CN105647849A - Cell complex culture method based on SIS (small intestinal submucosa) - Google Patents
Cell complex culture method based on SIS (small intestinal submucosa) Download PDFInfo
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- CN105647849A CN105647849A CN201610104099.9A CN201610104099A CN105647849A CN 105647849 A CN105647849 A CN 105647849A CN 201610104099 A CN201610104099 A CN 201610104099A CN 105647849 A CN105647849 A CN 105647849A
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Abstract
The invention discloses a cell complex culture method based on SIS (small intestinal submucosa). The method comprises steps as follows: the SIS is prepared with an abraham method, HUVECs (human umbilical vein endothelial cells) are cultured with a primary culture method, the SIS is cut into small pieces in the equal size, the HUVECs are inoculated to a cell culture plate containing the SIS, and the cell culture plate is put in a 5% CO2 cell incubator with the temperature of 37 DEG C for culture. According to the cell complex culture method based on the SIS, materials are easy to obtain, the HUVECs can be proliferated in quantity on the SIS and are closely arranged, and a basis is provided for research of artificial blood vessel materials with higher tissue compatibility.
Description
Technical field
The present invention relates to a kind of cell compound culture method based on SIS, belong to technical field of cell biology.
Background technology
The adoptable main material of current artificial blood vessel has: terylene, politef, polypropylene, silicone rubber etc., but the histocompatibility of existing artificial blood vessel material is poor, and easily cause infection, inflammation, therefore the better artificial blood vessel material of histocompatibility is the direction that people study and find.
SIS (submucous layer of small intestine) is a kind of membrane material with induction/guide tissue regeneration function, it is used for reconstruction bladder, urethra, esophagus, bone and cartilage and has good histocompatibility, the method that the existing SIS of employing builds, as substrate, the tissue engineering material being used for esophagus, bone and cartilage at present; HUVECs (Human umbilical vein endothelial cells) is a kind of vascular endothelial cell taking from neonatal umbilical cord, it it is the common used material of vascular endothelial cell experiment in vitro, but it belongs to the cell of low growth, when cultivating in vitro, survival and appreciation rate are relatively low, carry out the compatibility of SIS and HUVECs, and HUVECs propagation on SIS, characteristic research, the tissue engineering material carrying out artificial blood vessel is studied significant.
Summary of the invention
The technical problem to be solved is the defect overcoming prior art, a kind of cell compound culture method based on SIS is provided, HUVECs a large amount of propagation close-packed arrays on SIS can be made, and provide basis for the artificial blood vessel material that research histocompatibility is higher.
For solving above-mentioned technical problem, the present invention provides a kind of cell compound culture method based on SIS, comprises the following steps:
The preparation of A, SIS: adopt abraham method to prepare submucous layer of small intestine;
The original cuiture of B, HUVECs: take the fresh umbilical cord of neonate, after phosphate buffer rinses, by syringe lavation umbilical vein, again umbilical cord one end folder is closed, inject digestive enzyme solution from other end syringe to described umbilical vein, draw Digestive system with syringe after digestion and collect, in described Digestive system, add the culture fluid containing serum terminate digestion, it is inoculated in culture bottle after piping and druming several times, puts 37 DEG C of 5%CO2Cell culture incubator is cultivated;
C, HUVECs and SIS compound criteria: take the SIS obtained in step A and make the SIS fritter that size is impartial, put in Tissue Culture Plate after ultra violet lamp sterilization, after soaking 1-3 days with cell culture fluid, add the Ox blood serum of 5-20%, put in 37 DEG C of cell culture incubators and hatch 12-16h;Take the HUVECs cultivated in step B, after adding digestive enzyme solution digestion, collect Digestive system and add the culture fluid termination digestion containing serum, by Digestive system collected after centrifugation HUVECs cell, and make cell suspension with the cell culture fluid containing 10-20% Ox blood serum, plant to the Tissue Culture Plate being placed with SIS after cell suspension is diluted, put into 37 DEG C of 5%CO2Cultivate in cell culture incubator, within every 1-4 days, change liquid once.
