WO2018107484A1

WO2018107484A1 - Decellurization method for swine cornea, decellularized cornea thereof, and use method for dried lamellar cornea

Info

Publication number: WO2018107484A1
Application number: PCT/CN2016/110461
Authority: WO
Inventors: 李志寒; 董晓鸥; 詹晓亮; 刘靖; 李洁
Original assignee: 厦门大开生物科技有限公司
Priority date: 2016-12-16
Filing date: 2016-12-16
Publication date: 2018-06-21
Also published as: CN109069263B; CN109069263A

Abstract

A decellurization method for swine cornea, employing the following steps: S1 drying treatment: drying a pretreated cornea to a water content of 5% to 20%; S2 enzyme treatment: employing DMEM for configuring a Benzonase solution; adding the cornea to the enzyme solution, and, when air bubbles are removed from the surface of the cornea, same is placed in a shaker incubator for a shaking treatment for no more than 4 h; S3 cleaning: treating the cornea by shaking and washing in a cleaning solution for no less than 5 times, the time of shaking and washing being no greater than 1 h each time. Impact on the transparency of the cornea is minimized.

Description

Porcine corneal decellularization method and its decellularized cornea and lamellar dry cornea use method

The present invention relates to a method for decellularizing a porcine cornea as an artificial cornea, a decellularized cornea using the porcine cornea as a material, and a method of using a decellularized lamellar cornea.

Background technique

Corneal blindness is the second most common blind eye disease in China. Corneal transplantation is the only effective treatment for re-exposure, but the lack of corneal donor material seriously affects patients with corneal transplantation. After more than ten years of efforts by Chinese scientists, artificial corneas made of porcine cornea have replaced human corneas and have taken the lead in clinical trials worldwide.

The existing research results show that the artificial cornea prepared from porcine cornea is of good biocompatibility. In particular, the porcine cornea has a tissue structure, biophysical properties and optical properties similar to those of the human cornea, which is the accepted conclusion of the best choice for corneal substitutes. Progress in domestic research shows that porcine cornea has been used as an important alternative source for human corneal transplant materials. At present, some detached porcine corneal products have been clinically staged and have certain clinical effects. In order to solve the serious shortage of corneal donors for transplantation in China, it provides an excellent solution for the hope of rehabilitating millions of patients suffering from corneal blindness in China.

From the perspective of tissue engineering, the primary problem to be solved by using porcine cornea as a substitute for human cornea transplantation is to reduce the rejection rate of porcine cornea as a heterologous corneal transplant. Therefore, decellularization is an indispensable process for preparing artificial corneas with cornea as a raw material.

Currently widely used in the decellularization method of biological materials: chemical detergent (SDS, Triton X-100, hypotonic Tris solution, EDTA, sodium deoxycholate, sodium orthovanadate), biological enzyme treatment (aprotinin) , nuclease, trypsin, physical assisted means (liquid nitrogen repeated freeze-thaw, ultrasound, cryo-electrophoresis, UV) three ways or a combination thereof. Compared with the chemical detergent decellularization and physical assisted decellularization methods, the bio-enzyme decellularization method has a better decellularization effect. However, each of the enzymes used in the prior art has different effects on the cells, and therefore, in order to obtain a better decellularization effect, different enzymes are also required to be used in combination.

In addition, in order to achieve a better decellularization effect, the cornea is often treated differently by different methods, such as repeated freezing and thawing, and swelling treatment in hypertonic or hypotonic solution. Through these treatment methods, the cell membrane of the stromal cells can be destroyed, and the biological enzymes used thereafter are more likely to enter.

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Replacement page (Article 26) The stromal cells are introduced, and the nucleic acids in the cells are further digested, thereby achieving the purpose of removing the corneal antigen component. However, the rationale for any of the above pretreatment methods involves disrupting the cornea prior to enzymatic treatment, and cell debris can be released from the interstitial space of the cornea after enzymatic treatment. At the same time, however, this will inevitably loosen the original collagen fiber structure of the tissue, thereby losing the original collagen fiber arrangement. At the same time, the moisture content inside the cornea is greatly improved. Therefore, in the subsequent enzyme treatment step, the biological enzyme solution is difficult to enter the corneal tissue, usually by prolonging the enzyme treatment time and increasing the amount of the enzyme to achieve the desired decellularization effect. Prolonging the prolonged enzyme treatment time will inevitably increase the probability of damage to the original collagen fiber structure of the cornea. Increasing the amount of enzyme will increase the amount of enzyme residues in the cornea, which will also damage the transparency of the cornea.

In another conventional freeze-thaw method in the prior art, after repeated freeze-thaw treatment, large irregular voids due to ice crystals may appear in the corneal stroma layer, although the purpose of loose collagen tissue is achieved. However, these irregular voids cause irreversible damage to the original collagen arrangement. This has no major impact on other organizations that have no transparency requirements and special requirements for regular collagen fiber alignment. However, the cornea differs from all other tissues in its distinctive features: transparency, which is the basis for the cornea to perform its physiological functions. Therefore, from the perspective of corneal transparency maintenance, artificial corneal preparation must strive for a regular arrangement of collagen fibers to maintain a good transparency of the cornea. Therefore, finding an ideal method of decellularization is especially necessary to maintain the corneal transparency.

