WO2005021014A1 - Preparation d'un amnios biologique conservable a temperature ambiante - Google Patents
Preparation d'un amnios biologique conservable a temperature ambiante Download PDFInfo
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- WO2005021014A1 WO2005021014A1 PCT/CN2004/001010 CN2004001010W WO2005021014A1 WO 2005021014 A1 WO2005021014 A1 WO 2005021014A1 CN 2004001010 W CN2004001010 W CN 2004001010W WO 2005021014 A1 WO2005021014 A1 WO 2005021014A1
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- amniotic membrane
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
Definitions
- the invention relates to a method for preparing a biological amniotic membrane of a medical device dressing that can be stored at normal temperature. Background technique
- Amniotic membrane has attracted the attention of scholars at home and abroad due to its unique characteristics.
- Amniotic membrane refers to the basement membrane layer of human placenta. It is a collagen tissue layer without blood vessels and cell structure. It is rich in IV, V collagen, and basic fibroblast growth factor. (BFGF) and stem cell growth factor (HGF), which can easily make epithelial cells migrate and grow, promote epithelial differentiation and proliferation, and play an important role in epithelial wound healing.
- Amniotic membrane contains various protein suppressors, which exert anti-inflammatory effects by inhibiting corresponding proteases. It also contains TGF-, a component that inhibits cytokine expression and regulates apoptosis, which can avoid excessive proliferation of collagen fibers and prevent adhesions.
- amniotic membranes used in clinical practice are either fresh amniotic membranes or amniotic membranes stored at a deep low temperature (80 ° C). Fresh amniotic membrane often cannot guarantee the urgent need for clinical use, and the cryopreserved amniotic membrane has a shorter storage time and is inconvenient to transport. Summary of the invention
- An object of the present invention is to provide a method for preparing a biological amniotic membrane that is simple to handle, can be stored at room temperature, has a long shelf life, is convenient to transport, and can meet clinical needs.
- the preparation method of the biological amniotic membrane that can be stored at normal temperature of the present invention is as follows: the amniotic membrane of the placenta and the chorion are bluntly peeled off and washed to obtain the amniotic epithelial cell layer on top, supported by a nitrocellulose filter paper, and dried by vacuum
- the amniotic membrane has a water content ranging from 1% to 20%, that is, a biological amniotic membrane suitable for storage at room temperature is obtained.
- the preparation method of the biological amniotic membrane which can be stored at normal temperature according to the present invention can be frozen before being dried in vacuum.
- the method for preparing the biological amniotic membrane that can be stored at normal temperature is as follows: the washed amniotic membrane is treated with 0.1% to 30% glycerol, and placed in a freeze dryer working room at 0 ° C ⁇ 5656 to prefreeze 1 ⁇ 10 hours, turn on the condensation chamber to cool, so that the temperature of the cold trap reaches -10 ° C ⁇ 56 ° C, turn on the vacuum pump, and vacuum
- the method for preparing the biological amniotic membrane that can be stored at normal temperature according to the present invention, the optimal temperature range of the freeze dryer workshop: 0- ⁇ -30 ° C, the optimal prefreezing time range: 1 ⁇ 2 hours, the optimal temperature of the cold trap Range: -30 ° C-45 ° C, best vacuum degree lPa ⁇ 40Pa.
- the storage time of the amniotic membrane is extended to 2 to 3 years, and the storage condition is storage at normal temperature. Therefore, transportation is convenient and can meet clinical needs at any time.
- the present invention adopts a vacuum drying method, so that after fresh amniotic membranes are vacuum dried, they can be stored at room temperature for a long time. Because the storage method is not limited by temperature, it is conducive to the large-scale production and storage of amnion dressings, and provides sufficient amnion dressings for patients with ocular surface diseases to relieve clinical pressure.
- Placental tissues of healthy cesarean women were selected, and prenatal serological examinations were performed in accordance with relevant national standards to exclude hepatitis B, C, syphilis and acquired immunodeficiency syndrome.
- the amniotic membrane and chorion of the placenta were bluntly peeled off, washed, and the amniotic epithelial cell layer was obtained.
- the amniotic membrane layer was supported by nitrocellulose filter paper, immersed in 8% glycerin, and the material was placed in a working chamber using a freeze dryer.
- the dried amniotic membrane can be stored at normal temperature by vacuum packaging and sterilization.
- the tissue structure of amniotic membrane stored at room temperature for one year compared with fresh amniotic membrane
- the biological safety and toughness of the amniotic membrane stored for one year at room temperature can meet the requirements of clinical application.
- Placental tissues of healthy cesarean women were selected, and prenatal serological examinations were performed in accordance with relevant national standards to exclude hepatitis B, C, syphilis and acquired immunodeficiency syndrome.
