CN107596446B - Decellularization kit for maintaining tissue ultrastructure and nutrition microenvironment - Google Patents
Decellularization kit for maintaining tissue ultrastructure and nutrition microenvironment Download PDFInfo
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- CN107596446B CN107596446B CN201710877917.3A CN201710877917A CN107596446B CN 107596446 B CN107596446 B CN 107596446B CN 201710877917 A CN201710877917 A CN 201710877917A CN 107596446 B CN107596446 B CN 107596446B
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Abstract
The invention relates to a decellularization kit for maintaining a tissue ultrastructure and a nutrition microenvironment, which consists of solution A and solution B, wherein the solution A is a mixed solution of ethanol and Triton, and the solution B is a hexyl- β -D-glucopyranoside aqueous solution.
Description
Technical Field
The invention relates to the field of tissue engineering materials, in particular to a decellularization kit for maintaining a tissue ultrastructure and a nutrition microenvironment.
Background
Various organs and tissues in the body can be considered to be composed of two parts: cells and extracellular components, also known as extracellular matrix (ECM). Most immunogenic substances are present inside the cell. Compared with single or composite artificially synthesized biological molecular material, the extracellular matrix after cell removal not only has extracellular environment more similar to that of natural organism, but also retains the biological activity to the maximum extent, and is more and more valued and applied in tissue engineering.
The preparation of extracellular matrix is to remove immunogenic components existing in cells by a method of removing tissue cells to obtain the extracellular matrix which can be used as a substitute and a scaffold material of corresponding tissues. The composition and characteristics of various tissues and organs in vivo are different, and the cell removal schemes suitable for the tissues and organs are respectively provided.
At present, the preparation method of the acellular matrix is various and mainly comprises a physical method, a chemical method and a biological treatment method. The physical method mainly removes cells through physical actions such as freeze thawing, liquid high pressure, ultrasonic waves, electric shock and the like, and the basic principle of the method is to destroy the cell membrane structure of cells in tissues, the cell membrane structure changes to cause the cells to generate adverse biochemical reactions, the continuous treatment leads the cells to die, and then the cells in the tissues are removed through cleaning of solution and removal of nucleic acid and lipid. The chemical method is to use chemical reagent to break cells to achieve the purpose of removing cells. Specific chemical agents such as acids, bases, detergents and the like can penetrate various layers of tissues, dissolve bilayer phospholipids of cell membranes and even destroy cell membrane proteins to cause cell death and disruption, and then wash away cell debris and antigenic substances by steps such as shaking washing. Biological processes are primarily directed to the lysis of cells using enzymatic reagents. For example, in decellularization methods, trypsin, a serine protease that selectively hydrolyzes peptide chains of proteins consisting of the carboxyl groups of lysine or arginine, is commonly used, and the action of trypsin hydrolyzes intercellular proteins to disperse cells.
However, the above-mentioned decellularization reagents and methods have the following general problems: (1) the prepared biological scaffold can not maintain the three-dimensional space form, a large amount of important protein is lost, and the microstructure and the nutrition microenvironment of the original tissue are damaged. (2) The reagents used are toxic and not easily removable, and the prepared bioscaffold risks tissue toxicity and immune irritation, requiring tedious post-processing to remove residual detergent. (3) The reagents used are independent of each other, the treatment time is long, the efficiency is low, the effects cannot be exerted synergistically, and a simple and convenient kit is lacked. (4) Does not have the bacteriostatic and antiseptic effects, and the prepared biological stent needs further sterilization treatment.
At present, no effective technical method is seen, and the common diseases existing in the preparation process of the acellular matrix can be simultaneously solved.
Disclosure of Invention
In order to overcome the defects of the existing acellular treatment reagent and method and solve common diseases existing in the preparation process of the acellular matrix, the invention provides a non-toxic and simple and convenient acellular kit for maintaining the tissue ultrastructure and the nutrition microenvironment. Therefore, the core of the invention is to provide a safe and efficient cell removal kit.
The inventor also finds that when the novel decellularization kit is used for treating tissues, various decellularized biological scaffolds derived from animal tissues or plant tissues can be prepared by one operation or a combination of multiple operations of pouring, washing or infiltrating.
Therefore, the decellularization kit for maintaining the tissue ultrastructure and the nutrition microenvironment consists of a solution A and a solution B, wherein the solution A is a mixed solution of ethanol and Triton, the mass percentage of the ethanol is 85-99.9%, the mass percentage of the Triton is 0.1-15%, the solution B is an aqueous solution of hexyl- β -D-glucopyranoside, the mass percentage of hexyl- β -D-glucopyranoside is 1-65%, and the steps of treating the tissue by using the decellularization kit for preparing the decellularized biological scaffold comprise:
(1) treating the tissue with liquid A which is 50-100 times of the volume of the tissue;
(2) washing the tissue treated in (1) with 0.01M phosphate buffer solution in an amount corresponding to 100 times the volume of the tissue;
(3) treating the tissue treated in the step (2) with liquid B which is 50-100 times of the volume of the tissue;
(4) washing the tissue treated in (3) with 0.01M phosphate buffer solution in an amount corresponding to 100 times the volume of the tissue to obtain a decellularized biological scaffold.
