WO2018107482A1 - 一种脱细胞猪角膜的制备方法及其脱细胞板层角膜和用法 - Google Patents

一种脱细胞猪角膜的制备方法及其脱细胞板层角膜和用法 Download PDF

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WO2018107482A1
WO2018107482A1 PCT/CN2016/110458 CN2016110458W WO2018107482A1 WO 2018107482 A1 WO2018107482 A1 WO 2018107482A1 CN 2016110458 W CN2016110458 W CN 2016110458W WO 2018107482 A1 WO2018107482 A1 WO 2018107482A1
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cornea
lamellar
porcine
preparing
corneal
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PCT/CN2016/110458
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English (en)
French (fr)
Inventor
董晓鸥
刘靖
李志寒
李洁
詹晓亮
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厦门大开生物科技有限公司
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Priority to CN201680077703.1A priority Critical patent/CN109475663B/zh
Priority to PCT/CN2016/110458 priority patent/WO2018107482A1/zh
Publication of WO2018107482A1 publication Critical patent/WO2018107482A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

Definitions

  • the invention relates to a preparation method of acellular porcine cornea, and a lamellar decellularized cornea which can be directly used for human corneal transplantation, and a method for using the same. Background technique
  • Corneal blindness is the second most common blind eye disease in China. Corneal transplantation is the only effective treatment for patients with corneal blindness. However, the lack of corneal donor material seriously affects corneal transplantation. Artificial corneas made from porcine corneas, which have been developed by Chinese scientists for more than ten years, can replace human corneas and take the lead in clinical trials worldwide.
  • 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.
  • some detached porcine corneal products have been clinically staged and have certain clinical effects.
  • it provides an excellent solution for the hope of rehabilitating millions of patients suffering from corneal blindness in China.
  • the cornea Located at the front of the eyeball, the cornea is a highly structured, relatively cell-free, transparent collagenous tissue.
  • the existing lamellar acellular corneal stroma contains only the front elastic layer and the matrix layer. After necessary decellularization, the immunogenicity (DNA) in the stromal layer is removed, and the immune rejection of porcine corneal transplantation is reduced. Inactivation by virus and sterilization can reduce the animal-derived virus and bacterial infection in corneal transplantation, so as to achieve the necessary biological indicators that the lamellar cornea can be used for transplantation.
  • the structural feature of the corneal stroma is that the three-helix type I collagen is ordered and parallel to each other. This arrangement constitutes the structural basis of the physical properties of the corneal elastic mechanical strength and its transparency. This important feature of the cornea can induce the orderly and uniform growth of the recipient corneal stromal cells after implantation, keeping the cornea transparent.
  • the height of the corneal stroma layer arrangement is an important factor in realizing the necessary biophysical properties of the lamellar cornea.
  • the current decellularization method replaces the page (Article 26) Damage to the ordered arrangement of the matrix layer can be caused to varying degrees.
  • the physical properties such as the elastic mechanical strength of the cornea and its transparency are inevitably destroyed.
  • the transparency of the cornea greatly detracts from the healing power of the cornea.
  • Enzymatic methods are the most effective of the various methods of corneal decellularization. Enzymes used in existing enzymatic methods mainly include: lipases, nucleases, proteases, etc.; different enzymes target specific cellular components. The defects: First, the complete decellularization effect cannot be achieved; Secondly, the enzymes listed above, especially proteases, also have obvious destructive effects on the corneal extracellular matrix, resulting in a decrease in the transparency of the cornea.
  • the enzymatic decellularization method is now carried out by directly immersing the enzyme solution in the cornea after pretreatment. Because the water content of the cornea inevitably increases during the pretreatment process, the enzyme solution is difficult to enter the cornea after directly immersing the cornea in the enzyme solution, which greatly affects the degradation of the cells in the corneal stroma. In many cases, in order to ensure the decellularization effect, the method of prolonging the enzymatic treatment time is often used, which increases the probability of destruction of the physical properties such as the transparency of the cornea.
  • Sterilization of the cornea is an indispensable step in the preparation of the cornea. Its function is to completely kill harmful microorganisms such as bacteria and viruses in the cornea.
  • irradiation sterilization technology has introduced sterilization of the cornea.
  • the transparency and biomechanical properties of the cornea decreased significantly after irradiation sterilization.
  • the American International Organization Library conducted a human corneal irradiation sterilization test and analysis of experimental data comparing the transparency of the cornea before and after irradiation, and pointed out that the human corneal donor can be irradiated and sterilized under normal temperature conditions, and the gamma ray can be The molecular bond of collagen in the extracellular matrix is changed, and the physical and chemical properties of the cornea are changed after irradiation, resulting in significant changes in the transparency, toughness, and hydrophilicity of the cornea after irradiation compared with the cornea under normal physiological conditions. Irradiation sterilization is not conducive to the clinical sterilization of corneas.” Research conclusions.
  • the removal of cellular components in the stromal layer, the reduction of immune rejection, and the sterilization of the two key steps of reducing the infection of the animal-borne virus inevitably destroy the corneal elasticity.
  • Physical properties such as mechanical strength and transparency, especially the transparency of the cornea.
  • the current method proposed by the researchers is to perform the necessary drying treatment on the cornea after decellularization in order to obtain a dry cornea.
  • vacuum drying is a commonly used drying method.
  • the applicant has conducted research and statistical analysis on a large amount of experimental data obtained by all the existing vacuum drying methods, and found that the adverse effect of the vacuum drying method on the artificial cornea is that the drying process is too intense relative to the cornea, thereby causing collagen tissue. Irregular changes in the alignment disrupt the highly regular collagen arrangement in the original corneal stroma, which greatly affects the transparency of the cornea.
  • any one of the treatment steps inevitably causes damage to the regular arrangement in the corneal stroma, and thus it is extremely disadvantageous to the "transparency" of the prepared cornea. influences.
  • the damage of corneal transparency caused by the preparation process will also affect the transplantation effect to a large extent. It is necessary to provide a method to minimize the damage to the regularly arranged collagen structures in the corneal stroma layer to ensure that the cornea after the complex preparation process still has excellent transparency, achieving the market for artificial corneal products.
  • the object of the present invention is to provide a method for preparing a porcine decellularized lamellar cornea, which can maximize the biological parameters necessary for ensuring strict transplantation conditions in key steps such as necessary decellularization treatment and sterilization treatment. Limiting the damage to the regular arrangement of corneal collagen fibers. Maintaining physical properties such as corneal transparency and its elastic mechanical strength, especially to minimize damage to corneal transparency.
  • Another object of the present invention is to provide a method for preparing a corneal decellularized lamellar cornea, which can minimize the damage of the corneal collagen structure during the drying process of the cornea, and the corneal product maintains a smooth and smooth appearance. Conducive to the growth of corneal cells.
  • a further object of the present invention is to provide a method for preparing a porcine acellular cell layer cornea and a drying angle thereof
  • the membrane has a product form that is convenient for storage, transportation and use. Therefore, the defects of the cornea products in the prior art are different depending on the preparation methods thereof, the quality is different, and the products are difficult to be standardized, and the technical requirements for mass production are achieved. Promote the development of ophthalmology and medical care in China, and solve many problems that cannot be effectively treated due to the lack of corneal donors.
  • the object of the present invention is achieved by the present invention, which provides a method for preparing a porcine decellularized lamellar cornea, at least as follows: Sl, pretreatment: Pretreatment of fresh porcine cornea includes the following treatment process: S1.1 taking fresh Porcine cornea, removing the epithelial layer after cleaning; S1.2 preparing a lamellar cornea; the lamellar cornea includes only the front elastic layer and the matrix layer; S1.3 is cleaned;
  • S3 Decellularization treatment: The cornea is subjected to decellularization treatment after drying: S3.1 Enzyme treatment: Configure the versatile nuclease (Benzonase®) solution in DMEM medium; add the dried cornea to the above enzyme solution; The shock in the box is not less than 1 hour; S3.2 cleaning: the cornea is added to the cleaning solution, placed in a shaking incubator to wash and wash, to obtain acellular cornea;
  • S3.1 Enzyme treatment Configure the versatile nuclease (Benzonase®) solution in DMEM medium; add the dried cornea to the above enzyme solution; The shock in the box is not less than 1 hour;
  • S3.2 cleaning the cornea is added to the cleaning solution, placed in a shaking incubator to wash and wash, to obtain acellular cornea;
  • Sterilization treatment Sterilization with cobalt 60 irradiation, the irradiation dose is not more than 25kgy.
  • the enzymatic treatment using Benzonase® is not required, and it is not necessary to use a combination of various enzymes, which is quite good.
  • Cellular effect, HE staining without cell nucleus, DAPI without staining, corneal DNA residue less than lOOng/mgo, and the decellularization method of the present invention can maximize the arrangement of regular collagen fibers whose cornea is very close to the natural cornea.
  • the cornea is dried before the decellularization treatment, and the osmotic pressure difference between the cornea and the enzyme solution is increased by reducing the corneal water content, so that the biological enzyme is more easily infiltrated into the cornea, and the enzyme is greatly improved.
  • the efficiency which shortens the enzyme processing time. Therefore, the damage caused by the enzymatic treatment process on the structure of the corneal collagen fibers is minimized, and the technical effect of maintaining corneal transparency is achieved.
  • the translucency of the cornea ensures that it is over 80% in the wavelength range of 380-780 nm.
  • the fresh lamellar cornea prepared in step S1.2 has a thickness of from 300 um to 700 um.