- the amniotic membrane and chorion of the placenta were bluntly peeled off, washed, and the amniotic epithelial cell layer was obtained, supported by nitrocellulose filter paper, immersed in 10% glycerin, and then the material was placed in a working room with a freeze dryer.
- Placental tissues of healthy cesarean women were selected, and prenatal serological examinations were performed in accordance with relevant national standards to exclude hepatitis B, C, syphilis and acquired immunodeficiency syndrome.
- the amniotic membrane and chorion of the placenta were bluntly peeled off, washed, and the amniotic epithelial cell layer was obtained, supported by nitrocellulose filter paper, immersed with 0.1% glycerol, and the material was set to work by using a freeze dryer.
- the dried amniotic membrane can be stored at room temperature by vacuum packing and sterilizing.
- Placental tissues of healthy cesarean women were selected, and prenatal serological examinations were performed in accordance with relevant national standards to exclude hepatitis B, C, syphilis and acquired immunodeficiency syndrome.
- the amniotic membrane and chorion of the placenta were bluntly peeled off and washed to obtain the amniotic epithelial cell layer on top, supported by nitrocellulose filter paper, immersed in 8% glycerin, and the material was vacuumed at room temperature using a vacuum dryer. After drying for 5 hours, the vacuum degree can reach 4 ⁇ 20Pa.
- the dried amniotic membrane can be stored at normal temperature by vacuum packaging and sterilizing.
- Placental tissues of healthy cesarean women were selected, and prenatal serological examinations were performed in accordance with relevant national standards to exclude hepatitis B, C, syphilis and acquired immunodeficiency syndrome.
- the amniotic membrane and chorion of the placenta were bluntly peeled off and washed to obtain the amniotic epithelial cell layer on top, supported by nitrocellulose filter paper, immersed in 5% glycerin, and the material was vacuumed at room temperature using a vacuum dryer. After drying for 10 hours, the vacuum degree can reach 2 ⁇ 10Pa.
- the dried amniotic membrane can be stored at normal temperature by vacuum packaging and sterilizing.
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Description
一种可常温保存的生物羊膜的制备方法 技术领域
本发明涉及一种可常温保存的医疗器械敷料生物羊膜的制备方法。 背景技术
羊膜由于自身独有的特性受到国内外学者关注，羊膜是指人类胎盘的基 底膜层， 是无血管、 无细胞结构的胶原组织层， 富含 IV、 V型胶原、 碱性成 纤维细胞生长因子（BFGF)、干细胞生长因子 (HGF)， 易使上皮细胞移行长入、 促进上皮的分化、 增殖， 在上皮创伤愈合中起重要作用。 羊膜含各种蛋白抑 制因子， 通过抑制相应蛋白酶而发挥抗炎作用。 同时还含有抑制细胞因子表 达和调节细胞凋亡成分 TGF- ， 可避免胶原纤维过度增生， 预防粘连。 作 为一种新的生物材料， 近年来国内外临床上广泛用于烧伤创面及眼科疾病的 辅助治疗， 其疗效非常显著。 从国内外文献资料报导看， 临床上所用羊膜均 为新鲜羊膜， 或采用深低温（一 80Ό )保存的羊膜。 新鲜羊膜往往不能保证 临床急需， 深低温保存的羊膜保存时间较短， 运输不方便。 发明内容
本发明的目的在于： 针对上述问题， 提供一种处理方法简便、 可常温保 存、 保存期长、 运输方便、 能满足临床需要的生物羊膜的制备方法。
