CN111001040A - Preparation method of extracellular matrix material of biological tissue - Google Patents
Preparation method of extracellular matrix material of biological tissue Download PDFInfo
- Publication number
- CN111001040A CN111001040A CN201911361707.4A CN201911361707A CN111001040A CN 111001040 A CN111001040 A CN 111001040A CN 201911361707 A CN201911361707 A CN 201911361707A CN 111001040 A CN111001040 A CN 111001040A
- Authority
- CN
- China
- Prior art keywords
- small intestine
- intestine submucosa
- tissue
- solution
- extracellular matrix
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
Abstract
The invention discloses a preparation method of a biological tissue extracellular matrix material, which comprises the following steps: primary treatment of raw materials: filtering and freezing small intestine of animal; removing raw materials: thawing the small intestine, and cutting it to leave small intestine submucosa tissue; virus inactivation treatment: putting the small intestine submucosa tissue into an inactivation solution in a stirring container for stirring to inactivate viruses; and (3) cell removal treatment: fixing the inactivated small intestine submucosa tissue on a negative conductive electrode sheet, and carrying out decellularization in an electrolyte by adding negative electricity by a double-electrode system; and then cleaning the decellularized small intestine submucosa tissue to obtain the small intestine submucosa extracellular matrix material. The preparation method disclosed by the invention is simple, the steps of decellularization by using the biological transferase and multiple times of cleaning in the traditional method are omitted, and particularly, the raw material is frozen and only a decellularization process is completed by one step. The process is simple and the effect is good. The test result shows that the morphology of the extracellular matrix is kept intact and has no cell residue.
Description
Technical Field
The invention belongs to the technical field of preparation of biological materials, and particularly relates to a preparation method of a biological tissue extracellular matrix material.
Background
Cells are very fragile organisms that must be supported by an extracellular matrix to survive. Therefore, extracellular matrix materials play an important role in tissue engineering.
The components of the extracellular matrix are often retained between species and are tolerated by xenoreceptors, i.e. extracellular matrices of animal origin (porcine, bovine or ovine) etc. are acceptable for human consumption. Currently, extensive research and applications have been made in tissue engineering and regenerative applications from different tissues, including skin, heart valves, blood vessels, nerves, tendons, ligaments, small intestine submucosa.
However, it is a technical difficulty how to remove all the cells and nuclear materials efficiently and safely while preserving the complex network structure of the extracellular matrix. At present, various methods for obtaining decellularized extracellular matrix from animal sources exist, but the steps are complicated, and the decellularization and the cleaning are carried out by biological protease and various chemicals.
Disclosure of Invention
Aiming at the problems, the invention provides a preparation method of a biological tissue extracellular matrix material, which can prepare an extracellular matrix by simple steps, and the morphology of the extracellular matrix is completely kept without cell residues.
The invention discloses a preparation method of a biological tissue extracellular matrix material, which comprises the following steps:
step (1), the primary treatment of raw materials comprises: draining small intestine of animal, and freezing;
and (2) removing the raw materials, comprising the following steps: thawing the small intestine, and cutting it to leave small intestine submucosa tissue;
and (3) performing virus inactivation treatment, which comprises the following steps: putting the small intestine submucosa tissue into an inactivation solution in a stirring container, and stirring to inactivate viruses, wherein the inactivation solution is a mixed solution of peroxide and ethanol;
and (4) performing cell removal treatment, which comprises the following steps: fixing the inactivated small intestine submucosa tissue on a negative conductive electrode sheet, and carrying out decellularization in an electrolyte by adding negative electricity by a double-electrode system; and then cleaning the decellularized small intestine submucosa tissue to obtain the small intestine submucosa extracellular matrix material.
Preferably, step (5), the fixing and forming, comprises: flatly paving one or more layers of small intestine submucosa extracellular matrix materials on a mould by using the mould, and fixing and shaping by using an upper bottom plate and a lower bottom plate; and (6) drying treatment, comprising: and (3) putting the small intestine submucosa extracellular matrix material fixed between the two bottom plates into a sterilized ventilation oven for drying.
Preferably, step (7) is included after step (6), and the sterilization resolution includes sterilization with ethylene oxide; the analysis is then performed in an analysis chamber.
Preferably, in step (1), the freezing is carried out at-20 ℃ to-80 ℃. In the step (3), the volume ratio of the inactivation solution to the small intestine submucosa tissue is 20: 1-30: 1; and/or the concentration of the peroxide is 0.1-10% (v/v), and the concentration of the ethanol is 0.1-8% (v/v).