  • the concentration of the versatile nuclease (Benzonase®) solution is from 100 to 1000 U/mg (the U is the unit of activity of the enzyme). Since the versatile nuclease is affected by many factors such as the batch, mode of transport and storage time of the manufacturer, the degree of activity of the enzyme also changes, so the enzyme dissolves.
  • the concentration of the liquid is selected within the above range depending on the degree of activity of the enzyme, and the concentration of the enzyme solution should be increased as the enzyme activity is lowered.
  • the cleaning solution is distilled water or sodium chloride solution or a buffer solution having a pH of 6.0 to 8.0.
  • the oscillating treatment temperature of the cornea in the enzyme solution is the oscillating treatment temperature of the cornea in the enzyme solution
  • the cornea is oscillated in the enzyme solution at a frequency of 50-100 beats per minute.
  • a lower oscillation frequency can greatly reduce the degree of damage to the original collagen arrangement of the cornea.
  • the temperature of the cornea during the cleaning process is controlled at 5-20 °C. It is kept at a constant temperature throughout the cleaning process to avoid denaturation of corneal collagen due to excessive temperature.
  • the cobalt 60 is irradiated and sterilized by low temperature irradiation; the cornea is placed in an insulated container filled with a refrigerant for irradiation sterilization; and the cornea is made by a refrigerant
  • the irradiation was kept at a low temperature below 0 °C throughout the irradiation.
  • the refrigerant may be any one of ice or dry ice or liquid nitrogen.
  • the temperature of the corneal low temperature irradiation sterilization shall not exceed 0 °C.
  • the cornea is individually sealed in a single piece prior to irradiation sterilization, and the cornea after sealing and packaging is placed in a low temperature refrigerant for irradiation.
  • the cornea is terminally sterilized in the sealed packaging state until clinical, which is beneficial to maintain the cornea's sterilizing state in the transportation and preservation of the cornea.
  • the cornea is dried after the decellularization treatment to prepare a dried cornea having a moisture content of 5-20%, which is advantageous for mass production of the cornea as a product and Save, transport market commodity attributes.
  • the drying process can be vacuum dried.
  • the vacuum drying is a drying method in which the pressure is gradually reduced from high to low.
  • the decompression range value in the gradual depressurization is a normal pressure to an ultimate vacuum.
  • the gradual drying under reduced pressure is not more than 24 hours.
  • the temperature in the vacuum drying chamber is controlled between 0 ° C and 30 ° C.
  • the invention provides a decellularized pig lamellar cornea, which is composed of a front elastic layer and a matrix layer of a porcine cornea; the matrix layer maintains a regular arrangement structure of collagen fibers; the corneal DNA residue is not more than 100 ng/mg.
  • the light transmittance of the cornea is not less than 80% in the visible light range.
  • Another acellular porcine lamellar cornea comprises a front elastic layer of a porcine cornea and a matrix layer; the matrix layer maintains a regular arrangement of collagen fibers; and the corneal DNA residue is not more than 100 ng/mg.
  • the cornea In the visible light range, the cornea has a light transmittance of not less than 80%; and a dried cornea having a water content of not more than 20%.
  • the method for using the decellularized plate layer dried cornea provided by the present invention takes out the cornea from the sterilized sealed package, and is immersed in physiological saline for 15-30 minutes, and is directly used for the heterogeneous keratoplasty.
  • a bio-enzymatic decellularization method is still employed, but in the present invention, enzymatic treatment using Benzoin® is selected.
  • the technical effect of the present invention is quite remarkable: First, the decellularization effect of the present invention is excellent, corneal HE staining has no cell nucleus, DAPI has no staining, and corneal DNA residue is less than 100 ng/mg.
  • the decellularization method of the present invention maximizes the regular arrangement of the corneal collagen fibers. As shown in Figs. 1A to 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 natural cornea.
  • the cornea is dried before the decellularization treatment, so that the cornea entering the enzyme solution has a lower water content, so that the biological enzyme is more easily infiltrated into the cornea, so that the enzymatic hydrolysis efficiency of the enzyme is greatly enhanced.
  • the enzyme treatment time is shortened while ensuring the decellularization effect.
  • the damage caused by the enzymatic treatment process on the structure of the corneal collagen fibers is minimized.
  • the light transmittance of the cornea treated by the decellularization method of the present invention can be ensured to be 80% or more in the wavelength of 380 to 780 nm.
  • the gradual decompression drying method adopted in the invention effectively overcomes the excessively dry drying of the vacuum drying, makes the vacuum drying process more gentle, and minimizes the corneal stromal layer during the drying process.
  • the drying treatment method used in the preparation method of the present invention is applied to the preparation of the dried cornea, and the obtained dried cornea has a smooth and flat appearance, and thus another significant clinical effect is epithelial cell attachment after transplantation.
  • the proliferation rate is fast and the effect is good.
  • Figure 1 is a flow chart of Embodiment 1 of the present invention.
  • FIG. 1A is a flowchart of preprocessing S1 in Embodiment 1 of the present invention.
  • Embodiment 1B is an embodiment of S2 drying treatment in Embodiment 2 of the present invention.
  • 1C is another embodiment of the S2 drying process in Embodiment 2 of the present invention.
  • Figure 2 is a flow chart of Embodiment 2 of the present invention.
  • FIG. 2A is a third embodiment of the drying process of S2 and S5 in Embodiment 2 of the present invention.
  • Fig. 3 is a photograph of the corneal product of the decellularized and dried lamellar layer of the pig of the present invention.
  • Fig. 4 Photograph of corneal HE staining of the decellularized and dried lamellar layer of the pig of the present invention.
  • Figure 5A is a cross-sectional arrangement of the collagen arrangement of the human cornea
  • Figure 5C is a cross-sectional arrangement of collagen of the porcine cornea after decellularization treatment of the present invention.
  • FIG. 6 Photograph of the decellularized and dried lamellar cornea of the present invention after transplantation into the New Zealand white rabbit.
  • Figure 7A is a pre-operative photo of a clinical transplant of the present invention.
  • Figure 7B is a photograph of the 3 day after clinical transplantation of Figure 7A;
  • Figure 7C is a photograph of Figure 2A after 2 months of clinical transplantation
  • Figure 7D is a photograph of the 6 months after clinical transplantation in Figure 7A;
  • Figure 7E is a photo of Figure 1A after 1 year of clinical transplantation.
  • the preparation method of a porcine acellular cell lamellar cornea provided in the first embodiment is at least four treatment processes. among them:
  • the first step S1 is to pretreat the porcine cornea as shown in Fig. 1A.
  • the pretreatment should at least include the following processing: S1.1 taking fresh porcine cornea, after cleaning
  • the rear elastic layer and the back skin layer are removed to form a lamellar cornea that retains only the front elastic layer and the matrix layer; an alternative embodiment for making fresh
  • the thickness of the lamellar cornea is 300um ⁇ 700um.
  • the present invention can be used to prepare lamellar corneas of different thicknesses in this range, so that it is necessary to accurately select the corneal thickness for different transplant cases.
  • the second step S2 of the first embodiment is a drying process, and the pre-treated lamellar cornea is dried; the S2 drying treatment of the first embodiment is to reduce the water content in the cornea as much as possible before the enzyme treatment, so as to facilitate the enzyme.
  • the solution is incorporated into the cornea to increase the speed and effectiveness of the enzyme treatment. Therefore, the S2 drying treatment method can be any conventional drying method which is available.
  • the drying process used in the S2 step can not be too intense, otherwise it will cause irreparable damage to the collagen arrangement of the cornea.
  • An alternative drying method specifically for the present embodiment 1 can employ a conventional natural drying method. However, since the natural drying method has defects in which temperature is difficult to control, the natural drying method is suitable for use in small batches or under test conditions. It takes a long time to dry naturally, but considering that the drying time is too long, it will adversely affect the cornea's collagen denaturation, so the drying process of naturally drying should not exceed 24 hours. Since the S2 link is an intermediate treatment step, the requirement for corneal drying is to reduce the water content of the cornea as much as possible.
  • a preferred drying method embodiment of this embodiment 1 employs a vacuum drying method.
  • a drying method in which the pressure is gradually reduced from high to low is employed in the first embodiment.
  • the decompression range in the gradual depressurization is a normal pressure to an ultimate vacuum, and the gradual depressurization drying time is not more than 24 hours.
  • the pressure reduction range was from 80 kPa to 0.3 kPa, and the drying time under reduced pressure was 12 hours.
  • the gradual depressurization is an operation of pressure gradient decompression of the vacuum regulating system.
  • the decompression gradients are 80kpa, 60kpa, 40kpa, 20kpa, and 0.3kpa for 2 hours, 2 hours, 2 hours, 1 hour, 1 hour, and drying time of 8 hours, corneal moisture content.
  • the decompression gradients are 80kpa, 60kpa, 40kpa, 20kpa, and 0.3kpa for 2 hours, 2 hours, 2 hours, 1 hour, 1 hour, and drying time of 8 hours, corneal moisture content.
  • the temperature in the vacuum drying chamber is controlled between 0 ° C and 30 ° C.
  • the decompression range is from atmospheric pressure to ultimate vacuum, and the time for drying under reduced pressure is 12 hours, and the temperature in the vacuum drying chamber is controlled at 0 ° C to 30 ° °C.
  • the gradual decompression is specifically carried out in this test by means of continuous decompression, and the pressure is reduced to the maximum vacuum of the apparatus over a period of time.
  • a continuous pressure reduction operation was performed for a period of time by controlling the vacuum adjustment system, and the pressure reduction curve is as shown in Fig. 1C.
