CN108048391B - One-way amnion decellularization method - Google Patents
One-way amnion decellularization method Download PDFInfo
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Abstract
The invention relates to a unidirectional amnion decellularization method, which comprises the following steps: a) stripping and cleaning fresh amnion; b) flatly laying and fixing the amnion obtained in the step a) on a support body with a plane; c) placing the amnion treated in the step b) into a decellularization solution for treatment; d) washing the amnion treated in the step c), and then putting the amnion into a nuclease solution for treatment; e) washing the amnion treated in the step d) to obtain the human acellular amnion. Compared with the prior art, the invention combines the structural characteristics of the amnion, adopts a one-way acellular method to protect the amnion matrix layer, ensures that the amnion is spread and spread flatly, the epithelial surface is fully contacted with the acellular solution, is favorable for completely removing amnion epithelial cells, and the cells with very small matrix layer are removed because the tissue permeability is contacted with a small amount of acellular solution. The cell removing method is simple and convenient to operate, ensures the stability of the process and is beneficial to large-scale production.
Description
Technical Field
The invention relates to the technical field of tissue engineering, in particular to a unidirectional amnion decellularization method.
Background
Amnion, i.e. human placental mucosa, is the innermost layer of the fetal membrane, is tightly attached to the chorion, has a normal amniotic membrane thickness of 0.02-0.5mm, is a smooth and semitransparent film, has certain toughness, and has no blood vessels, nerves and lymphatic vessels, and consists of a single epithelial cell layer, a compact basement membrane, a non-vascular matrix and other components. The amniotic membrane matrix contains a large amount of collagen, fibronectin, laminin and other components, and has the characteristics of growth factor, extremely low immunogenicity, antibiosis, inflammation reduction, fibrosis resistance, neovascularization inhibition and the like, so that the amniotic membrane matrix has more than 100 years of application history in the medical field, and has the main applications of ocular surface disease repair, tympanic membrane injury repair, surface covering after burn and the like.
The amnion used clinically at present is mainly fresh amnion, deep-frozen preserved amnion and acellular amnion (also called amnion acellular matrix). Amnion with epithelial cells preserved at deep low temperature (-80 ℃) is mostly adopted abroad, and amnion with epithelial cells preserved at dry normal temperature which is clinically applied at home has complex technical requirements, and because of the existence of the amniotic epithelial cells, in ophthalmology, amnion implantation possibly has complications such as graft rejection, graft falling, displacement dissolution, infection and the like, and the safety of the amnion implantation has certain risk. The amnion acellular matrix does not contain amnion epithelial cells, and retains amnion basement membrane and matrix layer, and the main components of the amnion acellular matrix are type III, type IV and type V collagen, proteoglycan and glycoprotein, which are natural extracellular matrices and are good tissue engineering biological carrier materials. However, as an allogeneic biological tissue scaffold material, the main clinical problem is to overcome the immunogenicity brought by the inherent cells.
The common amnion decellularization treatment method adopts a surfactant or a surfactant combined with mechanical force scraping, pancreatin enzymolysis and a physical method.
Chinese patent publication No. CN103114073B discloses a method for removing cells from human amniotic membrane, which uses surfactant triton x-100 in combination with pancreatic lipase and dnase, and removes cells from the amniotic membrane by soaking and shaking, but because the amniotic membrane is thin, it is very easy to curl (or twist) in solution to form vacuoles, and floats on the upper surface of the solution, and cannot effectively contact the solution, and cannot achieve complete cell removal effect, and has different degrees of damage to the mechanical and chemical properties of the amniotic membrane substrate.
Chinese patent publication No. CN1594555A discloses a method and device for completely decellularizing amnion by spreading and fixing amnion as a 3D matrix tissue engineering scaffold, which comprises soaking amnion in trypsin, spreading the soaked amnion on a fixing device, and rotating the trypsin solution by a rotator to further decellularize. The trypsin has a destructive effect on extracellular matrix and is not easy to remove, secondary pollution may be generated in the preparation process, and the protein structure and the toughness of the amniotic membrane matrix can be damaged by excessive enzymolysis due to the adoption of soaking enzyme treatment and then spreading enzyme treatment. In addition, the fixing device is complicated and not suitable for mass production.