The experimental data prove that the enzyme treatment method is very important for the selection of enzymes, and the difference in the decellularization effect achieved by different biological enzymes is also relatively large. In addition, in the enzymatic treatment decellularization method, it is necessary to appropriately select the important parameters such as different enzymes, the time of selection of the enzyme treatment, and the concentration of the enzyme solution, so as to achieve a better decellularization effect. Applicants have discovered through the decellularization test of various enzyme treatment methods listed in the prior art that the currently disclosed biological enzyme decellularization treatment method cannot achieve the effect of removing corneal cells while keeping the cornea transparent. Therefore, finding an ideal method of decellularization is especially necessary to maintain the transparency of porcine cornea. Summary of the invention

The object of the present invention is a method for decellularization of an animal-derived cornea, in particular, a method for decellularizing an artificial cornea derived from porcine cornea, and a decellularized cornea using porcine cornea as a material. In order to ensure the decellularization requirement of the artificial cornea, the special collagen fiber arrangement structure of the cornea is maximized, so that the effect of the decellularization treatment on the transparency of the cornea is minimized.

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Replacement page (Article 26) Another object of the present invention is to provide a corneal decellularization method which appropriately selects the kind of biological enzyme for decellularization treatment and achieves a better decellularization effect. The DNA residue of the artificial corneal products derived from porcine cornea conforms to the strict domestic standards for DNA residues of animal-derived biological materials.

A further object of the present invention is to provide a corneal decellularization method, which is easy to perform bio-enzymatic degradation in the whole process of enzymatic treatment of the cornea without excessive pretreatment of the corneal material, thereby maximizing The structure of the collagen fibers in the corneal stroma layer is regularly arranged to maintain a good decellularization effect under the premise of keeping the cornea transparent.

The method for decellularizing porcine cornea provided by the present invention comprises pretreating fresh porcine cornea to prepare a full-layer or lamellar cornea, and decellularizing the cornea by using a biological enzyme; wherein the decellularization treatment comprises at least the following steps: : Drying treatment: Drying the cornea, increasing the osmotic pressure of the enzyme solution to the corneal tissue, and accelerating the absorption of the enzyme solution by the cornea; S2: Enzymatic treatment: Configuring the pluripotent nuclease solution using DMEM medium; adding the dried cornea to the above The enzyme solution is placed in a shaking incubator for a period of not less than 1.0 hour; S3: Washing: the cornea is added to the washing solution, and placed in a shaking incubator for shaking washing treatment to obtain a decellularized porcine cornea.

The technical effect of the present invention is quite remarkable. First, the decellularization method of biological enzyme treatment is still employed in the present invention, but in the present invention, the versatile nuclease is selected for enzymatic treatment. The experiment proves that the decellularization effect of the pluripotent nuclease has the advantages of good decellularization effect and high efficiency compared with the various enzymes and combinations thereof disclosed in the prior art, and is beneficial to achieve better decellularization effect. The corneal can maintain the regular arrangement of the original collagen fibers to the maximum extent, thereby achieving the object of the present invention.

Secondly, a large number of experiments have proved that the DMEM medium used in the present invention is provided with an enzyme solution which minimizes the destruction of the original collagen arrangement structure of the corneal stroma layer.

Third, the present invention performs a drying treatment on the cornea before performing the decellularization treatment. In contrast to prior art processes such as freeze-thaw or swelling of the cornea prior to enzymatic treatment, the present invention allows the corneal collagen fibers to remain substantially regularly arranged prior to drying by drying prior to enzymatic treatment. It is particularly important that the corneal moisture content after drying is low, and the osmotic pressure of the enzyme solution to the corneal tissue having a low water content is increased. In the subsequent enzyme treatment, the rate of absorption of the enzyme solution by the cornea is accelerated. The effect is that the biological enzyme is more likely to penetrate into the cornea, so that the enzyme treatment efficiency is greatly improved, thereby shortening the processing time of the enzyme, reducing the amount of the enzyme, and thus minimizing the enzymatic treatment process on the corneal collagen. Damage caused by fiber structure.

The HE cell staining of the pig acellular cornea obtained by the decellularization method of the present invention has no cell nucleus, DAPI is not stained, and the residual amount of corneal DNA is not more than 100 ng/mg. Maximize corneal collagen fibers

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Replacement page (Article 26) The regular arrangement of the structure. As shown in Fig. 1A to Fig. 1C, the corneal electron microscope structure obtained by the method of the present invention shows that the structure of the collagen fibers is extremely close to that of the native cornea.

In an alternative embodiment of the invention, in the S1 drying treatment step, the corneal drying treatment is preferably carried out to a water content of not more than 30%.

In an alternative embodiment of the invention, the concentration of the pluripotent nuclease solution is 100 U - 25000 U / In a preferred embodiment of the invention, the omnipotent nuclease is optimally selected to be a versatile nuclease produced by Merck & Co., USA. (Benzonase®); The concentration of the Benzonase® solution is 100 U 1000 U/ml. The versatile nuclease (Benzonase®) selected by the present invention can greatly reduce the amount of enzyme, thereby reducing the amount of enzyme remaining in the cornea after decellularization.

