell layer can be individually adjusted according to actual needs, so that the nutrition capsule can be realized and controlled in terms of technology.

Further, the cornea tissue to be preserved is preserved inside the capsule ball outer shell of the nutrition capsule. The corneal tissue comprises corneal stroma, stromal lens, corneal endothelium, or corneal epithelium. The cornea tissue comprises a cornea stroma lens, and can also comprise other cornea isolated tissues such as cornea stroma, cornea endothelium, cornea epithelium and the like, and also comprises other isolated organ tissues. Typically corneal tissue is maintained in an aqueous simulated layer or in a lubricating layer.

Further, the nutrient substances in the cornea tissue three-dimensional preservation nutrient capsule comprise sodium chondroitin sulfate, sodium hyaluronate, dextran and the like, other commonly used nutrient substances in cornea preservation liquid such as sodium alginate, sodium ascorbate, trehalose, dextran, polyvinyl alcohol, chitosan, polyethylene glycol and the like can be added, and the nutrient substances can be flexibly selected.

Further, the cornea basal medium added in the cornea tissue stereoscopic preservation nutrition capsule of the invention can comprise DMEM/F-12, DMEM, MEM or DME, and other components such as diabody and the like can be added in the medium.

The invention also provides a preparation method of the nutritional capsule for long-term three-dimensional preservation in cornea tissue, which comprises the following steps:

(1) Adding chondroitin sulfate into the cornea basic culture medium, and then adding nutrient substances for mixing to form an aqueous solution simulation layer and a lubricating layer;

(2) Adding sodium alginate into the product obtained in the step (1) to form a pregel solution;

(3) And adding calcium ions into the pregelatinized solution, forming a lipid simulation layer after gelling, and obtaining the nutritional capsule for long-term three-dimensional preservation in the cornea tissue.

More specifically, the preparation method of the nutritional capsule provided by the invention comprises the following steps:

(1) Taking a cornea basic culture medium as a mother solution, adding 0.1-3% of chondroitin sulfate, 1% of diab, 0.01-0.5% of sodium ascorbate and 0.2-2% of trehalose into the mother solution, and completely dissolving the substances to obtain a water liquid simulation layer and a lubricating layer;

(2) Adding sodium alginate into the product obtained in the step (1) until the final concentration is 0.5-3 wt% to form a pregel solution;

(3) Dropwise adding 50-300 mM CaCl into the pregel solution ₂ The aqueous solution is glued for 10-280 s, and the nutritional capsule for long-term three-dimensional preservation in the cornea tissue is obtained。

The invention makes the nutrient preservation solution containing sodium alginate and calcium ion quickly diffuse and coordinate to form a three-dimensional buffer capsule, and the chondroitin sulfate and the surface of the cornea matrix lens generate electrostatic action to form a lubricating layer, thus playing the roles of three-dimensional preservation and nutrient supply. Experiments show that the three-layer structure of the lipid simulation layer, the water liquid simulation layer and the lubricating layer can be well obtained by adopting the gel forming time of between 10 and 280 seconds, and the three-layer structure is highly similar to the structural composition of human tears.

Further, the concentration of sodium alginate in the pregelatinized solution in step (2) of the present invention is 1wt%. The inventor researches find that the viscosity of the pregel obtained by adopting the sodium alginate solution with the concentration in the step (2) is optimal, the obtained nutrition capsule has the best supporting property, and the obtained stereoscopic spherical capsule shell has the best uniformity. Corresponding nutritional capsules can also be obtained with sodium alginate of other concentrations, but the support and uniformity of the capsule shell are not as good as at 1% concentration.

Further, the CaCl in step (3) ₂ The concentration of the aqueous solution was 100mM and the gel time was 60s. The research of the inventor shows that under the above gel forming time, the obtained nutrition capsule has the best thickness, the stereoscopic spherical capsule has the best stability, and the degradation time is only 10-13min, thereby more meeting the requirements of clinical practical operation.

Further, the products obtained in the step (1) to the step (3) are subjected to sterilization treatment, for example, a filter membrane with the wavelength of 450nm and a filter membrane with the wavelength of 220nm can be adopted for filtration sterilization to realize sterilization.

Further, the step (2) further comprises the step of placing the cornea tissue in a pregelatinized solution, and then performing the operation of the step (3) for wrapping the cornea tissue such as a corneal stroma lens inside the nutrition capsule for medium-long-term stereoscopic preservation.

The beneficial effects of the invention are as follows:

(1) The nutrient preservation solution containing sodium alginate and calcium ions are rapidly diffused and coordinated to form the shell of the three-dimensional buffer capsule ball, and the nutrient capsule structure capable of maintaining the stable state of human tears is further constructed, and the nutrient capsule has a structure similar to the composition of the human tears in structure, is used for preserving cornea matrix lenses, and plays a good role in three-dimensional preservation, nutrient supply and stable buffer; the light transmittance, cell activity and collagen fiber density of the cornea stroma lens after 28 days of preservation of the nutrition capsule can be well maintained, the nutrition capsule is obviously superior to the existing cornea preservation solution, is equivalent to that of a fresh cornea stroma lens, can well maintain the characteristics of cornea tissues, and can realize long-term preservation.