The mass percentage of ethanol in the solution A is 90-95%, the mass percentage of Triton is 1-10%, and the mass percentage of hexyl- β -D-glucopyranoside in the solution B is 10-45%.
The mass percentage of ethanol in the solution A is 92%, the mass percentage of Triton is 8%, and the mass percentage of hexyl- β -D-glucopyranoside in the solution B is 35%.
The temperature range of the tissue to be treated by the acellular kit is 10-42 ℃, the treatment time of the solution A is 1-24 hours, and the treatment time of the solution B is 12-72 hours.
The acellular biological scaffold is obtained by treating the tissue by using the acellular kit, the temperature range is 20-40 ℃, the treatment time of the solution A is 6-18 hours, and the treatment time of the solution B is 18-48 hours.
The decellularization biological scaffold is obtained by treating the tissue by using the decellularization kit, the temperature range is 37 ℃, the treatment time of the solution A is 12 hours, and the treatment time of the solution B is 36 hours.
The treatment refers to one or more operations of pouring, washing or infiltrating.
The above tissue is derived from animals or plants.
The decellularized biological scaffold obtained by treating the tissue by using the decellularized kit is used for tissue regeneration engineering.
① three reagents which can work synergistically are combined to prepare the kit, so that the acellular process is more convenient, safe and efficient ② the kit has good biocompatibility and biodegradability ③ the kit is applied to prepare a biological scaffold, the nutrition microenvironment and the microscopic three-dimensional morphological structure of the original tissue are effectively kept ④ the kit does not contain any toxic component and does not generate toxic or side reaction or immunogenicity caused by reagent residues, ⑤ the ethanol and the hexyl- β -D-glucopyranoside in the kit have sterilization and bacteriostasis effects, and the kit has the advantages of good biodegradation, no toxicity, no immunogenicity, no pollution to the environment and the like ⑥ the kit is more convenient to store, transport and apply.
Therefore, compared with the prior art, the acellular kit provided by the invention can simultaneously solve the common problem in the preparation process of the acellular matrix, and is safer and more efficient.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered as being isolated, and they may be combined with each other to achieve better technical effects.
EXAMPLE 1 preparation of a Decellularization kit
According to the component ratio of the solution A and the solution B in Table 1, the decellularization kit for the experimental group and the control group was prepared.
TABLE 1 compositions of the Decellularization kit
Note: "/" indicates that the component is 0.
Example 2 preparation of acellular bioscaffolds Using the acellular kit and quality evaluation
This example applied the decellularization kit of each group of example 1 to prepare a rat bone tissue decellularized biological scaffold.
[ preparation of decellularized biological scaffolds ] A number of rats were harvested and bone tissues of the rats were isolated. The decellularized biological scaffold of rat bone tissue was prepared by treating the tissue using the decellularized kit of example 1 according to the following procedure.
① treating the tissue with solution A50-100 times the volume of the tissue;
② washing ① -treated tissue with 0.01M phosphate buffer solution in an amount corresponding to 100 times the volume of the tissue;
③ treating ② treated tissue with liquid B in an amount of 50-100 times the volume of the tissue;
④ the decellularized bioscaffold was obtained by washing ③ -treated tissue with 0.01M phosphate buffer solution in an amount corresponding to 100 times the volume of the tissue.
The above process for treating cells was carried out at 25 ℃ for 48 hours in both solution A and solution B.
[ evaluation of the quality of decellularized biological scaffolds ] the quality of the decellularized biological scaffolds prepared by the decellularization kit of each group of example 1 was examined according to the following indexes.
(1) And (3) detecting the decellularization degree: and (3) DNA content detection, namely quantitatively detecting the DNA residual quantity of the decellularized biological scaffold obtained by the treatment of the decellularized kit.
(2) And (3) component retention detection, namely quantitatively detecting the retention conditions of laminin, fibronectin and elastin in the decellularized biological scaffold obtained by treatment of the decellularized kit by using E L ISA.
(3) And (3) toxicity detection: the cytotoxicity of the decellularized biological scaffold obtained by the treatment of the decellularized kit is detected through a CCK-8 cell proliferation experiment.
(4) And (3) detecting the structural integrity: and observing the structural integrity of the decellularized biological scaffold obtained by the treatment of the decellularized kit through a scanning electron microscope.