  • the continuous decompression method adopted in this test example can be realized by automatic control, which does not require manual operation, and is therefore suitable for mass production.
  • the pressure is gradually reduced to a maximum vacuum of the apparatus after about 12 hours by gradually depressurizing, thereby achieving the corneal drying requirement.
  • the drying process of the embodiment is gradually gentler, and the drying process is more gentle, thereby minimizing the regular arrangement damage of the collagen fibers in the matrix layer during the corneal drying process. .
  • the time is shorter, avoiding corneal protein changes in this dry phase.
  • S3 Decellularization: Desiccation of the cornea after drying: S3.1 Enzyme treatment: Configure the versatile nuclease (Benzonase®) solution in (DMEM) medium; add 0.5-3 ml of the above enzyme according to each cornea. Solution; first vortex to the surface of the cornea to remove air bubbles, remove the gas in the cornea; then oscillate in the shock incubator for not less than 1 hour;
  • DMEM versatile nuclease
  • 0.7 ml of the above-mentioned all-enzyme nuclease (Benzonase®) solution was added to each cornea, and vortexing was used to remove the air bubbles from the cornea surface.
  • Tests have shown that the bubbles on the corneal surface 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 corneal stroma layer substantially in the form of bubbles. Then, the oscillating treatment was carried out, and specifically oscillated for 2.5 to 3 hours in this test example.
  • the concentration of the benzoonase® solution is 100 1000 U/ml. Since the unitonase (Benzonase®) is affected by many factors such as the batch, mode of transport and storage time of the manufacturer, the degree of activity of the enzyme also changes. Therefore, the concentration of the enzyme solution should be selected according to the activity of the enzyme. The concentration of the solution should increase as the enzyme activity decreases.
  • the oscillating treatment temperature of the cornea in the enzyme solution is 15-37 ° C, which is slightly lower than the optimal temperature recommended by the Benzonase® manufacturer (35 °). C or so) to minimize the destruction of the regular collagen fiber arrangement in the corneal stroma by the enzymatic hydrolysis process.
  • the oscillation treatment temperature in this test example is controlled at about 25 °C.
  • the oscillation frequency of the cornea in the enzyme solution is controlled at a lower level of 50-100 times per minute. Specifically, in the test example, the oscillation frequency is selected to be 75 times per minute. When testing to demonstrate enzymatic treatment, the goal of selecting a lower oscillation frequency is to minimize the extent of damage to the original collagen arrangement of the cornea.
  • the S3.2 cleaning treatment is to clean the cell residue generated in the matrix layer during the S3.1 enzyme treatment.
  • the specific treatment method is to add not less than 5 ml of the cleaning solution to each cornea, and the shaking washing is not less than 5 times, each time not more than 30 minutes;
  • the temperature of the cornea during the cleaning process is controlled at 5-25 °C. Specifically, in the test example, the temperature during the cleaning process was controlled at about 15 ° C, and the temperature was kept constant throughout the cleaning process to avoid denaturation of corneal collagen due to excessive cleaning temperature.
  • the cornea is oscillated at a frequency of 100-160 times per minute during the washing process to promote the release of cellular components from the matrix layer.
  • the oscillation frequency was 150 times per minute in this test example.
  • Each shaking time is 15 minutes to 20 minutes.
  • the cleaning solution is a distilled water or sodium chloride solution or a buffer solution having a pH of 6.0 to 8.0.
  • distilled water or a buffer solution having a pH of 6.0 to 8.0 was used as the washing solution in this test example.
  • S4 Sterilization treatment.
  • the cornea is terminally sterilized, and the cornea should be sterilized to meet the relevant national sterilization standards.
  • cobalt 60 irradiation sterilization is used, and the irradiation dose is not more than 25 kgy. Based on existing research results, the use of irradiation sterilization inevitably adversely affects the important property of corneal transparency.
  • the cobalt 60 is irradiated and sterilized by low temperature irradiation; the cornea is placed in an insulated container filled with a refrigerant for irradiation sterilization; The cornea is kept at a low temperature below 0 °C throughout the irradiation.
  • the refrigerant may be any of ice or dry ice.
  • the refrigerant is dry ice. Since dry ice itself has a lower initial temperature (-78 ° C), it is not easy to heat up quickly in the insulated container, and it is easier to obtain and maintain a lower irradiation temperature, so dry ice is used as a corneal irradiation sterilization refrigerant. It is a preferred embodiment of the present invention. The cornea can be placed in dry ice first, and then the cornea is quickly lowered to a low temperature environment before starting.
  • the refrigerant is ice
  • the initial temperature of the ice should be selected from -18 to 25 ° C, even if the ice rises with the temperature of the irradiation environment, the ice The temperature was maintained at a low temperature of less than -5 ° C during the entire irradiation.
  • the temperature in the insulated container is at a low temperature environment much lower than 0 ° C, and further destruction of the collagen structure of the cornea during irradiation sterilization is effectively avoided.
  • the cornea is individually sealed and packaged before being irradiated, and the cornea after sealing and packaging is placed in a low temperature refrigerant for irradiation.
  • the cornea is terminally sterilized in a sealed package until clinical, which helps the cornea to maintain the cornea in a state of transportation and preservation.
  • the cornea is a non-dried cornea, which can be directly used for transplantation surgery. If it is necessary to store it for a period of time, it can be sealed in a non-dried cornea in the prior art, such as DMEM cell culture medium, and stored under low temperature in a sealed state.
  • the damage effect on the regular arrangement structure of the original collagen fibers of the cornea is first considered, and effective measures are taken as much as possible in the treatment method. Avoid or minimize the disadvantages of this damage.
  • Tests have shown that the artificial cornea obtained by the preparation method of the present invention maximizes the regular arrangement structure which is very close to the natural cornea, and minimizes the requirement of the national standard for immunogenic (DNA) residues.
  • the damage caused by the regular arrangement of the original collagen fibers in the corneal stroma layer during the preparation process is shown in Fig. 5 to Fig. 5C. Therefore, the cornea prepared by the present invention has physical properties such as elastic mechanical strength and transparency which are extremely similar to those of the human cornea, particularly the transparency of the cornea.
  • the obtained non-dried cornea treated by the decellularization method of the present invention has no cell nucleus by HE staining, no staining by DAPI, and the residual amount of corneal DNA is less than 100 ng/mg, and the light transmittance of the cornea can ensure 75% in the wavelength of 380-780 nm. the above.
  • a method for preparing a porcine acellular cell lamellar cornea provided in the second embodiment has at least five treatment processes. among them:
  • the first step S1 pretreatment and the second step S2 drying treatment are substantially the same as those of the first embodiment, and the specific embodiments can be selected within the range listed in the embodiment 1. Therefore
  • 0.5 ml of the above-mentioned all-enzyme nuclease (Benzonase®) solution was added to each cornea, and vortexing was first used to remove the air bubbles from the cornea surface. Specifically, in this test case, it is oscillated for 2.5 to 3 hours.
  • the concentration of the benzoonase® solution is 300 U 500 U/ml.
  • the concentration of the enzyme solution should be selected in the range of 300 U 500 U/ml depending on the degree of activity of the enzyme.
  • the oscillating treatment temperature of the cornea in the enzyme solution is 15-37 ° C, specifically the oscillating treatment temperature described in the test example is controlled at about 25 ° C.
  • the oscillation frequency is selected to be 65 times per minute.
  • the purpose of selecting a lower oscillation frequency is to minimize the degree of damage to the original collagen arrangement of the cornea.
  • the S3.2 cleaning treatment is to clean the cell residue generated in the matrix layer during the S3.1 enzyme treatment.
  • the specific treatment method is to add not less than 5 ml of the cleaning solution to each cornea, and the shaking washing is not less than 5 times, each time not more than 30 minutes;
  • the temperature of the cornea during the cleaning process is controlled at 5-25 °C. Specifically, in the test example, the temperature during the cleaning process was controlled at about 15 ° C, and the temperature was kept constant throughout the cleaning process to avoid denaturation of corneal collagen due to excessive cleaning temperature.
  • the cornea is oscillated at a frequency of from 100 to 160 times per minute during the washing process to promote the release of cellular components from the matrix layer.
  • the oscillation frequency is 100 times per minute.
  • the cleaning time is 10 minutes to 15 minutes per shake.
  • the cleaning solution is a distilled water or sodium chloride solution or a buffer solution having a pH of 6.0 to 8.0. Specifically, in this test example, 0.9% sodium chloride was used as the cleaning solution.
  • S4 Dry corneal preparation.
  • the cornea is dried after decellularization treatment to prepare a dried cornea having a water content of 5%-20%.
  • the dried cornea prepared in the second embodiment is advantageous for
  • the cornea has properties as a product in the mass production as well as preservation and transportation markets.
  • a drying method in which the pressure is gradually reduced from high to low in substantially the same manner as in the second step S2 of the foregoing step 2 can be employed.
  • the decompression range in the gradual depressurization is a normal pressure to an ultimate vacuum, and the gradual decompression drying time is not more than 12 hours.
  • the pressure reduction curve is shown in Fig. 1B and Fig. 1C.
  • the temperature for vacuum drying is controlled from 0 °C to 30 °C.
  • an alternative embodiment of the method for preparing a dried cornea of S4 of the second embodiment is as follows.
  • the pressure is reduced from 80 kPa by 5 steps in a gradient decompression manner.
  • the maximum vacuum to the equipment is 0.5kpa.