(2) The cornea matrix lens preserved by the nutrition capsule is used for treating complex ametropia or cornea diseases by transplanting human eye allogeneic lenses, the cornea matrix lens is safe and effective, the implanted lens is kept highly transparent, no shift and rejection phenomenon exists, and the optimal corrected vision of the postoperative eye of 70% of 3 months after the operation is increased by at least two rows compared with that before the operation. The nutrition capsule not only can provide a simple, safe and controllable strategy for three-dimensional preservation, transportation and reuse of cornea tissues, but also can provide a new idea for preservation of other living tissues or organs.

Drawings

FIG. 1 is a schematic representation of the preparation and characterization of a stereoscopic nutritional capsule with a highly similar structure to human tear fluid; a) Preparation and design of a three-dimensional nutrition capsule coated cornea tissue: 1. placing the stromal corneal lens directly into a 200 μla solution; 2. sucking out the solution A containing the micropores by using a 1mL pipetting gun; 3. transferring the solution A containing the stromal corneal lens into 3mL of solution B using a 1mL pipette; 4. forming hydrogel nutrition capsules to embed cornea tissues; 5. adding 3mL of culture solution containing nutrient solution, and preserving the nutrient capsules at 4 ℃; b) Photographing a physical object of the nutrition capsule embedded human cornea matrix lens; c) Viscosity and spherical efficiency of sodium alginate gel solutions with different concentrations; d) The thickness of the nutritional capsules at different gel times; e) Initial degradation time and complete degradation time of the nutritional capsule; f) Photography of the complete degradation process of the nutritional capsules; g) Observing the adsorption condition of the chondroitin sulfate on the lens by a confocal microscope; h) Embedding the human cornea stroma lens section view in the nutrition capsule; i) The shell structure and physical appearance observed by Scanning Electron Microscopy (SEM); j) Releasing live/dead staining images of cells after 5 days of culture in the nutritional capsules; k) The survival rate of CCK-8 cells cultured for 3d and 5d in the nutrition capsule group and the blank control group; con: blank control group; NC: a nutrition capsule group.

FIG. 2 is a diagram showing the cornea stroma lens (left) and different thickness and Ca in culture solution ²⁺ A nutrition capsule (right) with gel forming time, wherein the nutrition capsule after 300s of gel forming is of a solid structure; b, chart shows the live/dead staining of cornea lens preserved by nutrient capsules with different gel forming time between 3 days and 7 days; panel c shows quantitative analysis of live/dead staining of the corneal lenses preserved in the nutritional capsules at different gel formation times of 3 days and 7 days.

FIG. 3 shows the results of TUNEL staining and DAPI staining of nuclei of the corneal lenses preserved by different methods.

Fig. 4 is the average percentage of TUNEL positive cells for each treatment group.

FIG. 5 shows the live/dead staining of the stromal lenses of the cornea of each treatment group, with live cells (green) and dead cells (red) incubated with solutions of Calcein-AM (live cells) and homodimer-1 (dead cells), respectively.

FIG. 6 is an average percentage of live and dead cells in each treatment group; con: blank control group; NC: nutritional capsule groups, data expressed as mean standard deviation, P <0.05, P <0.0001.

FIG. 7 is a graph showing tissue structure and transmittance of a corneal stromal lens preserved in a nutritional capsule; a) HE staining, b) Masson staining, c) VG staining, showing histological changes of the corneal lenses at different storage times in the control and experimental groups; d) Observing the cross section of each group of collagen fibers by a transmission electron microscope; e) Average transmittance of each group of lenses; f) Comparison of collagen fiber density between control and experimental groups at 7d, 14d, 28 d; con: blank control group; NC: nutritional capsule groups, data expressed as mean standard deviation, P <0.05, P <0.0001.

FIG. 8 comparison of pre-operative and post-operative 1 month slit lamps with anterior ocular segment optical coherence tomography (AS-OCT) for nutrition capsule preservation of corneal lenses in patients with corneal disease.

In fig. 9, a is a schematic diagram of structural composition of human tears, and b is a schematic diagram of structural composition of a nutritional capsule.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be specifically described with reference to the following examples, which are provided for explaining and illustrating the present invention only and are not intended to limit the present invention. Some non-essential modifications and adaptations of the invention according to the foregoing summary will still fall within the scope of the invention.

Example 1

The embodiment provides a preparation method of a nutrition capsule, which comprises the following steps:

1. preparation of initial solution

Firstly, taking a cornea basic culture medium DMEM/F-12 (the components of which are 45% DMEM and 45% F-12) as a mother solution, adding chondroitin sulfate, diabody and trehalose to make the final concentration of the solution be 2.5wt%, 1wt% and 0.5wt%, after the solution is completely dissolved, adding sodium alginate to make the final concentration of the sodium alginate be 1wt%, and performing the above operations under aseptic conditions, and after the solution is completely dissolved, filtering and sterilizing the solution by using filter membranes of 450nm and 220nm respectively to obtain a component A solution.