In an alternative embodiment of the invention, the lamellar cornea comprises only the front elastic layer and the matrix layer. In an alternative embodiment of the present invention, the shaking treatment time in the shaking incubator in the S2 enzyme treatment step is preferably 2.0 5.0 hours.

In a preferred embodiment of the present invention, in the S2 enzyme treatment step, before the shaking treatment, the cornea is vortexed to the corneal surface to remove the air bubbles, and the corneal gas is discharged by vortexing to form a void. It is beneficial to accelerate the absorption rate of the enzyme, thereby shortening the enzymatic decellularization time.

Embodiment of the present invention an alternative embodiment, the enzyme solution cornea shock treatment temperature is 20 ° C ~ 30 ° _C; the cornea in the enzyme solution oscillation frequency of 50 to 100 times per minute.

In an alternative embodiment of the present invention, in the S3 cleaning process step, the washing shock frequency is 100-160 times per minute; and each shaking washing time is no more than 30 minutes.

In an alternative embodiment of the invention, the temperature of the cornea during the washing process is controlled at 5 V to 25 °C.

In an alternative embodiment of the invention, the washing solution of the cornea is distilled water or sodium chloride solution or a buffer solution having a pH of 6.0 to 8.0.

In an alternative embodiment of the invention, the method of drying the cornea is a dry method; drying to the dryness set by the cornea.

In an alternative embodiment of the invention, the corneal drying method is a vacuum drying method.

In a preferred embodiment of the present invention, the cornea drying method is a vacuum drying method in which the pressure is gradually reduced from high to low; and the pressure reduction in the gradually decreasing pressure ranges from normal pressure to near-limit vacuum. The gradual depressurization may be stepwise depressurized in a gradient within a reduced pressure range.

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Replacement page (Article 26) In an alternative embodiment of the invention, the corneal vacuum drying method has a drying time of no more than 24 hours.

The present invention provides a decellularized porcine cornea obtained by the decellularization method of the present invention; the acellular porcine cornea comprises a full-thickness corneal or lamellar cornea. Wherein, the DNA residue of the acellular porcine cornea is not more than 100 ng/mg.

The invention provides a decellularized pig layer dry cornea obtained by the decellularization method of the invention; the pig lamellar cornea comprises only a front elastic layer and a matrix layer; and the decellularized dried pig layer is dried The corneal water content is not more than 20%, and the light transmittance is more than 70%. The light transmittance is a light transmittance measured in a wavelength range of 380 nm to 780 nm. The DNA residue of the acellular porcine lamellar cornea is not more than 100 ng/mg.

The invention provides a method for using a decellularized lamellar dried cornea, and the dehydrated cell layer dried cornea is taken out from the sealed package, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for the heterogeneous corneal transplantation.

The test proves that the acellular porcine cornea and the decellularized lamellar dry cornea provided by the present invention have no cell nucleus stained by corneal HE, no staining of DAPI, and residual corneal DNA are less than 100 ng/mg. It can completely maintain the regular arrangement of corneal collagen fibers. As shown in Fig. 1A to Fig. 1C, the corneal electron microscope structure obtained by the decellularization method of the present invention shows that the structure of the collagen fibers is extremely close to that of the human cornea and the porcine cornea before the acellular treatment.

As shown in FIG. 2 to FIG. 3, the dried cornea of the porcine corneal acellular layer provided by the present invention minimizes the damage caused by the enzymatic treatment process on the structure of the corneal collagen fiber during the decellularization process, and thus, the present invention The transmittance of the dried lamellar film provided was 85% in the wavelength range of 380 to 780 nm.

The clinical corneal transplantation case shown in Figs. 5A to 5D demonstrates that the pig acellular cell layer dried cornea provided by the present invention has an excellent transplantation effect. In order to solve the serious shortage of corneal donors for transplantation in China, an excellent solution has been provided, which has brought hope to millions of patients suffering from corneal blindness in China. DRAWINGS

The invention and its specific embodiments and their effects are briefly described below with reference to the accompanying drawings, which are merely illustrative of some specific experimental examples of the invention, and not all of the invention.

Figure 1A is a cross-sectional arrangement of a collagen arrangement of a human cornea;

Figure 1B Cross-sectional arrangement of porcine corneal collagen without decellularization;

1C is a cross-sectional arrangement of collagen of a porcine cornea after decellularization treatment of the present invention;

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Replacement page (Article 26) Figure 2 is a photograph of a corneal product of a decellularized and dried lamellar layer of the present invention.

Fig. 3 is a photograph of a corneal HE staining of a decellularized and dried lamellar layer of the present invention.

Fig. 4 is a photograph of the post-transplantation of the decellularized lamellar cornea of the present invention to the New Zealand white rabbit.