Chinese patent publication No. CN107233623A discloses a method for preparing acellular amniotic membrane for use in tissue engineering skin bioscaffolds. Rinsing and disinfecting the amnion, and then soaking the amnion in DMEM and glycerol for 6 months at-80 ℃; then repeatedly freezing and thawing the soaked amnion for 2-4 times from-80 ℃ to 37 ℃, and then washing out DMEM and glycerol to incubate in trypsin-EDTA overnight; the incubated amniotic membrane was then subjected to stop digestion with serum-containing DMEM, followed by washing with PBS and lyophilization to obtain acellular amniotic membrane. The physical decellularization method of repeated freeze thawing and the chemical decellularization method of pancreatin digestion are combined, and the process steps are adjusted to be adaptive, so that the cells in the amnion are promoted to be efficiently and thoroughly removed, mechanical damage is avoided, and more cytokines in the amnion are retained. However, the method disclosed in this patent is too long in cycle time and cumbersome in steps.
The main cells of the amnion are positioned on the amnion epithelial layer, the basement membrane has no cells, the matrix layer has only a few cells, and the simple soaking and shaking treatment is adopted, although the cells on the amnion epithelial layer are removed, the matrix layer is also subjected to the treatment of a cell-removing reagent, which obviously damages the structure and components of the basement membrane and the matrix layer. The acellular treatment is not only a simple selection of an acellular method, but also a treatment mode without damaging the matrix by combining the structural characteristics of the used tissues. No solution is provided in the prior art as to how to remove only amniotic epithelial cells and protect the amniotic basement membrane and matrix layer structure.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a unidirectional amnion decellularization method.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a unidirectional amnion decellularization method, which comprises the following steps:
a) stripping and cleaning fresh amnion;
b) flatly laying and fixing the amnion obtained in the step a) on a support body with a plane;
c) placing the amnion treated in the step b) into a decellularization solution for treatment;
d) washing the amnion treated in the step c), and then putting the amnion into a nuclease solution for treatment;
e) washing the amnion treated in the step d) to obtain the human acellular amnion, namely the human amniotic extracellular matrix scaffold material.
In one embodiment of the invention, the fresh amniotic membrane is healthy human placental mucosa.
The health refers to negative detection of hepatitis B virus, hepatitis C virus, chlamydia, human immunodeficiency virus and syphilis.
In one embodiment of the present invention, the exfoliation is performed by separating the amniotic membrane and chorion from the placenta, then exfoliating and removing the chorion, and removing the remaining gel-like substance with sterile forceps.
The amniotic membrane and chorion are separated from the placenta, and the chorion is then stripped off and the remaining gelatinous material is removed with sterile forceps using conventional biological methods.
In one embodiment of the present invention, the washing in step a) refers to washing the stripped amnion with sterile 0.9% sodium chloride injection at 4-37 ℃ until the amnion is bloodless and transparent.
In one embodiment of the present invention, the method for fixing the amnion obtained in step a) on a support with a plane surface in step b) comprises: the amnion is laid on the plane of the support body, and the edge of the laid amnion is fixed in the groove on the side surface of the support body by the elastic ring.
The elastic ring refers to a medical elastic ring, and comprises but is not limited to an elastic medical rubber ring, a medical rubber band, a medical latex tube and the like.
The shape of the support body comprises a cuboid, a cube, a trapezoid, a cylinder, a round table, an ellipsoid and the like,
the support is preferably a shape without any corner, and if the support is a shape with a corner, the corner is changed from a right angle to an arc.
The support body is made of medical grade plastics or medical grade stainless steel.
In one embodiment of the invention, the amniotic membrane obtained in step a) is laid flat on a support having a flat surface, and the amniotic membrane hair side (i.e., the matrix layer of the amniotic membrane) is laid flat down on the support surface.
In one embodiment of the present invention, the amnion treated in step b) is placed in a decellularization solution, and alternative embodiments include: placing the amnion which is spread on the support body after the treatment of the step b) into a covered container containing a cell-removing solution for treatment, wherein the covered container is medical grade plastic or medical grade stainless steel. When the amniotic membrane is treated, the amniotic membrane and a support body are put into a cell removal solution together, the support body aims to ensure that the amniotic membrane is in a flat state, the amniotic membrane hair surface (namely a matrix layer of the amniotic membrane) is not directly contacted with the solution, and only a small amount of the solution permeates into the matrix layer of the amniotic membrane through an amniotic epithelial layer.
