JP4977345B2 - Dry amniotic membrane and method for drying amniotic membrane - Google Patents
Dry amniotic membrane and method for drying amniotic membrane Download PDFInfo
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Description
本発明は乾燥羊膜及び羊膜の乾燥処理方法に関し、更に詳細には人を含む動物の胎児を包む生羊膜を乾燥処理して得た乾燥羊膜及び羊膜の乾燥処理方法に関する。 The present invention relates to a dried amnion and a method for drying amnion, and more particularly to a dried amnion obtained by drying a raw amnion that encloses a fetus of an animal including a human and a method for drying amnion.
角膜内皮の移植に必要な角膜内皮様シートを得るために、採取した角膜幹細胞を羊膜上に播種し培養することが行なわれている（例えば、下記特許文献１）。
かかる羊膜としては、無菌状態で得た人を含む動物の胎児を包む生羊膜、特に人の帝王切開分娩で得られた胎盤から採取された生羊膜を、直ちに使用することが最も好ましい。
しかし、好適な生羊膜は使用したいときに常に入手できるとは限らず、予め入手した好適な生羊膜を保存することが必要である。
かかる羊膜の保存としては、特許文献１の段落番号[００２６]及び[００２７]には、生羊膜を保存液に浸漬して−８０℃で冷凍保存し、使用の際に、冷凍された羊膜を室温で解凍して使用することが記載されている。
As such an amniotic membrane, it is most preferable to immediately use a raw amniotic membrane that envelops the fetus of an animal including a human obtained under aseptic conditions, particularly a raw amniotic membrane collected from a placenta obtained by cesarean delivery of a human.
However, a suitable raw amniotic membrane is not always available when it is desired to use it, and it is necessary to preserve a suitable raw amniotic membrane obtained in advance.
For preservation of such amniotic membrane, paragraphs [0026] and [0027] of Patent Document 1 describe that the raw amniotic membrane is immersed in a preservation solution and stored frozen at −80 ° C., and the frozen amniotic membrane is stored at the time of use. It is described that it is used after thawing at room temperature.
上記特許文献１に記載された羊膜の冷凍保存方法によれば、生羊膜を簡便に保存することができる。
しかしながら、冷凍保存された羊膜の保存期間は３月程度であり、保存期間を経過した羊膜は焼却処分される。
しかも、生羊膜を冷凍する際に、羊膜を構成する細胞内の水が凍って大きな氷結晶が生成されると、生成された氷結晶によって細胞膜が破壊されることがあるため、細胞中に細胞膜を破壊するような大きな氷結晶を生成させることなく冷凍することを要し、生羊膜が冷凍するまでの降温速度等に格別の注意が必要である。
また、冷凍羊膜を−８０℃もの冷凍温度に常に維持することは、所定の設備を必要とするため、冷凍羊膜の保存及び運搬は容易ではない。
これに対し、生羊膜の組織を維持して乾燥した乾燥羊膜を得ることができれば、その管理維持及び運搬には特別の注意を払うことなく容易に行なうことができる。
そこで、本発明の課題は、生羊膜の組織を維持して乾燥され且つ長時間の保存を容易に行なうことができる乾燥羊膜及び羊膜の乾燥処理方法を提供することにある。
According to the amniotic membrane cryopreservation method described in Patent Document 1, the raw amniotic membrane can be easily stored.
However, the storage period of frozen amniotic membrane is about three months, and the amniotic membrane after the storage period has been incinerated.
Moreover, when freezing the raw amniotic membrane, if the water in the cells that make up the amniotic membrane freezes to produce large ice crystals, the cell membrane may be destroyed by the generated ice crystals. It is necessary to freeze without generating large ice crystals that would break down, and special attention should be paid to the temperature drop rate until the raw amniotic membrane is frozen.
In addition, it is not easy to store and transport the frozen amniotic membrane because it is necessary to always maintain the frozen amniotic membrane at a freezing temperature of -80 ° C.
On the other hand, if a dry amniotic membrane can be obtained while maintaining the structure of the raw amniotic membrane, its maintenance and transportation can be easily performed without paying special attention.
Accordingly, an object of the present invention is to provide a dry amniotic membrane that can be dried while maintaining the tissue of the raw amniotic membrane and can be stored for a long time, and a method for drying the amniotic membrane.
本発明者は、前記課題を解決すべく、先ず、生羊膜を冷凍乾燥することを試みたところ、得られた乾燥羊膜を緩衝液に浸漬して再水和しても、細胞の萎縮が著しくて細胞培養には到底使用できないことが判明した。
本発明者は、生羊膜の細胞組織を保持しつつ乾燥処理すべく種々検討を重ねた結果、処理槽内に載置した生羊膜を、処理槽内に設けた遠赤外線ヒータによって連続して加温して、処理槽内を減圧状態とする減圧操作と、この生羊膜に処理槽外に設けたマイクロ波加熱装置からもマイクロ波を照射して羊膜を加温しつつ、減圧状態の処理槽内を復圧する復圧操作とを、複数回繰り返すことによって、生羊膜の細胞組織を保持しつつ乾燥できることを見出し、本発明に到達した。
In order to solve the above problems, the present inventor first tried to freeze-dry raw amniotic membrane, and even when the obtained dried amniotic membrane was immersed in a buffer solution and rehydrated, cell atrophy was markedly reduced. Thus, it was found that it cannot be used for cell culture.
The present inventor has made various studies for drying treatment while maintaining the cellular tissue of the raw amniotic membrane. As a result, the raw amniotic membrane placed in the treatment tank is continuously added by a far infrared heater provided in the treatment tank. Depressurization operation in which the inside of the treatment tank is depressurized by heating and the treatment tank in a depressurized state while heating the amniotic membrane by irradiating the raw amniotic membrane with microwaves from the microwave heating device It has been found that by repeating the decompression operation for restoring the pressure inside a plurality of times, it can be dried while retaining the cell tissue of the raw amniotic membrane, and the present invention has been achieved.
