CN101879333A - Preparation method of acellular organism stent - Google Patents
Preparation method of acellular organism stent Download PDFInfo
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- CN101879333A CN101879333A CN2010102014405A CN201010201440A CN101879333A CN 101879333 A CN101879333 A CN 101879333A CN 2010102014405 A CN2010102014405 A CN 2010102014405A CN 201010201440 A CN201010201440 A CN 201010201440A CN 101879333 A CN101879333 A CN 101879333A
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- blood vessel
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Abstract
The invention discloses a preparation method of an acellular organism stent, belonging to the technical field of biology. The preparation method comprises a preparation method of an acellular organism stent by only using a decontaminating agent and a preparation method of an acellular organism stent by combining the decontaminating agent and proteinase; no cell is reminded in the blood vessel tubular tissue which is subject to acellular processing; the blood vessel tubular tissue which is subject to acellular processing and is fixed by Genipin is always in a tubular shape, and a large amount of extracellular matrix component is remained in the tissue, i.e. collagen and elastic fibers which perform an important role on maintaining the elasticity and strength of the blood vessel and the conglutination, migration, proliferation and differentiation of cells planted thereon. The invention provides a novel method and source for building tissue engineered blood vessels having physiology medical function or the abundant organism stent of other tubular pipes.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the preparation method of acellular organism stent.
Background technology
From the body organ transplantation is treatment organ defect the most effective medical procedure, but is insoluble practical problem in the organ transplantation therapeutic process from body organ origin deficiency always.Seed cell is planted on the biodegradable timbering material, external structure has the engineered organ of certain vital activity and corresponding physiological medical science function, as substitute from the body organ, transplant the histoorgan of repairing pathological changes or disappearance, faced in the time of can fundamentally solving the organ transplantation treatment from the insufficient contradiction of body organ origin.
Support is one of three elements (cell, material and biotic factor) that make up in engineered organ.The component of timbering material, structure, interface, aperture, intensity and elasticity, the biomechanical characterization of the engineered organ of decision prebuild also plays important regulatory role to adhesion, migration, increment and the differentiation of planting cell thereon simultaneously.Biomaterial is because of having the favorable tissue compatibility and implant safety, and the configuration that is complementary with the prebuild histoorgan, and is used as timbering material widely, and is used for the structure of engineered organ.
Collagen protein, elastic fibers, hyaluronic acid and proteoglycan etc. are the extracellular matrix composition, it also is the key component of biologic bracket material, determining the adhesion increment and the differentiation of the biomechanical characterization and the plantation seed cell thereon of biologic bracket material, but the cell component that biologic bracket material Central Plains pre-exists, may bring out the host after the transplanting, produce immunological rejection, need therefrom be removed.
Go the cytoskeleton material because of the main component that keeps extracellular matrix and with the similar configuration of natural tissues, become one of preferred material that makes up engineered organ.The engineered blood vessel of cytoskeleton material construction is removed in utilization, as from the substitute of body blood vessel, be faced with in the time of can fundamentally solving the blood vessel transplantation treatment from the body blood vessel insufficient contradiction of originating.The method of acellular organism support is different, and effect also is not quite similar.Invent a kind of simple and easy to do, go cell effect reliable, and the cell blood vessel biological support that goes that can keep the extracellular matrix Main Ingredients and Appearance is to make up at present the technical bottleneck that engineered blood vessel at first solves.
Summary of the invention
The object of the present invention is to provide a kind of preparation method that is used to make up the acellular organism stent of engineered blood vessel.
The present invention includes independent preparation method of acellular organism stent of detergent and detergent-protease associating preparation method of acellular organism stent, wherein:
1. the independent preparation method of acellular organism stent of detergent comprises the following steps:
1. under the aseptic condition, get fresh or frozen blood vessel tubular tissue (umbilical blood vessels, neck blood vessel, ventral aorta or great saphenous vein).
2. with containing antibiotic normal saline, or the inside and outside blood of phosphate buffer flush away tube chamber.
3. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to tube chamber is that 1% sodium lauryl sulphate and concentration are 0.02% Hydrazoic acid,sodium salt.