In one embodiment of the present invention, the decellularization solution comprises a main component and a preparation solution, wherein the main component is TrypLETMA combination of one or more of Select Enzyme (a trypsin substitute), trypsin, neutral protease, non-ionic detergent, zwitterionic detergent;
the preparation solution is sterile 0.9% sodium chloride injection, phosphate buffer solution, D-Hanks solution or HEPES solution.
The non-ionic detergent is selected from Triton X-100, Tween-80 and the like.
The zwitterionic detergent is selected from 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt (CHAPS), dodecyl dimethyl hydroxypropyl sulfobetaine and the like.
In the present invention, the main component of the decellularizing agent comprises TrypLETMSelect Enzyme (a trypsin substitute), trypsin, neutral protease, nonionic detergent, zwitterionic detergent, etc., which can remove cells by destroying the cell membrane structure (enzymes hydrolyze membrane proteins and destroy the connection between cell membrane and tissue matrix to remove cells), and detergents destroy the protein-protein, protein-lipid, lipid-lipid on cell membraneThe cell is removed by encapsulating cell membrane proteins, lipids, etc. to form micelles).
In one embodiment of the present invention, the condition for placing the amnion treated in step b) into the decellularization solution is: shaking at 4-37 deg.C for 1min-48 h. The purpose of shaking during the cell removal treatment is to ensure the sufficient uniform mixing of the solution and the sufficient contact of the solution and the tissue.
In one embodiment of the present invention, the amnion treated in step c) is washed and then treated with a nuclease solution, and the optional embodiments include: the amniotic membrane, which is still laid flat on the support after treatment with the decellularization solution, is placed in a covered container containing a nuclease solution for treatment. The container with the cover is made of medical grade plastic or medical grade stainless steel.
In one embodiment of the invention, the nuclease solution is a totipotent nuclease solution or a broad-spectrum nuclease solution, or a mixed solution of dnase and rnase,
the totipotent Nuclease solution or the broad-spectrum Nuclease solution is selected from Super Nuclease, Benzonase Nuclease, Biolonase Nuclease, etc.
In the invention, the nuclease solution comprises totipotent nuclease solution or broad-spectrum nuclease solution, or mixed solution of DNA enzyme and RNA enzyme, and the like, and the nuclease has the same action and decomposes DNA and RNA released by cells.
The cell removing reagent has the functions of destroying cell membranes in tissues and other membrane structures in cells, breaking the connection between the cell membranes and tissue matrixes and releasing components in the cells. The nuclease solution acts to break down DNA and RNA released from the cells. The acellular reagent selected by the invention is a mild reagent, has little damage to a basement membrane and a matrix layer of a tissue, has a compact structure of the basement membrane and a relatively loose structure of the matrix layer, and further protects the structure and components of the matrix layer by adopting a one-way treatment mode. The nuclease is a targeting enzyme, only digests DNA and RNA which are specific substrates, and does not damage the structure and components of a basement membrane and a matrix layer.
The concentration of the cell removing agent and the nuclease solution is an important index, TrypLETM Selecthe concentration of t Enzyme is 0.001-5% (v/v), preferably 0.005-0.25% (v/v); (ii) a trypsin concentration of 0.001% to 5% (v/v), preferably 0.005% to 0.25% (v/v); the concentration of neutral protease is 0.001% -5% (v/v), preferably 0.005% -0.25% (v/v); non-ionic detergents (e.g., Triton X-100, Tween-80, etc.) at concentrations of 0.001% -5% (v/v), preferably 0.005% -0.5% (v/v); zwitterionic detergents (3- [3- (cholamidopropyl) dimethylamino)]Propanesulfonic acid inner salt (CHAPS), dodecyl dimethyl hydroxypropyl sulfobetaine, etc.) concentration is 0.001% -5% (v/v), preferably 0.005% -0.5% (v/v). The concentration of the totipotent (or broad-spectrum) nuclease solution is 0.1-2000u/ml, preferably 1-200 u/ml; the concentration of the mixed solution of the DNase and the RNase is 50-10000u/ml, preferably 200-8000 u/ml.