すなわち、本発明は、人を含む動物の胎児を包む生羊膜を、生羊膜の細胞組織を破壊することなく乾燥処理して得た乾燥羊膜であって、無菌状態の乾燥大気中で保存できるように脱水乾燥されていると共に、水又は緩衝液に浸漬して再水和した際、生羊膜と同様の、上皮細胞、基底膜及び結合組織が保持されていることを特徴とする乾燥羊膜にある。
また、本発明は、人を含む動物の胎児を包む生羊膜を乾燥処理する羊膜の乾燥処理方法において、該生羊膜を乾燥する乾燥装置として、羊膜を載置した処理槽内を減圧状態とする減圧手段と、減圧状態の前記処理槽内に載置した羊膜を加温する加温手段と、前記処理槽内の減圧状態を大気圧方向に復圧する復圧手段とを具備する乾燥装置を用い、前記加温手段により、羊膜を、羊膜の細胞組織を破壊することのない温度に加温しつつ、前記処理槽内を前記減圧手段により減圧する減圧工程と、前記加温手段により、羊膜を、羊膜の細胞組織を破壊することのない温度に加温しつつ、前記復圧手段により前記処理槽内を大気圧もしくは大気圧に近い圧力まで上昇させる復圧工程とを交互に複数回繰り返して、羊膜をその構成する上皮細胞、基底膜及び結合組織を破壊することなく脱水・乾燥することを特徴とする羊膜の乾燥処理方法でもある。
That is, the present invention is a dry amniotic membrane obtained by subjecting a raw amniotic membrane wrapping a fetus of an animal including a human to a dry amniotic membrane without destroying the cell tissue of the raw amniotic membrane , and can be stored in a sterile dry atmosphere. It is a dry amniotic membrane characterized in that it retains epithelial cells, basement membrane and connective tissue similar to raw amniotic membrane when rehydrated by immersion in water or buffer. .
Further, the present invention provides a method for drying amniotic membrane for drying a raw amniotic membrane that encloses a fetus of an animal including a human. Using a drying apparatus comprising a decompression means, a heating means for heating the amniotic membrane placed in the treatment tank in the decompressed state, and a return pressure means for restoring the decompressed state in the treatment tank in the atmospheric pressure direction. The depressurization step of depressurizing the inside of the treatment tank by the depressurization means while heating the amniotic membrane to a temperature that does not destroy the cell tissue of the amniotic membrane by the heating means, and the amniotic membrane by the heating means And a re-pressure step of alternately raising the inside of the treatment tank to atmospheric pressure or a pressure close to atmospheric pressure by the re-pressure device while heating to a temperature that does not destroy the cell tissue of the amniotic membrane, alternately and repeatedly. The epithelial cells that make up the amniotic membrane, the base And even drying process of the amniotic membrane, which comprises dehydrating and drying without destroying the connective tissues.
かかる本発明において、乾燥羊膜として、人由来の乾燥羊膜とすることによって、再生医療に用いられる細胞シート状態の細胞培養に好適に用いることができる。この乾燥羊膜を、乾燥剤が封入された滅菌パック中に密閉保存することによって長期間の保存を可能にできる。
また、本発明において、加温手段としては、遠赤外線ヒータ及びマイクロ波照射装置の少なくとも一方を好適に用いることができる。遠赤外線ヒータから発せられる遠赤外線及びマイクロ波照射装置から発せられるマイクロ波は、減圧雰囲気中に載置されている羊膜を昇温できるからである。かかる加温手段の設定温度を５０℃以下とすることによって、羊膜を構成する細胞組織の破壊を可及的に少なくできる。
更に、乾燥に処する生羊膜としては、人由来の生羊膜を好適に用いることができ、生羊膜を処理槽内にシート状に広げて載置するによって、生羊膜の脱水を容易に行なうことができる。
かかる処理槽内を復圧操作によって復圧したとき、処理槽内の圧力を大気圧よりも低圧とすることによって、次の減圧操作によって処理槽内を早期に最高減圧到達圧力とすることができる。
尚、乾燥の終了を、羊膜が載置された処理槽内の最高減圧到達圧力と羊膜が載置されていない処理槽を減圧したときの最高減圧到達圧力とが等しくなったときとすることにより、乾燥処理の終了を一定とすることができる。
In the present invention, the dried amniotic membrane can be suitably used for cell culture in a cell sheet state used for regenerative medicine by using a human-derived dried amniotic membrane. The dried amniotic membrane can be stored for a long period of time by hermetically storing it in a sterilized pack containing a desiccant.
Moreover, in this invention, at least one of a far-infrared heater and a microwave irradiation apparatus can be used suitably as a heating means. This is because the far infrared rays emitted from the far infrared heater and the microwaves emitted from the microwave irradiation device can raise the temperature of the amniotic membrane placed in the reduced pressure atmosphere. By setting the temperature of the heating means to 50 ° C. or less, the destruction of the cell tissue constituting the amniotic membrane can be minimized.
Furthermore, as the raw amnion to be dried, a human-derived raw amnion can be suitably used, and the raw amnion can be easily dehydrated by spreading and placing the raw amnion in a sheet shape in the treatment tank. it can.
When the inside of such a treatment tank is restored by a decompression operation, the pressure inside the treatment tank can be made lower than the atmospheric pressure, so that the inside of the treatment tank can be quickly reached the maximum pressure reduction pressure by the next decompression operation. .
The end of drying is determined when the maximum pressure reduction pressure in the processing tank on which the amniotic membrane is placed is equal to the maximum pressure reduction pressure when the processing tank on which the amniotic membrane is not placed is decompressed. The end of the drying process can be made constant.
本発明に係る乾燥羊膜は、無菌状態の乾燥大気中で保存でき、凍結羊膜に比較して、その保存性及び取扱性を向上できると共に、長期間の保存を可能にできる。また、乾燥羊膜は、生羊膜の細胞組織が実質的に破壊されることなく保持できるため、水又は緩衝液に浸漬して再水和することによって、ほぼ生羊膜様の羊膜を得ることができ、細胞の培養や、皮膚欠損損傷の治療等に用いることができる。
また本発明に係る羊膜の乾燥処理方法によれば、人を含む動物の胎児を包む生羊膜を乾燥処理する羊膜の乾燥処理方法において、該生羊膜を乾燥する乾燥装置として、羊膜を載置した処理槽内を減圧状態とする減圧手段と、減圧状態の前記処理槽内に載置した羊膜を加温する加温手段と、前記処理槽内の減圧状態を大気圧方向に復圧する復圧手段とを具備する乾燥装置を用い、前記加温手段により、羊膜を、羊膜の細胞組織を破壊することのない温度に加温しつつ、前記処理槽内を前記減圧手段により減圧する減圧工程と、前記加温手段により、羊膜を、羊膜の細胞組織を破壊することのない温度に加温しつつ、前記復圧手段により前記処理槽内を大気圧もしくは大気圧に近い圧力まで上昇させる復圧工程とを交互に複数回繰り返すようにしたので、羊膜をその構成する上皮細胞、基底膜及び結合組織を破壊することなく脱水・乾燥させることができる。
The dried amniotic membrane according to the present invention can be stored in aseptically dried air, and can improve its storage and handling properties as compared with a frozen amniotic membrane, and can be stored for a long period of time. In addition, the dried amniotic membrane can retain the cell structure of the raw amniotic membrane without substantial destruction, so that it can be obtained by immersing it in water or a buffer solution and rehydrating it. It can be used for cell culture , treatment of skin defect damage, and the like.