Preferably, in step (3), the washing of the inactivated small intestine submucosa tissue comprises: firstly, cleaning solution is adopted in an ultrasonic cleaning machine, the power of ultrasonic wave is less than 3500W, the cleaning solution is PBS solution with the PH value of 7.2-7.4, the temperature of the PBS solution is 20-37 ℃, and the volume ratio of the PBS solution to the small intestine submucosa tissue is 20: 1-50: 1; and then ultrasonically cleaning the small intestine submucosa tissue cleaned by PBS with deionized water, wherein the temperature of the deionized water is 20-37 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue is 20: 1-50: 1.
Preferably, in the step (4), the range of negative electricity is-0.5 to-10V, the power-up time is 5 to 30 minutes, and the electrolyte is PBS solution; and/or fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet by using a conductive clip.
Preferably, in step (4), the small intestine submucosa tissue after decellularization is washed with PBS.
Preferably, in the step (5), the mold is a mesh plate with holes; and/or, in the step (6), the temperature of the ventilation oven is between 10 and 40 ℃, and the drying time is 2 to 8 hours.
Preferably, in step (7), the sterilization conditions are: firstly, preserving heat in a heat preservation box with the temperature of 10-40 ℃, and then introducing ethylene oxide with the concentration of 200-900mg/L for sterilization; and/or, carrying out the analysis in an analysis chamber, and controlling the temperature to be between 10 and 19 ℃.
Compared with the prior art, the embodiment of the invention has the following advantages:
the preparation method is simple, eliminates the steps of decellularization by using biological transferase and multiple times of cleaning in the traditional method, particularly carries out freezing treatment on raw materials, and only needs one step to complete the decellularization process. The process is simple and the effect is good. The test result shows that the morphology of the extracellular matrix is kept intact and has no cell residue.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is the small intestinal submucosa of control swine or bovine or ovine treated with PBS. The dots represent cells remaining in the tissue.
FIG. 2 is a view of porcine or bovine or ovine small intestine submucosa inactivated and decellularized using peracetic acid in combination with an ethanol solution according to the present invention. The picture is without cell nucleus, and represents that extracellular matrix material has no cell residue.
Fig. 3 is a front sheet structure of small intestine submucosa tissue of pig, cow or sheep under a scanning electron microscope, and the picture can clearly see that the material surface has no cell residue and the tissue morphology is well preserved.
FIG. 4 shows a side-sectioned structure of a small intestine submucosa tissue of a pig, a cow, or a sheep under a scanning electron microscope, in which the multilayer structure is obtained by physical compression.
FIG. 5 is a flow chart of the method of the present invention for preparing extracellular matrix material for biological tissue.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.
FIG. 5 is a flow chart of the method of the present invention for preparing extracellular matrix material for biological tissue. As shown in the figure, the invention is a preparation method for an extracellular matrix material of a biological tissue, comprising the following steps:
step (1), the primary treatment of raw materials comprises: the small intestine of the animal was drained and frozen.
For example, fresh pig, cattle or sheep intestines within four hours of slaughter are washed clean with deionized water, drained using a filter device (e.g., a strainer), and frozen at-20 ℃ to-80 ℃, e.g., for 4 to 24 hours.
In the invention, the small intestine is frozen, the fiber structure of the material can be kept after freezing, the material cannot expand too much to lose elasticity due to subsequent treatment, and a better base material is provided for the subsequent treatment of a sample; but also can provide a natural sterile environment for material treatment, thereby not only improving the subsequent virus killing function, but also reducing the risk of bacterial infection.
And (2) removing the raw materials, comprising the following steps: the small intestine is thawed and cut to leave small intestine submucosa tissue.
For example, the small intestine of a pig or cow or sheep is thawed to a temperature at which it can be cut, and then it is divided into several small sections, for example 10cm, and the unwanted tissue is removed, and the whole operation can be carried out, for example, on an ice board or on a heat-insulating board with a temperature of 0 deg.f. After the sarcolemma and serosa are removed, the remaining tissue is the small intestine submucosa tissue.
And (3) performing virus inactivation treatment, which comprises the following steps: putting the small intestine submucosa tissue into an inactivation solution in a stirring container, and stirring to inactivate viruses, wherein the inactivation solution is a mixed solution of peroxide and ethanol;
for example, porcine or bovine or ovine small intestine submucosa tissue is placed in an inactivation solution for virus inactivation. The inactivation solution may be a mixture of peroxide and ethanol. For example, peroxides include peracetic acid, perpropionic acid, perbenzoic acid, hydrogen peroxide, and the like. The concentration of the peroxide is 0.1-10% (v/v), and the concentration of the ethanol is 0.1-8% (v/v). The volume ratio of the inactivation solution to the tissue is 20: 1-30: 1. The intestinal submucosa tissue is placed into the inactivation solution in a stirring vessel and stirred, for example, at a stirring speed of 100-500 rpm for 15-40 minutes.