Figure 5A is a pre-operative photo of a clinical transplant of the present invention;

Figure 5B is a photograph of the 3 day after clinical transplantation of Figure 5A;

Figure 5C is a photograph of Figure 2A after 2 months of clinical transplantation;

Figure 5D is a photograph of Figure 6A after 6 months of clinical transplantation;

Figure 5E is a photograph of Figure 1A after 1 year of clinical transplantation. detailed description

The technical solutions of the present invention will be described in detail below with reference to the embodiments of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention. Example 1

In the present embodiment, the method for decellularizing the porcine cornea is to pretreat the fresh porcine cornea, and then to prepare a whole layer or a lamellar cornea, and then decellularize the cornea with a biological enzyme. The decellularization treatment includes at least the following steps:

S1: drying treatment: drying the cornea to increase the osmotic pressure of the enzyme solution to the corneal tissue, and accelerating the absorption of the enzyme solution by the cornea; the difference between the drying treatment in the present invention and the dehydration treatment in the prior art is that the present invention The moisture content in the cornea after drying treatment should be lower than the moisture content of the fresh cornea (in the art, dehydration treatment refers to the removal of water from the cornea during the preparation process, and the cornea retains substantially the same water content as the fresh cornea after dehydration. rate). In the first embodiment, the cornea drying treatment is preferably such that the water content is not more than 30%. In a specific test example of this Example 1, the cornea was dried to a moisture content of 15% ± 3%. In the cornea in the dry state, there is basically no excessive loosening of the collagen matrix structure of the corneal stroma due to swelling, and excessive irregular pores are formed due to repeated freezing and thawing, thereby effectively maintaining the inner layer of the matrix. Regular collagen fiber alignment structure.

S2: Enzyme treatment: The versa nuclease solution is configured by using DMEM medium; the cornea after drying is added to the above enzyme solution; and the shaking treatment time is not less than 1.0 hour in the shaking incubator;

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Replacement page (Article 26) Because the omnipotent nuclease is affected by many factors such as the manufacturer, product batch, transportation mode and storage time, the degree of enzyme activity varies greatly. Therefore, the concentration of the enzyme solution should be selected according to the activity level of the enzyme. The concentration of the enzyme solution should be Increase with the decrease of enzyme activity.

In a preferred embodiment of the present invention, in a preferred embodiment of the present invention, the omnipotent nuclease is selected from Merck & Co., Inc. to produce a versatile nuclease (Benzonase®), the versatile nuclease.

The concentration of the (Benzonase®) solution is 100 U 1000 U/ml. The versatile nuclease (Benzonase®) selected in the present embodiment can greatly reduce the amount of enzyme, thereby reducing the effect of residual enzymes in the cornea after decellularization. In particular, the solvent of the all-enzyme nuclease (Benzonase®) enzyme solution in the DMEM medium can minimize the damage of the collagen structure of the cornea while ensuring the decellularization effect of the enzyme.

In a specific test example of the preferred embodiment of the first embodiment, the DMEM medium was selected to have a concentration of 300 U 500 U/ml of a versatile nuclease (Benzonase®) enzyme solution. It has been found that, in this specific test case, the DNA residue of the cornea obtained by decellularization of the enzyme solution can reach a level of about 50 ng/mg.

In an alternative embodiment of the first embodiment, the shaking treatment time in the shaking incubator in the S2 enzyme processing step is preferably 2.0 5.0 hours.

In another preferred embodiment of the first embodiment, in the S2 enzyme treatment step, before the shaking treatment, the cornea is vortexed to the corneal surface to remove the air bubbles, and the corneal gas is discharged as soon as possible by vortexing. The formation of pores is more conducive to shortening the enzyme treatment time.

In a specific test example of the first embodiment, 0.5 0.8 ml of the above enzyme solution was added to each cornea, and vortexing was first used to remove the air bubbles from the cornea surface. Tests have shown that the bubbles on the surface of the cornea have been removed in a very short time (usually no more than 1 minute). At this time, the gas contained in the cornea is discharged from the surface of the cornea in the form of bubbles. Then, it was placed in a shaking incubator for shaking treatment, and the shaking treatment time was about 3.0 hours.

In an alternative embodiment of the first embodiment, the corneal is subjected to an oscillating treatment temperature of 20 to 30 ° C in the enzyme solution; this temperature range is suitable for the enzymatic hydrolysis of cells in the stromal layer by the biological enzyme used in the present invention. More fully. In the specific test example of the first embodiment, the oscillating treatment temperature was controlled at about 25 °C.

In an alternative embodiment of this embodiment 1, the oscillation frequency of the cornea in the enzyme solution is controlled at a lower level of 50-100 times per minute. In a preferred specific test example of the first embodiment, the oscillation frequency is selected to be 75 times per minute. When testing the enzyme treatment, choose a lower oscillation frequency.

Replacement page (Article 26) In order to reduce the degree of damage to the original collagen fiber arrangement of the cornea.

S3: Washing: The enzyme-treated cornea is added to the washing solution, and placed in a shaking incubator for shaking washing to obtain a decellularized porcine cornea.

In an alternative embodiment of the first embodiment, the washing oscillation frequency is 100-160 times per minute; each shaking washing time is no more than 30 minutes. The temperature of the cornea during the washing process is controlled within the range of 5 °C to 25 °C. Specifically, in a preferred test example of the present embodiment, the temperature during the cleaning process is controlled at about 15 ° C, and the temperature is kept constant throughout the cleaning process to avoid denaturation of the corneal collagen protein due to excessive temperature.