Subsequently, 100mM CaCl was prepared with sterile water ₂ Aqueous solution (B component solution) and 100mM sodium citrate aqueous solution (C component solution), and after dissolution, filtration-sterilization was performed through 450nm and 220nm filters.

2. Preparation of nutrient capsules

The 1mLA component solution was removed and added dropwise to 3mL of the B component solution, allowed to stand for gel formation for 60s to form a hydrogel capsule, followed by removal of the surplus B component and rapid washing with 3mLPBS for 3 times to remove the surplus B component, to obtain a nutritional capsule.

3mL of the nutrient-containing solution was added as a medium, and the medium was stored at 4℃for further use.

Example 2

A nutritional capsule was prepared according to the method of example 1, wherein the concentration of chondroitin sulfate, diabody, trehalose was sequentially adjusted to 0.1%, 0.5%, 0.2%, while 0.01% sodium ascorbate was added, and after dissolution, 0.5% sodium alginate was added thereto, with the other method unchanged.

Example 3

A nutritional capsule was prepared according to the method of example 1, wherein the concentration of chondroitin sulfate, diabody, trehalose was sequentially adjusted to 3%, 2%, 1%, while 0.3% sodium ascorbate was added, and 3% sodium alginate was added thereto after dissolution, the other method being unchanged.

Comparative example 1

In patent document CN 104046587A, a calcium alginate microgel bead (which is a solid structure) containing chondroitin sulfate is reported, so this comparative example has searched for the preparation of a nutrition capsule into a solid structure for preserving a corneal stroma lens, and the specific preparation method of the solid nutrition capsule is as follows: referring to the preparation method of example 1, when a pregelatinized solution containing a corneal stromal lens was added to a calcium ion solution, the capsule became solid when the gel time was prolonged to 300 s.

The solid nutritional capsule obtained after 300s of gel formation was used for preservation of the corneal stroma lens, and the long-term preservation effect thereof was examined.

Comparative example 2

The preservation solution described in example 4 of patent document CN 113907066a (in 1000mL of preservation solution: 38mg of chondroitin sulfate, 4.5g of HEPES buffer, 40mg of tobramycin, 200g of glutamine, which is a cell nutrient component, and the balance being MEM culture solution containing an acid-base modifier, pH 7.6) was used for medium-long term preservation of a corneal stromal lens, and the effects of the preservation solution on the fiber structure and transparency of the corneal stromal lens and the activity of the corneal stromal cells after 14 days and 28 days of preservation were examined.

Comparative example 3

The preservation method (a colorless transparent gel-like solution composed of 3% sodium hyaluronate and 4% sodium chondroitin sulfate and physiological buffer balance salt) employed in the examples in patent document CN 110384088A was used for medium-long term preservation of a corneal stromal lens, and the effects of the preservation method on the fibrous structure and transparency of the corneal stromal lens and the activity of the corneal stromal cells after 14 days and 28 days of preservation were examined.

Comparative example 4

The preservation solution described in the example of patent document CN 104094925B (comprising 200g/L of glycerin, 5g/L of hyaluronic acid, 25g/L of chondroitin sulfate, 5g/L of N-acetylcysteine as an antioxidant, and 30mmol/L of phosphate buffer) was used for medium-long term preservation of a corneal stromal lens, and the effects of the preservation method on the fiber structure and transparency of the corneal stromal lens and the activity of the corneal stromal cells after 14 days and 28 days of preservation were examined.

Comparative example 5

The preservation solution formulation described in the example of patent document CN 109699631A (2.0 g of low melting agarose and 0.1g of dextran (molecular weight 40 kDa) were dispersed in 80ml of 0.2mol/L PBS buffer, heated and stirred until the mixture was completely swelled, 0.1g of glycerin was added, stirred uniformly, and the PBS buffer was diluted to 100ml to a final pH of 7.2 to 7.4) for medium-long term preservation of a corneal stromal lens, and the effects of the preservation method on the fiber structure and transparency of the corneal stromal lens and the activity of the corneal stromal lens after 14 days and 28 days of preservation were examined.

Positive control example 1

Glycerol is used as preservation solution for medium-long term preservation of corneal stroma lens, and is used as positive control group I.

Positive control example 2

Optisonl-GS (Bausch & Lomb Inc., bridgewater, NJ, USA) was used as a preservative fluid for medium-long term preservation of stromal corneal lenses as a positive control group two.

Positive control example 3

DMEM medium (Gibco, thermo Fisher Scientific, USA) was used as a preservation solution for medium-long term preservation of the stromal corneal lenses as a positive control group three.