Further, according to the method for drying amniotic membrane according to the present invention, in the method for drying amniotic membrane for drying raw amniotic membrane wrapping fetuses of animals including humans, the amniotic membrane is placed as a drying device for drying the raw amniotic membrane. Depressurizing means for depressurizing the inside of the treatment tank, heating means for heating the amniotic membrane placed in the depressurized state of the treatment tank, and decompression means for returning the depressurized state of the treatment tank to the atmospheric pressure direction A depressurization step of depressurizing the inside of the treatment tank by the depressurization means while heating the amniotic membrane to a temperature that does not destroy the cell tissue of the amniotic membrane by the heating means, A restoring pressure step of raising the inside of the treatment tank to atmospheric pressure or a pressure close to atmospheric pressure by the restoring pressure means while heating the amniotic membrane to a temperature that does not destroy the cell tissue of the amniotic membrane by the heating means. And repeat multiple times alternately Since, it is possible to dehydrated and dried without destroying the amniotic epithelial cells that configuration, the basement membrane and connective tissue.
本発明に係る羊膜の乾燥処理方法に用いる乾燥装置の一例の概略図を図１に示す。図１に示す乾燥装置では、処理槽１０内に回転テーブル１２が配設されており、回転テーブル１２は処理槽１０外に載置されたモータ１６によって回転する。
かかる処理槽１０の減圧手段として、真空ポンプ１８が処理槽１０外に配設されており、処理槽１０と真空ポンプ１８とを接続する減圧配管２２の途中に電磁弁２０が設けられている。
更に、処理槽１０の復圧手段として、減圧状態の処理槽１０内に外気を吸引して復圧すべく、吸込む外気を濾過するフィルター２４と電磁弁２６とが設けられた復圧配管２８が処理槽１０に接続されている。
また、処理槽１０の回転テーブル１２上に載置された羊膜の加温手段として、処理槽１０内に遠赤外線ヒータ１４が配設されていると共に、回転テーブル１２上に載置された羊膜にマイクロ波を照射できるように、処理槽１０の外側にマイクロ波照射装置３０が設けられている。かかる加温手段の設置温度は生羊膜を構成する細胞組織を破壊することのない温度、好ましくは５０℃以下とする。
FIG. 1 shows a schematic diagram of an example of a drying apparatus used in the method for drying amniotic membrane according to the present invention. In the drying apparatus shown in FIG. 1, a rotary table 12 is disposed in the processing tank 10, and the rotary table 12 is rotated by a motor 16 placed outside the processing tank 10.
As a pressure reducing means for the processing tank 10, a vacuum pump 18 is disposed outside the processing tank 10, and an electromagnetic valve 20 is provided in the middle of a pressure reducing pipe 22 that connects the processing tank 10 and the vacuum pump 18.
Further, as a pressure recovery means for the processing tank 10, a return pressure pipe 28 provided with a filter 24 and an electromagnetic valve 26 for filtering the outside air to be sucked in to recover the pressure by sucking the outside air into the processing tank 10 in a reduced pressure state. It is connected to the tank 10.
Further, as a means for heating the amniotic membrane placed on the turntable 12 of the treatment tank 10, a far infrared heater 14 is disposed in the treatment tank 10, and the amniotic membrane placed on the turntable 12 A microwave irradiation device 30 is provided outside the processing tank 10 so that microwaves can be irradiated. The installation temperature of the heating means is a temperature that does not destroy the cell tissue constituting the raw amniotic membrane, preferably 50 ° C. or less.
図１に示す乾燥装置を用いて生羊膜を乾燥処理する際に、回転テーブル１２上に載置する羊膜としては、人を含む動物の胎児を包む生羊膜を載置する。この生羊膜としては、人由来の生羊膜、特に帝王切開分娩した胎盤等から無菌状態で分離した生羊膜を好適に用いることができる。
かかる生羊膜は、処理槽１０内の回転テーブル１２上にシート状に広げて載置する。特に、生羊膜を、処理槽１０内の回転テーブル１２上に広げた吸水紙上にシート状に広げて載置することが好ましい。生羊膜の脱水を容易に行なうことができるからである。
この様に、生羊膜を載置した回転テーブル１２をモータ１６によって連続回転しつつ、加温手段としての遠赤外線ヒータ１４によって生羊膜を連続的に加温する。
When the raw amniotic membrane is dried using the drying apparatus shown in FIG. 1, as the amniotic membrane to be placed on the rotary table 12, the raw amniotic membrane that wraps the fetus of an animal including a human is placed. As this raw amnion, a raw amnion derived from humans, particularly a raw amnion separated aseptically from a placenta or the like delivered from a caesarean section can be preferably used.
Such raw amniotic membrane is spread and placed on the rotary table 12 in the treatment tank 10 in a sheet shape. In particular, it is preferable that the raw amniotic membrane is spread and placed in a sheet form on a water absorbent paper spread on the rotary table 12 in the treatment tank 10. This is because the raw amnion can be easily dehydrated.
In this way, while the rotary table 12 on which the raw amniotic membrane is placed is continuously rotated by the motor 16, the raw amniotic membrane is continuously heated by the far infrared heater 14 as a heating means.
遠赤外線ヒータ１４によって処理槽１０内の羊膜を連続的に加温しつつ、減圧手段としての真空ポンプ１８を駆動し且つ電磁弁２０を開放にして、処理槽１０内を減圧状態とする。この際に、復圧手段としての電磁弁２６は閉じている。
処理槽１０内が減圧状態となると、処理槽１０内が生羊膜を構成する細胞組織を破壊することのない温度、好ましくは５０℃以下であっても、水の沸点低下によって、羊膜内の水を蒸発除去できる。
かかる処理槽１０内の減圧曲線を図２に示す。図２に示すグラフの横軸は時間であり、縦軸は処理槽１０内の圧力を示す。
図２では、減圧曲線Ａが、生羊膜を載置した処理槽１０内を大気圧から減圧を開始した減圧曲線である。かかる減圧曲線Ａから明らかな様に、処理槽１０内の圧力は、減圧開始直後では、その減圧速度が速いものの、減圧開始から暫く経過すると、処理槽１０内の減圧速度が低下する。この現象は、羊膜内の水の蒸発によって、水の蒸発潜熱によって羊膜温度が低下して、羊膜内の水の蒸発速度が著しく低下することによる。
While the amniotic membrane in the processing tank 10 is continuously heated by the far-infrared heater 14, the vacuum pump 18 as the pressure reducing means is driven and the electromagnetic valve 20 is opened to make the processing tank 10 in a reduced pressure state. At this time, the electromagnetic valve 26 as the pressure restoring means is closed.
When the inside of the treatment tank 10 is in a reduced pressure state, even if the temperature in the treatment tank 10 does not destroy the cell tissue constituting the raw amniotic membrane, preferably 50 ° C. or less, Can be removed by evaporation.
