阅读说明：本技术 一种供临床使用的脱细胞角膜基质的制备方法 (Preparation method of acellular corneal stroma for clinical use ) 是由 王海利 张晋南 宇咏梅 唐小琪 马百双 甘良波 于 2019-11-04 设计创作，主要内容包括：本发明公开了一种供临床使用的脱细胞角膜基质的制备方法,该方法包括去除角膜上皮层之后削切得到预定厚度的板层角膜,还包括将削切后的板层角膜密封后置于液氮中冷冻、将冷冻后的板层角膜置于水浴中解冻、将解冻后的板层角膜放入脱细胞试剂中进行振荡处理、将振荡处理后的板层角膜置于等渗液中进行超声处理、将脱细胞后的板层角膜放入软化液中静置,冷冻、解冻以及在脱细胞试剂中振荡处理有利于脱除细胞,在软化液中静置有利于角膜胶原微观结构的恢复,再放入水中振荡处理,得到透明性好,微观胶原结构接近于人体角膜并且不需经过其他处理而直接能供临床使用的脱细胞角膜基质。(The invention discloses a preparation method of acellular corneal stroma for clinical use, which comprises the steps of removing corneal epithelium layer, cutting to obtain lamellar cornea with preset thickness, sealing the cut lamellar cornea, freezing in liquid nitrogen, unfreezing the frozen lamellar cornea in water bath, placing the unfrozen lamellar cornea into an acellular reagent for oscillation treatment, placing the lamellar cornea after oscillation treatment in isotonic solution for ultrasonic treatment, placing the lamellar cornea after acellular treatment in a softening solution for standing, freezing, unfreezing and oscillating in the acellular reagent to facilitate the acellular treatment, the obtained acellular corneal stroma is good in transparency, the microcosmic collagen structure is close to the cornea of a human body, and the acellular corneal stroma can be directly used clinically without other treatment.)

1. A method of preparing an acellular corneal stroma for clinical use, comprising: the method for cutting a lamellar cornea with a predetermined thickness after removing the corneal epithelium layer is characterized by further comprising the following steps:

a) sealing the cut cornea and then freezing the cornea in liquid nitrogen;

b) thawing the frozen cornea in water bath;

c) placing the thawed cornea into a decellularization reagent for oscillation treatment;

d) placing the cornea after the oscillation treatment in isotonic solution for ultrasonic treatment to remove cells;

e) placing the cornea after decellularization into softening liquid for standing, and then placing the cornea into water for oscillation treatment; the softening liquid has the function of promoting the recovery of the microstructure of the corneal collagen.

2. The method of claim 1, wherein the decellularizing reagent comprises 0.2-1.0% neutral protease and 0.01-0.20% EDTA.

3. The method according to claim 1, wherein the freezing of step a) is performed for a period of 5-60 min.

4. The method of claim 3, wherein the temperature of the water bath of step b) is 20-37 ℃ and the duration of the cornea in the water bath is 10-30 min.

5. The method according to claim 1, wherein the oscillation process of step c) is: shaking in cell-removing reagent at 4-45 deg.C for 10-120 min.

6. The method of claim 1, wherein the isotonic solution of step d) is physiological saline or/and PBS buffer; or, the frequency of the ultrasound in the step d) is 10-100kHz, and the duration of the ultrasound is 8-15 min.

7. The method according to claim 1, wherein steps a) and b) are repeated alternately 1-5 times, or steps c) and d) are repeated alternately 1-8 times.

8. The method of any of claims 1-7, wherein the softening solution comprises polyvinyl pyrrolidone.

9. The method of claim 8, wherein the softening solution comprises one or both of hyaluronic acid and L-aspartic acid.

10. The decellularization method of claim 9, wherein: the temperature of the softening liquid in the step e) is 2-37 ℃, the standing time of the step e) is 10-60min, and the oscillation treatment time of the step e) is 1-10 min.

Technical Field

The invention belongs to the technical field of tissue engineering, and particularly relates to a preparation method of an acellular corneal stroma for human clinical use.