  • the vacuum regulating system adjusts the pressure of the first stage to gradually decrease to the pressure of the latter stage, that is, the pressure in the control vacuum drying chamber is gradually reduced from the previous gradient pressure value to the next one. Pressure gradient value.
  • the decompression curve is as shown in Fig. 2B.
  • the cornea has a water content of 10% to 20% and a light transmittance of 84% to 87%.
  • the drying method shown in Fig. 2B is also applicable to the S2 drying treatment before decellularization in Example 1 and Example 2.
  • the above-described gradual vacuum drying method of this Example 2 is milder than the vacuum decompression method of the prior art. Therefore, the obtained dried cornea ensures that the moisture content of the dried cornea is in the range of 5% to 20%, and the light transmittance is more than 80%.
  • the dried cornea prepared in the second embodiment has a smooth surface and no hyacous protrusions or fine folds visible to the naked eye, and another significant clinical effect brought about by this feature is that the postoperative epithelial cells adhere and proliferate quickly. .
  • the moisture content of the dried cornea should reach 0 to 20%.
  • the treatment procedure S5 refers to the terminal sterilization treatment of the dried cornea, and the sterilization of the cornea should meet the national sterilization standards.
  • cobalt 60 irradiation is used for sterilization, and the irradiation dose is not more than 25 kgy.
  • the cobalt 60 is irradiated and sterilized by low temperature irradiation; the cornea is placed in an insulated container filled with a refrigerant for irradiation sterilization; The cornea is kept at a low temperature below 0 °C throughout the irradiation.
  • the refrigerant is the same as the embodiment 1 described above except for the ice (including the cold storage agent) or the dry ice in the first embodiment. The detailed description will not be repeated in this example 2.
  • the cornea is first subjected to irradiation sterilization.
  • the cornea is sealed and packaged in a low-temperature refrigerant for irradiation.
  • the cornea is terminally sterilized in a sealed package until it is opened in the clinic.
  • a large number of experiments have proved that the dry cornea with lower corneal moisture content is sterilized by low temperature irradiation under the same conditions as the non-dried cornea.
  • the physicochemical properties of the cornea after sterilization are small, and the transparency, toughness and hydrophilicity are basically No change occurs, and the adverse effects of irradiation on the physical and chemical properties of the cornea under normal temperature conditions are effectively avoided.
  • the acellular porcine layer is dried by the above preparation method, and the residual corneal DNA is not more than 100 ng / mg, as shown in FIG.
  • the invention comprises a lamellar dried cornea composed of a front elastic layer and a matrix layer of the porcine cornea; the matrix layer maintains a regularly arranged collagen fiber structure, as shown in the electron micrograph of FIG. 5 to FIG. 5C, the cornea obtained by the preparation method of the invention and the human
  • the collagen structure of the cornea and the non-decellularized porcine cornea is extremely close; the dry cornea having a water content of not more than 20% is in the visible light range, and the cornea has a light transmittance of not less than 80%, as shown in FIG.
  • the dried cornea of the present invention is excellent not only in transparency but also in product properties having high surface flatness.
  • a large number of trials have shown that a flat corneal surface facilitates the attachment and proliferation of epithelial cells.
  • the better flatness of the pre-corneal elastic layer is very beneficial to increase the growth rate and growth quality of epithelial cells after corneal implantation.
  • the method for using the decellularized lamellar dried cornea of the present invention is relatively simple and easy to operate.
  • the cornea is taken out from the sterilized sealed package before surgery, and immersed in physiological saline for 15-30 minutes, and directly used for xenogeneic keratoplasty.