A decompression curve in the treatment tank 10 is shown in FIG. The horizontal axis of the graph shown in FIG. 2 is time, and the vertical axis shows the pressure in the processing tank 10.
In FIG. 2, the depressurization curve A is a depressurization curve in which depressurization is started from the atmospheric pressure in the treatment tank 10 in which the raw amniotic membrane is placed. As is apparent from the decompression curve A, the pressure in the treatment tank 10 is high immediately after the start of decompression, but the pressure reduction speed in the treatment tank 10 decreases after a while from the start of decompression. This phenomenon is caused by the evaporating rate of water in the amniotic membrane due to the evaporation of water in the amniotic membrane, resulting in a decrease in the amniotic membrane temperature due to the latent heat of water evaporation.
この羊膜の温度低下を、図１に示す乾燥装置では、遠赤外線ヒータ１４からの遠赤外線の照射のみによって防止できず、マイクロ波照射装置３０から回転テーブル１２上に載置された、未乾燥状態の羊膜にマイクロ波を所定時間照射して加温する。
かかる遠赤外線の照射とマイクロ波の照射との際に、減圧手段としての真空ポンプ１８の駆動を停止すると共に、電磁弁２０を閉じ、復圧手段としての電磁弁２６を開放し、処理槽１０内に外気を吸引して、図２の減圧曲線Ａに示す様に、処理槽１０内を復圧する。かかる復圧によって、減圧雰囲気中に載置されている羊膜に、マイクロ波照射装置３０からのマイクロ波のエネルギーが集中することに起因する羊膜温度の過剰昇温を防止できる。
この復圧は、羊膜温度が過剰昇温されることを防止するためのものであり、減圧曲線Ａに示す様に、大気圧まで復圧することは必要ではなく、羊膜の温度を所定温度に昇温できれば、大気圧以下であってもよい。この場合の羊膜温度も、羊膜を構成する細胞組織を破壊することのない温度とすることは勿論のことである。
かかるマイクロ波の照射時間は、予め実験的に好適な照射時間を求めておくことが好ましい。
処理槽１０内の羊膜にマイクロ波を所定時間照射して加温した後、復圧手段としての電磁弁２６を閉じ、減圧手段としての真空ポンプ１８を再駆動し且つ電磁弁２０を開放して処理槽１０内を、再度、加温された羊膜温度で羊膜中の水分を更に蒸発除去できる減圧状態とする。
In the drying apparatus shown in FIG. 1, the temperature decrease of the amniotic membrane cannot be prevented only by irradiation with far infrared rays from the far infrared heater 14, and is in an undried state placed on the rotary table 12 from the microwave irradiation apparatus 30. The amnion is heated by microwave irradiation for a predetermined time.
At the time of irradiation with far infrared rays and microwave irradiation, the driving of the vacuum pump 18 as the decompression means is stopped, the electromagnetic valve 20 is closed, the electromagnetic valve 26 as the return pressure means is opened, and the treatment tank 10 The outside air is sucked in, and the inside of the processing tank 10 is decompressed as shown in a decompression curve A in FIG. Such return pressure can prevent an excessive increase in the amniotic membrane temperature caused by the concentration of microwave energy from the microwave irradiation device 30 on the amniotic membrane placed in a reduced-pressure atmosphere.
This decompression is intended to prevent the amniotic membrane temperature from excessively rising, and as shown in the decompression curve A, it is not necessary to restore the pressure to the atmospheric pressure, and the amniotic membrane temperature is raised to a predetermined temperature. As long as it can be warmed, it may be below atmospheric pressure. It goes without saying that the amniotic temperature in this case is also set to a temperature that does not destroy the cell tissue constituting the amniotic membrane.
It is preferable that the irradiation time of such a microwave is obtained in advance by an experimentally suitable irradiation time.
After heating the amniotic membrane in the treatment tank 10 by microwave irradiation for a predetermined time, the electromagnetic valve 26 as the decompression means is closed, the vacuum pump 18 as the decompression means is redriven, and the electromagnetic valve 20 is opened. The inside of the processing tank 10 is again brought into a reduced pressure state in which moisture in the amniotic membrane can be further evaporated and removed at the heated amniotic membrane temperature.
かかる処理槽１０内の羊膜に遠赤外線を照射しつつ減圧する減圧操作と、羊膜に遠赤外線の照射とマイクロ波の照射とを含む処理槽１０内を復圧する復圧操作とを、図２に示す様に、複数回繰り返すことによって、処理槽１０内に載置された生羊膜を、その細胞組織を破壊することなく乾燥できる。
かかる乾燥が進行すると、羊膜が載置された処理槽１０内の最高減圧到達圧力が、羊膜が載置されていない処理槽１０を減圧したときの最高減圧到達圧力に近づく。このため、羊膜が載置された処理槽１０内の最高減圧到達圧力と、羊膜が載置されていない処理槽１０を減圧したときの最高減圧到達圧力とが等しくなったとき、経験的に羊膜の乾燥が終了したものと判断できる。この様に、最高減圧到達力によって羊膜の乾燥終了を判断することにより、乾燥程度の略等しい乾燥羊膜を得ることができる。
かかる乾燥羊膜の保存は、無菌状態の乾燥大気中で保存でき、具体的には乾燥剤が封入された滅菌パック中に密閉して保存できる。その有効保存期間は、保存条件によって１年以上とすることは可能である。
尚、図１に示す乾燥装置には、加温手段として、遠赤外線ヒータ１４とマイクロ波照射装置３０とを具備しているが、その出力によっては遠赤外線ヒータ１４及びマイクロ波照射装置３０の一方を設けることができる。
FIG. 2 shows a depressurization operation for depressurizing the amniotic membrane in the treatment tank 10 while irradiating it with far infrared rays, and a decompression operation for restoring the pressure in the treatment tank 10 including far infrared irradiation and microwave irradiation to the amniotic membrane. As shown, by repeating a plurality of times, the raw amniotic membrane placed in the treatment tank 10 can be dried without destroying the cell tissue.
As this drying progresses, the maximum pressure reduction ultimate pressure in the treatment tank 10 on which the amniotic membrane is placed approaches the maximum pressure reduction ultimate pressure when the treatment tank 10 on which the amniotic membrane is not placed is decompressed. For this reason, when the maximum pressure reduction ultimate pressure in the processing tank 10 in which the amniotic membrane is placed is equal to the maximum pressure reduction ultimate pressure when the processing tank 10 in which the amniotic membrane is not placed is decompressed, the amniotic membrane is empirically determined. It can be determined that the drying of the product is finished. Thus, by determining the end of drying of the amniotic membrane based on the maximum pressure attainment, it is possible to obtain a dried amniotic membrane having substantially the same degree of drying.
Such dry amniotic membrane can be stored in aseptically dried air, and specifically, sealed in a sterile pack containing a desiccant. The effective storage period can be one year or longer depending on the storage conditions.