Background

The second leading cause of corneal blindness is keratopathy, of which 80% of patients can avoid blindness by corneal transplantation. But the donation of the cornea is less in China, so that the allogenic cornea donation is deficient, and the appearance of the artificial cornea brings hope to patients with the cornea disease. With the advent of tissue engineering, people have focused on natural animal corneal materials. Foreign cells and antigen substances of heterogeneous corneas are removed by a decellularization method, so that heterogeneous cell-free corneal extracellular matrix materials are obtained. The tissue engineering cornea material needs to meet three most basic requirements in clinical application, namely the thoroughness of removing heterogeneous corneal cells, the transparency of the cornea and the physiological radian of the cornea. When the tissue engineering cornea material is transplanted to a patient, if the foreign cells are not completely removed, immunological rejection reaction is generated, so that the transplanting effect is poor; if the damage to the corneal microstructure is large in the decellularization process, the transparency of the transplanted cells is not recovered well, the vision of a patient is still affected, and the life of the patient is not improved; thus, the main factors affecting the post-corneal transparency are: whether xenogeneic cells are completely removed, whether the cornea has the original collagen microstructure and whether the cornea has the original physiological radian will determine whether the corneal material can be successfully applied clinically.

The methods commonly used today for decellularization include chemical, physical and biological enzymatic methods. The conventional single chemical method or physical method for decellularizing needs long-time action to achieve the decellularizing effect, and the long-time action causes the microstructure of the corneal collagen to be damaged. Patent document 201510074946.7 (patent name: a method for preparing artificial cornea) prepares lamellar cornea using lamellar knife, drills corneal lamellae of a prescribed size, and performs decellularization by freeze-thawing and ultrasonic-endonuclease degradation. In the method, the lamellar cornea is obtained by using the lamellar cutter, so that the flatness of the section cannot be ensured, the joint between the grafted lamellar cutter and a graft bed is affected, and the postoperative transparency is recovered. Patent document 201410230365.3 (patent name: a lamellar corneal stroma scaffold and a method for producing the same) subjects an animal cornea to hypotonic swelling, repeated freeze-thawing, and enzymatic digestion treatment using a buffer system of dnase and rnase in this order. Patent document 201610080864.8 (patent name: method for preparing bovine corneal stroma using fresh bovine cornea and method for application) comprises soaking bovine corneal stroma in hypotonic solution, taking out corneal epithelial cells to obtain bovine corneal stroma comprising a pre-elastic layer and a specific stromal layer, repeatedly freezing and thawing, rinsing with buffer solution, soaking in decellularization reagent to remove cells, rinsing with ultrapure water, soaking in dehydrating agent, and sterilizing to obtain bovine corneal stroma. Both of the above two patent techniques use hypotonic solutions that cause the cornea to swell easily, are more destructive to the corneal stromal structure, and do not incubate to soften the decellularized cornea.

Disclosure of Invention

Aiming at the defects and limitations of the prior art, the invention provides a method for preparing acellular corneal stroma which has good transparency and microscopic collagen structure close to the human cornea and can be used clinically by adopting a physical method and a low-toxicity reagent. The method comprises the following steps: skiving a lamellar cornea of predetermined thickness after removal of the corneal epithelium layer, further comprising:

a) sealing the cut cornea and then freezing the cornea in liquid nitrogen;

b) thawing the frozen cornea in water bath;

c) placing the thawed cornea into a decellularization reagent for oscillation treatment;

d) placing the cornea after the oscillation treatment in isotonic solution for ultrasonic treatment to remove cells;

e) placing the cornea after decellularization into softening liquid for standing, and then placing the cornea into water for oscillation treatment; the softening liquid has the function of promoting the recovery of the microstructure of the corneal collagen.

In a non-limiting embodiment of the present invention, the predetermined thickness is 200-500 μm, and those skilled in the art can determine the thickness of the lamellar cornea according to the requirements of subsequent treatment and clinical use. The applicant concluded through a large number of experiments that the cornea formed by the method is convenient for subsequent treatment and can meet the clinical requirements when the thickness is 200-500 μm.

In a non-limiting embodiment of the invention, the sealing is performed by placing the cornea in a sealed container to prevent contamination during the freezing process.

