CN1311072C - Preparation method of dried active amnion - Google Patents
Preparation method of dried active amnion Download PDFInfo
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- CN1311072C CN1311072C CNB2005100468563A CN200510046856A CN1311072C CN 1311072 C CN1311072 C CN 1311072C CN B2005100468563 A CNB2005100468563 A CN B2005100468563A CN 200510046856 A CN200510046856 A CN 200510046856A CN 1311072 C CN1311072 C CN 1311072C
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Abstract
The present invention relates to a method for preparing dried active amnions. In the method, healthy placenta tissues are adopted for carrying out germicidal processing for separating amnions from chorions; spongy layers are scraped off; afterwards, epithelial surfaces are upwards laid and fixed on nitrocellulose paper; paper sheets with amnions are sheared into proper sizes and are incubated in the mixed liquid of 0.05% to 0.5% of trypsin and 0.01% to 0.1% of ethylene diamine tetraacetic acid or 0.05% to 0.5% of trypsin or 0.01% to 0.1% of ethylene diamine tetraacetic acid liquid at 37 DEG C for 1/2 hour to 4 hours; a cell slicker or a cotton rod is used for removing epithelial cells; the amnions are washed by physiological saline repeatedly and are put in a freeze dryer after the fact that epithelial surfaces have no cell under microscopes is verified; the amnions and the nitrocellulose paper are frozen and dried together under the vacuum condition and are packed in vacuum at room temperature; finally, the amnions are irradiated by gamma rays. The method has the advantages that the cells in the amnions are removed, effective substrate active components in the cells are simultaneously reserved, the dried active amnions can be widely applied to amnion covering operations, amnion transplantation operations, amnion filling implantation operations, etc. in ophthalmology, and the dried active amnions can also be used as new tissue engineering materials for the cultivation and the transplantation of epitheliums and endothelia.
Description
Affiliated technical field
The present invention relates to medical amniotic membrane preparations, especially a kind of preparation method who takes off the cell dried active amnion with long preservation period.
Background technology
Amnion is to develop from cytotrophoblast, is the internal layer of human two-layer fetal membrane, is basilar membrane the thickest in the human body.The no blood vessel of amnion self and neural, lymphatic vessel, its antigenicity lower (mainly coming from cellular constituent wherein).The application of amnion starts from 1910 the earliest, and Divis is used for transplanting to fetal membrane (amnion+chorion) first and achieves success.Then, amnion is widely used in various operations as burned skin graft, artificial vaginal mucosa sheath etc., carries out amnion transplantation up to nineteen ninety-five Kim and Tseng at the anterior corneal surface of chemical burn mould shape, and amnion transplantation causes oculistic attention gradually.1997, Ma etc. studied the feasibility of amnion transplantation from the molecular biology angle, and the mechanism of action of amnion is mainly: 1. basilar membrane effect for stem cell provides " fertile soil ", provides shiny surface to be beneficial to the cell growth; 2. wherein various albumen compositions can promote epithelium differentiation, hyperplasia; 3. strengthen the albumen adhesivity, promote the epithelial proliferation reparation; 4. the proteolytic enzyme arrestin enzyme that contains of amnion and bring into play it and press down scorching effect; 5. amnion contains the composition that suppresses cytokine-expressing and natural death of cerebral cells; 6. can avoid the keratocyte that inflammatory cytokine brings out and the hyperplasia of collegen filament, thereby suppress local inflammation, promote the surface healing and do not stay scar.
In recent years, along with the continuous development of people to amnion research, amnion transplantation by broader applications in the treatment ophthalmic diseases.Amnion covering operation, Transplantation of amniotic membrane, amnion filling, implantation etc. have improved the treatment success ratio of illness in eye such as comprising eye surface diseases, glaucoma, sclera disease.
The amnion that uses clinically mostly is fresh amnion or profound hypothermia preservation amnion at present, is difficult to accomplish ready access upon use, the convenient preservation.Though but patent publication No. is the freeze-drying amnions of the bioamnion goods of CN1522568A for the normal temperature preservation, it does not slough the cell of amnion, brings difficulty to clinical application, and can not carry out epithelium, endothelium cultivation transplanting as the organizational engineering material.
Summary of the invention
The objective of the invention is to: at the problems referred to above, but but the broader applications that a kind of prolonged preservation, ready access upon use be provided in the preparation method of clinical dried active amnion goods.