In the present embodiment, a sodium chloride solution having a concentration of 0.9% was used as a washing solution to obtain a decellularized porcine cornea. In a specific test example of the first embodiment, a cleaning solution consisting of 5 ml of a 0.9% sodium chloride solution was added to each cornea, and placed in a shaking incubator for 5 times in a shock washing process, and the oscillation frequency was per minute. 150 times, each time the shock washing process is 15 minutes.

In the present invention, the cleaning treatment after deoxidation by enzymatic treatment aims to completely remove the components other than the scaffold from the corneal stroma layer, such as the heteroprotein, the depleted cell residue, the enzyme residue and the like. The pores in the cornea are cleared by the residual cells. When the original collagen arrangement in the cornea is not destroyed or the extent of damage is small, the porosity and pore position and pores generated in the cornea after removal are removed. The size and the corneal protocells remain basically the same, which is very conducive to the orderly growth of human cells after transplantation.

In another embodiment of the first embodiment, the washing solution of the cornea may also be distilled water or a buffer solution having a pH of 6.0 to 8.0.

In the invention, a full-thickness corneal or lamellar cornea is produced during the porcine corneal pretreatment process, and any conventional manufacturing method can be employed. In a specific embodiment of this embodiment 1, a lamellar cornea comprising only the front elastic layer and the matrix layer is prepared prior to the decellularization treatment.

In the present invention, the S1 drying treatment before the enzyme treatment exerts an important influence on the enzymatic treatment decellularization effect of the cornea, and therefore, when the drying method is selected, a conventional drying method which has less damage to the corneal collagen arrangement should be selected. In an alternative embodiment of the first embodiment, a drying method in which the drying process is mild is selected to be dried or vacuum dried.

In a preferred embodiment of the first embodiment, a vacuum drying method in which the pressure is gradually reduced from high to low is employed, and the pressure reduction in the gradual depressurization ranges from atmospheric pressure to ultimate vacuum. Further, the gradual depressurization may be stepwise decompressed in a gradient within a reduced pressure range. The gradual decompression can make the vacuum drying process of the cornea more

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Replacement page (Article 26) Mild, minimizes damage to the corneal collagen array during dryness. (The vacuum vacuum drying method adopted in the present invention has been separately applied, and the case will not be described again).

In an alternative embodiment of this embodiment 1, the corneal vacuum drying method has a drying time of no more than 24 hours. In a preferred embodiment of this embodiment 1, the vacuum drying time is about 12 hours.

A decellularized porcine cornea obtained by the decellularization method of this Example 1 is a porcine lamellar cornea composed of only a front elastic layer and a matrix layer. Tests have shown that the DNA residue of the pig decellularized cornea obtained by the preferred embodiment of the present embodiment is about 50 ng/mg and the light transmittance is more than 80%; it can be directly applied to the heterogeneous corneal transplantation after sterilization.

Using the decellularized pig lamellar cornea obtained by the decellularization method of Example 1, the dried cornea obtained by drying treatment after decellularization treatment, in a dry state having a water content of not more than 20%, the light transmittance More than 85%.

Wherein the light transmittance can be measured by a conventional detecting method, specifically, the light transmittance measured in the wavelength range of 380 nm to 780 nm in the present embodiment.

In the method for using the cell dried cornea of the present invention, the decellularized layer dried cornea is taken out from the sealed package, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for the heterogeneous keratoplasty.

5A to FIG. 5E are a group of photographs of a human corneal transplantation of a porcine acellular cell layer dried cornea according to Example 1 of the present invention, which are respectively a photograph of the preoperative case of FIG. 5A, and FIG. 5B is a postoperative 3 days; 5C is 2 months after surgery; Figure 5D is 6 months after surgery; Figure 5E is 1 year after surgery. Compared with the existing documented clinical effects, the lamellar dry cornea proposed in the first embodiment has been in a transparent state 3 days after the operation, and the corneal epithelium is basically repaired, and no obvious rejection reaction is observed. The cornea was completely restored to transparency, no neovascularization occurred, and no rejection was observed. 1 year after surgery, it was completely transparent, basically achieving the same effect as human corneal transplantation, and the corrected visual acuity was 1.0. Example 2

In the present embodiment, the method of decellularizing the porcine cornea, the fresh porcine cornea is pretreated, and after the whole layer or lamellar cornea is prepared, the cornea is decellularized by using the biological enzyme. The decellularization treatment includes at least the following steps:

S1: Drying treatment: The cornea is dried to accelerate the absorption of the enzyme solution by the cornea; in a specific test example of the second embodiment, the cornea is dried to a water content of 5% ± 3%.

S2: Enzyme treatment: Configure the versa nuclease solution in DMEM medium; add the cornea after drying

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Replacement page (Article 26) The enzyme solution is placed in a shaking incubator for a period of not less than 1.0 hour. In the preferred embodiment of the second embodiment, the versatile nuclease is selected from the company Merck to produce a versatile nuclease (Benzonase®). In a specific test example of the present embodiment, a versatile nuclease (Benzonase®) enzyme solution having a concentration of 500 U 1000 U/ml in a DMEM medium was selected. It has been found that, in this specific test case, the DNA residue of the cornea obtained after decellularization using the enzyme solution can reach a level of about 20 ng/mg.