In addition, although the far-infrared heater 14 and the microwave irradiation apparatus 30 are provided as a heating means in the drying apparatus shown in FIG. 1, depending on the output, one of the far-infrared heater 14 and the microwave irradiation apparatus 30 is provided. Can be provided.
図１に示す乾燥装置を用いて得られた乾燥羊膜の表面についての走査電子顕微鏡写真を図３に示す。図３（ａ）〜（ｃ）は、乾燥羊膜表面の異なる部分の走査電子顕微鏡写真であり、平坦で起伏、断裂に乏しく一定の構造を保持していた。図３（ｂ）には、線で囲んだ部分が1個の細胞と考えられる部分であり、鱗状に細胞が存在することが観察される。 但し、図３（ｂ）の反対側面である図３（ａ）（ｃ）では、明瞭な構造が観察されず、上皮下に存在する結合組織が基質成分を含めきちんと保持されているためと考えられる。
これに対し、生羊膜を凍結乾燥した乾燥羊膜の表面についての走査電子顕微鏡写真を図４に示す。図４（ａ）〜（ｄ）は、生羊膜をリン酸緩衝液（ＰＢＳ）で洗浄した後に凍結乾燥して得た乾燥羊膜についてのものであり、図４（ａ）（ｂ）は乾燥羊膜の一面側のものであり、その反対面側を図４（ｃ）（ｄ）に示す。
図４（ａ）（ｂ）からは、細胞が鱗状に配列しているようにも見えるが、皺が多いために明確に区別できない。
更に、図４（ｃ）（ｄ）からは、膠原繊維（ＣＦ）がシート状に存在していることが観察される。
また、図４（ｅ）（ｆ）は、生羊膜を蒸留水で洗浄した後に凍結乾燥して得た乾燥羊膜についてのものであり、図４（ｅ）は乾燥羊膜の一面側のものであり、その反対側面を図４（ｆ）に示す。
図４（ｅ）からは細胞に大小の穴が開いていることが観察され、図４（ｆ）からは細胞及び基質成分のすべてが洗い流され、膠原繊維のみが残存していることが観察される。
この様に、生羊膜を凍結乾燥して得た乾燥羊膜は、図１に示す乾燥装置を用いて生羊膜を乾燥処理して得た乾燥羊膜に比較して、上皮、結合組織とも保存が不完全である。
FIG. 3 shows a scanning electron micrograph of the surface of the dried amniotic membrane obtained using the drying apparatus shown in FIG. 3 (a) to 3 (c) are scanning electron micrographs of different portions of the dried amniotic membrane surface, which are flat and have a uniform structure with little undulation and tearing. In FIG. 3 (b), the part surrounded by a line is a part considered as one cell, and it is observed that the cells exist in a scale shape. However, in FIGS. 3 (a) and 3 (c), which are the opposite sides of FIG. 3 (b), a clear structure is not observed, and the connective tissue existing in the subepithelium is properly retained including the matrix component. It is done.
On the other hand, the scanning electron micrograph about the surface of the dry amniotic membrane which freeze-dried the raw amniotic membrane is shown in FIG. 4 (a) to 4 (d) relate to a dry amnion obtained by washing a raw amniotic membrane with a phosphate buffer (PBS) and then freeze-drying, and FIGS. 4 (a) and 4 (b) show a dried amnion. 4 (c) and 4 (d) show the opposite surface side.
From FIGS. 4 (a) and 4 (b), it seems that the cells are arranged like a scale, but it cannot be clearly distinguished because there are many wrinkles.
Further, from FIGS. 4C and 4D, it is observed that collagen fibers (CF) are present in a sheet form.
FIGS. 4 (e) and 4 (f) are for a dry amnion obtained by lyophilization after washing a raw amnion with distilled water, and FIG. 4 (e) is for one side of the dried amnion. The opposite side surface is shown in FIG.
From FIG. 4 (e), it is observed that the cells have large and small holes, and from FIG. 4 (f), it is observed that all the cells and matrix components are washed away, and only the collagen fibers remain. The
As described above, the dried amniotic membrane obtained by freeze-drying the raw amniotic membrane is less preserved in the epithelium and connective tissue than the dried amniotic membrane obtained by drying the raw amniotic membrane using the drying apparatus shown in FIG. Is complete.
図１に示す乾燥装置を用いて生羊膜を乾燥処理して得た乾燥羊膜をリン酸緩衝液（ＰＢＳ）に浸漬して再水和した羊膜の顕微鏡写真を図５（ａ）に示す。この顕微鏡写真は、乾燥羊膜をリン酸緩衝液（ＰＢＳ）に浸漬して再水和した羊膜を、通常の光学顕微鏡標本作成方法に準拠して作成した標本を、光学顕微鏡を用いて組織像を撮影したものである。かかる標本は、再水和した羊膜を１０％ホルマリン固定液で固定し、アルコール脱水、キシレン透徹、パラフィン包埋した後、１〜２μｍの切片標本を作成し、その後、切片標本をヘマトキシリン−エオジン染色（Ｈ−Ｅ染色）を施して得たものである。
更に、参照のために、図５（ｄ）に、生羊膜の顕微鏡写真を示す。この顕微鏡写真も、図５（ａ）に示す顕微鏡写真と同様にして撮影したものである。
図５（ａ）の顕微鏡写真と図５（ｄ）の顕微鏡写真とを比較すると、両者には、上皮細胞（Ｅｎ）、結合組織（Ｃｔ）及び間葉系の細胞（矢印Ｍ）が認められ、両者は略同様の組織像である。
FIG. 5 (a) shows a micrograph of the amniotic membrane obtained by rehydrating the dried amniotic membrane obtained by drying the raw amniotic membrane using the drying apparatus shown in FIG. 1 in a phosphate buffer solution (PBS). This photomicrograph shows a sample of an amniotic membrane that has been rehydrated by immersing the dried amniotic membrane in phosphate buffered saline (PBS) in accordance with a normal optical microscope specimen preparation method. It was taken. Such specimens were prepared by fixing rehydrated amniotic membrane with 10% formalin fixative, dehydrating with alcohol, penetrating xylene, and embedding with paraffin, and preparing 1-2 μm section specimens, and then sectioning the specimens with hematoxylin-eosin staining (H-E dyeing).
Further, for reference, a micrograph of the raw amniotic membrane is shown in FIG. This photomicrograph was also taken in the same manner as the photomicrograph shown in FIG.
When comparing the micrograph of FIG. 5 (a) with the micrograph of FIG. 5 (d), epithelial cells (En), connective tissue (Ct), and mesenchymal cells (arrow M) are observed in both. Both are substantially similar tissue images.