Cleaning can then be carried out: the cleaning is performed with a cleaning liquid, and the cleaning may be performed with an ultrasonic cleaning machine, for example, and the power of the ultrasonic wave is 3500W or less. The cleaning solution is PBS solution with the pH value of 7.2-7.4, the temperature of the PBS solution is 20-37 ℃, and the volume ratio of the PBS solution to the small intestine submucosa tissue of the pig, the cattle or the sheep is 20: 1-50: 1. The washing is performed for 3 to 5 times, each time for 10 to 30 minutes. Then, the cleaned tissue was ultrasonically cleaned with deionized water. The temperature of the deionized water is 20-37 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue of the pig, the cattle or the sheep is 20: 1-50: 1. The washing is performed for 3 to 5 times, each time for 10 to 30 minutes.
And (4) performing cell removal treatment, which comprises the following steps: fixing the inactivated small intestine submucosa tissue on a negative conductive electrode sheet, and carrying out decellularization in an electrolyte by adding negative electricity by a double-electrode system; and then cleaning the decellularized small intestine submucosa tissue to obtain the small intestine submucosa extracellular matrix material.
A two-electrode system refers to a circuit system having positive and negative conductive electrode sheets. For example, the inactivated small intestine submucosa tissue is secured to a negative conductive electrode sheet using a conductive clip. Thus, in a two-electrode system, small intestine submucosa tissue can be negatively charged. The range of negative electricity is-0.5 to-10V, the power-up time is 5 to 30 minutes, and the electrolyte is PBS solution. The conductive electrode plate can be a platinum electrode, a silver electrode, a copper electrode or a stainless steel electrode. The decellularized tissue can be washed with PBS.
Then, the method can comprise a step (5) of fixing and forming, comprising: one or more layers of small intestine submucosa extracellular matrix materials are paved on the mould by utilizing the mould, and are fixed and molded by an upper bottom plate and a lower bottom plate.
For example, the mold is a perforated mesh plate. The periphery of the reticular plate is provided with a pressing frame, and the size of the pressing frame can be determined to be different according to different finished products. One or more layers of small intestine submucosa extracellular matrix materials can be laid on the mould according to requirements, and then fixed and formed by a bottom plate above and below the mould respectively.
And (6) drying treatment, comprising: and (3) putting the small intestine submucosa extracellular matrix material fixed between the two bottom plates into a sterilized ventilation oven for drying.
For example, the temperature of the ventilated oven is between 10 ℃ and 40 ℃ and the drying time is between 2 hours and 8 hours.
Thereafter, the method may further include step (7) of sterilization analysis, including sterilization with ethylene oxide, for example, under the following sterilization conditions: firstly, preserving heat in a heat preservation box with the temperature of 10-40 ℃, for example, preserving heat for 1-4 hours, and then introducing ethylene oxide with the concentration of 200-900mg/L for sterilization, for example, for 8-24 hours; then the analysis is carried out in an analysis chamber, the temperature can be controlled at 10-19 ℃ for 15-20 days.
Some examples are shown below to facilitate a better understanding of the method of the invention.
Example one
Firstly, the small intestine of a fresh pig, cow or sheep slaughtered within four hours is cleaned by deionized water, is placed in a filter screen for filtration, and is frozen at the temperature of minus 30 ℃ for 20 hours.
The small intestine of the pig or cow or sheep is then thawed to a temperature at which it can be cut, divided into 10cm pieces, the unwanted tissue is removed, and the entire operation is carried out on an ice plate. After the sarcolemma and serosa are removed, the remaining tissue is the small intestine submucosa tissue.
Then, the small intestine submucosa tissue is put into an inactivation solution in a stirring container to be stirred for virus inactivation, wherein the inactivation solution is a mixed solution of peroxide and ethanol, and the peroxide comprises peroxyacetic acid. The peroxide concentration was 1% (v/v) and the ethanol concentration was 0.5% (v/v). The volume ratio of inactivation solution to tissue was 22: 1. The small intestine submucosa tissue was placed in the inactivation solution in a stirred vessel and stirred at 200rpm for 15 minutes.