In a non-limiting embodiment of the present invention, the freezing of step a) is performed by sealing the cornea and then placing the cornea in liquid nitrogen for 5-60 min; the skilled person can select the specific time required for freezing according to the specific amount of the cornea, the freezing time is too short, which may not perform cell freezing crystallization, and further the purpose of cell disruption is achieved, which may result in incomplete cell disruption, the freezing time is too long, which may result in corneal collagen fiber damage, and further may result in difficult recovery of the cornea basic structure, and the production cost is increased.

In a non-limiting embodiment of the present invention, the thawing in step b) refers to a process of thawing the frozen cornea into a state similar to the pre-frozen state, and a person skilled in the art can thaw the frozen cornea by a thawing method commonly used in the art, for example, placing the cornea in a room temperature air for thawing. In a non-limiting embodiment of the invention, the cornea is preferably thawed by placing it in a water bath.

In a non-limiting embodiment of the invention, the preferred temperature of the water bath is 20-37 deg.C, too high a temperature will denature the cornea, and too low a temperature will not achieve the goal of effectively disrupting corneal cells.

In a non-limiting embodiment of the invention, in addition to the temperature of the water bath, the duration of placing the frozen cornea in the water bath will also affect the fragmentation of the corneal cells and thus the removal of the corneal cells, and too short a time in the water bath will not have the thawing effect, and too long a time in the water bath will cause the cornea to bubble and make the basic structure of the cornea difficult to recover, and the duration of placing the frozen corneal lamellae in the water bath is preferably 10-30 min.

In a non-limiting embodiment of the invention, the cells are frozen by step a) and thawed by step b), the freezing and thawing facilitating cell disruption and thus facilitating removal of the cells. Repeated alternating repetition of steps a) and b) is more advantageous for cell disruption and removal, but too many repetitions will result in destruction of the corneal basic structure. Preferably, steps a) and b) are repeated alternately 1 to 5 times.

In a non-limiting embodiment of the invention, the cornea after the treatment in step c) is first placed in an acellular agent for oscillation treatment and then placed in an isotonic solution for ultrasound treatment. The decellularization reagent can be selected from decellularization reagents commonly used in the prior art in the field, such as the decellularization reagent disclosed in patent document CN 104001217A. The isotonic fluid is a solution having an osmotic pressure parameter that does not alter the structure of the cornea.

In a non-limiting preferred embodiment of the invention, the decellularising agent of step c) preferably comprises 0.2-1.0% (w/v) neutral protease and 0.01-0.20% (w/v) EDTA or its sodium salt. The effect of the neutral protease is to destroy the cell structure and accelerate the cell lysis, the effect of the EDTA is to combine metal ions and destroy the adhesion of cells to ECM, and when the two are used together, the EDTA can promote the cell removal effect of the neutral protease and is beneficial to keeping the basic structure of the corneal collagen fiber from being damaged.

In the non-limiting embodiment of the present invention, the cornea is first treated by shaking in the acellular agent in step c), and then treated by ultrasonication in the isotonic solution in step d), so that the basic structure of the cornea is maintained in a mild environment. Repeating steps c) and d) is more favorable for thorough removal of cells, but the basic structure of the cornea is damaged by repeating the steps for too many times. Preferably, steps c) and d) are repeated 1 to 8 times.

In a non-limiting preferred embodiment of the invention, the oscillation treatment of step c) is preferably: shaking in cell-removing reagent at 4-45 deg.C for 10-120 min. As is well known to those skilled in the art, the shorter the contact time between the cornea and the enzyme is, the more beneficial the basic structure is, the more the applicant has concluded through many experiments that the contact time between the cornea and the enzyme can be effectively shortened by immersing the cornea in the cell-removing liquid while performing the oscillation treatment, and then performing the ultrasound treatment in the isotonic liquid, and the basic structure of the collagen fiber of the cornea can be maintained while achieving the purpose of removing the cells.

In a non-limiting preferred embodiment of the invention, the isotonic fluid is a solution having an osmotic pressure parameter that does not alter the structure of the cornea. In a non-limiting preferred embodiment of the invention, the isotonic solution is preferably physiological saline or/and PBS buffer.