これに対し、生羊膜を凍結乾燥して得た乾燥羊膜の組織像を図５（ｂ）（ｃ）に示す。この顕微鏡写真も、図５（ａ）に示す顕微鏡写真と同様にして撮影したものである。図５（ｂ）は、採取した生羊膜を蒸留水で洗浄してから凍結乾燥して得た乾燥羊膜をリン酸緩衝液（ＰＢＳ）に浸漬して再水和したものである。また、図５（ｃ）は、採取した生羊膜をそのまま凍結乾燥して得た乾燥羊膜をリン酸緩衝液（ＰＢＳ）に浸漬して再水和したものである。
図５（ｂ）に示す組織像では、図５（ａ）（ｄ）に示す組織像に比較して、著しく萎縮しており、上皮組織（Ｅｎ）は濃縮し、結合組織内には細胞がほとんど認められない。また、図５（ｃ）に示す組織像では、図５（ｂ）に示す組織像よりも更に細胞が萎縮している。
On the other hand, the tissue image of the dried amnion obtained by freeze-drying the raw amnion is shown in FIGS. This photomicrograph was also taken in the same manner as the photomicrograph shown in FIG. FIG. 5 (b) shows a dried amnion obtained by washing a collected raw amnion with distilled water and then freeze-drying it in a phosphate buffer solution (PBS) for rehydration. FIG. 5 (c) shows a dried amnion obtained by lyophilizing the collected raw amnion as it is and dipped in a phosphate buffer (PBS) for rehydration.
In the tissue image shown in FIG. 5 (b), the tissue images shown in FIGS. 5 (a) and 5 (d) are markedly atrophied, the epithelial tissue (En) is concentrated, and cells are present in the connective tissue. Almost not recognized. Further, in the tissue image shown in FIG. 5C, the cells are further contracted than in the tissue image shown in FIG.
図１に示す乾燥装置を用いて生羊膜を乾燥処理して得た乾燥羊膜は、生羊膜の基本的組織である、基底膜及び結合組織が保持されているため、水又は緩衝液に浸漬して再水和した羊膜は、角膜内皮細胞の培養に用いることができる。更に、かかる羊膜は、火傷や創傷等による皮膚欠損の治療にも用いることができる。 The dry amniotic membrane obtained by drying the raw amniotic membrane using the drying apparatus shown in FIG. 1 retains the basement membrane and connective tissue, which are the basic tissues of the raw amniotic membrane, so it is immersed in water or a buffer solution. The rehydrated amniotic membrane can be used for culturing corneal endothelial cells. Furthermore, such amniotic membrane can also be used to treat skin defects due to burns, wounds, and the like.
（１）生羊膜の採取
予め同意を得た妊婦の帝王切開分娩で排出された胎盤を、直ちに無菌生理食塩水によって脱落膜や血餅等を除去洗浄して生羊膜を採取した。採取した生羊膜は直ちに生理食塩水と共にスピッツ内に密閉して冷蔵保存した。
(1) Collection of raw amniotic membrane The placenta excreted by cesarean section delivery of a pregnant woman who had obtained consent in advance was immediately removed by removing decidua and blood clots with sterile physiological saline, and the raw amniotic membrane was collected. The collected raw amnion was immediately sealed in a spitz together with physiological saline and stored refrigerated.
（２）生羊膜の乾燥
図１に示す乾燥装置を用いて生羊膜の乾燥を行なった。この乾燥装置では、マイクロ波照射装置３０としては、出力１．５ＫＷのマグネトロンを用いた。また、遠赤外線ヒータ１４の温度設定を５０℃とし、遠赤外線を羊膜に対して乾燥開始から終了まで連続照射した。更に、処理槽１０内に羊膜を載置していないとき、真空ポンプ１８による最高減圧到達圧力を０．４ｋＰａとなるように設定した。
かかる図１に示す乾燥装置によって先に採取した羊膜を乾燥する際に、シワにならないように広げた吸水紙としてのクッキングペーパ上に、スピッツから取り出した生羊膜（５０ｇ）をシワのないように広げ、これらをトレイ上に載置した。更に、このトレイを処理槽１０内の回転テーブル１２上に載置した後、回転テーブル１２を回転した。この回転テーブル１２は、乾燥開始から終了まで連続回転した。
次いで、遠赤外線ヒータ１４をＯＮとして、真空ポンプ１８を駆動すると共に電磁弁２０を開けて処理槽１０内を減圧する減圧操作を開始した。減圧開始から暫くすると減圧速度が低下してきたため、最高減圧到達圧力が０．９０ｋＰａに到達したとき、真空ポンプ１８を停止すると共に電磁弁２０を閉じ、電磁弁２６を開いて、フィルター２４によってゴミや細菌が濾過された空気を処理槽１０内に導入する復圧操作を開始し、処理槽１０内の圧力を４．５３ｋＰａに復圧した。
かかる復圧操作の開始と同時に、マイクロ波照射装置３０としてのマグネトロンをＯＮとしてマイクロ波を回転テーブル１２上の羊膜に照射する加温操作を施した。
(2) Drying of raw amniotic membrane The raw amniotic membrane was dried using the drying apparatus shown in FIG. In this drying apparatus, a magnetron having an output of 1.5 KW was used as the microwave irradiation apparatus 30. Moreover, the temperature setting of the far-infrared heater 14 was set to 50 ° C., and the far-infrared ray was continuously irradiated on the amniotic membrane from the start to the end of drying. Furthermore, when the amniotic membrane was not placed in the treatment tank 10, the maximum pressure reduction achieved by the vacuum pump 18 was set to 0.4 kPa.
When the amniotic membrane previously collected by the drying apparatus shown in FIG. 1 is dried, the raw amniotic membrane (50 g) taken out from Spitz is not wrinkled on the cooking paper as water absorbent paper spread so as not to be wrinkled. They were spread and placed on a tray. Furthermore, after placing this tray on the turntable 12 in the processing tank 10, the turntable 12 was rotated. The turntable 12 continuously rotated from the start to the end of drying.
Next, the far-infrared heater 14 was turned on, the vacuum pump 18 was driven, the electromagnetic valve 20 was opened, and a pressure reducing operation for reducing the pressure inside the processing tank 10 was started. Since the decompression speed has decreased for a while after the start of decompression, when the maximum decompression ultimate pressure reaches 0.90 kPa, the vacuum pump 18 is stopped and the solenoid valve 20 is closed and the solenoid valve 26 is opened. A return pressure operation for introducing air in which bacteria were filtered into the treatment tank 10 was started, and the pressure in the treatment tank 10 was restored to 4.53 kPa.
Simultaneously with the start of the re-pressure operation, the magnetron as the microwave irradiation device 30 was turned on, and a heating operation was performed to irradiate the amniotic membrane on the rotary table 12 with microwaves.