Then, the cleaning solution was used in an ultrasonic cleaning machine, and the power of ultrasonic waves was 3000W. The washing solution is PBS solution with pH value of 7.3, the temperature of the PBS solution is 23 ℃, and the volume ratio of the PBS solution to the porcine, bovine or ovine small intestine submucosa tissue is 25: 1. Washing was carried out 4 times for 15 minutes each. Then, the cleaned tissue was ultrasonically cleaned with deionized water. The temperature of the deionized water is 23 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue of the pig, the cattle or the sheep is 25: 1. Washing was carried out 4 times for 15 minutes each.
Then, fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet, and negatively charging in electrolyte by using a double-electrode system, wherein the negative charge is-0.8V, and the charging time is 10 minutes; and then washing the decellularized small intestine submucosa tissue by PBS to obtain the small intestine submucosa extracellular matrix material.
Then, one or more layers of the small intestine submucosa extracellular matrix material are laid on the mould by using the mould, and are fixed and molded by using an upper bottom plate and a lower bottom plate.
The small intestine submucosa extracellular matrix material secured between the two substrates was then dried in a sterile, vented oven. The temperature of the ventilation oven is 20 ℃, and the drying time is 3 hours.
Then, firstly, preserving the heat for 1.5 hours in a heat preservation box with the temperature of 20 ℃, and then introducing ethylene oxide with the concentration of 300mg/L for sterilization for 10 hours; then, the solution was analyzed in an analysis chamber, and the temperature was controlled at 12 ℃ for 15 days.
Example two
Firstly, the small intestine of a fresh pig, cow or sheep slaughtered within four hours is cleaned by deionized water, is placed in a filter screen for filtration, and is frozen at-40 ℃ for 16 hours.
The small intestine of the pig or cow or sheep is then thawed to a temperature at which it can be cut, divided into 15cm pieces, the unwanted tissue is removed, and the entire operation is carried out on an ice plate. After the sarcolemma and serosa are removed, the remaining tissue is the small intestine submucosa tissue.
Then, the small intestine submucosa tissue is put into an inactivation solution in a stirring container to be stirred for virus inactivation, wherein the inactivation solution is a mixed solution of peroxide and ethanol, and the peroxide comprises peroxypropionic acid. The peroxide concentration was 3% (v/v) and the ethanol concentration was 2% (v/v). The volume ratio of inactivation solution to tissue was 24: 1. The small intestine submucosa tissue was placed in the inactivation solution in a stirred vessel and stirred at 300rpm for 20 minutes.
Then, the cleaning was carried out in an ultrasonic cleaning machine using a cleaning liquid, and the power of ultrasonic waves was 2800W. The washing solution is PBS solution with pH value of 7.35, the temperature of the PBS solution is 26 ℃, and the volume ratio of the PBS solution to the porcine, bovine or ovine small intestine submucosa tissue is 35: 1. Washing was carried out 4 times for 15 minutes each. Then, the cleaned tissue was ultrasonically cleaned with deionized water. The temperature of the deionized water is 26 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue of the pig, the cattle or the sheep is 35: 1. Washing was carried out 4 times for 15 minutes each.
Then, fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet, and negatively charging in electrolyte by using a double-electrode system, wherein the negative charge is-2V, and the charging time is 15 minutes; and then washing the decellularized small intestine submucosa tissue by PBS to obtain the small intestine submucosa extracellular matrix material.
Then, one or more layers of the small intestine submucosa extracellular matrix material are laid on the mould by using the mould, and are fixed and molded by using an upper bottom plate and a lower bottom plate.
The small intestine submucosa extracellular matrix material secured between the two substrates was then dried in a sterile, vented oven. The temperature of the ventilation oven is 25 ℃, and the drying time is 4 hours.
Then, firstly, preserving the heat for 2 hours in a heat preservation box with the temperature of 25 ℃, and then introducing ethylene oxide with the concentration of 500mg/L for sterilization for 10 hours; then, the solution was analyzed in an analysis chamber, and the temperature was controlled at 14 ℃ for 18 days.
Example III
Firstly, the small intestine of a fresh pig, cow or sheep slaughtered within four hours is cleaned by deionized water, is placed in a filter screen for filtration, and is frozen at-50 ℃ for 10 hours.
The small intestine of the pig or cow or sheep is then thawed to a temperature at which it can be cut, and divided into 12cm pieces, the unwanted tissue is removed, and the entire operation is carried out on an ice plate. After the sarcolemma and serosa are removed, the remaining tissue is the small intestine submucosa tissue.