In the non-limiting embodiment of the present invention, the frequency and duration parameters of the ultrasound in step d) have similar reasoning as those of the water bath, and too high or too low frequency and too short or too long duration of the ultrasound affect the removal effect of the corneal cells, the preferred frequency of the ultrasound is 10-100kHz, and the preferred duration of the ultrasound is 8-15 min.

In the non-limiting embodiment of the present invention, the softening liquid in step e) can protect the lamellar cornea from swelling in the softening liquid, promote the recovery of the collagen structure of the cornea, make the cornea more transparent, and improve the clinical application effect.

In the non-limiting above embodiment of the present invention, the softening liquid of step e) contains polyvinyl pyrrolidone. The softening liquid containing the polyvinylpyrrolidone can protect the lamellar cornea from swelling in the softening liquid, promote the recovery of a corneal collagen structure, make the cornea more transparent and improve the clinical application effect.

In a non-limiting preferred embodiment of the invention, the softening solution contains 0.1 to 5% polyvinyl pyrrolidone.

In the non-limiting embodiment of the present invention, the softening liquid contains polyvinylpyrrolidone, and preferably further contains one or two of hyaluronic acid and L-aspartic acid.

In a non-limiting preferred embodiment of the invention, the softening liquid preferably comprises the following components: contains 0.1-5% of polyvinyl pyrrolidone, 0.1-3% of hyaluronic acid, 0.1% -4% of L-aspartic acid and the balance of water. The softening liquid containing the polyvinylpyrrolidone, the hyaluronic acid and the aspartic acid can protect lamellar corneas from swelling in the softening liquid, better promote the recovery of corneal collagen structures, make the corneas more transparent and improve the clinical application effect.

In a non-limiting preferred embodiment of the invention, the temperature of the softening liquid in step e) is preferably 2 to 37 ℃, and the duration of the standing is preferably 10 to 60 min; the duration of the shaking treatment of step e) is preferably 1 to 10 min. In a non-limiting preferred embodiment of the invention, the shaking process may be repeated 1-5 times.

In a non-limiting embodiment of the invention, the cornea of step a) is a porcine cornea. However, as is well known to those skilled in the art, the technical solution provided by the present invention is to treat the mammalian corneal cells, and is not related to the types of mammalian cells, so that the mammalian corneal cells can be treated by the method, and the acellular corneal stroma which can be used clinically without other treatments can be obtained.

Compared with the prior art, the method uses mild neutral protease and EDTA as a decellularization reagent, and is beneficial to maintaining the microstructure of the cornea; the cornea is immersed in the acellular fluid for oscillation treatment, and then is subjected to ultrasonic treatment in the isotonic fluid, so that the contact time between the cornea and enzyme is effectively shortened, and the basic structure of corneal collagen fibers can be maintained while the purpose of acellular removal is achieved; according to the invention, the cornea after being decellularized is placed in the softening liquid for standing, so that the recovery of the cornea collagen microstructure can be effectively promoted; thereby obtaining the acellular corneal stroma which can be directly used clinically without other treatments.

Drawings

FIG. 1 is a flow chart of a method for preparing an acellular corneal stroma for clinical use according to the present invention

FIG. 2 photograph of acellular cornea produced according to example 8 of the present invention placed on white paper printed with black letter A

FIG. 3 is a photograph of a human body transplanted with a decellularized cornea obtained in example 10 of the present invention after surgery

Detailed Description

Non-limiting embodiments of the present invention are further described below in conjunction with the appended drawings. The following embodiments are merely illustrative, and should not be construed as limiting the technical scope of the present invention.

The lamellar cornea of the invention is a cornea which is obtained by removing the corneal epithelium layer, comprises a front elastic layer and a matrix layer and can be used for clinical transplantation, and if not specially marked, the cornea and the lamellar cornea of the invention have the same meaning; if not specially marked, the test environment of the embodiment of the invention is an environment meeting the cleanliness of three types of medical instruments; the water used in the embodiment of the invention is water for injection; the percentage content units of the components of the cell-removing reagent and the components of the softening liquid in the embodiment of the invention are mass-volume ratios.