In one embodiment of the present invention, the conditions for washing the amnion treated in step c) and then treating the amnion in the nuclease solution are as follows: shaking at 37 deg.C for 1-48 hr.
In one embodiment of the present invention, the washing conditions in step d) and step e) are: and (3) shaking and cleaning the treated amnion by using sterile 0.9% sodium chloride injection at the temperature of 4-37 ℃.
The amnion washed in step e) needs to be taken off from the support, and the specific preservation modes include freeze drying preservation, preservation in glycerol at-80 ℃ or 2-8 ℃, preservation in culture medium at-80 ℃ and the like.
In a second aspect of the invention, an auxiliary support body for unidirectional amnion decellularization treatment is provided, wherein the support body comprises a plane serving as a support surface and a groove located on the side surface of the support surface, and the groove is used for fixing the edge of the laid amnion in the groove on the side surface of the support body through an elastic ring.
The shape of the support body comprises a cuboid, a cube, a trapezoid, a cylinder, a round table, an ellipsoid and the like,
the support is preferably a shape without any corner, and if the support is a shape with a corner, the corner is changed from a right angle to an arc.
The support body can be made of medical grade plastics or medical grade stainless steel.
The elastic ring refers to a medical elastic ring, and comprises but is not limited to an elastic medical rubber ring, a medical rubber band, a medical latex tube and the like.
When the support body provided by the invention is used, the amnion is flatly laid on the plane of the support body, and the edge of the laid amnion is fixed in the groove on the side surface of the support body by the elastic ring.
The support is not limited to a rigid material such as medical grade plastic or medical grade stainless steel, but may be a soft medical grade thin film material, and the amnion is laid flat on the surface of the material, and the tissue edge is fixed by a suture or by pressing a rigid strip (or magnetic strip) to fix the tissue edge.
In a third aspect of the invention, a human amniotic extracellular matrix scaffold material is provided, which is obtained by the unidirectional amniotic acellular method of the first aspect of the invention.
Compared with the prior art, the invention has the following beneficial effects:
(1) the amniotic membrane matrix layer is protected by combining the structural characteristics of the amniotic membrane and adopting a unidirectional acellular method, so that the amniotic membrane is laid flat and unfolded, the epithelial surface is fully contacted with an acellular solution, the complete removal of amniotic epithelial cells is facilitated, and cells with a very small matrix layer are removed due to the fact that the tissue permeability is contacted with a small amount of acellular solution.
(2) The acellular method is more beneficial to the removal of amniotic epithelial cells by adopting a flat laying mode, specifically protects a basement membrane and a matrix layer structure, adopts a mild acellular reagent, and preserves the components and the structure of the amniotic membrane to a greater extent.
(3) The cell removing method is simple and convenient to operate, ensures the stability of the process and is beneficial to large-scale production.
Drawings
FIG. 1 is a schematic structural diagram of an auxiliary support for unidirectional amnion decellularization treatment.
Figure 2 is a picture (200-fold) of HE staining of fresh amniotic membrane tissue.
FIG. 3 is a photograph (200 times) of HE staining of an amniotic tissue after decellularization, showing that the amniotic epithelial surface cells are completely removed after the treatment of the present invention, and there is no obvious damage to the stromal structure.
Detailed Description
The invention uses the unidirectional decellularization device to ensure that the amniotic membrane is decellularized more thoroughly, greatly improves the stability of batch production, and overcomes the defects of insufficient decellularization and matrix damage caused by simple soaking and shaking in the prior art.
The invention relates to a unidirectional amnion decellularization method, which comprises the following steps:
a) stripping and cleaning fresh amnion;
b) flatly laying and fixing the amnion obtained in the step a) on a support body with a plane;
c) placing the amnion treated in the step b) into a decellularization solution for treatment;
d) washing the amnion treated in the step c), and then putting the amnion into a nuclease solution for treatment;
e) washing the amnion treated in the step d) to obtain the human acellular amnion, namely the human amniotic extracellular matrix scaffold material.
The fresh amnion is healthy human placental mucosa. The health refers to negative detection of hepatitis B virus, hepatitis C virus, chlamydia, human immunodeficiency virus and syphilis.
The stripping is to separate the amnion and chorion from placenta, then strip and remove chorion, and remove the residual gelatinous substance with sterile forceps.