Then, the small intestine submucosa tissue is placed into an inactivation solution in a stirring container to be stirred for virus inactivation, wherein the inactivation solution is a mixed solution of peroxide and ethanol, and the peroxide comprises perbenzoic acid. The peroxide concentration was 5% (v/v) and the ethanol concentration was 4% (v/v). The volume ratio of inactivation solution to tissue was 26: 1. The small intestine submucosa tissue was placed in the inactivation solution in a stirred vessel and stirred at 350rpm for 30 minutes.
Then, the cleaning was carried out in an ultrasonic cleaning machine using a cleaning liquid at an ultrasonic power of 2500W. The washing solution is PBS solution with pH value of 7.37, the temperature of the PBS solution is 30 ℃, and the volume ratio of the PBS solution to the porcine, bovine or ovine small intestine submucosa tissue is 40: 1. Washing was carried out 4 times for 15 minutes each. Then, the cleaned tissue was ultrasonically cleaned with deionized water. The temperature of the deionized water is 30 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue of the pig, the cattle or the sheep is 40: 1. Washing was carried out 4 times for 15 minutes each.
Then, fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet, and negatively charging in electrolyte by using a double-electrode system, wherein the negative charge is-4V, and the charging time is 20 minutes; and then washing the decellularized small intestine submucosa tissue by PBS to obtain the small intestine submucosa extracellular matrix material.
Then, one or more layers of the small intestine submucosa extracellular matrix material are laid on the mould by using the mould, and are fixed and molded by using an upper bottom plate and a lower bottom plate.
The small intestine submucosa extracellular matrix material secured between the two substrates was then dried in a sterile, vented oven. The temperature of the ventilation oven is 30 ℃, and the drying time is 5 hours.
Then, firstly, preserving heat for 3 hours in a heat preservation box with the temperature of 30 ℃, and then introducing ethylene oxide with the concentration of 600mg/L for sterilization for 15 hours; then, the solution was analyzed in an analysis chamber, and the temperature was controlled at 15 ℃ for 18 days.
Example four
Firstly, cleaning fresh pig, cattle or sheep small intestine slaughtered within four hours by using deionized water, placing the cleaned pig, cattle or sheep small intestine in a filter screen for filtering, and placing the cleaned pig, cattle or sheep small intestine in a freezing environment at-60 ℃ for 10 hours.
The small intestine of the pig or cow or sheep is then thawed to a temperature at which it can be cut, and divided into 8cm pieces, the unwanted tissue is removed, and the entire operation is carried out on an ice plate. After the sarcolemma and serosa are removed, the remaining tissue is the small intestine submucosa tissue.
Then, the small intestine submucosa tissue is placed into an inactivation solution in a stirring container to be stirred for virus inactivation, wherein the inactivation solution is a mixed solution of peroxide and ethanol, and the peroxide comprises hydrogen peroxide. The peroxide concentration was 7% (v/v) and the ethanol concentration was 6% (v/v). The volume ratio of inactivation solution to tissue was 27: 1. The small intestine submucosa tissue was placed in the inactivation solution in a stirred vessel and stirred at 400rpm for 30 minutes.
Then, the cleaning was carried out in an ultrasonic cleaning machine using a cleaning liquid at an ultrasonic power of 2500W. The washing solution is PBS solution with pH value of 7.38, the temperature of the PBS solution is 32 ℃, and the volume ratio of the PBS solution to the porcine, bovine or ovine small intestine submucosa tissue is 42: 1. Washing was carried out 4 times for 15 minutes each. Then, the cleaned tissue was ultrasonically cleaned with deionized water. The temperature of the deionized water is 33 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue of the pig, the cattle or the sheep is 42: 1. Washing was carried out 4 times for 15 minutes each.
Then, fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet, and negatively charging in electrolyte by using a double-electrode system, wherein the negative charge is-6V, and the charging time is 22 minutes; and then washing the decellularized small intestine submucosa tissue by PBS to obtain the small intestine submucosa extracellular matrix material.
Then, one or more layers of the small intestine submucosa extracellular matrix material are laid on the mould by using the mould, and are fixed and molded by using an upper bottom plate and a lower bottom plate.
The small intestine submucosa extracellular matrix material secured between the two substrates was then dried in a sterile, vented oven. The temperature of the ventilation oven is 35 ℃, and the drying time is 5 hours.
Then, firstly, preserving the heat for 4 hours in a heat preservation box with the temperature of 30 ℃, and then introducing ethylene oxide with the concentration of 700mg/L for sterilization for 15 hours; then, the solution was analyzed in an analysis chamber, and the temperature was controlled at 15 ℃ for 18 days.
Example five
Firstly, cleaning fresh pig, cattle or sheep small intestine slaughtered within four hours by using deionized water, placing the cleaned pig, cattle or sheep small intestine in a filter screen for filtering, and placing the cleaned pig, cattle or sheep small intestine in a freezing environment at-70 ℃ for freezing for 6 hours.