本发明的可常温保存的生物羊膜的制备方法为：将胎盘的羊膜与绒毛膜 钝性剥离，洗净，得到的羊膜上皮细胞层在上，下面用硝酸纤维素滤纸托着， 经真空干燥使羊膜含水量范围在 1 %〜20%， 即得适宜在常温保存的生物羊 膜。
本发明所述的可常温保存的生物羊膜的制备方法，在真空干燥前可进行 冷冻处理。
本发明所述的可常温保存的生物羊膜的制备方法为： 洗净的羊膜经用 0. 1 %〜30%的甘油处理， 置冷冻干燥机工作室经 0°C 〜一 56¾预冻 1〜10小 时， 开启冷凝室制冷， 使冷阱温度达到 -10°C 〜一 56 ， 开启真空泵， 真空
确认本
冷冻干燥， 使羊膜水份含量达到 1 %〜20%， 再包装、 灭菌， 即可。
本发明所述的可常温保存的生物羊膜的制备方法，冷冻干燥机工作室最 佳温度范围： 0 - ^一 30°C, 最佳预冻时间范围： 1〜2小时， 冷阱最佳温度 范围： -30°C - 45°C, 最佳真空度 lPa〜40Pa。
本发明的生物羊膜的制备方法， 将羊膜经过真空干燥后， 使羊膜的保存 时间延长到 2〜3年， 保存条件为常温保存。 因此， 运输很方便， 可随时满 足临床需要。 本发明采用真空干燥法， 使新鲜羊膜经真空干燥后， 可在常温 下长期保存。 由于保存方法不受温度的限制， 有利于羊膜敷料规模化生产、 保存， 为眼表疾病患者提供足够的羊膜敷料， 缓解临床的压力。 具体实施方式
以下结合实施例对本发明进行详细说明。
实施例 1 :
选用健康剖宫产产妇的胎盘组织，产前血清学检査按照国家相关标准进 行， 排除乙肝、 丙肝、 梅毒及获得性免疫缺陷综合症。 将胎盘的羊膜与絨毛 膜钝性剥离， 洗净， 得到的羊膜上皮细胞层在上， 下面用硝酸纤维素滤纸托 着， 用 8%的甘油浸没， 采用冷冻干燥机， 将物料置工作室经 0 °C 〜一 30°C 预冻 1〜2小时， 开启冷凝室制冷， 使冷阱温度达 -35 "C 〜一 45°C时开启真 空泵， 真空冷冻干燥 3〜8小时， 使真空度达到 4Pa〜20Pa， 干燥后的羊膜经 真空包装、 灭菌即可在常温下保存。 通过用电镜观察和免疫组化试验对比， 常温保存壹年的羊膜与新鲜羊膜比较其组织结构上没有发生明显变化。常温 保存壹年的羊膜其生物安全性及韧性等均能达到临床应用的要求。
实施例 2:
选用健康剖宫产产妇的胎盘组织，产前血清学检查按照国家相关标准进 行， 排除乙肝、 丙肝、 梅毒及获得性免疫缺陷综合症。 将胎盘的羊膜与绒毛 膜钝性剥离， 洗净， 得到的羊膜上皮细胞层在上， 下面用硝酸纤维素滤纸托 着， 用 10%的甘油浸没， 釆用冷冻干燥机， 将物料置工作室经 0 〜一 30°C 预冻 1〜2小时， 开启冷凝室制冷， 使冷阱温度达 -10 °C '- ^一 56°C时开启真 空泵， 真空冷冻干燥 8小时， 使真空度达到 4Pa〜30Pa， 干燥后的羊膜经真
空包装、 灭菌即可在常温下保存。
实施例 3:
选用健康剖宫产产妇的胎盘组织，产前血清学检查按照国家相关标准进 行， 排除乙肝、 丙肝、 梅毒及获得性免疫缺陷综合症。 将胎盘的羊膜与绒毛 膜钝性剥离， 洗净， 得到的羊膜上皮细胞层在上， 下面用硝酸纤维素滤纸托 着，用 0. 1%的甘油浸没，采用冷冻干燥机，将物料置工作室经 0°C 〜一 30QC, 预冻 2小时，开启冷凝室制冷，使冷阱温度达 - 35 °C 〜一 45°C时开启真空泵， 真空冷冻干燥 5小时， 使真空度达到 6〜30Pa， 干燥后的羊膜经真空包装、 灭菌即可在常温下保存。
实施例 4:
选用健康剖宫产产妇的胎盘组织，产前血清学检査按照国家相关标准进 行， 排除乙肝、 丙肝、 梅毒及获得性免疫缺陷综合症。 将胎盘的羊膜与绒毛 膜钝性剥离， 洗净， 得到的羊膜上皮细胞层在上， 下面用硝酸纤维素滤纸托 着， 用 8%的甘油浸没， 采用真空干燥机， 将物料在常温下真空干燥 5小时， 使真空度达到 4〜20Pa，干燥后的羊膜经真空包装、灭菌即可在常温下保存。 实施例 5:
选用健康剖宫产产妇的胎盘组织，产前血清学检査按照国家相关标准进 行， 排除乙肝、 丙肝、 梅毒及获得性免疫缺陷综合症。 将胎盘的羊膜与绒毛 膜钝性剥离， 洗净， 得到的羊膜上皮细胞层在上， 下面用硝酸纤维素滤纸托 着， 用 5%的甘油浸没， 采用真空干燥机， 将物料在常温下真空干燥 10小时， 使真空度达到 2〜10Pa,干燥后的羊膜经真空包装、灭菌即可在常温下保存。
Claims
1. 一种可常温保存的生物羊膜的制备方法， 其特征在于： 将胎盘的羊膜与絨 毛膜钝性剥离， 洗净， 得到的羊膜上皮细胞层在上， 经真空干燥使羊膜水 份含量范围在 1 %〜20%， 即得适宜在常温保存的生物羊膜。
2. 根据权利要求 1所述的可常温保存的生物羊膜的制备方法， 其特征在于： 羊膜真空干燥前进行了冷冻处理。
3. 根据权利要求 1或 2所述的可常温保存的生物羊膜的制备方法， 其特征在 于：洗净的羊膜经用 0. 1 %〜30%的甘油处理，置冷冻干燥机工作室经 0°C〜 一 56°C预冻 1〜10小时， 开启冷凝室制冷， 使冷阱温度达到一 10°C〜一 56 °C， 开启真空泵， 真空冷冻干燥， 使羊膜水份含量达到 1 %〜15 %， 再包 装、 灭菌， 即可。
4. 根据权利要求 3所述的可常温保存的生物羊膜的制备方法， 其特征在于： 冷冻干燥机工作室最佳温度范围： 0 °C 〜一 30°C，预冻最佳时间范围： 1一 2小时， 冷阱最佳温度范围： 一 30UC 〜一 45°C， 最佳真空度 2Pa〜15Pa， 真 空冷冻干燥最佳时间范围： 3〜10小时， 羊膜最佳含水量范围： 5 %〜12 %。
5. 根据权力要求 3所述的可常温保存的生物羊膜的制备方法， 其特征在于： 真空冷冻干燥时间 1〜72小时。
6. 根据权力要求 3所述的可常温保存的生物羊膜的制备方法， 其特征在于： 真空冷冻干燥时，真空度范围 lPa〜40Pa。
7. 根据权力要求 3所述的可常温保存的生物羊膜的制备方法， 其特征在于： 包装， 采用真空包装。
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| CNB2004800239337A CN100496238C (zh) | 2003-09-03 | 2004-09-02 | 一种可常温保存的生物羊膜的制备方法 |