The versatile nuclease (Benzonase®) selected in the DMEM medium in the present embodiment can greatly reduce the amount of the enzyme, thereby reducing the effect of the enzyme residue in the cornea after decellularization. The destruction of the original collagen arrangement of the cornea can be minimized while ensuring the decellularization effect of the enzyme.

In an alternative embodiment of the second embodiment, the shaking treatment time in the shaking incubator in the S2 enzyme treatment step is preferably 2.0 5.0 hours.

In another preferred embodiment of the second embodiment, in the S2 enzyme treatment step, before the shaking treatment, the cornea is vortexed to the corneal surface for air bubble removal, and the corneal gas is discharged as soon as possible by vortexing. It is more conducive to shortening the enzyme treatment time.

In a specific test example of the second embodiment, 1.0 2.0 ml of the above enzyme solution was added to each cornea, and vortexing was first used to remove the air bubbles from the cornea surface. Tests have shown that the bubbles on the surface of the cornea have been removed in a very short time (usually no more than 1 minute). At this time, the gas contained in the cornea is discharged from the surface of the cornea in the form of bubbles. Then, it was placed in a shaking incubator for shaking treatment, and the shaking treatment time was about 5.5 6.0 hours.

In an alternative embodiment of the present embodiment, the corneal is subjected to an oscillating treatment temperature of 15 to 37 ° C in the enzyme solution _; this temperature range is suitable for the enzymatic hydrolysis of cells in the stromal layer by the biological enzyme used in the present invention. More effective. In the specific test example of the second embodiment, the oscillating treatment temperature is controlled at 25 ° C to 30 ° C.

In an alternative embodiment of the present embodiment 1, the oscillation frequency of the cornea in the enzyme solution is controlled at a low level of 50-100 times per minute. In a preferred specific test example of the second embodiment, the frequency is selected to be 50 times per minute. In the specific test example, when the shaking enzyme treatment time is appropriately increased, a lower oscillation frequency is selected, the purpose of which is to reduce the degree of destruction of the original collagen fiber arrangement of the cornea by the enzyme shaking treatment.

S3: Washing: The enzyme-treated cornea is added to the washing solution, and placed in a shaking incubator for shaking washing to obtain a decellularized porcine cornea. In an alternative embodiment of the second embodiment, 8 ml of the cleaning solution is added to each cornea and placed in a shaking incubator for 5 times in a shaking incubator, each shaking washing time.

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Replacement page (Article 26) After 30 minutes, the decellularized porcine cornea was obtained. In one test example of the second embodiment, the cleaning solution is a buffer solution having a pH of 6.0 to 8.0. The temperature of the cornea during the cleaning process is controlled at around 20 °C. And the temperature is kept constant throughout the cleaning process. The cornea oscillates at a frequency of 100 times per minute during the washing process.

The cornea before the decellularization treatment in the present Example 2 was formed into a lamellar cornea including only the front elastic layer and the matrix layer.

In the present invention, the corneal drying treatment has an important influence on the enzymatic treatment decellularization effect of the cornea, and in the present embodiment 2, a conventional air drying method in which the corneal collagen fiber arrangement is less damaged is selected. Tests have shown that the drying method can maximize the regular arrangement of the ultrastructure of the collagen fibers in the matrix layer. In the second embodiment, the lamellar corneal stroma sheet is affixed to the ultra-clean culture dish and dried in a desiccant containing a desiccant for about 16 24 hours to achieve the corneal moisture content set in the present embodiment. Not more than 30% level.

The decellularized porcine cornea obtained by the decellularization method of the second embodiment is a porcine lamellar cornea composed of only the front elastic layer and the stromal layer, and has a good decellularization effect, and the corneal DNA residue detection is not more than 20 ng. /mg, corneal light transmittance after enzyme treatment is greater than 75%; can be applied to xenogeneic corneal transplantation after sterilization.

The dried cornea obtained by the acellular cell layer obtained by the decellularization method of the present invention, which is dried after the decellularization treatment, has a light transmittance greater than that in a dry state having a water content of not more than 20%. 85%.

The light transmittance of the present invention can be controlled by a conventional method, specifically in the present embodiment,

Light transmittance measured in the wavelength range of 380 nm to 780 nm.

In the method for using the decellularized dried cornea of the present invention, the decellularized dried cornea is taken out from the sealed package, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for the heterogeneous keratoplasty. Example 3

The decellularization method of the porcine cornea proposed in the third embodiment pretreats the fresh porcine cornea to prepare a full-thickness cornea, and after drying, the cornea is decellularized by using a biological enzyme. The decellularization treatment includes at least the following steps:

S1: Drying treatment: The pretreated cornea is dried and dried to a corneal water content of about 30%; after drying, the cornea is substantially in a dry corneal state.