The small intestine of the pig or cow or sheep is then thawed to a temperature at which it can be cut, and divided into 8cm pieces, the unwanted tissue is removed, and the entire operation is carried out on an ice plate. After the sarcolemma and serosa are removed, the remaining tissue is the small intestine submucosa tissue.
Then, the small intestine submucosa tissue is placed into an inactivation solution in a stirring container to be stirred for virus inactivation, wherein the inactivation solution is a mixed solution of peroxide and ethanol, and the peroxide comprises hydrogen peroxide. The peroxide concentration was 8% (v/v) and the ethanol concentration was 7% (v/v). The volume ratio of inactivation solution to tissue was 28: 1. The small intestine submucosa tissue was placed in the inactivation solution in a stirred vessel and stirred at 500rpm for 35 minutes.
Then, the cleaning was carried out in an ultrasonic cleaning machine using a cleaning liquid at an ultrasonic power of 2500W. The washing solution is PBS solution with pH value of 7.4, the temperature of the PBS solution is 35 ℃, and the volume ratio of the PBS solution to the porcine, bovine or ovine small intestine submucosa tissue is 45: 1. Washing was carried out 4 times for 15 minutes each. Then, the cleaned tissue was ultrasonically cleaned with deionized water. The temperature of the deionized water is 37 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue of the pig, the cattle or the sheep is 45: 1. Washing was carried out 4 times for 15 minutes each.
Then, fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet, and negatively charging the tissues in electrolyte by using a double-electrode system, wherein the size of the negative charge is-8V, and the charging time is 30 minutes; and then washing the decellularized small intestine submucosa tissue by PBS to obtain the small intestine submucosa extracellular matrix material.
Then, one or more layers of the small intestine submucosa extracellular matrix material are laid on the mould by using the mould, and are fixed and molded by using an upper bottom plate and a lower bottom plate.
The small intestine submucosa extracellular matrix material secured between the two substrates was then dried in a sterile, vented oven. The temperature of the ventilation oven is 40 ℃, and the drying time is 3 hours.
Then, firstly, preserving the heat for 3.5 hours in a heat preservation box with the temperature of 40 ℃, and then introducing ethylene oxide with the concentration of 800mg/L for sterilization for 10 hours; then, the solution was analyzed in an analysis chamber, and the temperature was controlled at 18 ℃ for 20 days.
The results of one detection process are shown below.
1. Porcine, bovine or ovine intestinal submucosa residual cells measured by fluorescence microscopy
The prepared small intestine submucosa of swine or bovine or ovine is fixed with 10% formalin. Tissues were cut into 1CM square pieces and stained with 4' 6 diamidino-2-phenylindole Dihydrochloride (DAPI) dye. DAPI stained nuclei and developed blue.
FIG. 1 is the small intestinal submucosa of control swine or bovine or ovine treated with PBS. The dots represent cells remaining in the tissue.
FIG. 2 is a view showing the submucosa of the small intestine of a pig or a cow or a sheep, which has been subjected to inactivation treatment with a solution of peracetic acid and ethanol and then decellularized by the method of the present invention. There were no nuclei in the pictures. Representing no cell residue of extracellular matrix material.
2. Photograph of small intestine submucosa tissue of pig, cattle or sheep under Scanning Electron Microscope (SEM)
Fig. 3 is a front sheet structure of small intestine submucosa tissue of pig, cow or sheep under a scanning electron microscope, and the picture can clearly see that the material surface has no cell residue and the tissue morphology is well preserved.
FIG. 4 shows a side-sectioned structure of a small intestine submucosa tissue of a pig, a cow, or a sheep under a scanning electron microscope, in which the multilayer structure is obtained by physical compression. The picture shows that the submucosal tissue structure is well preserved and the fibrous and porous structures are clearly visible.
Therefore, the preparation method disclosed by the invention is very simple, the steps of decellularization by using biological transferase and multiple times of cleaning in the traditional method are omitted, and particularly, the raw materials are subjected to freezing treatment, and the decellularization process is completed only by one step. The process is simple and the effect is good. The test result shows that the morphology of the extracellular matrix is kept intact and has no cell residue.
It is to be understood that the above-described embodiments of the present invention are merely illustrative of or explaining the principles of the invention and are not to be construed as limiting the invention. Therefore, any modification, equivalent replacement, improvement and the like made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. Further, it is intended that the appended claims cover all such variations and modifications as fall within the scope and boundaries of the appended claims or the equivalents of such scope and boundaries.