Experimental example 1

The nutritional capsules prepared in the examples were physically characterized and the effect of different preparation conditions on the nutritional capsules was examined

Characterization method

(1) The viscosity of the sodium alginate gel mother solution with different concentrations is measured by a rheometer (TA-DHR-2), and the balling effect of the mother solution with different viscosities is observed by photographing.

(2) Rhodamine is added into the component B, and laser confocal is used for detecting the formation thickness of capsules with different gel forming times.

(3) The initial degradation time and the complete degradation time of the nutrition capsule are monitored in real time through in-vitro photographing observation and a microscope, and the three-dimensional structure and the physical appearance of the nutrition capsule are observed through GelMA photosensitive gel embedding cutting and SEM scanning electron microscope. In addition, chondroitin sulfate (Chs-Rho) labeled with Rhodamine B isothiocyanate (rhodamine B isothiocyanate) was incubated with the lens for 1 hour, and washed with physiological saline for 2 hours to wash off unadsorbed Chs-Rho, and observed with a laser confocal (zeiss, germany) lens.

(II) characterization results

1. A nutritional capsule was prepared according to the method of example 1, and the effect of sodium alginate concentrations of 0.5%, 1%, 1.5% and 2% on the stereoscopic sphericity of the nutritional capsule was examined, and the results showed that: sodium alginate solution with concentration of 1% is more uniform than 0.5%, 1.5% and 2% concentration; although the initial viscosities of the 2% and 1.5% pregel solutions were found to be higher from the viscosities of the pregel solutions, reaching 4.27 Pa.S and 1.17 Pa.S, respectively, and easy to form teardrop capsules, the lower viscosity of the 0.5% pregel solution was only 0.059 Pa.S, and the resulting capsules were less supportive, but the 1% concentration pregel solution was more uniform in sphering. Therefore, sodium alginate with concentration of 1% is used as the pregel solution in the subsequent experiments.

2. Rhodamine is added to the calcium ion solution and a confocal microscope is used to observe the relationship between the calcium ion permeation time and the thickness of the formed capsules. As can be seen from the d plot in FIG. 1, a film thickness of about 224.26 μm was formed when the pregelatinized solution was contacted with the calcium ion solution for 10s, and continued to increase over time, reaching 303.44 μm when the gel was formed for 60 s.

The initial degradation time and the final degradation time of the nutritional capsules in 100mM sodium citrate solution were further examined, and the overall degradation process of the gelled 60s nutritional capsules was examined by in vitro rough photographs and photomicrographs. From panels e and f of FIG. 1, it can be seen that the surface of the nutritional capsule began to crack at 7min, the solution slowly oozed out of the capsule for 9min, and the capsule was substantially degraded for 13 min. Compared with other crosslinking time, the crosslinked 60s gel is more stable, and the degradation operation time is more in line with the clinical practical operation.

3. For more visual observation of the capsule structure, the nutrition capsules were embedded in 20% photosensitive GelMA solution by 10mW/cm ² 365 The nm UV light source was crosslinked for 2min to allow the gel solution to fully solidify and separated by a surgical blade, as shown in the h plot of FIG. 1, the shell structure of the microspheres was clearly seen, and the corneal lens remained in its original structure. In addition, the inner wall of the capsule and the structure of the capsule were observed by SEM, and it can be seen from the i-plot in fig. 1 that the inner wall of the capsule exhibits a concavo-convex wrinkled structure, and the shell thickness was about 24 μm.

4. To verify the electrostatic adsorption of chondroitin sulfate to lenses, lenses were immersed in rhodamine (Rho) -labeled chondroitin sulfate Chs-Rho, and it was found from the g-plot in fig. 1 that Chs (chondroitin sulfate) had a significant adsorption to lenses, and that Chs were able to penetrate the entire lenses as clearly seen in the 3D plot.

5. To verify the biocompatibility of the nutritional capsules, human Corneal Stromal Cells (HCSCs) were encapsulated in the nutritional capsules, released into the culture medium for 3 and 5 days of in vitro culture after one week of preservation, and the effects of nutritional capsule preservation on cells were examined by live staining and CCK8 experiments. The results of the live-dead staining in the j-plot in FIG. 1 show that the vast majority of cells survived the 3 and 5 day culture process with no statistical differences between groups. Consistent with the results of live-dead staining, CCK8 experimental results showed that each group of cells continued to proliferate during 5 days of in vitro culture, and that the differences in cell viability between day 3 and day 5 of the experimental group and the control group were not statistically significant (p=0.59) and (p=0.08) (k panels in fig. 1). These results demonstrate that the nutritional capsules have good biocompatibility and can provide a good three-dimensional storage environment for the corneal tissue lens.

Experimental example 2

A nutrition capsule was prepared as described in example 1, and different gel forming times (30 s, 60s, 120s, 180s, 300 s) were set to examine the effect of different gel forming times on the structure of the nutrition capsule and on the preservation effect of the activity of the keratinocytes.