Concrete technical scheme is as follows: the preparation method of dried active amnion is: select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with blade or slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into suitable size then.Again in the mixed solution of 0.05～0.5% trypsinase and 0.01～0.1%EDTA or 0.05～0.5% trypsinase or 0.01～0.1%EDTA liquid 37 ℃ hatched 1/2-4 hour, slough epithelial cell with cell sleaker or cotton rod, normal saline flushing repeatedly, microscopically confirm that epithelial surface is acellular.Insert Freeze Drying Equipment, together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.Every batch of product sampling carrying out bacterium cultivation.
The amnion such as the bamboo paper sample of above method preparation, translucent, about 20 microns of thickness, tension stress and line shear force performance are good after the rehydration, and the transparency increases when dry.
The preparation method of dried active amnion of the present invention, sloughed entamniotic cell, greatly reduced the antigenicity of amnion, kept its effective matrix activeconstituents simultaneously, can wider model be used in the operations such as ophthalmology amnion covering operation, Transplantation of amniotic membrane, amnion filling, implantation, and can be used as novel organizational engineering material and transplant in order to the cultivation of epithelium, endothelium.After the lyophilize, the preservation condition that makes amnion is normal temperature (dry shady and cool place), and validity period can reach 1 year, has accomplished ready access upon use, the convenient preservation.Aseptic manipulation and gammairradiation sterilization have overcome fresh or the contaminated possibility of cryopreservation amnion.Amniotic membrane preparations of the present invention and cellulose nitrate paper together freeze-drying are preserved, and clip is convenient during use.
Embodiment
The present invention is described in detail below in conjunction with embodiment, but embodiments of the present invention are not limited thereto.
Embodiment 1:
Select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with the slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into 6cm * 2.5cm size then.Again in 0.02%EDTA liquid 37 ℃ hatched 3 hours, slough epithelial cell with the cell sleaker, normal saline flushing repeatedly, microscopically confirm that epithelial surface is acellular.Together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.
Embodiment 2:
Select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with the slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into 6cm * 2.5cm size then.Again in 0.25% trypsin solution 37 ℃ hatched 2 hours, help to slough epithelial cell with the cell sleaker, normal saline flushing repeatedly, microscopically confirms that epithelial surface is acellular.Together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.
Embodiment 3:
Select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with the slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into 4cm * 2.5cm size then.Again in 0.05%EDTA liquid 37 ℃ hatched 3 hours, help to slough epithelial cell with the cell sleaker, normal saline flushing repeatedly, microscopically confirms that epithelial surface is acellular.Together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.
Embodiment 4:
Select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with the slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into 6cm * 2.5cm size then.Again in 0.25% trypsinase and 0.02%EDTA mixed solution 37 ℃ hatched 1 hour, help to slough epithelial cell with the cell sleaker, normal saline flushing repeatedly, microscopically confirms that epithelial surface is acellular.Together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.
Embodiment 5:
Select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with the slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into 6cm * 2.5cm size then.Again in 0.2% trypsinase and 0.05%EDTA mixed solution 37 ℃ hatched 1 hour, help to slough epithelial cell with the cell sleaker, normal saline flushing repeatedly, microscopically confirms that epithelial surface is acellular.Together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.
Embodiment 6:
Select for use health to cut open the placenta tissue that the puerpera is produced in the palace, antenatal Serological testing is got rid of hepatitis B virus (HBV), hepatitis C virus (HCV) cytomegalovirus, treponema pallidum and immunodeficiency virus (HIV) according to relevant national standard and is infected.Obtain promptly to operate down behind the placenta from c-section or aseptic the delivering a child of natural labor, make following sterilising treatment: with the bloodstain on the clean placenta of normal saline flushing surface aseptic; Soak 5～10 minutes (concentration of microbiotic mixed solution is for containing 50ug/ml penicillin, 50ug/ml Streptomycin sulphate, 100ug/ml Xin Meisu and 2.5ug/ml amphotericin B) of placenta with containing antibiotic stroke-physiological saline solution.
Amnion is separated from chorion, scraped off spongy layer with the slide glass side as far as possible, epithelial surface tiles up and is fixed in (Bio-Tissue company provides) on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into 6cm * 2.5cm size then.Again in 0.2% trypsin solution 37 ℃ hatched 2.5 hours, help to slough epithelial cell with the cell sleaker, normal saline flushing repeatedly, microscopically confirms that epithelial surface is acellular.Together with the freeze-drying of cellulose nitrate paper, at room temperature row is vacuum-packed under the vacuum condition.Last gammairradiation.