4. the ligation blood vessel gets the other end, and above-mentioned blood vessel tubular tissue is placed in the beaker that contains 1% sodium lauryl sulphate+0.02% Hydrazoic acid,sodium salt.
5. put into stirring rod in beaker, beaker is placed on the magnetic stirring apparatus, start magnetic stirring apparatus, adjusting rotating speed is 30 rev/mins, rotates 12 hours continuously under the room temperature.
6. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, sodium lauryl sulphate that flush away is residual and sodium azide solution.
7. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to intravascular stent is 0.3% genipin (genipin) solution, the ligation support other end places intravascular stent in the above-mentioned genipin solution, fixes 4 hours under 4 ℃.
8. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, the genipin that flush away is residual.
9. after histology is qualified, lyophilization behind the oxirane disinfection, is stored in-20 ℃ the refrigerator standby.
2. detergent-protease associating preparation method of acellular organism stent comprises the following steps:
1. under the aseptic condition, get fresh or frozen blood vessel tubular tissue (umbilical blood vessels, neck blood vessel, ventral aorta or great saphenous vein).
2. with containing the inside and outside blood of antibiotic normal saline or phosphate buffer flush away tube chamber.
3. an end of ligation blood vessel, inject the ethylenediaminetetraacetic acid that concentration with the phosphate buffer preparation is 0.25% trypsin and 0.02% or the Digestive system of its sodium salt with syringe to tube chamber, the other end of ligation blood vessel, aseptic down, be placed under the room temperature digestion 30 minutes.
4. remove ligature, wash away Digestive system residual in the blood vessel with phosphate buffer or normal saline.
5. the two ends with blood vessel are connected with peristaltic pump, in the connection tube of peristaltic pump in advance implantation concentration be that 1% sodium lauryl sulphate and concentration are 0.02% Hydrazoic acid,sodium salt.
6. start peristaltic pump, adjusting rotating speed is 25-30 rev/min, continous perfusion 3 hours.
7. take out intravascular stent, with phosphate buffer or the whole tube chamber of normal saline flushing, sodium lauryl sulphate that flush away is residual and sodium azide solution.
8. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to support is 0.3% genipin (genipin) solution, the ligation support other end will prop up and be placed in the above-mentioned genipin solution, fix 4 hours under 4 ℃.
9. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, the genipin that flush away is residual.
10. after histology is qualified, lyophilization behind the oxirane disinfection, is stored in-20 ℃ the refrigerator standby.
The present invention's detergent, or detergent and protease associating, from vascular tissues such as umbilical blood vessels, neck blood vessel, ventral aorta, great saphenous vein, remove the cell composition, thereby obtained to have kept the acellular organism stent of matter composition (collagen protein and elastic fibers) between the extracellular is main.
The present invention provides a kind of new method and source for making up engineered blood vessel or the competent organism stent of other pipe with physiological medical science function.As seen HE dyeing contain a large amount of cell residue (see figure 1)s without the umbilical blood vessels that goes the cell processing, and respectively through not seeing cell and examine residual in the above-mentioned umbilical blood vessels that goes the cell processing.The umbilical blood vessels that goes cellization after genipin is fixing presents tubular structure all the time simultaneously, and remains with a large amount of extracellular matrix compositions in the tissue: collagen protein and elastic fibers (seeing Fig. 2 and Fig. 3).These collagen protein and elastic fibers are to keeping elasticity of blood vessels and intensity, and adhesion, migration, increment, the differentiation of plantation cell thereon all will play a significant role.
Description of drawings
Fig. 1 is through the painted umbilical blood vessels photo of HE
Fig. 2 is through the painted acellular organism stent photo of Masson
Fig. 3 is through the painted acellular organism stent photo of orcein
The specific embodiment
1. the independent preparation method of acellular organism stent of detergent comprises the following steps:
1. under the aseptic condition, get fresh or frozen umbilical blood vessels, neck blood vessel, ventral aorta or great saphenous vein.
2. use the normal saline that contains penicillin, streptomycin or amphotericin, or the inside and outside blood of phosphate buffer flush away tube chamber.
3. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to tube chamber is that 1% sodium lauryl sulphate and concentration are 0.02% Hydrazoic acid,sodium salt.
4. the ligation blood vessel gets the other end, and above-mentioned blood vessel tubular tissue is placed in the beaker that contains 1% sodium lauryl sulphate+0.02% Hydrazoic acid,sodium salt.
5. put into stirring rod in beaker, beaker is placed on the magnetic stirring apparatus, start magnetic stirring apparatus, adjusting rotating speed is 30 rev/mins, rotates 12 hours continuously under the room temperature.
6. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, sodium lauryl sulphate that flush away is residual and sodium azide solution.
7. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to intravascular stent is 0.3% genipin solution, the ligation support other end places intravascular stent in the above-mentioned genipin solution, fixes 4 hours under 4 ℃.
8. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, the genipin that flush away is residual.
9. after histology is qualified, lyophilization behind the oxirane disinfection, is stored in-20 ℃ the refrigerator standby.
2. detergent-protease associating preparation method of acellular organism stent comprises the following steps:
1. under the aseptic condition, get fresh or frozen umbilical blood vessels, neck blood vessel, ventral aorta or great saphenous vein.
2. with normal saline that contains penicillin, streptomycin or amphotericin or the inside and outside blood of phosphate buffer flush away tube chamber.
3. an end of ligation blood vessel, inject the ethylenediaminetetraacetic acid that concentration with the phosphate buffer preparation is 0.25% trypsin and 0.02% or the Digestive system of its sodium salt with syringe to tube chamber, the other end of ligation blood vessel, aseptic down, be placed under the room temperature digestion 30 minutes.
4. remove ligature, wash away Digestive system residual in the blood vessel with phosphate buffer or normal saline.
5. the two ends with blood vessel are connected with peristaltic pump, in the connection tube of peristaltic pump in advance implantation concentration be that 1% sodium lauryl sulphate and concentration are 0.02% Hydrazoic acid,sodium salt.
6. start peristaltic pump, adjusting rotating speed is 25-30 rev/min, continous perfusion 3 hours.
7. take out intravascular stent, with phosphate buffer or the whole tube chamber of normal saline flushing, sodium lauryl sulphate that flush away is residual and sodium azide solution.
8. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to support is 0.3% genipin solution, the ligation support other end will prop up and be placed in the above-mentioned genipin solution, fix 4 hours under 4 ℃.
9. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, the genipin that flush away is residual.
10. after histology is qualified, lyophilization behind the oxirane disinfection, is stored in-20 ℃ the refrigerator standby.
In the painted umbilical blood vessels of HE, there is a large amount of nucleus residual as seen from Figure 1.
In the painted acellular organism stent of Masson, leave a large amount of collagen protein as seen from Figure 2.
In the painted acellular organism stent of orcein, leave a large amount of elastic fiberss as seen from Figure 3.
Claims (2)
1. preparation method of acellular organism stent is characterized in that comprising independent preparation method of acellular organism stent of detergent and detergent-protease associating preparation method of acellular organism stent, wherein:
(1) the independent preparation method of acellular organism stent of detergent comprises the following steps:
1. under the aseptic condition, get fresh or frozen blood vessel tubular tissue.
2. with containing antibiotic normal saline, or the inside and outside blood of phosphate buffer flush away tube chamber.
3. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to tube chamber is that 1% sodium lauryl sulphate and concentration are 0.02% Hydrazoic acid,sodium salt.
4. the ligation blood vessel gets the other end, and above-mentioned blood vessel tubular tissue is placed in the beaker that contains 1% sodium lauryl sulphate+0.02% Hydrazoic acid,sodium salt.
5. put into stirring rod in beaker, beaker is placed on the magnetic stirring apparatus, start magnetic stirring apparatus, adjusting rotating speed is 30 rev/mins, rotates 12 hours continuously under the room temperature.
6. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, sodium lauryl sulphate that flush away is residual and sodium azide solution.
7. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to intravascular stent is 0.3% genipin solution, the ligation support other end places intravascular stent in the above-mentioned genipin solution, fixes 4 hours under 4 ℃.
8. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, the genipin that flush away is residual.
9. after histology is qualified, lyophilization behind the oxirane disinfection, is stored in-20 ℃ the refrigerator standby.
(2) detergent-protease associating preparation method of acellular organism stent comprises the following steps:
1. under the aseptic condition, get fresh or frozen blood vessel tubular tissue.
2. with containing the inside and outside blood of antibiotic normal saline or phosphate buffer flush away tube chamber.
3. an end of ligation blood vessel, inject the ethylenediaminetetraacetic acid that concentration with the phosphate buffer preparation is 0.25% trypsin and 0.02% or the Digestive system of its sodium salt with syringe to tube chamber, the other end of ligation blood vessel, aseptic down, be placed under the room temperature digestion 30 minutes.
4. remove ligature, wash away Digestive system residual in the blood vessel with phosphate buffer or normal saline.
5. the two ends with blood vessel are connected with peristaltic pump, in the connection tube of peristaltic pump in advance implantation concentration be that 1% sodium lauryl sulphate and concentration are 0.02% Hydrazoic acid,sodium salt.
6. start peristaltic pump, adjusting rotating speed is 25-30 rev/min, continous perfusion 3 hours.
7. take out intravascular stent, with phosphate buffer or the whole tube chamber of normal saline flushing, sodium lauryl sulphate that flush away is residual and sodium azide solution.
8. an end of ligation blood vessel, injecting concentration with the phosphate buffer preparation with syringe to support is 0.3% genipin solution, the ligation support other end will prop up and be placed in the above-mentioned genipin solution, fix 4 hours under 4 ℃.
9. take out intravascular stent, remove ligature, with phosphate buffer or the whole tube chamber of normal saline flushing, the genipin that flush away is residual.
10. after histology is qualified, lyophilization behind the oxirane disinfection, is stored in-20 ℃ the refrigerator standby.
2. by the described preparation method of acellular organism stent of claim 1, it is characterized in that described blood vessel tubular tissue is umbilical blood vessels, neck blood vessel, ventral aorta or great saphenous vein.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102014405A CN101879333A (en) | 2010-06-17 | 2010-06-17 | Preparation method of acellular organism stent |
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| CN2010102014405A CN101879333A (en) | 2010-06-17 | 2010-06-17 | Preparation method of acellular organism stent |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425905A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Blood vessel material and preparation method and application thereof |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903382A (en) * | 2005-07-29 | 2007-01-31 | 芮钢 | Method for preparing heterogenic bone with cell-removing matrix |
| CN101085376A (en) * | 2007-06-07 | 2007-12-12 | 四川省肿瘤医院 | Biological tissue material for artificial esophagus and preparation method thereof |
| CN101185775A (en) * | 2007-12-27 | 2008-05-28 | 南京市儿童医院 | Preparation method for pig blood vessel acellular bracket by chemical and physical combination |
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2010
- 2010-06-17 CN CN2010102014405A patent/CN101879333A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1903382A (en) * | 2005-07-29 | 2007-01-31 | 芮钢 | Method for preparing heterogenic bone with cell-removing matrix |
| CN101085376A (en) * | 2007-06-07 | 2007-12-12 | 四川省肿瘤医院 | Biological tissue material for artificial esophagus and preparation method thereof |
| CN101185775A (en) * | 2007-12-27 | 2008-05-28 | 南京市儿童医院 | Preparation method for pig blood vessel acellular bracket by chemical and physical combination |
Non-Patent Citations (1)
| Title |
|---|
| 吴春根: "人脐动脉脱细胞支架的制备及其生物相容性", 《中国组织工程研究与临床康复》, vol. 11, no. 26, 1 July 2007 (2007-07-01), pages 5083 - 2 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11994343B2 (en) | 2019-03-14 | 2024-05-28 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| CN113425905A (en) * | 2020-03-23 | 2021-09-24 | 成都中科奥格生物科技有限公司 | Blood vessel material and preparation method and application thereof |
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Application publication date: 20101110 |