Claims (10)
1. A method for preparing an extracellular matrix material for biological tissue, comprising:
step (1), the primary treatment of raw materials comprises: draining small intestine of animal, and freezing;
and (2) removing the raw materials, comprising the following steps: thawing the small intestine, and cutting it to leave small intestine submucosa tissue;
and (3) performing virus inactivation treatment, which comprises the following steps:
putting the small intestine submucosa tissue into an inactivation solution in a stirring container, and stirring to inactivate viruses, wherein the inactivation solution is a mixed solution of peroxide and ethanol;
and (4) performing cell removal treatment, which comprises the following steps:
fixing the inactivated small intestine submucosa tissue on a negative conductive electrode sheet, and carrying out decellularization in an electrolyte by adding negative electricity by a double-electrode system; and then cleaning the decellularized small intestine submucosa tissue to obtain the small intestine submucosa extracellular matrix material.
2. The method of claim 1, further comprising:
and (5) fixing and forming, comprising: flatly paving one or more layers of small intestine submucosa extracellular matrix materials on a mould by using the mould, and fixing and shaping by using an upper bottom plate and a lower bottom plate;
and (6) drying treatment, comprising: and (3) putting the small intestine submucosa extracellular matrix material fixed between the two bottom plates into a sterilized ventilation oven for drying.
3. The method of claim 2, comprising step (7) after step (6), sterilizing the solution, including sterilizing with ethylene oxide; the analysis is then performed in an analysis chamber.
4. The method according to claim 1, wherein in step (1), the freezing is carried out at-20 ℃ to-80 ℃.
5. The method according to claim 1, wherein in the step (3), the volume ratio of the inactivation solution to the small intestine submucosa tissue is 20: 1-30: 1; and/or the concentration of the peroxide is 0.1-10% (v/v), and the concentration of the ethanol is 0.1-8% (v/v).
6. The method according to claim 1, wherein in step (3), the washing of the inactivated small intestine submucosa tissue comprises: firstly, cleaning solution is adopted in an ultrasonic cleaning machine, the power of ultrasonic wave is less than 3500W, the cleaning solution is PBS solution with the PH value of 7.2-7.4, the temperature of the PBS solution is 20-37 ℃, and the volume ratio of the PBS solution to the small intestine submucosa tissue is 20: 1-50: 1; and then ultrasonically cleaning the small intestine submucosa tissue cleaned by PBS with deionized water, wherein the temperature of the deionized water is 20-37 ℃, and the volume ratio of the deionized water to the small intestine submucosa tissue is 20: 1-50: 1.
7. The method of claim 1, wherein in the step (4), the range of negative electricity is-0.5 to-10V, the electrification time is 5 to 30 minutes, and the electrolyte is PBS solution; and/or fixing the inactivated small intestine submucosa tissues on a negative conductive electrode sheet by using a conductive clip.
8. The method of claim 1, wherein in step (4), the decellularized small intestine submucosa tissue is washed with PBS.
9. The method of claim 2, wherein in step (5), the mold is a perforated mesh plate; and/or, in the step (6), the temperature of the ventilation oven is between 10 and 40 ℃, and the drying time is 2 to 8 hours.
10. The method according to claim 3, wherein in step (7), the sterilization conditions are: firstly, preserving heat in a heat preservation box with the temperature of 10-40 ℃, and then introducing ethylene oxide with the concentration of 200-900mg/L for sterilization; and/or, carrying out the analysis in an analysis chamber, and controlling the temperature to be between 10 and 19 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911361707.4A CN111001040A (en) | 2019-12-24 | 2019-12-24 | Preparation method of extracellular matrix material of biological tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911361707.4A CN111001040A (en) | 2019-12-24 | 2019-12-24 | Preparation method of extracellular matrix material of biological tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111001040A true CN111001040A (en) | 2020-04-14 |
Family
ID=70118678