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| CNB031508383A CN1241474C (zh) | 2003-09-03 | 2003-09-03 | 生物羊膜保存处理方法 |
| CN03150838.3 | 2003-09-03 | ||
| CN200410075361.9 | 2004-08-27 | ||
| CN 200410075361 CN1644047A (zh) | 2004-08-27 | 2004-08-27 | 一种可常温保存的生物羊膜的制备方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3139936A4 (en) * | 2014-05-07 | 2017-11-15 | Osiris Therapeutics, Inc. | Immunocompatible amniotic membrane products |
| US9956248B2 (en) | 2010-02-18 | 2018-05-01 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
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|---|---|---|---|---|
| WO1998037903A1 (en) * | 1997-02-28 | 1998-09-03 | Tseng Scheffer C G | Grafts made from amniotic membrane; methods of separating, preserving, and using such grafts in surgeries |
| WO2001008716A1 (en) * | 1999-08-02 | 2001-02-08 | Ann Pinfold | Method of preservation of amnion and uses thereof |
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2004
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1998037903A1 (en) * | 1997-02-28 | 1998-09-03 | Tseng Scheffer C G | Grafts made from amniotic membrane; methods of separating, preserving, and using such grafts in surgeries |
| WO2001008716A1 (en) * | 1999-08-02 | 2001-02-08 | Ann Pinfold | Method of preservation of amnion and uses thereof |
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|---|---|---|---|---|
| US10646519B2 (en) | 2010-02-18 | 2020-05-12 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
| US9956248B2 (en) | 2010-02-18 | 2018-05-01 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
| US10258650B2 (en) | 2010-02-18 | 2019-04-16 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible chorionic membrane products |
| US10265344B2 (en) | 2010-02-18 | 2019-04-23 | Osiris Therapeutics, Inc. | Immunocompatible chorionic membrane products |
| US10272116B2 (en) | 2010-02-18 | 2019-04-30 | Osiris Therapeutics, Inc. | Immunocompatible amniotic membrane products |
| US10576104B2 (en) | 2010-02-18 | 2020-03-03 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible amniotic membrane products |
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| US11590172B2 (en) | 2010-02-18 | 2023-02-28 | Osiris Therapeutics, Inc. | Immunocompatible chorionic membrane products |
| US11590173B2 (en) | 2010-02-18 | 2023-02-28 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
| US11638725B2 (en) | 2010-02-18 | 2023-05-02 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible chorionic membrane products |
| US11986498B2 (en) | 2010-02-18 | 2024-05-21 | Osiris Therapeutics, Inc. | Therapeutic products comprising vitalized placental dispersions |
| EP3139936A4 (en) * | 2014-05-07 | 2017-11-15 | Osiris Therapeutics, Inc. | Immunocompatible amniotic membrane products |
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