かかる遠赤外線ヒータ１４とマグネトロンとによる加温操作を３分間施した後、マグネトロンをＯＦＦにして、遠赤外線ヒータ１４をＯＮとしつつ減圧操作を再開した。再開した減圧操作によって処理槽１０内を０．６２ｋＰａまで減圧状態とした後、処理槽１０内を４．６３ｋＰａに復圧する復圧操作と、遠赤外線ヒータ１４とマグネトロンとによる３分間の加温操作とを施した。かかる減圧操作、加温操作及び復圧操作を合計で６回施して羊膜の乾燥を終了した。
この乾燥終了は、第５回目の減圧操作による処理槽１０内の最高減圧到達圧力と、処理槽１０内に羊膜を載置していないときの最高減圧到達圧力とによって判断した。すなわち、第６回目の減圧操作の最高減圧到達圧力が０．４０ｋＰａに到達し、処理槽１０内に羊膜を載置していないときの最高減圧到達圧力と等しくなったため、乾燥終了と判断した。
乾燥を終了した乾燥羊膜は、処理槽１０に載置した生羊膜５０ｇに対して１ｇに脱水乾燥されており、乾燥剤が封入された滅菌パック中に密閉して保存した。
After performing the heating operation with the far infrared heater 14 and the magnetron for 3 minutes, the magnetron was turned off and the decompression operation was restarted while the far infrared heater 14 was turned on. After reducing the pressure in the processing tank 10 to 0.62 kPa by the restarted pressure reducing operation, the pressure reducing operation for returning the pressure in the processing tank 10 to 4.63 kPa, and the heating operation for 3 minutes by the far infrared heater 14 and the magnetron And gave. This decompression operation, heating operation, and decompression operation were performed a total of 6 times to finish drying the amniotic membrane.
The end of this drying was judged by the maximum pressure reduction ultimate pressure in the treatment tank 10 by the fifth decompression operation and the maximum pressure reduction ultimate pressure when no amniotic membrane was placed in the treatment tank 10. That is, the maximum reduced pressure attainment pressure of the sixth decompression operation reached 0.40 kPa and became equal to the maximum reduced pressure attainment pressure when no amniotic membrane was placed in the treatment tank 10, so it was determined that the drying was finished.
The dried amniotic membrane after drying was dehydrated and dried to 1 g with respect to 50 g of raw amniotic membrane placed in the treatment tank 10, and sealed and stored in a sterilized pack containing a desiccant.
（３）乾燥羊膜の状態
得られた乾燥羊膜の両面を走査電子顕微鏡によって観察したところ、図３（ａ）〜（ｃ）に示す様に、平坦で起伏、断裂に乏しく一定の構造を保持していた。
また、この乾燥羊膜をリン酸緩衝液（ＰＢＳ）に浸漬して再水和した羊膜を、通常の光学顕微鏡標本作成方法に準拠して作成した標本を、光学顕微鏡を用いて観察したところ、図５（ａ）に示す様に、生羊膜と略同様に、上皮細胞（Ｅｎ）、結合組織（Ｃｔ）及び間葉系の細胞（矢印Ｍ）が認められた。
(3) State of dried amniotic membrane When both sides of the obtained dried amniotic membrane were observed with a scanning electron microscope, as shown in FIGS. 3 (a) to 3 (c), a flat structure with little undulation and tearing was maintained. It was.
In addition, when the amnion rehydrated by immersing this dried amniotic membrane in phosphate buffer (PBS) was observed using an optical microscope, the sample was prepared in accordance with a normal optical microscope preparation method. As shown in FIG. 5 (a), epithelial cells (En), connective tissue (Ct), and mesenchymal cells (arrow M) were observed in substantially the same manner as the raw amniotic membrane.
実施例１で得られた乾燥羊膜を用いた欠損損傷治癒に対する効果を検討した。
（１）欠損損傷の作成及び治療
７匹のマウス（C57BL/6 ♂、体重42〜46ｇ）の各々の背部を剃毛したのち、径3mm のデルマパンチで円形の欠損損傷を４個作成した。いずれの損傷も真皮が完全に消失し、皮下組織にまで達するものであって、Shea及びNPUAPの分類によるStage IIからStage IIIに相当する。
各個体の背部に形成した４個の欠損損傷のうち、１個の欠損損傷にはフィルム（ドレッシング剤）に貼着した乾燥羊膜による被覆を行ない、他の１個の欠損損傷にはフィルム（ドレッシング剤）に貼着した止血剤含有ガーゼによる被覆を行った。残りの２個の欠損損傷のうち、１個の欠損損傷にはフィルム（ドレッシング剤）のみの被覆を行ない、他の１個の欠損損傷は無処置とした。
ここで用いた乾燥羊膜は、実施例１で得た乾燥羊膜を、乾燥剤が封入された滅菌パック中に密閉して１月間保管した乾燥羊膜であって、乾燥状態のままで用いた。
欠損損傷を被覆した水準は、いずれも被覆物の付着は良好であった。特に、乾燥羊膜を用いた水準では、乾燥羊膜が平坦で吸着性がよく、付着が容易であったため、創傷部位全域を完全に被覆することができた。
治療開始後７日目に創傷治癒の状態を観察すべく材料の採取を行った。
尚、この間、被覆物の交換は一切行わず、消失した場合はそのままの状態とした。
The effect on defect damage healing using the dried amniotic membrane obtained in Example 1 was examined.
(1) Creation and treatment of defect damage After shaving the back of each of seven mice (C57BL / 6 ♂, body weight 42-46 g), four circular defect damages were created with a 3 mm diameter derma punch. In any case, the dermis completely disappears and reaches the subcutaneous tissue, and corresponds to Stage II to Stage III according to the Shea and NPUAP classification.
Of the four defect damages formed on the back of each individual, one defect damage is covered with a dry amniotic membrane affixed to a film (dressing agent), and the other defect damage is a film (dressing). Coating with a hemostatic gauze affixed to the agent. Of the remaining two defect lesions, one defect lesion was covered only with a film (dressing agent), and the other defect defect was not treated.
The dry amniotic membrane used here was a dry amniotic membrane obtained by sealing the dried amniotic membrane obtained in Example 1 in a sterilized pack containing a desiccant for one month, and was used in a dry state.
The level of covering the defect damage was good for the coating. In particular, at the level using dry amniotic membrane, the dry amniotic membrane was flat, adsorbable and easy to attach, so that the entire wound site could be completely covered.
On the 7th day after the start of treatment, the material was collected to observe the state of wound healing.
During this time, the covering was not changed at all, and when it disappeared, it was left as it was.