  • the dried cornea provided by the present invention is undoubtedly the best product state of the corneal product, which is convenient for storage and transportation. It can realize the quality identity and performance stability of dry corneal products in the market segments such as storage and transportation, and can achieve the effect of greatly prolonging the shelf life.
  • the dry cornea provided in the second embodiment has another important technical effect: the rehydration operation before the operation is simple, and the rehydration time is short.
  • the water content of the cornea can meet the surgical requirements, and the water content of the cornea after rehydration can be properly controlled, which is very important for controlling or shortening the corneal recovery time.
  • the present invention can completely standardize the rehydration operation before surgery, and achieve the problem of effectively controlling the water content of the cornea rehydration.
  • FIG. 7A to Figure 7E After 2 months of menstrual lamellar keratoplasty, the cornea was transparent and non-rejective. Postoperative visual acuity 0.6
  • the present invention achieves a good grafting effect in a large number of animal tests, as well as in all clinical corneal transplants currently performed.
  • Replacement page (Article 26)
  • the acellular cell layer dried cornea of the invention is composed of a biological material, and can solve the problem of human body rejection better than the synthetic eye implant material, and can be used for refractive correction application through a simple operation human eye. To achieve permanent refractive correction.

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Abstract

一种猪脱细胞板层角膜的制备方法及其猪脱细胞板层角膜，制备方法为：预处理（S1）：对新鲜猪角膜的预处理,制作板层角膜；干燥处理（S2）：将预处理后板层角膜进行干燥处理；采用酶处理方法进行脱细胞（S3）：将干燥后角膜加入酶溶液；置于震荡培养箱中震荡处理；再将角膜置于震荡培养箱震荡洗涤处理，获得脱细胞角膜；灭菌处理（S4）。

Description

1猪 膜的制 方法及其脱细胞板层 膜和用、法 所属领域
本发明涉及一种脱细胞猪角膜的制备方法，及可以直接用于人角膜移植术的 以猪角膜为材料的板层脱细胞角膜及其使用方法。 背景技术
角膜盲是我国的第二大致盲性眼病，角膜移植是角膜盲患者复明唯一有效的 治疗手段。但角膜供体材料的匮乏严重影响角膜移植，经过中国科学家十余年努 力研发出的以猪角膜为原材料的人工角膜可以代替代人源角膜，并在全世界范围 内率先取得临床试验的成功。
现有的研究成果证明，以猪角膜为来源材料制备的人工角膜具有很好的生物 相容性。特别是猪角膜具有与人角膜高度近似的组织结构、生物物理学特性和光 学特性是角膜替代物的最佳选择的公认结论。而国内的研究成果所取得进展显示， 猪角膜已经被作为人角膜移植备用材料一个重要的替代来源之一。目前部分脱细 胞的猪角膜产品已经进行临床阶段， 并产生一定的临床效果。为解决国内移植用 角膜供体严重缺乏的现状提供了极好的解决方案，为国内数百万因角膜致盲的患 者带来复明的希望。
角膜位于眼球的前部，是一种结构高度规则、相对无细胞的透明胶原组织。 现有的板层脱细胞角膜基质仅包含有前弹力层和基质层。经过必要的脱细胞处理 去除基质层内的免疫原性 （DNA)， 降低猪角膜移植的免疫排斥反应。 再通过病 毒灭活， 灭菌处理降低角膜移植中的动物源性病毒、细菌感染， 从而达到板层角 膜可以用于移植的必要的生物学指标。
―角膜基质层的结构特点在于三股螺旋的 I型胶原有序且相互平行的排列，这 种排列方式构成了角膜弹性机械强度及其透明度的等物理学特性结构基础。角膜 的这个重要特征可以在植入后， 诱导受体角膜基质细胞有序、 均匀地长入， 使角 膜保持透明。
基于对上述板层角膜基质特点的分析， 角膜基质层排列结构的高度保持， 是实现板层角膜必要的生物物理学特性的重要因素。然而， 目前的脱细胞方法都 替换页 （细则第 26条) 会在不同程度上对基质层的有序排列造成损害。其结果是在达到移植条件的必要 的生物学指标的同时，都不可避免地破坏了角膜弹性机械强度及其透明度的等物 理学特性。 特别是角膜的透明度， 从而大大地减损了角膜的复明功效。
酶法是角膜脱细胞处理的各种方法中最有效地方法。 现有酶解法中使用酶 试剂主要有： 脂类酶、 核酸酶和蛋白酶等； 不同的酶类针对特定的细胞成分。 其 所存在的缺陷： 首先， 不能达到完全的脱细胞效果； 其二， 上述所列出的酶、 特 别是蛋白酶，对角膜细胞外基质也存在明显的破坏作用，导致角膜的透明度下降。
另外，现采用酶解脱细胞方法是，在角膜进行预处理后直接浸入酶溶液中。 由于在预处理过程中， 角膜的含水量不可避免地增大了，再将角膜直接浸入酶溶 液后， 酶溶液很难进入角膜内， 大大地影响了酶对角膜基质内细胞的降解效果。 很多情况下为保证脱细胞效果往往采用延长酶解处理时间的办法，增大了对角膜 的透明度的等物理学特性的破坏机率。
角膜的灭菌处理是角膜制备不可或缺的步骤， 其作用是完全杀灭角膜内的 细菌、病毒等有害微生物。 随着各种新的灭菌技术的成熟及广泛应用， 辐照灭菌 技术引入了角膜的灭菌处理。但是通过大量的角膜辐照灭菌试验结果来看，辐照 灭菌后， 角膜的透明度、 生物力学特性均有比较明显的下降。美国国际组织库在 进行的人角膜辐照灭菌试验，以及对比辐照前后角膜的透明度等试验数据分析后 指出， 常温条件下对人来源的角膜供体进行辐照灭菌，伽马射线可以使细胞外基 质中胶原的分子键发生变化，辐照后角膜的理化性能发生改变， 导致辐照后角膜 的透明度、 韧性、 亲水力与正常生理状态下角膜相比， 发生明显变化， 提出 "辐 照灭菌不利于角膜临床灭菌使用"研究结论。
综上所述， 现有的角膜制备方法中， 去除基质层内的细胞成分， 降低免疫 排斥反应， 灭菌处理降低动物源性病毒感染的两个关键步骤中， 都不可避免地破 坏了角膜弹性机械强度及其透明度的等物理学特性， 特别是角膜的透明度。
目前大量的专利文献以及相关文献中所公开的各种脱细胞猪板层角膜的制 备方法及其效果，大都是依据试验室中完成脱细胞处理之后的非干燥角膜状态下 取得的。并且因其制备方法的不同， 其试验数据或临床效果也不尽完全相同。毋 庸置疑， 目前公开现有技术，所称的角膜产品及其应用效果仅仅是建立在非干燥 角膜的试验数据状态上。而非干燥角膜存在最大的缺陷是极其不容易于保存和运
替换页 （细则第 26条) 输， 很难保证在生产、保存、运输及使用等市场运营的每个环节上产品质量同一 性和性能稳定性， 还不具备可以在市场上推广使用的条件。
目前研究人员提出的方法是对脱细胞之后的角膜进行必要干燥处理以求获 得一种干燥角膜。在其中所列出的诸多常规干燥方法中， 真空干燥是常用的干燥 方法。本申请人在对现有的所有真空干燥方法获得的大量的试验数据进行研究统 计分析后发现，真空干燥方法对人工角膜的不利影响的原因是相对于角膜其干燥 过程过于剧烈， 从而使得胶原组织排列发生不规则的改变，破坏了原角膜基质层 中高度规则的胶原排列， 从而大大地影响了角膜的透明度。
而现有技术文献中公开的其它干燥方法基本上不能适于角膜的干燥处理。如 自然晾干因时间过长，因环境温度以及较容易污染等问题较使得角膜基质内的胶 原蛋白变性， 角膜因此而丧失其应用的透明度。而冻干或者真空冻干的方法会使 得角膜内因结晶而产生不规则空隙并因此而破坏了角膜的规则的胶原排列。
不难证明的事实是， 在人工角膜的制备过程中， 任何一个处理环节都不可 避免对角膜基质层内的规则排列造成的损害， 并因此而对制备后的角膜的 "透明 度"造成极不利的影响。虽然可以有效地解决异体移植用器官的免疫排斥及动物 源性病毒感染等问题，但是因制备过程对角膜透明度的不同程度的损伤， 同样会 在很大程度上影响移植效果。有必要提供一种方法， 最大限度在减小对角膜基质 层内规则排列的胶原结构的破坏，以确保在通过复杂的制备处理后的角膜仍具有 极佳的透明度， 达到实现人工角膜产品的市场化的应用的目的。 发明内容
本发明的目的在于提供一种猪脱细胞板层角膜的制备方法，在必要的脱细胞 处理和灭菌处理等关键步骤中，在确保达到严格的移植条件所必需的生物学指标 的同时， 能最大限度地降低对角膜胶原纤维规则排列结构的破坏。保持角膜透明 度及其弹性机械强度等物理学特性， 特别是将对角膜透明度的破坏降到最低。