(5) Acute inflammatory response and biocompatibility detection: the decellularized biological scaffold prepared by the decellularized kit is implanted into the subcutaneous part of a rat in a subcutaneous implantation mode, and is taken out after 3 days to detect the inflammatory reaction and taken out after 2 months to detect the histocompatibility.
The indexes are divided into five grades from good to poor, and one grade is represented by a plus sign. The more "+" indicates the better evaluation index.
Experimental results the experimental results are summarized in table 2. As shown in Table 2, the experimental groups 1 to 8 all achieve effective cell removal, laminin, fibronectin and elastin can be effectively retained, the microstructure is completely retained, no residual toxicity exists, and the safety is high. In addition, each index of the experimental group 8 is the best among the experimental groups.
Compared with an experimental group, parameters of control groups 1-12 are reduced in different degrees, the acellular biological scaffold prepared by the control groups 1-2 has high toxicity, the control groups 3-4 have a large amount of cell membranes remained in the scaffold, and the acellular degree is incomplete, and the control groups 5-12 use different components to replace hexyl- β -D-glucopyranoside of liquid B, so that the prepared acellular scaffold has high toxicity or large component loss of related proteins, and the control groups are obviously different from the experimental groups 1-8.
The experimental results show that the acellular kit has good capacity of maintaining the tissue ultrastructure and the nutrition microenvironment, and the components of the formula of the acellular kit are irreplaceable.
TABLE 2 quality evaluation of decellularized bioscaffolds of each group
Example 3 examination of the Effect of temperature and treatment time on the quality of the decellularized bioscaffold prepared
[ Experimental methods ] different decellularization kits were prepared according to the formulation of experimental group 8 in example 1, and the influence of different temperatures and treatment times on the decellularized biological scaffold of rat bone tissue was prepared and evaluated by the method of example 2, according to the settings of Table 3.
TABLE 3 optimal use temperature and time detection packet for kits
| Group of | Service time of liquid A (hour) | Service time (hour) of B liquid | Preparation temperature (. degree.C.) |
| Experimental group 8a | 24 | 12 | 37 |
| Experimental group 8b | 24 | 12 | 42 |
| Experimental group 8c | 24 | 12 | 10 |
| Experimental group 8d | 24 | 12 | 20 |
| Experimental group 8e | 24 | 12 | 40 |
| Experimental group 8f | 1 | 12 | 37 |
| Experimental group 8g | 24 | 72 | 37 |
| Experimental group 8h | 1 | 72 | 37 |
| Experimental group 8i | 24 | 12 | 37 |
| Experimental group 8j | 12 | 36 | 37 |
| Experimental group 8k | 18 | 48 | 37 |
| Experimental group 8l | 18 | 36 | 37 |
| Experimental group 8m | 12 | 48 | 37 |
| Experimental group 8n | 24 | 12 | 37 |
[ Experimental results ] the experimental results are shown in Table 4. From the results of the experimental groups, the optimal conditions are as follows: the optimal temperature is 37 ℃, the treatment time of the solution A is 12 hours, and the treatment time of the solution B is 36 hours.
TABLE 4 evaluation of decellularized bioscaffolds
| Group of | Residual amount of DNA | Laminin (ng/g) | Fibronectin (ng/g) | Elastin (ng/g) | Microstructure |
| Experimental group 8a | 0.03％ | ＞120 | ＞120 | ＞30 | ++++ |
| Experimental group 8b | 0.03％ | ＞100 | ＞100 | ＞20 | ++ |
| Experimental group 8c | 0.03％ | ＞100 | ＞100 | ＞20 | ++ |
| Experimental group 8d | 0.03％ | ＞100 | ＞100 | ＞20 | ++ |
| Experimental group 8e | 0.03％ | ＞100 | ＞100 | ＞20 | ++ |
| Experimental group 8f | 23.67％ | ＞100 | ＞100 | ＞20 | ++ |
| Experimental group 8g | 0.03％ | ＞80 | ＞80 | ＞15 | ++ |
| Experimental group 8h | 17.03％ | ＞100 | ＞100 | ＞20 | ++ |
| Experimental group 8i | 0.03％ | ＞100 | ＞100 | ＞20 | ++++ |
| Experimental group 8j | 0.03％ | ＞130 | ＞130 | ＞40 | +++++ |
| Experimental group 8k | 0.03％ | ＞100 | ＞100 | ＞20 | +++ |
| Experimental group 8l | 0.03％ | ＞100 | ＞100 | ＞20 | ++++ |
| Experimental group 8m | 0.03％ | ＞100 | ＞100 | ＞20 | +++ |
| Experimental group 8n | 0.03％ | ＞100 | ＞100 | ＞20 | ++++ |
The above detailed description is specific to possible embodiments of the invention, and all embodiments are not intended to limit the scope of the invention, and all equivalent implementations or modifications without departing from the scope of the invention should be included in the scope of the invention.