（２）欠損損傷治癒の肉眼及び触診による観察
乾燥羊膜、止血剤含有ガーゼ、フィルムのみの水準のいずれについても、体表面からの観察では概ね順調に治癒しているように見受けられた。
しかし、フィルムのみの水準及び止血剤含有ガーゼの水準では、創傷治癒の状態は個体差が大きく、７日目でも出血がみられるものがある一方、完全に完治したものも観察された。更に、フィルムや止血剤含有ガーゼが付着した部位近傍には、強い掻痒感のためと考えられる、新たに擦過傷が生じているものが見られた。
他方、乾燥羊膜の水準では、いずれの個体も創傷面が乾燥し、開口部が縮小していた。
(2) Observation of defect damage healing with the naked eye and palpation The dry amnion, the hemostatic agent-containing gauze, and the film-only level seemed to heal almost smoothly when observed from the body surface.
However, at the level of the film alone and the level of the hemostatic agent-containing gauze, the wound healing state was greatly different among individuals, and although bleeding was observed even on the 7th day, completely cured cases were observed. Furthermore, in the vicinity of the site where the film and the hemostatic agent-containing gauze were adhered, there were some newly scratched pieces considered to be due to a strong itching sensation.
On the other hand, at the level of dry amniotic membrane, the wound surface of each individual was dry and the opening was reduced.
次に、各水準について、創傷開口部の径および創傷周囲の硬結の範囲を測定し、その結果を図６に示した。図６において、創傷開口部の径を1)に示し、創傷周囲の硬結の範囲を2)に示した。
図６に示す様に、創傷開口部の径は、乾燥羊膜、止血剤含有ガーゼ、フィルムのみの各水準は、無処置の水準に比較して、いずれも縮小している。
しかし、触診したところ、止血剤含有ガーゼ、フィルムのみ、及び無処置の水準のすべてにおいて、創傷部位では開口部周囲を取り巻くように硬結部位が存在するのが確認された。
これに対し、乾燥羊膜の水準では、すべての個体において硬結を触知することができなかった。
Next, for each level, the diameter of the wound opening and the range of consolidation around the wound were measured, and the results are shown in FIG. In FIG. 6, the diameter of the wound opening is shown in 1), and the range of consolidation around the wound is shown in 2).
As shown in FIG. 6, as for the diameter of the wound opening, each level of the dry amnion, the hemostatic agent-containing gauze, and the film alone is reduced compared to the level of no treatment.
However, upon palpation, it was confirmed that there was a consolidation site around the opening at the wound site in all of the hemostatic agent-containing gauze, the film alone, and the untreated level.
In contrast, at the level of dry amniotic membrane, induration was not palpable in all individuals.
この様に、乾燥羊膜は、止血剤含有ガーゼに比較して、欠損損傷に対する付着性が良好で、損傷に対する刺激性も少なく、瘢痕形成を起こし難く、且つ過度の組織収縮を引き起こさない。このため、乾燥羊膜は、欠損損傷の治療にも有効である。 Thus, dry amniotic membrane has better adhesion to defect damage, less irritation to damage, less scar formation, and does not cause excessive tissue contraction, compared to gauze containing hemostatic agents. For this reason, dry amniotic membrane is also effective in treating defect damage.
１０ 処理槽
１２ 回転テーブル
１４ 遠赤外線ヒータ
１６ モータ
１８ 真空ポンプ
２０，２６ 電磁弁
２２ 減圧配管
２４ フィルター
２８ 復圧配管
３０ マイクロ波照射装置
DESCRIPTION OF SYMBOLS 10 Treatment tank 12 Rotary table 14 Far-infrared heater 16 Motor 18 Vacuum pump 20, 26 Solenoid valve 22 Pressure-reduction piping 24 Filter 28 Pressure-reduction piping 30 Microwave irradiation apparatus
Claims (10)
無菌状態の乾燥大気中で保存できるように脱水乾燥されていると共に、
水又は緩衝液に浸漬して再水和した際、生羊膜と同様の、上皮細胞、基底膜及び結合組織が保持されていることを特徴とする乾燥羊膜。 It is a dry amniotic membrane obtained by subjecting a raw amniotic membrane wrapping a fetus of an animal including a human to a dry amniotic membrane without destroying the cellular tissue of the raw amniotic membrane ,
It is dehydrated and dried so that it can be stored in a sterile dry atmosphere.
A dry amniotic membrane characterized by retaining epithelial cells, basement membrane and connective tissue similar to raw amniotic membrane when rehydrated by immersion in water or buffer.
該生羊膜を乾燥する乾燥装置として、羊膜を載置した処理槽内を減圧状態とする減圧手段と、減圧状態の前記処理槽内に載置した羊膜を加温する加温手段と、前記処理槽内の減圧状態を大気圧方向に復圧する復圧手段とを具備する乾燥装置を用い、
前記加温手段により、羊膜を、羊膜の細胞組織を破壊することのない温度に加温しつつ、前記処理槽内を前記減圧手段により減圧する減圧工程と、前記加温手段により、羊膜を、羊膜の細胞組織を破壊することのない温度に加温しつつ、前記復圧手段により前記処理槽内を大気圧もしくは大気圧に近い圧力まで上昇させる復圧工程とを交互に複数回繰り返して、羊膜をその構成する上皮細胞、基底膜及び結合組織を破壊することなく脱水・乾燥することを特徴とする羊膜の乾燥処理方法。 In a method for drying treatment of amniotic membrane for drying raw amniotic membrane wrapping fetuses of animals including humans,
As a drying apparatus for drying the raw amniotic membrane, a depressurizing means for depressurizing the treatment tank in which the amniotic membrane is placed, a heating means for heating the amniotic membrane placed in the depressurized treatment tank, and the treatment Using a drying device having a pressure-reducing means for restoring the reduced pressure state in the tank to the atmospheric pressure direction,
While the amniotic membrane is heated to a temperature that does not destroy the cell tissue of the amniotic membrane by the heating means, the depressurization step of depressurizing the inside of the treatment tank by the pressure reducing means, and the amniotic membrane by the heating means, While repeatedly heating to a temperature that does not destroy the cell tissue of the amniotic membrane, the return pressure step of raising the inside of the treatment tank to atmospheric pressure or a pressure close to atmospheric pressure by the return pressure means is repeated several times alternately, A method for drying amniotic membrane, comprising dehydrating and drying the amniotic membrane without destroying epithelial cells, basement membrane and connective tissue constituting the amniotic membrane.
5. The end of drying is defined as when the maximum pressure reduction ultimate pressure in the treatment tank on which the amniotic membrane is placed is equal to the maximum pressure reduction ultimate pressure when the treatment tank on which the amniotic membrane is not placed is decompressed. The drying process method as described in any one of -8.
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| PCT/JP2006/316269 WO2007023750A1 (en) | 2005-08-26 | 2006-08-18 | Dried amnion and method for drying treatment of amnion |
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| US8883210B1 (en) | 2010-05-14 | 2014-11-11 | Musculoskeletal Transplant Foundation | Tissue-derived tissuegenic implants, and methods of fabricating and using same |
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