The experimental method comprises the following steps: the corneal stroma lens extracted by SMILE surgery was placed in 200. Mu.LA solution, and then solution A containing the corneal stroma lens was transferred into 3ml solution B using a 1ml pipette to form a hydrogel nutritional capsule. The effect of different gel formation times on human corneal stromal cell activity was observed. The corneal stromal lenses were encapsulated in nutritional capsules of different gel formation times (30 s, 60s, 120s, 180s, 300 s), excess solution B was removed, and the hydrogels were washed 3 times with 3ml fbs. 3mL of cornea culture medium containing nutrient components was added, and the nutrient capsules were stored at 4 ℃. Staining was performed with Live/read kit (japan Dojindo Laboratorise) and photographed on days 3 and 7 using fluorescence microscopy (zeiss germany). The nutrient capsules with different gelling time are placed in a culture solution, and are respectively stored for 3 days and 7 days at the temperature of 4 ℃ and then are stained by Live/read. The experimental results are shown in FIG. 2.

(II) experimental results: as can be seen from the results of fig. 2, the number of viable cells of the corneal stroma lens in the nutritional capsule with the gel time of 60s is significantly greater than that of the cornea stroma lens preserved in the other nutritional capsule with the gel time, and the characteristics of the cornea stroma lens preserved by the method are significantly better than those of the cornea stroma lens preserved in the solid capsule. The main reasons are analyzed: 1) The longer the crosslinking time, the higher the calcium ion concentration, the greater the toxicity to cells; 2) The longer the crosslinking time, the more obvious the shrinkage of the capsule shell, and the physical tension in the crosslinking shrinkage process is the main reason for influencing the cell activity and the cornea light transmittance; 3) As the crosslinking time increases, the degradation time increases, resulting in a gradual decrease in the number of viable cells within the corneal stroma lens, and the corneal collagen structure is easily destroyed.

Experimental example 3

The experimental example examines the preservation effect of the nutrition capsule and different preservation methods after the cornea stroma lens is preserved for a medium and long time.

Experimental methods

1. Human cornea stroma cell culture

Experiments were performed using Human Corneal Stromal Cells (HCSCs) grown in DMEM/F-12 medium with a 10% mixture of antibiotics (diabodies) of fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin. Wherein the HCSC is derived from the department of otorhinolaryngology of the university of Compound denier of China, and the DMEM/F-12, fetal bovine serum, penicillin and streptomycin are all derived from Gibco, thermo Fisher Scientific, USA.

2. Nutritional capsule microbiological compatibility test

The cell-containing suspension (5X 10) was injected through a 1mL syringe ⁴ Cell concentration) was slowly dropped into 100mM calcium chloride solution, crosslinked for 1min to form cell gel microbeads, washed with DMEM/F-12 basal medium, and resuspended in 1mL fresh medium. The vials containing the calcium alginate coated cells were refrigerated at 4℃for 7 days, after storage for 7 days, the calcium alginate beads coated cells were washed with PBS and then added to the C-component solution to dissolve, after 10min of dissolution, centrifuged at 1000 rpm for 5 min and the deposited cells were resuspended in medium. In addition, the calcium alginate microbeads free of cells were stored in a complete medium containing nutrients, stored for 7 days, and the calcium alginate microbeads were removed and then the cells were cultured with a soak solution. Cells were cultured with complete medium for 3 and 5 days after release of calcium alginate-coated cells or with calcium alginate soak for 3 and 5 days, 3X 10 total cells per well ⁴ Individual cells were seeded in 96-well plates; CCK8 detection (Biyun biotechnology, china) was performed using a microplate reader (Tecan,the Optical Density (OD) was measured by Switzerland and the differently treated cells were stained with Live/read kit (Dojindo Laboratorise, japan), observed and photographed using a fluorescence microscope (zeiss, germany).

3. Biological property study of nutrition capsule-coated cornea stroma lens

(1) Comparison of different preservation methods of corneal stroma lenses

The nutrition capsules obtained in the examples of the present invention were used to preserve the stromal corneal lenses and compared with the methods in the comparative examples and the positive comparative examples to examine the long-term preservation effect of various preservation methods on stromal corneal lenses. The specific method comprises the following steps:

experiments were performed in different groups, positive control group one: 99% anhydrous glycerin (positive comparative example 1); positive control group two: optisonl-GS (positive comparative example 2); positive control group three: DMEM medium (positive comparative example 3); experimental group (taking the nutritional capsules obtained in example 1 as an example). The cornea stroma lens which needs to be preserved is taken out, washed twice in Phosphate Buffer (PBS), randomly divided into different groups, added into the preservation solution for preservation, and placed at 4 ℃ for 7 days, 14 days and 28 days respectively.

Fresh lenses (within 1 hour of post-operative removal) were used as blank control groups and compared to these stored corneal tissue lenses. And compared with the schemes of comparative examples 1 to 5, respectively.