The amnion of above method preparation carries out immunohistochemical experiment and shows that holostrome exists I type, III type, IV type and collagen type v and fiber adhesion element in the lyophilize amnion, and VII Collagen Type VI and Kallinin are present in the basilar membrane side.Close with position, the quantity of activeconstituents in the fresh amnion.
Choose 5 12～14 age in week body weight at New Zealand's large ear rabbit (male and female are not limit) of 1.5～2.5kg, the quiet notes general anesthesia of 15g/L Veronal sodium, 3.0mm place otch in corneal limbus, make the flaggy pocket, the amniotic membrane preparations of implanting top method preparation is in rabbit corneal matrix, do not do stitching, postoperative erythromycin ophthalmic ointment routine is coated with the art eye.In the time of one month, slit lamp observation sees that to implant the amnion consistency good, transparent, does not see that local cells infiltration, corneal edema and new vessel grow into; The visible keratocyte of pathological examination is grown in implanting amnion.Confirmed this dried active amnion excellent biological compatibility.
Claims (1)
1, a kind of preparation method of dried active amnion is characterized in that selecting for use healthy placenta tissue, carries out sterilising treatment, amnion is separated from chorion, scrape off spongy layer, epithelial surface tiles up and is fixed on the cellulose nitrate paper then, will have the scraps of paper of amnion to be cut into suitable size then; Again in the mixed solution of 0.05～0.5% trypsinase and 0.01～0.1%EDTA or 0.05～0.5% trypsinase or 0.01～0.1%EDTA liquid 37 ℃ hatched 1/2-4 hour, slough epithelial cell with cell sleaker or cotton rod, normal saline flushing repeatedly, microscopically confirm that epithelial surface is acellular; Insert Freeze Drying Equipment, together with the freeze-drying of cellulose nitrate paper, at room temperature carry out vacuum packaging under the vacuum condition; Last gammairradiation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100468563A CN1311072C (en) | 2005-07-12 | 2005-07-12 | Preparation method of dried active amnion |
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| CNB2005100468563A CN1311072C (en) | 2005-07-12 | 2005-07-12 | Preparation method of dried active amnion |
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| Publication Number | Publication Date |
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| CN1757717A CN1757717A (en) | 2006-04-12 |
| CN1311072C true CN1311072C (en) | 2007-04-18 |
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| CNB2005100468563A Active CN1311072C (en) | 2005-07-12 | 2005-07-12 | Preparation method of dried active amnion |
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| US8372437B2 (en) | 2006-08-17 | 2013-02-12 | Mimedx Group, Inc. | Placental tissue grafts |
| WO2009033160A1 (en) | 2007-09-07 | 2009-03-12 | Surgical Biologics | Placental tissue grafts and improved methods of preparing and using the same |
| CN101889569B (en) * | 2010-07-23 | 2013-05-22 | 马吉献 | Method for drying and storing biological amnion |
| KR20240042159A (en) * | 2011-02-14 | 2024-04-01 | 미메딕스 그룹 인크. | Micronized placental tissue compositions and methods for making and using the same |
| CN102225217A (en) * | 2011-06-23 | 2011-10-26 | 天津世纪康泰生物医学工程有限公司 | Preparation method of acellular dried active amnion |
| CN107335095A (en) * | 2017-06-05 | 2017-11-10 | 江西瑞诺健医学科技有限公司 | A kind of preparation method of medical multilayer placenta tissue implant |
| CN107714609A (en) * | 2017-11-08 | 2018-02-23 | 郑楠 | A kind of amnion basement membrane, amnion facial mask based on amnion basement membrane and preparation method thereof |
| CN108126246A (en) * | 2017-12-29 | 2018-06-08 | 山西医科大学 | Artificial skin construction method based on compound stem cell |
| CN110946879B (en) * | 2018-09-26 | 2021-05-11 | 成都清科生物科技有限公司 | Acellular biological amniotic membrane containing loose layer and preparation method thereof |
| US20210244845A1 (en) * | 2020-02-10 | 2021-08-12 | Leslie MARCELIN | Method Of Processing Amnion To Form A Suture |
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