S2: Enzymatic treatment: Configure the versatile nuclease (Benzonase®) solution in DMEM medium; add 2.0 -3.0 ml of the above enzyme solution to each cornea, first use vortex to the surface of the cornea.

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Replacement page (Article 26) In addition, the shock treatment is performed, and the shaking treatment time is approximately 7.5 8.0 hours.

S3. Washing: Add 10 ml of the washing solution to each cornea and place it in a shaking incubator for 3 times to wash the time for 20 minutes to obtain the decellularized porcine cornea. Specifically, the cleaning solution described in one of the test examples of Example 3 was distilled water.

In the third embodiment, a conventional corneal preparation method is used to form a full-thickness cornea.

In a preferred embodiment of this embodiment 3, the pluripotent nuclease Benzonase® solution has a concentration in the range of 100 400 U/ml. The oscillating treatment temperature of the cornea in the enzyme solution is controlled at 15 °C ~ 20 °C. The oscillating frequency of the cornea in the enzyme solution was controlled at 80 times per minute.

In Example 3, the temperature of the cornea during the cleaning treatment was controlled at about 20 °C. It is kept at a constant temperature throughout the cleaning process. The cornea oscillates at a frequency of 100 times per minute during the washing process.

In the third embodiment, the same gradual vacuum drying method as that of Example 1 was selected for the drying treatment method of S1. Drying time is 24 hours. Specifically, in the present embodiment, a vacuum drying method using gradient decompression is adopted, that is, the decompression mode in the closed drying chamber is at least two stages from high to low gradient decompression, and the pressure gradient of each stage lasts for a period of time. The final stage gradient pressure is then depressurized to the set maximum vacuum state to the dryness requirement of 20% corneal moisture content.

In the whole-layer acellular porcine cornea obtained by the decellularization method of Example 3, the detection of corneal DNA residue does not exceed 95 ng/mg. The light transmittance is greater than 70%; the background layer can be removed after sterilization to be used for xenogeneic corneal transplantation.

The dried cornea obtained by the acellular cell layer obtained by the decellularization method of the present invention, which is dried after the decellularization treatment, has a light transmittance greater than that in a dry state having a water content of not more than 20%. 70%.

Light transmittance measured in the wavelength range of 380 nm to 780 nm. Example 4

The decellularization method of the porcine cornea proposed in the fourth embodiment pretreats the fresh porcine cornea to prepare the lamellar cornea, and after drying, the cornea is decellularized by the biological enzyme. The decellularization treatment includes at least the following steps:

S1: Drying treatment: The pretreated cornea is dried and dried to a corneal water content of about 10%; after drying, the cornea is substantially in a dry corneal state.

S2: Enzyme treatment: In this example, 4 plants other than the versatile nuclease (Benzonase®) were selected.

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Replacement page (Article 26) The versatile nuclease provided by the manufacturer. The range of pluripotency nucleases can be selected based on the pluripotency nuclease activity. In this embodiment, a domestic versatile nuclease (for example, a biotechnology company in Shanghai) is used. In the specific test examples of the fourth embodiment, the concentration ranges selected according to the activity of different batches of versatile nuclease products provided by the manufacturer are: 1000 U ~ 5000 U / ml; 5000 U ~ 10000 U / ml; 10000 U ~ 15000 U / ml ; 15000U ~ 20000U / ml; 20000U ~ 25000U / ml. According to each cornea, 2.0 -3.0 ml of the above enzyme solution was added to the corneal surface by vortexing; then the shaking treatment was carried out, and the shaking treatment time was about 3.0 4.0 hours at a constant temperature of 25 °C. After testing, in the above specific test examples, the DNA residue of the cornea obtained by decellularization of the enzyme solution prepared by the versatile nuclease can reach a standard of not more than 100 ng/mg.

Tests have shown that the present invention selectively selects the DMEM medium to configure the pluripotent nuclease solution to substantially achieve an enhanced decellularization effect. By adjusting the concentration range of the versatile nuclease according to the versatile nuclease activity provided by different manufacturers, the standard effect of the DNA residue of the cornea is not more than 100 ng/mg. However, as the concentration of the versatile nuclease increases, the amount of enzyme is increased, and the amount of enzyme residues in the cornea also increases. This Example 4 increases the difficulty of removing the enzyme residue.

S3. Washing: 10 ml of the washing solution was added to each cornea and placed in a shaking incubator for 8 times. The shaking frequency was 150 times per minute, and the shaking time was 25 minutes each time to obtain the porcine cornea of the decellularized cells. Specifically, the cleaning solution described in one of the test examples of Example 4 was a sodium chloride solution having a concentration of 0.9%.

In the fourth embodiment, a conventional corneal preparation method is used to form a full-thickness cornea.

In the embodiment 4, the temperature during the cleaning process is controlled to be about 20 to 25 °C. It is kept at a constant temperature throughout the cleaning process. The cornea oscillates at a frequency of 100 times per minute during the washing process.

In the drying treatment method of S1 in the present Example 4, the same gradual vacuum drying method as in Example 1 was selected.

In the whole-layer acellular porcine cornea obtained by the decellularization method of the fourth embodiment, the detection of corneal DNA residue does not exceed the lOOng/mg standard. The light transmittance is greater than 70%; the background layer is removed after sterilization for heterogeneous corneal transplantation.