本发明的另一个目的在于提供一种猪脱细胞板层角膜的制备方法， 对角膜 进行干燥处理过程中， 最大程度地减少干燥过程对角膜胶原结构的破坏， 角膜产 品保持有平整光滑外观形态， 有利于角膜细胞的生长。
本发明的再一目的在于提供一种猪脱细胞板层角膜的制备方法及其干燥角
替换页 （细则第 26条) 膜， 使其具有方便保存、运输及使用的产品形态。从而克服现有技术中角膜产品 依赖其制备方法不同而质量不一、产品难以规范化生产的缺陷，达到批量生产的 技术要求。促进我国眼科医疗水平的发展，解决众多因缺乏角膜供体而不能得到 有效医治的问题。
本发明的目的是这样实现的，本发明提供的一种猪脱细胞板层角膜的制备方 法， 至少如下步骤： Sl、 预处理： 对新鲜猪角膜的预处理包括如下处理过程： S1.1 取新鲜猪角膜， 清洁后去除上皮层； S1.2 制备板层角膜； 所述板层角膜仅 包括前弹力层和基质层； S1.3 清洗干净；
S2: 干燥处理： 将制备的板层角膜进行干燥处理；
S3: 脱细胞处理： 将干燥处理后角膜进行脱细胞处理过程： S3.1 酶处理： 采用 DMEM培养基配置全能核酸酶 （Benzonase®) 溶液； 将干燥后角膜加入上 述酶溶液； 置于震荡培养箱中震荡不小于 1小时； S3.2清洗： 将角膜加入清洗溶 液中， 置于震荡培养箱震荡洗涤处理， 获得脱细胞角膜；
S4: 灭菌处理： 采用钴 60辐照灭菌， 辐照剂量不大于 25kgy。
由于在本发明中仍采用生物酶解的脱细胞方法，但在本发明中选择使用全能 核酸酶（Benzonase®)进行酶处理无需再对各种不同酶进行组合使用， 即可以取 得相当好的脱细胞效果， HE染色无细胞核， DAPI无染色， 角膜 DNA残留低于 lOOng/mgo其次，本发明脱细胞方法可以最大限度地保持了角膜与天然角膜极为 接近的规则的胶原纤维的排列结构。
另外，在本发明在进行脱细胞处理之前对角膜进行干燥处理，通过降低角膜 含水率， 增大了角膜与酶溶液间的渗透压差， 使得生物酶更容易渗入角膜内， 大 大地提高了酶的效率， 从而缩短酶处理时间。 因此， 最大限度地减小了酶处理过 程对角膜胶原纤维结构造成的损害，达到保持角膜透明度的技术效果。角膜的透 光率均可确保在 380-780nm波长内达到 80%以上。
本发明的一个可选择的实施方式中在 S1.2步骤中制备的新鲜板层角膜的厚度 为 300um~700um。
本发明的一个较佳的实施方式中所述全能核酸酶（Benzonase®)溶液的浓度为 100〜 1000U/mg (其 U为酶的活性单位）。由于全能核酸酶因产生厂家产品批次、 运输方式及保存时间等诸多因素的影响， 酶的活性程度也会发生变化， 因此酶溶
替换页 （细则第 26条) 液的浓度随酶的活性程度在上述范围内选择，酶溶液的浓度应当随酶活性的降低 而增高。
本发明的一个可选择的实施方式中，所述清洗溶液为蒸馏水或氯化钠溶液或 pH6.0至 8.0的缓冲溶液。
本发明的一个可选择的实施方式中， 角膜在酶溶液中的震荡处理温度
15-37°C， 略低于该全能核酸酶 （Benzonase®) 厂家推荐使用的最佳温度 （35 °C 左右）， 以最大程度地降低酶解过程对角膜基质层内胶原纤维规则排列的破坏。
本发明的一个可选择的实施方式中， 角膜在酶溶液中的震荡频率为每分钟 50-100次。试验证明的进行酶处理时，较低的震荡频率可以大大地减少对角膜原 有胶原排列的破坏程度。
在本发明中的一个选择实施方式中， 角膜在清洗处理过程中的温度控制在 5-20 °C。 并在全部的清洗过程基本上保持恒温状态， 以避免因温度过高而产生角 膜胶原蛋白的变性。
在本发明中的一个较佳的实施方式中， 所述钴 60辐照灭菌采用低温辐照； 将角膜置于填充有制冷剂的保温容器中进行辐照灭菌；通过制冷剂使得角膜在辐 照全过程中保持在低于 0°C的低温状态下。 其中， 所述的制冷剂可以是冰或干冰 或液氮任意一种。 所述角膜低温辐照灭菌全程中温度不得高于 0°C。
在本发明中的一个较佳的实施方式中，在进行辐照灭菌前， 先将角膜进行单 片密封包装，将密封包装后角膜置于低温制冷剂之中进行辐照。角膜在密封包装 状态下进行终端灭菌直至临床，有利于角膜在运输及保存等环节上保持角膜的灭 菌状态。
在本发明中的另一个较佳的实施例中，在脱细胞处理后对角膜进行干燥处理， 制备成含水率含水量为 5-20%的干燥角膜，有利于角膜具有作为产品在批量生产 以及保存、 运输市场商品属性。
在本发明中的一个较佳实施方式中，所述的干燥处理可采用真空干燥。在一 个较佳的干燥处理实施方式为所述真空干燥为压力自高向低的逐渐减压的干燥 方法。所述逐渐减压中的减压范围值为常压至极限真空。所述逐渐减压干燥的时 间不大于 24小时。
在本发明中的一个选择实施方式中， 真空干燥室内的温度控制在 0°C~30°C。
替换页 （细则第 26条) 本发明提供的一种脱细胞猪板层角膜， 由猪角膜的前弹力层和基质层构成； 所述基质层保持有胶原纤维的规则排列结构； 角膜 DNA残留不大于 100ng/mg。 在可见光范围内， 所述角膜的透光率不低于 80%。
本发明提供的另一种脱细胞猪板层角膜，由猪角膜的前弹力层和基质层构成； 所述基质层保持有胶原纤维的规则排列结构； 角膜 DNA残留不大于 100ng/mg。 在可见光范围内， 所述角膜的透光率不低于 80%; 含水率不大于 20%的干燥角 膜。
本发明提供的脱细胞板层干燥角膜的使用方法，从灭菌密封包装中取出角膜， 浸入生理盐水 15-30分钟后， 直接用于异种角膜移植术。
在本发明中仍采用生物酶解的脱细胞方法，但在本发明中选择使用全能核酸 酶（Benzonase®)进行酶处理。 本发明的技术效果相当显著： 首先， 本发明脱细 胞效果极佳，角膜 HE染色无细胞核， DAPI无染色，角膜 DNA残留低于 100ng/mg。 其次， 本发明脱细胞方法， 最大限度地保持了角膜胶原纤维的规则排列结构。如 图 1A〜图 1C所示， 本发明方法获得的角膜电镜结构显示胶原纤维的结构与天然 角膜极为接近。
在本发明技术效果，在进行脱细胞处理之前对角膜进行干燥处理，使得进入 酶溶液的角膜具有较低含水率，使得生物酶的更容易渗入角膜内，使得酶的酶解 效率被大大地增强了， 在保证脱细胞效果的同时， 缩短酶处理时间。最大限度地 减小了酶处理过程对角膜胶原纤维结构造成的损害。经本发明脱细胞方法处理的 角膜的透光率均可确保在在 380-780nm波长内达到 80%以上。
本发明中的采用的逐渐减压的干燥方法，有效地克服了真空干燥的干燥过于 剧烈的缺陷，使得真空干燥的过程变得更加温和， 最大限度地减小了干燥过程中 对角膜基质层的胶原纤维规则排列的破坏程度。本发明制备方法中所采用的干燥 处理方法，应用在制备干燥角膜时，所获得干燥角膜具有表面光滑平整的外观形 态，因此而带来的另一显著的临床效果是移植术后上皮细胞贴附和增殖速度快效 果好。 附图说明
下面结合附图对本发明及其具体实施方式及其效果作简单地介绍，下面的附
替换页 （细则第 26条) 图仅仅是选择本发明的一些具体试验例说明， 而非本发明的全部。
图 1 本发明实施例 1的流程图；
图 1A 本发明实施例 1中预处理 S1的流程图；
图 1B 本发明实施例 2中 S2干燥处理的一种实施方式；
图 1C 本发明实施例 2中 S2干燥处理的另一种实施方式；
图 2 本发明实施例 2的流程图；
图 2A本发明实施例 2中 S2和 S5干燥处理的第三种实施方式；
图 3 本发明猪脱细胞干燥板层角膜产品照片。
图 4 本发明猪脱细胞干燥板层角膜 HE染色照片。
图 5 本发明与其它角膜在电镜下胶原结构对比；
图 5A 人角膜的胶原排列横截面排列结构；
图 5B 未脱细胞处理的猪角膜胶原横截面排列结构；
图 5C 本发明经脱细胞处理后的猪角膜的胶原横截面排列结构；
图 6 本发明猪脱细胞干燥板层角膜向新西兰大白兔板层移植术后照片。 图 7A本发明一临床移植术前照片；
图 7B为图 7A临床移植术后 3天照片；
图 7C为图 7A临床移植术后 2个月照片；
图 7D为图 7A临床移植术后 6个月照片；
图 7E为图 7A临床移植术后 1年的照片
具体实施方式
下面将结合本发明实施例，对本发明技术方案进行清楚、完整地描述，显然， 所描述的实施例仅仅是本发明一部分实施例， 而不是全部的实施例。基于本发明 中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其 他实施例， 都属于本发明保护的范围。
实施例 1
如图 1所示，本实施例 1提供的一种猪脱细胞板层角膜的制备方法， 至少由 四个处理过程。 其中：
如图 1A所示， 第一步骤 S1是对猪角膜进行预处理， 如图 1A所示。具体在 本实施例中， 预处理至少应当包括如下处理过程： S1.1 取新鲜猪角膜， 清洁后
替换页 （细则第 26条) 去除上皮层； S1.2 制备板层角膜； 所述板层角膜仅包括前弹力层和基质层； S1.3 清洗干净； 本发明对新鲜猪角膜的预处理主要是完成板层角膜制取， 并进行必要 的清洗以备进行后续处理程序，因此本发明预处理看 S1.1 S1.3的每个处理方法 均可以采用任意一种常规的处理方法。
在本实施例 1 中 S1.2 步骤中可选择的实施方式中，去除后弹力层和后皮层， 构成仅保留前弹力层和基质层的板层角膜；其中一个可选择的实施方式制取新鲜 板层角膜厚度为 300um ~ 700um。本发明可在这个范围内制备成不同厚度规格板 层角膜， 以供不同移植病例有必要对角膜厚度进行精准选择需求。
本实施例 1的第二步骤 S2为干燥处理，将预处理后板层角膜进行干燥处理； 本实施例 1的 S2干燥处理是为了在酶处理前尽可能减少角膜内的含水量， 以利 于酶溶液参入角膜内， 提升酶处理的速度和效力。 因此 S2干燥处理方法可采用 现有的任意一种常规的干燥方法。
在本 S2环节中采用的干燥方法的干燥过程不能过于剧烈， 否则将对角膜的 胶原排列造成不可恢复的破坏。具体在本实施例 1的一个可选择的干燥方法可采 用常规的自然晾干方法。但是， 由于自然晾干法存在温度较难控制的缺陷， 因此 自然晾干法适用于小批量， 或者试验条件下的使用。 自然晾干所需时间比较长， 但是考虑到干燥时间太长会对角膜的产生胶原变性的不利影响，因此自然晾干的 干燥过程不得大于 24小时。 由于 S2环节是中间处理环节，对角膜的干燥要求是 尽可能地减少角膜的含水量。