(2) Corneal stromal cell activity assay

Apoptotic cells were detected IN each group of corneal tissue lenses under different storage conditions, and the IN situ cell death detection kit (Roche Applied Science, indianapolis, ind.) was used according to the instructions using the TUNEL method. Lenses were stained with Live/read kit (japan Dojindo Laboratories), and observed and photographed using a fluorescence microscope (zeiss, germany).

(3) Light transmittance and microstructure observations

Each set of lenses was then placed in a 96-well plate and surrounding liquid was blotted with a triangular sponge. Absorbance measurements were performed using a microplate reader (Tecan,swiss). The absorbance (A) is measured under the visible light wavelength, the range is 380nm to 780nm, a detection point is taken every 10nm, the absorbance of a continuous multi-point detection sample is repeatedly measured for 3 times, the numerical value is recorded, a blank pore plate is selected as a contrast, and the calculation formula of the transmittance (T) is as follows: t=10 ^-A 。

Fresh or stored lenses were removed and then rapidly cut into small pieces (1 mm ³ ) These sections were fixed at 2.5% glutaraldehyde for 2 hours and after fumigation the samples were burned with 0.1M osmium tetroxide for 0.5-2 hours to allow complete carbonization. Rinsing with 100% acetone for 5min, mixing with 100% acetone and epoxy resin solution, and standing at room temperature for 15min. The sample is placed in an oven And covering the surface of the sample with tissue surface acetone at 38 ℃ for 2-3 hours, adding mixed solution of epoxy resin and 100% acetone, placing the sample in a 38 ℃ oven for 2-3 hours, removing the mixed saturated solution as much as possible, replacing the mixed saturated solution with pure epoxy resin, and placing the sample in the 38 ℃ oven for 1 hour. Ultra-thin sections (70 nm) were cut using the Leica EM-UC7 (Leica biosystems, berlin, germany), and specimens were double stained with 3% uranyl acetate and lead citrate, followed by observation and imaging under a 120kV Tecnai G2 Spirit BioTWIN electron microscope (FEI, portland, US).

(4) Histology and immunohistochemical detection

The corneal lens sections were HE stained for histopathological observation, van Gieson (VG) stained and Masson stained for collagen structure observation, and immunohistochemistry was performed to examine HLA-DR, HLA-ABC and CD45 expression.

(II) results of experiments

1. Corneal stromal cell Activity assay after storage of each treatment group

The number of apoptosis and survival in the lenses after storage of each treatment group was observed using TUNEL and live dead staining. The experimental results of the experimental group, the positive control group and the blank control group are shown in fig. 3 to 6.

As can be seen from FIG. 3, tunel-positive cells were seen in each of the groups of lenses stored differently for 7 days, 14 days and 28 days under the conditions of storage of each of the treatment groups. After 7 days of storage, the experimental group (NC, nutrient capsule group) had no significant difference in Tunel positive cell rate compared to the blank control group (Con), whereas Tunel positive cell rates were significantly increased in positive control group one (Gan Youzu) (P < 0.0001), positive control group two (optisuol group) (p=0.018), and positive control group three (DEME group) (P < 0.0001). Day 14 and day 28, tunel positive cell rates were significantly higher for each group than for the blank (P < 0.0001) (fig. 4).

Figures 5 and 6 show that the results of dead-alive staining support the results of TUNEL analysis, indicating that more viable cells were present in the corneal tissue lens after nutrient capsule preservation compared to other preservation methods over different preservation times of 7-28 days. When the glycerol group is stored for 7 days, the living cells of the glycerol group are obviously reduced (P < 0.0001) compared with the blank control group, and the other groups have no obvious difference; when the cells were stored for 14 days, the live cells of the DMEM group (P=0.02) and the glycerol group (P < 0.0001) were significantly reduced compared with the blank control group, and the nutrition capsule group and the Optiosol group have no significant difference compared with the blank control group; when stored for 28 days, the nutrition-only capsule group had no significant difference from the control group, and the viable cells of the optissol group (p=0.03), DMEM group (P < 0.0001) and glycerol group (P < 0.0001) were significantly reduced compared to the blank control group.

Gan Youzu is prone to tissue edema after thawing. Optissol is capable of preserving corneal endothelial cells at 4 ℃ for up to 2 weeks, but has limited longer term preservation applications. DMEM has a single nutrient component, and it is difficult to maintain the ultrastructural and light transmittance characteristics of the natural corneal stroma collagen for a long period of time. The nutritional capsule group of the invention obviously reduces the storage cost, improves the survival rate of cells in a low-temperature storage state, and can store cornea tissues for a medium and long time.

The preservation protocol of the comparative example was further examined and the results of comparative examples 1 to 5 for medium-long term preservation of the stromal corneal lenses were compared with the nutrition capsule group (example 1), and the experimental results are shown in table 1 below.