Using the decellularized pig lamellar cornea obtained by the decellularization method of the fourth embodiment, the dried cornea obtained by drying after decellularization treatment, in a dry state having a water content of not more than 20%, the light transmission The rate is greater than 70%.

For the detailed explanation of the above embodiments, the purpose is only to explain the present invention, so as to facilitate

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Replacement page (Article 26) The invention may be better understood, but the description is not to be construed as limiting the invention in any way. In particular, the various features described in the different embodiments may be combined in any combination to form other embodiments. There are clear and concise descriptions that should be understood as being applicable to any one embodiment, and not limited to the described embodiments.

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Claims

m ^

A method for decellularizing a porcine cornea, pretreating a fresh porcine cornea, preparing a full-thickness or lamellar cornea, and decellularizing the cornea with a biological enzyme; the decellularization treatment comprises at least the following steps:

S1 : Drying treatment: Drying the cornea to accelerate the absorption of the enzyme solution by the cornea;

S3: Washing: Add the cornea to the washing solution, and shake it in a shaking incubator to obtain the decellularized porcine cornea.

2. The porcine corneal de-scouring method according to claim 1, wherein the lamellar cornea comprises only a front elastic layer and a matrix layer.

The porcine corneal decellularization method according to claim 1, wherein in the S1 drying treatment step, the cornea drying treatment is preferably carried out to a water content of not more than 30%.

The porcine corneal decellularization method according to claim 1, wherein in the S2 enzyme treatment step, the concentration of the pluripotent nuclease solution is 100 U 25000 U/ml.

The porcine corneal decellularization method according to claim 1, wherein in the S2 enzyme treatment step, the _pluripotent nuclease is preferably a versatile nuclease (Be _nz0nase ® ).

The porcine corneal decellularization method according to claim 5, wherein the concentration of the benzoonase® solution is 100 U 1000 U/ml.

The porcine corneal decellularization method according to claim 1, wherein in the S2 enzyme treatment step, the shaking treatment time in the shaking incubator is preferably 2.0 5.0 hours.

8. The porcine corneal decellularization method according to claim 1, wherein in the S2 enzyme treatment step, the cornea is vortexed to the corneal surface for bubble removal before the oscillating treatment.

The porcine corneal decellularization method according to claim 1, wherein in the S2 enzyme treatment step, the cornea is subjected to an oscillating treatment temperature of 20 ° C to 30 ° C in the enzyme solution.

The decellularization method of porcine cornea according to claim 1, wherein in the S2 enzyme treatment step, the cornea is oscillated in the enzyme solution at a frequency of 50 to 100 times per minute.

11. The method for decellularizing porcine cornea according to claim 1, wherein in the S3 washing treatment step, the washing oscillation frequency is 100-160 times per minute; and each shaking washing time is not more than 30 minutes.

The method for decellularizing a porcine cornea according to claim 1, wherein said cornea is washed

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Replacement page (Article 26) The temperature during the process is controlled from 5 °C to 25 °C.

The method for decellularizing porcine cornea according to claim 1, wherein the washing solution of the cornea is distilled water or sodium chloride solution or a buffer solution having a pH of 6.0 to 8.0.

The method for decellularizing porcine cornea according to claim 1, wherein the method of drying the cornea is an air drying method; and drying to a dryness set by the cornea.

The method for decellularizing porcine cornea according to claim 1, wherein the cornea drying method is a vacuum drying method.

The method for decellularizing porcine cornea according to claim 15, wherein the corneal drying method is a vacuum drying method in which a pressure is gradually reduced from high to low; Atmospheric pressure to near the ultimate vacuum.

The method for decellularizing a porcine cornea according to claim 15 or 16, wherein the gradual decompression is gradually reduced in a gradient in a reduced pressure range.

The method for decellularizing porcine cornea according to claim 15 18, wherein the corneal vacuum drying method has a drying time of not more than 24 hours.

A decellularized porcine cornea, characterized in that said acellular porcine cornea is obtained by the decellularization method according to any of the preceding claims; said acellular porcine cornea comprises a full-thickness corneal or lamellar cornea.

The acellular porcine cornea according to claim 20, wherein the DNA residue of the acellular porcine cornea is not more than 100 ng/mg.

A decellularized porcine lamellar dried cornea obtained by the decellularization method according to any one of claims 1 to 18; wherein the porcine lamellar cornea comprises only a front elastic layer and a stromal layer; The dried corneal moisture content of the treated pig layer is not more than 20%, and the light transmittance is more than 70%.

The acellular pig lamellar cornea according to claim 23, wherein said light transmittance is a light transmittance measured in a wavelength range of 380 _n m to 780 nm.

The acellular pig lamellar cornea according to claim 23, wherein the DNA residue of the acellular porcine lamellar cornea is not more than 100 ng/mg.

A method for using a decellularized lamellar dried cornea according to claim 21, wherein the decellularized lamellar layer is dried, and after being sterilized, the physiological saline is rehydrated for 15-30 minutes, and then directly used for xenogeneic corneal transplantation. Surgery.

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