The described method adopting abraham method to prepare submucous layer of small intestine is: take pig small intestine, placenta percreta and the muscle layer of small intestinal is removed after cleaning, small intestinal after processing is soaked 12-18h in the solution containing 100mmol/L ethylenediaminetetraacetic acid and 10mmol/LNaOH, with deionized water, the small intestinal after immersion is cleaned up, again with containing 1mmol/LHCl and 1mmol/LNaCl solution soaks 5��10h, in the phosphate buffer containing 1mmol/LNaCl, 12-18h is soaked with deionized water after cleaning, in phosphate buffer, 1-4h is soaked with deionized water after cleaning, use deionized water rinsing 2h again, prepared SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 6-10h, 1-4h is cleaned with the phosphate buffer containing 0.05% Hydrazoic acid,sodium salt, gamma-rays illumination-based disinfection is used after lyophilization.
Described digestive enzyme solution is the trypsin solution of 0.25%.
Collagen type-I it is covered with in advance in described culture bottle in step B.
Tissue Culture Plate described in step C is 6 orifice plates, and the inoculum density of described cell suspension is 2��5 �� 105/ hole.
SIS fritter described in step C is sized to 2.5cm �� 2.5cm.
What the present invention reached has the beneficial effects that:
1.SIS mainly has I, III collagen type to constitute, and has good cell compatibility, can promote to promote the adhesion of HUVECs, growth and differentiation, and therefore HUVECs can be grown to the close cellular layer of arrangement;
2.SIS has no antigen, is not result in immunoreation for transplanting, and HUVECs derives from fetal cord, and material is easier to obtain, and therefore using SIS and HUVECs as the tissue engineering material of artificial blood vessel, has better histocompatibility.
Therefore, a kind of cell compound culture method based on SIS provided by the present invention, material is easier to obtain, it is possible to makes HUVECs a large amount of propagation close-packed arrays on SIS, and provides basis for the artificial blood vessel material that research histocompatibility is higher.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described. Following example are only for clearly illustrating technical scheme, and can not limit the scope of the invention with this.
Embodiment one:
A kind of cell compound culture method based on SIS, comprises the following steps:
The preparation of A, SIS: adopt abraham method to prepare submucous layer of small intestine;
The original cuiture of B, HUVECs: take the fresh umbilical cord of neonate, after phosphate buffer rinses, by syringe lavation umbilical vein, again umbilical cord one end folder is closed, inject digestive enzyme solution from other end syringe to described umbilical vein, draw Digestive system with syringe after digestion and collect, in described Digestive system, add the culture fluid containing serum terminate digestion, it is inoculated in culture bottle after piping and druming several times, puts 37 DEG C of 5%CO2Cell culture incubator is cultivated;
C, HUVECs and SIS compound criteria: take the SIS obtained in step A and make the SIS fritter that size is impartial, put in Tissue Culture Plate after ultra violet lamp sterilization, after soaking 1 day with cell culture fluid, add the Ox blood serum of 5%, put in 37 DEG C of cell culture incubators and hatch 16h;Take the HUVECs cultivated in step B, after adding digestive enzyme solution digestion, collect Digestive system and add the culture fluid termination digestion containing serum, by Digestive system collected after centrifugation HUVECs cell, and make cell suspension with the cell culture fluid containing 10% Ox blood serum, plant to the Tissue Culture Plate being placed with SIS after cell suspension is diluted, put into 37 DEG C of 5%CO2Cultivate in cell culture incubator, within every 1 day, change liquid once.
The described method adopting abraham method to prepare submucous layer of small intestine is: take pig small intestine, placenta percreta and the muscle layer of small intestinal is removed after cleaning, small intestinal after processing is soaked 12h in the solution containing 100mmol/L ethylenediaminetetraacetic acid and 10mmol/LNaOH, with deionized water, the small intestinal after immersion is cleaned up, again with containing 1mmol/LHCl and 1mmol/LNaCl solution soaks 5h, in the phosphate buffer containing 1mmol/LNaCl, 12h is soaked with deionized water after cleaning, in phosphate buffer, 1h is soaked with deionized water after cleaning, use deionized water rinsing 2h again, prepared SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 6h, 1h is cleaned with the phosphate buffer containing 0.05% Hydrazoic acid,sodium salt, gamma-rays illumination-based disinfection is used after lyophilization.
Described digestive enzyme solution is the trypsin solution of 0.25%.