TABLE 1 Effect of different preservation methods on cornea preservation Effect

Note that: mild oedema/cavitation bubbles: < 30% oedema/cavitation compared to fresh tissue;

moderate oedema/cavitation bubbles: 30% -50% oedema/cavitation compared to fresh tissue;

severe edema/cavitation bubbles: > 50% edema/cavitation occurred compared to fresh tissue.

As can be seen from Table 1, the preservation methods of comparative examples 2 to 5 were used for preservation of the stromal corneal lens, and after long-term preservation, the transparency of the stromal corneal lens and the activity of stromal corneal cells were both significantly reduced, and the fibrous structure of the stromal corneal lens was also significantly destroyed, and the preservation effect was significantly lower than that of the nutrition capsule preservation group.

2. Cornea lens tissue structure and light transmittance detection after preservation of nutrition capsules

And detecting the light transmittance of the lens by using an enzyme-labeled instrument. As can be seen from panel e in fig. 7, the average light transmittance value of the glycerol group was significantly reduced (p=0.013) after 7 days compared to the control group, and the optiosol, DMEM and nutrient capsule groups maintained lenses with no significant difference from fresh lenses. The difference of the lens light transmittance after 14 days of storage of the nutrition capsule group compared with the control group is not statistically significant (P=0.12), the light transmittance of other 3 groups is lower than that of the control group, and the difference is statistically significant (P < 0.05). At 28 days, each group had significantly lower light transmittance than the control group (all P < 0.05).

In addition, HE, MASSON, VG staining and transmission electron microscopy were used to observe the protective effect of the nutritional capsule on the lens tissue structure. HE. The Masson and VG staining structures show that the fresh lens cornea collagen fibers are regularly arranged under a light microscope, the cornea denatured tissues with linearly deep stained edges are visible, and obvious damage is not seen in the deep parts of the tissues. After the preservation for 7 days, the lens collagen fibers preserved by the nutrition capsule group and the Optiosol group are arranged in parallel, and are regular and clear in boundary. Gan Youzu and DMEM group part of the collagen fibers showed edema, and a small amount of oval cavitation bubbles were seen in the middle zone of the lens. After 28 days of storage, the surfaces of the nutrient capsule group and the Optiosol group lens are relatively smooth and slightly irregular, a small amount of oval cavitation bubbles can be seen in the middle area of the lens, and part of bubbles are mutually communicated. The edges of the DMEM group and the glycerol group lens are rough, collagen tissues are loose and disordered as a whole, fibers are arranged in an unparallel mode, large cavitation bubbles are visible in the center, and partial cavitation bubbles are mutually fused.

The lens collagen fiber density was measured using a transmission electron microscope. The d plot in fig. 7 shows that there was no significant difference in collagen fibril diameter of the lenses between all the preservation experimental groups and the control group. After 7 days and 14 days of storage, the average corneal collagen fibril numbers of all experimental groups were not significantly different from that of the control group (P > 0.05). After 2 days of preservation, the average number of corneal collagen fibrils in all the preservation experimental groups is obviously reduced compared with that in the control group, and the difference is statistically significant. At day 28, the mean fiber density was significantly higher in the nutritional capsule group than in the DMEM group (p=0.016) and the glycerol group (P < 0.001), with no significant difference compared to the optiosol group (f-panel in fig. 7). Compared with other experimental groups, the nutrition capsule is more compact in arrangement of cornea collagen fibers of the lens group, fewer in cavitation bubbles and clear in lens edge, and is beneficial to maintaining original collagen fiber structure and transparency of the cornea lens for a long time.

The results prove that the cornea lens tissue after the nutrition capsule is preserved has good light transmittance and tissue structure.

3. Corneal stroma lens immunogenicity detection after preservation of nutritional capsules

Experiments show that HLA-DR, HLA-ABC and CD45 are less expressed or not expressed after each lens group is preserved, which indicates that the immunogenicity of the corneal stroma lens is reduced or even eliminated after the lens group is preserved.

Experimental example 4

The experimental example examined the effect of the corneal stroma lens stored in the operation and maintenance capsule for treating keratopathy by the corneal lens transplantation operation.

Experimental methods

The method for transplanting and treating 10 cases of keratopathy patients (including corneal dystrophy, corneal degeneration and laser postoperative corneal dilatation of myopia) by adopting the lenses stored by wrapping the nutrition capsules comprises the following steps:

before lens implantation surgery, the nutrient capsules placed in the culture medium are taken out, the lens capsules are soaked in 3mL of C solution to dissolve the capsule shells, the capsule shells are washed 3 times by 3mLPBS, the stored donor cornea lenses are released from the nutrient capsules, the lenses are taken out, washed by PBS, and then washed by normal saline for use.

The postoperative follow-up time is 1 day, 1 week, 1 month and 3 months, respectively, and the examination comprises slit-lamp biological microscope, uncorrected Distance Vision (UDVA), subjective refraction, corrected Distance Vision (CDVA), corneal topography Pentacam anterior ocular segment tomographic scan (Oculus) ) And anterior ocular segment AS-OCT.