The step a) of cleaning refers to cleaning the stripped amnion by using sterile 0.9% sodium chloride injection at the temperature of 4-37 ℃ until the amnion has no blood filaments and is transparent.
The method for flatly fixing the amnion obtained in the step a) on the support body with the plane in the step b) comprises the following steps: the amnion is laid on the plane of the support body, and the edge of the laid amnion is fixed in the groove on the side surface of the support body by the elastic ring. The elastic ring refers to a medical elastic ring, and comprises but is not limited to an elastic medical rubber ring, a medical rubber band, a medical latex tube and the like. The shape of the support body comprises a cuboid, a cube, a trapezoid body, a cylinder, a circular truncated cone, an ellipsoid and the like, the support body is preferably in a shape without edges, and if the support body is a body with edges, the edges are changed from right angles to arc shapes. The support body is made of medical grade plastics or medical grade stainless steel.
Spreading the amnion obtained in the step a) on a support body with a plane, and spreading the amnion hair surface (namely the amnion matrix layer) downwards and flatly on the surface of the support body.
Placing the amnion treated in step b) into a decellularization solution for treatment, wherein the optional embodiment comprises: placing the amnion which is spread on the support body after the treatment of the step b) into a covered container containing a cell-removing solution for treatment, wherein the covered container is medical grade plastic or medical grade stainless steel.
The cell removing solution comprises a main component and a preparation solution, wherein the main component is TrypLETMA combination of one or more of Select Enzyme (a trypsin substitute), trypsin, neutral protease, non-ionic detergent, zwitterionic detergent;
the preparation solution is sterile 0.9% sodium chloride injection, phosphate buffer solution, D-Hanks solution or HEPES solution.
The non-ionic detergent is selected from Triton X-100, Tween-80 and the like.
The zwitterionic detergent is selected from 3- [3- (cholamidopropyl) dimethylamino ] propanesulfonic acid inner salt (CHAPS), dodecyl dimethyl hydroxypropyl sulfobetaine and the like.
The conditions for placing the amnion treated in the step b) into a decellularization solution are as follows: shaking at 4-37 deg.C for 1min-48 h.
Washing the amnion treated in step c), and then putting the amnion into a nuclease solution for treatment, wherein the optional embodiment comprises the following steps: the amniotic membrane, which is still laid flat on the support after treatment with the decellularization solution, is placed in a covered container containing a nuclease solution for treatment. The container with the cover is made of medical grade plastic or medical grade stainless steel.
The Nuclease solution is a totipotent Nuclease solution or a broad-spectrum Nuclease solution, or a mixed solution of DNase and RNAse, and the totipotent Nuclease solution or the broad-spectrum Nuclease solution is selected from Super Nuclease, Benzonase Nuclease, Biolonase Nuclease and the like.
The nuclease solution comprises totipotent nuclease solution or broad-spectrum nuclease solution, or mixed solution of DNA enzyme and RNA enzyme.
Washing the amnion treated in the step c), and then putting the amnion into a nuclease solution for treatment under the following conditions: shaking at 37 deg.C for 1-48 hr.
The washing conditions in step d) and step e) are as follows: and (3) shaking and cleaning the treated amnion by using sterile 0.9% sodium chloride injection at the temperature of 4-37 ℃.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1:
taking placenta of healthy parturient, stripping amnion from the placenta, washing with 0.9% physiological saline for several times in biological safety cabinet, stripping with sterile medical forceps to remove residual chorion substance, and removing blood components. The amnion is laid on a sterile square body one-way acellular device (20x20x2cm, shown in figure 1, 1 refers to a plane, and 2 refers to a groove on the side surface in figure 1) (the matrix layer is downward), the edge of the amnion is wrapped on the groove of the device by using a sterile elastic medical latex tube for fixation, and the unfixed amnion is cut off along the groove by using sterile medical scissors. Placing the fixed amnion into TrypLETMIn the Select Enzyme solution, the mixture was shaken at 37 ℃ for 2 hours. Washing with sterile normal saline for 5 times, placing the fixed amnion into Super nucleic solution, and treating for 3h at 37 deg.C. And finally, shaking and cleaning the obtained product for 5 times by using sterile physiological saline to obtain the human amniotic extracellular matrix scaffold material.