在本实施例 1的一个较佳的干燥方法实施方式是采用真空干燥方法。为避免 现有技术中现有的真空干燥法过于剧烈的缺陷，具体在本实施例 1中采用一种压 力自高向低的逐渐减压的干燥方法。所述逐渐减压中的减压范围值为常压至极限 真空， 所述逐渐减压干燥的时间不大于 24小时。
如图 1B所示， 在实施例的一个具体的试验例中， 减压范围值为 80 kpa ~ 0.3kpa, 减压干燥的时间 12小时。本试验例中， 所述逐渐减压是通过对真空调节 系统进行压力梯度减压的操作。如图 2A所示，减压梯度为 80kpa、60 kpa、40 kpa、 20 kpa、 0.3kpa持续时间分别为 2小时、 2小时、 2小时、 1小时、 1小时， 干燥 时间 8小时， 角膜含水率大大地被降低了。
在本发明中的一个选择实施方式中， 真空干燥室内的温度控制在 0°C~30°C。
替换页 （细则第 26条) 如图 1C所示， 在实施例 1的另一个具体的试验例中， 减压范围值为常压至 极限真空， 减压干燥的时间 12小时， 真空干燥室内的温度控制在 0°C~30°C。 本 试验例中，所述逐渐减压是具体在本试验中采用连续减压的方式，在一时间段内 将压力降至设备最大真空度。本试验例中通过控制真空调节系统，在一段时间内 进行连续减压操作， 减压曲线如图 1C所示。 本试验例所采用的连续减压方式可 通过自动控制方式实现， 无需进行手动操作， 因此适用于大批量生产。
在本实施例 1较佳的逐渐减压真空干燥方法，经过逐渐减压的方式，经过大 约 12个小时左右将压力逐步降低至设备最大真空度， 从而达到角膜干燥要求。 与现有直接进入最大真空环境中进行干燥的方法相比，本实施例逐渐减压的方式 干燥过程更为温和，因而最大限度地减小了角膜干燥过程中对基质层的胶原纤维 规则排列破坏。与自然晾干相比， 时间比较短， 避免在这个干燥环节造成角膜的 蛋白变。
S3 : 脱细胞处理： 将干燥处理后角膜进行脱细胞处理过程： S3.1 酶处理： 采用 （DMEM)培养基配置全能核酸酶（Benzonase®)溶液； 按照每片角膜加入 0.5-3毫升上述酶溶液； 先涡旋震荡至角膜表面气泡去除， 排除角膜内的气体； 再置于震荡培养箱中震荡不小于 1小时；
具体在本实施例 1的一个试验例中， 按照每片角膜加入 0.7毫升上述全能核 酸酶（Benzonase®)溶液，先采用涡旋震荡方式至角膜表面气泡去除。试验证明， 在很短的时间内角膜表面的气泡即已经去除， （通常不会超出 1分钟的时间）。而 此时角膜内所含气体基本上以气泡形式从角膜基质层内排出。然后进行震荡处理， 具体在本试验例中震荡处理 2.5~3小时。
在本实施例 1的较佳的实施方式中， 所述全能核酸酶（Benzonase®)溶液的 浓度为 100 1000U/毫升。由于全能核酸酶（Benzonase®)因产生厂家产品批次、 运输方式及保存时间等诸多因素的影响， 酶的活性程度也会发生变化， 因此酶溶 液的浓度应当随酶的活性程度进行选择，酶溶液的浓度应当随酶活性的降低而增 高。
在实施例 1的一种可选择的实施方式中， 角膜在酶溶液中的震荡处理温度 15-37°C， 略低于该全能核酸酶 （Benzonase®) 厂家推荐使用的最佳温度 （35 °C 左右）， 以最大程度地降低酶解过程对角膜基质层内规则胶原纤维排列的破坏。
替换页 （细则第 26条) 具体在本试验例所述震荡处理温度控制在 25 °C左右。
在实施例 1的一个选择实施方式中，角膜在酶溶液中的震荡频率控制在每分 钟 50-100次较低的水平。 具体在本试验例中， 所述的震荡频率选择为每分钟 75 次。试验证明的进行酶处理时，选择一个较低的震荡频率的目的是最大限度在减 少对角膜原有胶原排列的破坏程度。
S3.2清洗处理作用是将 S3.1酶处理过程中在基质层内产生的细胞残留清洗 出来。具体处理方法是按照每片角膜加入不低于 5毫升的清洗溶液，震荡洗涤不 少于 5次， 每次不大于 30分钟；
在本实施例 1的一个选择实施方式中，角膜在清洗处理过程中的温度控制在 5-25 °C。具体在本试验例中清洗处理过程中的温度控制在 15 °C左右，并在全部的 清洗过程基本上保持恒温， 以避免因清洗温度过高而产生角膜胶原蛋白的变性。
本实施例 1的一个较佳的实施方式，角膜在洗涤过程中的震荡频率为每分钟 100-160次， 以促进细胞成分从基质层中游离出来。 具体在本试验例中震荡频率 为每分钟 150次。 每次震荡清洗时间 15分钟〜 20分钟。
本实施例 1的一个可选择的实施方式中，所述清洗溶液为蒸馏水或氯化钠溶 液或 pH6.0至 8.0的缓冲溶液。 具体在本试验例中采用蒸馏水或 pH6.0至 8.0的 缓冲溶液作为清洗溶液。
S4: 灭菌处理。 本处理程序中指角膜有终端灭菌处理， 通过灭菌处理角膜应 当达到国家相关的灭菌的标准。 具体在本实施例 1中采用钴 60辐照灭菌， 辐照 剂量不大于 25kgy。基于现有的研究成果显示， 采用辐照灭菌不可避免地对角膜 的透明度这个重要特性产生不利的影响。因此，在本发明中的一个较佳的实施方 式中， 所述钴 60辐照灭菌采用低温辐照； 将角膜置于填充有制冷剂的保温容器 中进行辐照灭菌； 通过制冷剂使得角膜在辐照全过程中保持在低于 0°C的低温状 态下。
本实施例 1 的一个可选择的实施方式中， 所述的制冷剂可以是冰或干冰任 意一种。 具体在本实施例的一个试验例中， 所述的制冷剂采用干冰。 由于干冰本 身具有较低的初始温度（-78°C )， 保温容器内不易很快地升温， 并较容易获得并 保持较低的辐照温度，因此以干冰作为角膜辐照灭菌的制冷剂是本发明的较佳实 施方式。可先将角膜先置于干冰中，待角膜迅速降至低温环境温度后再开始进行
替换页 （细则第 26条) 辐照。
在本实施例的另一个试验例中，所述的制冷剂采用冰，冰的初始温度应当选 择于 -18〜- 25°C， 即使冰随辐照环境温度的上升而有所升高， 冰的温度在全部辐 照过程中的保持在低于 -5°C的低温状态。从而使得在保温容器内的温度处于远低 于 0°C的低温环境，有效地避免了在辐照灭菌过程中对角膜的胶原结构的进一步 破坏。
在本实施例 1的一个较佳的实施方式中，在进行辐照灭菌前， 先将角膜进行 单片密封包装，将密封包装后角膜置于低温制冷剂之中进行辐照。角膜在密封包 装状态下进行终端灭菌直至临床，有利于角膜在运输及保存等环节上保持角膜的 灭菌状态。特别是在本实施例中， 经过脱细胞处理后角膜为非干燥角膜， 可以直 接用于移植手术。如需保存一段时间后再使用， 可以采用现有技术中对于非干燥 角膜的密封包装， 如 DMEM细胞培养基中保存， 密封状态下的低温保存。
综上所述， 由于在本实施例中针对角膜制备的所有处理方法中， 首先考虑到 其对角膜原有地胶原纤维的规则排列结构的破坏影响，并在处理方法中尽可能地 采取有效措施避免或者最大限度地减少该破坏的不利因素。试验证明，本发明的 制备方法获得的人工角膜最大限度地保持了与天然角膜极为接近的规则的排列 结构， 在达到国家标准对免疫原性 （DNA) 残留的要求的情况下， 最大限度地 减少了制备过程中对角膜基质层内原有的胶原纤维的规则排列结构造成的损害， 如图 5〜图 5C所示。 因此， 本发明制备的角膜具有与人角膜极其近似的弹性机械 强度、透明度的等物理学特性， 特别是角膜的透明度。经本发明脱细胞方法处理 的获得的非干燥角膜， HE染色无细胞核， DAPI无染色， 角膜 DNA残留量低于 lOOng/mg, 角膜的透光率均可确保在 380-780nm波长内达到 75%以上。
实施例 2
如图 2所示，本实施例 2提供的一种猪脱细胞板层角膜的制备方法， 至少五 个处理过程。 其中：
第一步骤 S1预处理和第二步骤 S2干燥处理与实施例 1中 S1和 S2程度大 致相同， 其中， 具体的实施方式可在实施例 1所列出的范围内进行选择。故而本
替换页 （细则第 26条) 实施例 2中对第一步骤 SI预处理和第二步骤 S2干燥处理方法不再赘述。
S3 : 脱细胞处理， 将干燥处理后角膜进行脱细胞处理过程： S3.1 酶处理： 采用达 DMEM培养基配置全能核酸酶 （Benzonase®) 溶液； 按照每片角膜加入
0.5-3毫升上述酶溶液； 先涡旋震荡至角膜表面气泡去除， 排除角膜内的气体； 再置于震荡培养箱中震荡不小于 1小时；
具体在本实施例 2的一个试验例中， 按照每片角膜加入 0.5毫升上述全能核 酸酶（Benzonase®)溶液， 先采用涡旋震荡方式至角膜表面气泡去除。 具体在本 试验例中震荡处理 2.5~3小时。
在本实施例 2的较佳的实施方式中， 所述全能核酸酶（Benzonase®)溶液的 浓度为 300U 500U/毫升。酶溶液的浓度应当随酶的活性程度在 300U 500U/毫升 范围进行选择。
在实施例 2的一种可选择的实施方式中， 角膜在酶溶液中的震荡处理温度 15-37°C， 具体在本试验例所述震荡处理温度控制在 25 °C左右。
在实施例 2的一个具体试验例中， 所述的震荡频率选择为每分钟 65次。 试 验证明的进行酶处理时，选择一个较低的震荡频率的目的是最大限度在减少对角 膜原有胶原排列的破坏程度。
S3.2清洗处理作用是将 S3.1酶处理过程中在基质层内产生的细胞残留清洗 出来。具体处理方法是按照每片角膜加入不低于 5毫升的清洗溶液，震荡洗涤不 少于 5次， 每次不大于 30分钟；
在本实施例 2的一个选择实施方式中，角膜在清洗处理过程中的温度控制在 5-25 °C。具体在本试验例中清洗处理过程中的温度控制在 15 °C左右，并在全部的 清洗过程基本上保持恒温， 以避免因清洗温度过高而产生角膜胶原蛋白的变性。
本实施例 2的一个较佳的实施方式，角膜在洗涤过程中的震荡频率为每分钟 100-160次， 以促进细胞成分从基质层中游离出来。 在具体试验例中震荡频率为 每分钟 100次。 每次震荡清洗时间 10分钟〜 15分钟。
本实施例 2的一个可选择的实施方式中，所述清洗溶液为蒸馏水或氯化钠溶 液或 pH6.0至 8.0的缓冲溶液。具体在本试验例中采用 0.9%氯化钠作为清洗溶液。
S4:干燥角膜制备，在本处理程序中，将脱细胞处理后对角膜进行干燥处理， 制备成含水率含水量为 5%-20%的干燥角膜。本实施例 2制备的干燥角膜有利于