In addition, various modifications, additions and substitutions in other forms and details may occur to those skilled in the art within the scope and spirit of the invention as disclosed in the claims. It is understood that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention as disclosed in the accompanying claims.
Claims (9)
1. The acellular kit for maintaining the tissue ultrastructure and the nutrition microenvironment is characterized by comprising a solution A and a solution B, wherein the solution A is a mixed solution of ethanol and Triton, the mass percent of the ethanol is 85-99.9%, the mass percent of the Triton is 0.1-15%, the solution B is an aqueous solution of hexyl- β -D-glucopyranoside, the mass percent of hexyl- β -D-glucopyranoside is 1-65%, and the step of preparing the acellular biological scaffold by treating the tissue by using the acellular kit comprises the following steps:
(1) treating the tissue with liquid A which is 50-100 times of the volume of the tissue;
(2) washing the tissue treated in (1) with 0.01M phosphate buffer solution in an amount corresponding to 100 times the volume of the tissue;
(3) treating the tissue treated in the step (2) with liquid B which is 50-100 times of the volume of the tissue;
(4) washing the tissue treated in (3) with 0.01M phosphate buffer solution in an amount corresponding to 100 times the volume of the tissue to obtain a decellularized biological scaffold.
2. The decellularization kit for maintaining the tissue ultrastructure and the nutrition microenvironment according to claim 1, wherein the mass percentage of ethanol in the solution A is 90% -95%, the mass percentage of Triton is 1% -10%, and the mass percentage of hexyl- β -D-glucopyranoside in the solution B is 10% -45%.
3. The decellularization kit for maintaining the tissue ultrastructure and the nutrient microenvironment according to claim 2, wherein the mass percentage of ethanol in the solution A is 92%, the mass percentage of Triton is 8%, and the mass percentage of hexyl- β -D-glucopyranoside in the solution B is 35%.
4. The decellularization kit for maintaining tissue ultrastructure and nutrient microenvironment of claim 1, wherein: the temperature range of the tissue to be treated by the acellular kit is 10-42 ℃, the treatment time of the solution A is 1-24 hours, and the treatment time of the solution B is 12-72 hours.
5. The decellularization kit for maintaining tissue ultrastructure and nutrient microenvironment according to claim 4, wherein: the acellular biological scaffold is obtained by treating the tissue by using the acellular kit, the temperature range is 20-40 ℃, the treatment time of the solution A is 6-18 hours, and the treatment time of the solution B is 18-48 hours.
6. The decellularization kit for maintaining tissue ultrastructure and nutrient microenvironment according to claim 5, wherein: the decellularization kit is applied to treat tissues to obtain the decellularized biological scaffold, the temperature range is 37 ℃, the treatment time of the solution A is 12 hours, and the treatment time of the solution B is 36 hours.
7. The decellularization kit for maintaining tissue ultrastructure and nutrient microenvironment of claim 1, wherein: the treatment refers to one operation or a combination of operations of perfusion, flushing or infiltration.
8. The decellularization kit for maintaining tissue ultrastructure and nutrient microenvironment of claim 1, wherein: the tissue is from animal or plant.
9. A decellularization kit for maintaining a tissue ultrastructure and a nutrient microenvironment according to any one of claims 1 to 8, wherein: the decellularized biological scaffold obtained by treating the tissue by using the decellularized kit is used for tissue regeneration engineering.
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| EP1244396B1 (en) * | 1999-12-29 | 2005-11-09 | Children's Medical Center Corporation | Methods for organ decellularization |
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| IL139708A0 (en) * | 2000-11-15 | 2002-02-10 | Amiel Gilad | Process of decellularizing biological matrices and acellular biological matrices useful in tissue engineering |
| GB2375771A (en) * | 2001-05-24 | 2002-11-27 | Univ Leeds | Decellularisation of tissue implant material |
| GB201207781D0 (en) * | 2012-05-03 | 2012-06-13 | Restoration Of Appearance And Function Trust | Extracellular matrix - synthetic skin scaffold |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1244396B1 (en) * | 1999-12-29 | 2005-11-09 | Children's Medical Center Corporation | Methods for organ decellularization |
| CN103025361A (en) * | 2010-05-03 | 2013-04-03 | 坦吉恩股份有限公司 | Smooth muscle cell constructs |
| CN102671242A (en) * | 2012-05-24 | 2012-09-19 | 东华大学 | Method for preparing accellular pericardial material |
| CN103536967A (en) * | 2012-07-10 | 2014-01-29 | 上海微创医疗器械（集团）有限公司 | Decellularization method for preparing extracellular matrix (ECM) support material |
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