Collagen type-I it is covered with in advance in described culture bottle in step B.
Tissue Culture Plate described in step C is 6 orifice plates, and the inoculum density of described cell suspension is 2 �� 105/ hole.
SIS fritter described in step C is sized to 2.5cm �� 2.5cm.
Embodiment two
A kind of cell compound culture method based on SIS, comprises the following steps:
The preparation of A, SIS: adopt abraham method to prepare submucous layer of small intestine;
The original cuiture of B, HUVECs: take the fresh umbilical cord of neonate, after phosphate buffer rinses, by syringe lavation umbilical vein, again umbilical cord one end folder is closed, inject digestive enzyme solution from other end syringe to described umbilical vein, draw Digestive system with syringe after digestion and collect, in described Digestive system, add the culture fluid containing serum terminate digestion, it is inoculated in culture bottle after piping and druming several times, puts 37 DEG C of 5%CO2Cell culture incubator is cultivated;
C, HUVECs and SIS compound criteria: take the SIS obtained in step A and make the SIS fritter that size is impartial, put in Tissue Culture Plate after ultra violet lamp sterilization, after soaking 3 days with cell culture fluid, add the Ox blood serum of 20%, put in 37 DEG C of cell culture incubators and hatch 12h; Take the HUVECs cultivated in step B, after adding digestive enzyme solution digestion, collect Digestive system and add the culture fluid termination digestion containing serum, by Digestive system collected after centrifugation HUVECs cell, and make cell suspension with the cell culture fluid containing 20% Ox blood serum, plant to the Tissue Culture Plate being placed with SIS after cell suspension is diluted, put into 37 DEG C of 5%CO2Cultivate in cell culture incubator, within every 4 days, change liquid once.
The described method adopting abraham method to prepare submucous layer of small intestine is: take pig small intestine, placenta percreta and the muscle layer of small intestinal is removed after cleaning, small intestinal after processing is soaked 18h in the solution containing 100mmol/L ethylenediaminetetraacetic acid and 10mmol/LNaOH, with deionized water, the small intestinal after immersion is cleaned up, again with containing 1mmol/LHCl and 1mmol/LNaCl solution soaks 10h, in the phosphate buffer containing 1mmol/LNaCl, 18h is soaked with deionized water after cleaning, in phosphate buffer, 4h is soaked with deionized water after cleaning, use deionized water rinsing 2h again, prepared SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 10h, 4h is cleaned with the phosphate buffer containing 0.05% Hydrazoic acid,sodium salt, gamma-rays illumination-based disinfection is used after lyophilization.
Described digestive enzyme solution is the trypsin solution of 0.25%.
Collagen type-I it is covered with in advance in described culture bottle in step B.
Tissue Culture Plate described in step C is 6 orifice plates, and the inoculum density of described cell suspension is 5 �� 105/ hole.
SIS fritter described in step C is sized to 2.5cm �� 2.5cm.
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the technology of the present invention principle; can also making some improvement and deformation, these improve and deformation also should be regarded as protection scope of the present invention.
Claims (6)
1. the cell compound culture method based on SIS, it is characterised in that: comprise the following steps:
The preparation of A, SIS: adopt abraham method to prepare submucous layer of small intestine;
The original cuiture of B, HUVECs: take the fresh umbilical cord of neonate, after phosphate buffer rinses, by syringe lavation umbilical vein, again umbilical cord one end folder is closed, inject digestive enzyme solution from other end syringe to described umbilical vein, draw Digestive system with syringe after digestion and collect, in described Digestive system, add the culture fluid containing serum terminate digestion, it is inoculated in culture bottle after piping and druming several times, puts 37 DEG C of 5%CO2Cell culture incubator is cultivated;
C, HUVECs and SIS compound criteria: take the SIS obtained in step A and make the SIS fritter that size is impartial, put in Tissue Culture Plate after ultra violet lamp sterilization, after soaking 1-3 days with cell culture fluid, add the Ox blood serum of 5-20%, put in 37 DEG C of cell culture incubators and hatch 12-16h; Take the HUVECs cultivated in step B, after adding digestive enzyme solution digestion, collect Digestive system and add the culture fluid termination digestion containing serum, by Digestive system collected after centrifugation HUVECs cell, and make cell suspension with the cell culture fluid containing 10-20% Ox blood serum, plant to the Tissue Culture Plate being placed with SIS after cell suspension is diluted, put into 37 DEG C of 5%CO2Cultivate in cell culture incubator, within every 1-4 days, change liquid once.