(II) results of experiments

Lenses stored in nutrition capsules for 28 days were removed and subjected to lens allograft for the treatment of corneal disease. Firstly, culturing microorganisms on a part of preserved lenses, and performing lens transplantation operation after confirming that the preserved lenses are not polluted by fungi and bacteria. In this experiment, 10 patients receiving lens allografts were enrolled, and 9-eye PTK-EP surgery, 1-eye LIKE surgery, was performed in 10 patients. All patients completed a follow-up for at least 3 months after surgery. Patients were blindly visited during and after surgery, and had no complications or immune rejection.

From FIG. 8, it is shown that the corneal epithelium starts to grow 1 day after PTK-EP surgery and the transplanted lens adheres well. 1 month after the operation, the cornea epithelium has been remodeled, and the lens remains in good condition. During the post-operative follow-up, the preservation lenses used in the surgery remain transparent. AS-OCT shows that the implanted lens shows low reflectivity and visible demarcation throughout the follow-up period. The implanted lens has no displacement, no wrinkles, and is tightly adhered to adjacent tissues. During the follow-up, the tissue density around the lens increases and the boundary between the lens and the original corneal tissue becomes progressively blurred. Taken together, these results demonstrate that lens reuse after storage in hydrogel nutritional capsules for allograft has good safety and efficacy.

As shown in fig. 9, it can be seen that the structure of the nutritional capsule constructed by the invention has high similarity with the structural composition of human tear, and has the function of maintaining the stable state of human tear when being used for preserving human cornea tissues such as cornea stroma lens, thus having multiple functions of good physical barrier, nutrition supply, surface lubrication and the like and being capable of realizing the medium-long-term preservation of cornea stroma lens.

Claims

1. The nutritional capsule for the long-term three-dimensional preservation of the cornea tissue is characterized by being of a capsule ball structure containing nutritional liquid inside, the capsule ball structure sequentially comprises a lipid simulation layer, a water liquid simulation layer and a lubricating layer from outside to inside, the lipid simulation layer comprises an outer shell layer formed by complexing sodium alginate and calcium ions through diffusion coordination, and the water liquid simulation layer comprises a cornea basic culture medium and nutritional substances; the lubricating layer comprises chondroitin sulfate; the diameter of the balls of the nutrition capsule is 1-20mm, and the thickness of the shell of the nutrition capsule is 10-500 mu m.

2. The long term stereoscopic preserving nutritional capsule in corneal tissue of claim 1, wherein the corneal tissue is preserved within a capsule globus-outer shell of the nutritional capsule, the corneal tissue comprising a corneal stroma, a stromal lens, a corneal endothelium or a corneal epithelium.

3. The stereoscopic preserving nutritional capsule for corneal tissue according to claim 1 or 2, wherein the nutritional substance comprises at least one of chondroitin sodium sulfate, sodium hyaluronate, dextran, sodium alginate, sodium ascorbate, trehalose, dextran, polyvinyl alcohol, chitosan, polyethylene glycol.

4. The long term stereoscopic preserving nutritional capsule in corneal tissue according to claim 1 or 2, wherein the corneal basal medium comprises DMEM/F-12, DMEM, MEM or DME.

5. A method of preparing a long-term stereoscopic preserving nutritional capsule in corneal tissue according to any one of claims 1 to 4, comprising the steps of:

6. The method of manufacturing according to claim 5, comprising the steps of:

(1) Taking a cornea basic culture medium DMEM/F-12 as a mother solution, sequentially adding 0.1-3% of chondroitin sulfate, 1% of diab, 0.01-0.5% of sodium ascorbate and 0.2-2% of trehalose into the mother solution, and completely dissolving;

(3) Dropwise adding 50-300 mM CaCl into the pregel solution ₂ The aqueous solution is glued for 10-280 s, and then the nutritional capsule for long-term three-dimensional preservation in the cornea tissue is obtained.

7. The method according to claim 6, wherein the final concentration of sodium alginate in the pregelatinized solution in step (2) is 1wt%.

8. The method of claim 6, wherein said CaCl in step (3) ₂ The concentration of the aqueous solution was 100mM and the gel time was 60s.

9. The method according to claim 6, wherein the products obtained in steps (1) to (3) are sterilized.

10. Use of a nutritional capsule for long-term stereoscopic preservation in corneal tissue according to any one of claims 1 to 4 and a nutritional capsule for long-term stereoscopic preservation in corneal tissue prepared by the method according to any one of claims 5 to 9 for preservation of corneal tissue, wherein the use is to preserve corneal tissue inside the nutritional capsule for medium-long term stereoscopic preservation of corneal tissue.

11. A method for the medium-long term stereoscopic preservation of corneal tissue using the nutritional capsule prepared by the method according to claim 5, wherein the corneal tissue is placed in the pregelatinized solution formed in step (2) and then subjected to the operation of step (3) for the medium-long term stereoscopic preservation of corneal tissue.