替换页 （细则第 26条) 角膜具有作为产品在批量生产以及保存、 运输市场商品属性。
在本实施例 2的 S4制备干燥角膜的方法的一种可选择实施方式， 可以采用 实施例 2中与前述第二步 S2中基本相同的压力自高向低的逐渐减压的干燥方法。 所述逐渐减压中的减压范围值为常压至极限真空，所述逐渐减压干燥的时间不大 于 12小时。其减压曲线如图 1B和图 1C所示。真空干燥的温度控制在 0°C~30°C。
如图 2B所示， 为本实施例 2的 S4制备干燥角膜的方法的一种可选择实施 方式， 在本实施方式中， 基本上采用梯度减压的方式， 通过 5 个梯级将压力从 80kpa降低到设备最大真空度 0.5kpa。 但是， 在本实施方式中， 压力梯度变化时 所述真空调节系统调节前一级压力挡逐渐降低到后一级压力挡，即控制真空干燥 室内的压力从上一个梯度压力值逐渐减至下一个压力梯度值。 减压曲线如图 2B 所示，通过本实施例 2上述三种具体实施方式的干燥角膜制备过程， 角膜的含水 率为 10%~20%， 透光率 84%~87%。 如图 2B所示干燥方法同样适用于实施例 1 和实施例 2中脱细胞前的 S2干燥处理。
采用本实施例 2 的上述逐渐减压干燥的方法相对于现有技术中的真空减压 方法而言， 其干燥过程更加温和。 因此所获得的干燥角膜， 确保干燥角膜的含水 率达到 5%~20%范围内， 透光率均大于 80%。 而本实施例 2所制备的干燥角膜 具有表面光滑平整，无肉眼可见的崎状突起或 细小褶皱， 而这个特征带来的另 一显著的临床效果是术后上皮细胞贴附和增殖速度快效果好。
由于本实施例 2提供的是干燥角膜的制备过程， 因此在 S4干燥角膜制备方 法中， 对干燥角膜的含水率应当达到 0~20%标准。
S5: 灭菌处理。 本处理程序 S5就指对干燥角膜有终端灭菌处理， 通过灭菌 处理角膜应当达到国家相关的灭菌的标准。 具体在本实施例 2中采用钴 60辐照 灭菌， 辐照剂量不大于 25kgy。 具体在本实施例 2中一个较佳的实施方式中， 所 述钴 60辐照灭菌采用低温辐照； 将角膜置于填充有制冷剂的保温容器中进行辐 照灭菌； 通过制冷剂使得角膜在辐照全过程中保持在低于 0°C的低温状态下。
本实施例 2的终端灭菌处理的可选择的实施方式中，所述的制冷剂除实施例 1中的冰（含蓄冷剂）或干冰， 其具体方式基本上与前述的实施例 1相同， 本实 例 2中不再重复进行具体说明。
在本实施例 2的一个较佳的实施方式中， 在进行辐照灭菌前， 先将角膜进行
替换页 （细则第 26条) 单片密封包装，将单片密封包装后角膜置于低温制冷剂之中进行辐照。角膜在密 封包装状态下进行终端灭菌直至临床时打开。大量试验证明，在角膜含水率较低 的干燥角膜相对于非干燥角膜，进行相同的条件下的低温辐照灭菌， 灭菌后的角 膜的理化性能变化小， 透明度、韧性和亲水力基本上不发生改变， 有效地避免了 常温状态下辐照对角膜理化性能产生的不利影响。
本发明通过上述制备方法所获得脱细胞猪板层干燥角膜，角膜 DNA残留不 大于 100ng /mg， 如图 4所示。 本发明由猪角膜的前弹力层和基质层构成的板层 干燥角膜;其基质层保持有规则排列的胶原纤维结构，如图 5〜图 5C电镜图显示， 本发明制备方法获得的角膜与人角膜以及未脱细胞的猪角膜的胶原结构极其接 近； 含水率不大于 20%的干燥角膜在可见光范围内， 所述角膜为透光率不低于 80%， 如图 3所示。 从图 3所示出的本发明干燥角膜图片可以看出， 本发明的干 燥角膜不仅透明度极佳， 同时具有的表面平整度高的产品特性。而大量的试验证 明， 平整的角膜表面有利于上皮层细胞的贴附与增殖。特别是角膜前弹力层的较 好的平整度极有利于提高角膜植入后上皮细胞的生长速度和生长质量。
本发明所述的脱细胞板层干燥角膜的使用方法相当简单容易操作，在术前从 灭菌密封包装中取出角膜， 浸入生理盐水 15-30分钟后， 直接用于异种角膜移植 术。
另外， 本发明提供的干燥角膜无疑是角膜产品最好的产品状态， 方便于保 存、运输。 可以实现在保存、运输等市场环节中干燥角膜产品的质量同一性和性 能稳定性， 可达到大大地延长保存期的效果。
而本实施例 2提供的干燥角膜具有另一个重要技术效果是， 手术前的复水 操作简单， 复水时间短。 在复水过程中， 在角膜的含水量即能达到手术要求， 又 能恰当地控制复水后角膜的含水量， 对于控制或者缩短角膜复明时间非常重要。 而本发明完全可以将手术前的复水操作规范化，达到有效地控制角膜复水含水量 的问题。
附图 6兔角膜行板层人工角膜移植术后 3月， 角膜完全恢复透明， 无排斥反 应。
图 7A〜图 7E人接受板层人工角膜移植术后 2月， 角膜透明无排斥反应。 术 后视力 0.6
。 本发明在大量的动物试验， 以及目前所做的所有角膜移植临床中均取得很好 有移植效果。 替换页 （细则第 26条) 本发明所述脱细胞猪板层干燥角膜由生物材料构成， 相对于人工合成的眼植 入材料而言可以很好解决人体的排异问题，可以通过简单的手术人眼进行屈光校 正应用， 以达到永久的屈光校正效果。
针对上述各实施方式的详细解释， 其目的仅在于对本发明进行解释， 以便于 能够更好地理解本发明，但是，这些描述不能以任何理由解释成是对本发明的限 制， 特别是， 在不同的实施方式中描述的各个特征也可以相互任意组合， 从而组 成其他实施方式， 除了有明确相反的描述，这些特征应被理解为能够应用于任何 一个实施方式中， 而并不仅局限于所描述的实施方式。
替换页 （细则第 26条)

Claims

^ ^ ^
1、 一种猪脱细胞板层角膜的制备方法， 至少如下步骤：
Sl、 预处理： 对新鲜猪角膜的预处理包括如下处理：
S1.1 取新鲜猪角膜， 去除上皮层；
S1.2 制备板层角膜； 所述板层角膜仅包括前弹力层和基质层；
S1.3 清洗干净；
S2: 干燥处理： 将预处理后板层角膜进行干燥处理；
S3: 脱细胞处理： 将干燥处理后角膜进行脱细胞处理：
S3.1 酶处理： 采用 DMEM培养基配置全能核酸酶 （Benzonase®) 溶液； 将干燥后角膜加入上述酶溶液； 置于震荡培养箱中震荡不小于 1小时；
S3.2清洗： 将角膜加入清洗溶液中， 置于震荡培养箱震荡洗涤处理， 获得 脱细胞角膜；
S4: 灭菌处理： 采用钴 60辐照灭菌， 辐照剂量不大于 25kgy。
2、 如权利要求 1所述的猪脱细胞板层角膜的制备方法， 其特征在于， 在 S1.2步 骤中取板层角膜厚度为 300um~700um。
3、 如权利要求 1所述的猪脱细胞板层角膜的制备方法， 其特征在于， 所述配制 溶剂为 DMEM培养基， 配制的全能核酸酶 （Benzonase®) 溶液的浓度为
Figure imgf000017_0001
4、 如权利要求 1所述的猪脱细胞板层角膜的制备方法， 其特征在于， 角膜的清 洗溶液为蒸馏水或氯化钠溶液或 pH6.0至 8.0的缓冲溶液。
5、 如权利要求 1所述的猪脱细胞板层角膜的制备方法， 其特征在于， 角膜在酶 溶液中的震荡处理温度 20-30°C。
6、 如权利要求 1所述猪脱细胞板层角膜的制备方法， 其特征在于， 角膜在酶溶 液中的震荡频率为每分钟 50-80次。
7、 如权利要求 1所述猪脱细胞板层角膜的制备方法， 其特征在于， 角膜在震荡 洗涤过程中的温度 10-20°C。
8、 如权利要求 1所述猪脱细胞板层角膜的制备方法， 其特征在于， 角膜在洗涤 过程中的震荡频率为每分钟 120 160次。
9、 如权利要求 4所述猪脱细胞板层角膜的制备方法， 其特征在于， 所述清洗用 替换页 （细则第 26条) 氯化钠溶液浓度为 0.9 %。
10、 如权利要求 1所述猪脱细胞板层角膜的制备方法， 其特征在于， 所述钴 60 辐照灭菌采用低温辐照； 将角膜置于填充有制冷剂的保温容器中进行辐照灭菌； 通过制冷剂使得角膜在辐照全过程中保持在低于 0°C的低温状态下。
11、 如权利要求 10所述猪脱细胞板层角膜的制备方法， 其特征在于， 所述的制 冷剂是冰、 干冰。
12、 如权利要求 10或 11所述猪脱细胞板层角膜的制备方法， 其特征在于， 所 述角膜低温辐照灭菌全程中温度不得高于 0°C。
13、 如权利要求 11至 12任意一种猪脱细胞板层角膜的制备方法， 其特征在于， 在进行辐照灭菌前， 先将角膜进行单片密封包装，将密封包装后角膜置于低温制 冷剂之中进行辐照。
14、如权利要求 1所述的猪脱细胞板层角膜的制备方法， 其特征在于， 在脱细胞 处理后对角膜进行干燥处理， 制备成含水率含水量为 5%-20%的干燥角膜。
15、 如权利要求 1或 14所述的猪脱细胞板层角膜的制备方法， 其特征在于， 所 述的干燥处理可采用真空干燥。
16、 如权利要求 15所述的猪脱细胞板层角膜的制备方法， 其特征在于， 所述真 空干燥为压力自高向低的逐渐减压的干燥方法。
17、 如权利要求 16所述的猪脱细胞板层角膜的制备方法， 其特征在于， 所述逐 渐减压中的减压范围值为 99~0.3kpa。
18、如权利要求 16或 17的猪脱细胞板层角膜的制备方法， 其特征在于， 所述逐 渐减压干燥的时间不大于 24小时。
19、 权利要求 1或 16述的猪脱细胞板层角膜的制备方法， 其特征在于， 密闭真 空干燥室内的温度控制在 0°C~30°C。
20、 如上述任意一权利要求制备方法所获得的脱细胞猪板层角膜， 其特征在于， 由猪角膜的前弹力层和基质层构成；所述基质层保持有胶原纤维的规则排列结构； 角膜 DNA残留不大于 100ng/mg。 在可见光范围内， 所述角膜的透光率不低于 70%。
21一种如上述权利要求 13至 18任意一制备方法所获得的脱细胞猪板层角膜， 其特征在于，所述板层角膜由猪角膜的前弹力层和基质层构成； 所述基质层保持
替换页 （细则第 26条) 有胶原纤维的规则排列结构；角膜 DNA残留不大于 lOOng/mg。在可见光范围内， 所述角膜为透光率不低于 70%， 含水率不大于 20%的干燥角膜。
22、 一种如权利要求 20所述的脱细胞板层干燥角膜的使用方法， 从灭菌密封包 装中取出角膜， 浸入生理盐水 15分钟复水后， 直接用于异种角膜移植术。
替换页 （细则第 26条)
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