2. a kind of cell compound culture method based on SIS as claimed in claim 1, it is characterized in that: the described method adopting abraham method to prepare submucous layer of small intestine is: take pig small intestine, placenta percreta and the muscle layer of small intestinal is removed after cleaning, small intestinal after processing is soaked 12-18h in the solution containing 100mmol/L ethylenediaminetetraacetic acid and 10mmol/LNaOH, with deionized water, the small intestinal after immersion is cleaned up, again with containing 1mmol/LHCl and 1mmol/LNaCl solution soaks 5��10h, in the phosphate buffer containing 1mmol/LNaCl, 12-18h is soaked with deionized water after cleaning, in phosphate buffer, 1-4h is soaked with deionized water after cleaning, use deionized water rinsing 2h again, prepared SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 6-10h, 1-4h is cleaned with the phosphate buffer containing 0.05% Hydrazoic acid,sodium salt, gamma-rays illumination-based disinfection is used after lyophilization.
3. a kind of cell compound culture method based on SIS as claimed in claim 1, it is characterised in that: described digestive enzyme solution is the trypsin solution of 0.25%.
4. a kind of cell compound culture method based on SIS as claimed in claim 1, it is characterised in that: it is covered with Collagen type-I in advance in the described culture bottle in step B.
5. a kind of cell compound culture method based on SIS as claimed in claim 1, it is characterised in that: Tissue Culture Plate described in step C is 6 orifice plates, and the inoculum density of described cell suspension is 2��5 �� 105/ hole.
6. a kind of cell compound culture method based on SIS as claimed in claim 1, it is characterised in that: SIS fritter described in step C is sized to 2.5cm �� 2.5cm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610104099.9A CN105647849A (en) | 2016-02-25 | 2016-02-25 | Cell complex culture method based on SIS (small intestinal submucosa) |
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| CN201610104099.9A CN105647849A (en) | 2016-02-25 | 2016-02-25 | Cell complex culture method based on SIS (small intestinal submucosa) |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101623518A (en) * | 2009-06-30 | 2010-01-13 | 中国人民解放军第二军医大学 | Anti-infection bio-derived hernia and body wall repair material, preparation and application thereof |
| CN102743791A (en) * | 2011-08-01 | 2012-10-24 | 四川大学华西医院 | Tissue repair material and preparation method and application thereof |
| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
| CN102861360A (en) * | 2011-10-21 | 2013-01-09 | 四川大学华西医院 | Nerve repair promoting material and preparation method and application thereof |
-
2016
- 2016-02-25 CN CN201610104099.9A patent/CN105647849A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101623518A (en) * | 2009-06-30 | 2010-01-13 | 中国人民解放军第二军医大学 | Anti-infection bio-derived hernia and body wall repair material, preparation and application thereof |
| CN102743791A (en) * | 2011-08-01 | 2012-10-24 | 四川大学华西医院 | Tissue repair material and preparation method and application thereof |
| CN102861360A (en) * | 2011-10-21 | 2013-01-09 | 四川大学华西医院 | Nerve repair promoting material and preparation method and application thereof |
| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
Non-Patent Citations (2)
| Title |
|---|
| STEPHEN F. BADYLAK等: "Small intestinal submucosa: a substrate for in vitro cell growth", 《JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION》 * |
| 薛庆善主编: "《体外培养的原理与技术》", 31 December 2001, 科学出版社 * |
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Inventor after: Wan Zhen Inventor after: Fang Fan Inventor after: Yin Zongming Inventor after: Wang Yiwen Inventor before: Han Lijun |
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Effective date of registration: 20170419 Address after: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215000 BioBAY No. 218 building A4 room 110 Applicant after: Suzhou Bai Bai Biotechnology Co., Ltd. Address before: Kolding road high tech Zone of Suzhou City, Jiangsu Province, No. 78 215000 Applicant before: JIANGSU SIBAI MEDICAL TECHNIQUE CO., LTD. |
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