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911361707.4A Pending CN111001040A (en) | 2019-12-24 | 2019-12-24 | Preparation method of extracellular matrix material of biological tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111001040A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114949330A (en) * | 2022-06-16 | 2022-08-30 | 国科温州研究院(温州生物材料与工程研究所) | Acellular fish skin matrix and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090269317A1 (en) * | 2008-04-29 | 2009-10-29 | Davalos Rafael V | Irreversible electroporation to create tissue scaffolds |
| CN103721296A (en) * | 2013-12-11 | 2014-04-16 | 陕西瑞盛生物科技有限公司 | Tissue regeneration-guidable biomembrane and preparation method thereof |
| CN103751842A (en) * | 2008-07-30 | 2014-04-30 | 米辛瑟斯有限公司 | Tissue scaffolds derived from forestomach extracellular matrix |
| US20140147830A1 (en) * | 2012-11-29 | 2014-05-29 | Omar Barakat | Compositions and methods related to organ or tissue decellularization |
| CN105209478A (en) * | 2013-03-15 | 2015-12-30 | 拜欧米特生物制剂有限责任公司 | Methods for making cytokine compositions from tissues using non-centrifugal methods |
| CN107397972A (en) * | 2016-05-20 | 2017-11-28 | 北京纳通科技集团有限公司 | A kind of preparation method of animal skin acellular matrix dressing and the dressing of gained |
-
2019
- 2019-12-24 CN CN201911361707.4A patent/CN111001040A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090269317A1 (en) * | 2008-04-29 | 2009-10-29 | Davalos Rafael V | Irreversible electroporation to create tissue scaffolds |
| CN103751842A (en) * | 2008-07-30 | 2014-04-30 | 米辛瑟斯有限公司 | Tissue scaffolds derived from forestomach extracellular matrix |
| US20140147830A1 (en) * | 2012-11-29 | 2014-05-29 | Omar Barakat | Compositions and methods related to organ or tissue decellularization |
| CN105209478A (en) * | 2013-03-15 | 2015-12-30 | 拜欧米特生物制剂有限责任公司 | Methods for making cytokine compositions from tissues using non-centrifugal methods |
| CN103721296A (en) * | 2013-12-11 | 2014-04-16 | 陕西瑞盛生物科技有限公司 | Tissue regeneration-guidable biomembrane and preparation method thereof |
| CN107397972A (en) * | 2016-05-20 | 2017-11-28 | 北京纳通科技集团有限公司 | A kind of preparation method of animal skin acellular matrix dressing and the dressing of gained |
Non-Patent Citations (2)
| Title |
|---|
| 曾敏: "基于脱细胞技术的肝脏组织工程研究进展", 《肝脏》 * |
| 赵宇: "脱细胞技术及其在组织工程中的应用研究进展", 《中国修复重建外科杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114949330A (en) * | 2022-06-16 | 2022-08-30 | 国科温州研究院(温州生物材料与工程研究所) | Acellular fish skin matrix and preparation method thereof |
| CN114949330B (en) * | 2022-06-16 | 2024-02-20 | 国科温州研究院(温州生物材料与工程研究所) | Acellular fishskin matrix and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Choudhury et al. | Decellularization systems and devices: State-of-the-art | |
| AU2012262311B2 (en) | Adipose tissue matrices | |
| US20220160937A1 (en) | Rapid allograft treatment systems and methods | |
| CN111084904B (en) | Cell removing method of tissue engineering scaffold | |
| EP2279765A1 (en) | A method for preparing the decellularized matrix | |
| CN112618799B (en) | Fish skin acellular dermal matrix and preparation method and application thereof | |
| CN112755247B (en) | Acellular dermal matrix and preparation method thereof | |
| CN106474547B (en) | A kind of biologic bracket material and preparation method thereof of suitable cell growth | |
| CN111084900A (en) | Preparation method and application of acellular fish skin matrix | |
| WO2022188531A1 (en) | Preparation method and apparatus for tissue engineering biomaterial | |
| CN111001040A (en) | Preparation method of extracellular matrix material of biological tissue | |
| CN109675112B (en) | Preparation method of human-derived acellular dermal matrix | |
| EP2872191B1 (en) | Methods for improved treatment of adipose tissue | |
| CN114796615B (en) | Cartilage acellular matrix and preparation method thereof | |
| WO2021056964A1 (en) | Biological tissue matrix material, preparation method therefor, and application thereof | |
| JP4189484B2 (en) | Treatment method of biological tissue by microwave irradiation | |
| CN111330076B (en) | Cell removing device of tissue engineering bracket | |
| CN215460543U (en) | Tissue engineering biomaterial preparation facilities | |
| Kumaresan et al. | Development of Human Umbilical cord based scaffold for tissue engineering application | |
| RU2686309C1 (en) | Method for producing osteoplastic material from bone tissue | |
| CN114286694B (en) | Animal fat-derived extracellular matrix and animal fat-derived extracellular matrix preservation solution | |
| TWI712687B (en) | Acellular organs, and methods of producing the same | |
| CN111110917A (en) | Method for preparing acellular biological tissue material | |
| KR102586304B1 (en) | Acellular nerve graft and method of making the same | |
| CN110755687A (en) | Method for decellularizing mammalian cornea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200414 |