Detection and evaluation:
(1) HE staining: the fresh amnion and the decellularized amnion are detected by adopting a conventional HE histological pathological section, and the result shows that the epithelial layer of the fresh amnion contains a row of cells (shown in figure 2), while the amnion treated by the invention has no cell residue (shown in figure 3).
(2) And (3) detecting the residual quantity of the DNA: weighing an appropriate amount of amnion sample, purifying DNA according to DNeasy Blood & Tissue Kit, and determining the DNA residue by using PicoGreen dsDNA Assay Kit. The content of fresh amnion DNA is 296.15 +/-21.37 ng/mg through detection, while the DNA residual quantity of the amnion treated by the method is 6.92 +/-0.56 ng/mg, and the method has significant difference.
(3) And (3) measuring the tensile strength: cutting fresh amnion and decellularized amnion into plate shape, breaking the plate shape on a universal material testing machine, and calculating the tensile strength of the amnion material. The tensile strength of the fresh amnion is 8.71 plus or minus 1.55MPa, and the tensile strength of the amnion treated by the method is 7.33 plus or minus 1.86 MPa. The tensile strength of the amnion treated by the invention is not obviously changed.
(4) And (3) measuring the content of the collagen: the amniotic membrane was lysed with 0.5M acetic acid containing 1mg/ml pepsin, hydroxyproline content was determined according to the Sircol Soluble Collagen Assay Kit, and finally Collagen content was calculated. The detection shows that the content of the fresh amnion collagen is 59.61 +/-0.64 percent, and the amnion collagen processed by the method is 78.53 +/-0.88 percent. The decellularization treatment removes cell protein, lipid and other components, retains main components of the amniotic membrane fiber, improves the purity of the collagen, and improves the content of the collagen.
(5) And (3) transparency determination: the transparency of the amniotic membrane was visually observed under light. The appearance of the acellular biological amnion prepared by the method is a semitransparent film.
Example 2:
taking placenta of healthy parturient, stripping amnion from the placenta, washing with 0.9% physiological saline for several times in biological safety cabinet, stripping with sterile medical forceps to remove residual chorion substance, and removing blood components. The amnion is laid on a sterile cylindrical one-way acellular device (the diameter is 15cm, the height is 2cm), the matrix layer is downward, the edge of the amnion is wrapped on a groove of the device by a sterile elastic medical rubber ring for fixation, and the unfixed amnion is cut off along the groove by sterile medical scissors. Placing the fixed amnion into neutral protease solution, and shaking at 37 deg.C for 30 min. Washing with sterile normal saline for 5 times, placing the fixed amnion into Super nucleic solution, and treating for 1h at 37 deg.C. And finally, shaking and cleaning the obtained product for 5 times by using sterile physiological saline to obtain the human amniotic extracellular matrix scaffold material.
Detection and evaluation: HE staining shows that the amnion treated by the method has no cell residue, the appearance is semitransparent, and the results of DNA content, collagen content and tensile strength are as follows:
the amnion nucleic acid treated by the method has low residue, and the mechanical property and the main structural components of the amnion matrix are well reserved.
Example 3:
taking placenta of healthy parturient, stripping amnion from the placenta, washing with 0.9% physiological saline for several times in biological safety cabinet, stripping with sterile medical forceps to remove residual chorion substance, and removing blood components. The amnion is laid on a sterile rectangular one-way acellular device (20x15x3cm) (with a downward matrix layer), the edge of the amnion is wrapped on a groove of the device by a sterile elastic medical rubber ring for fixation, and the unfixed amnion is cut off along the groove by sterile medical scissors. And (3) placing the fixed amnion into a CHAPS solution, and shaking at 37 ℃ for 12 h. After shaking and washing for 5 times by using sterile normal saline, the fixed amnion is put into Benzonase nucleic solution and is shaken for 8 hours at 37 ℃. And finally, shaking and cleaning the obtained product for 5 times by using sterile physiological saline to obtain the human amniotic extracellular matrix scaffold material.
Detection and evaluation: HE staining shows that the amnion treated by the method has no cell residue, the appearance is semitransparent, and the results of DNA content, collagen content and tensile strength are as follows:
the amnion nucleic acid treated by the method has low residue, and the mechanical property and the main structural components of the amnion matrix are well reserved.
Example 4:
taking placenta of healthy parturient, stripping amnion from the placenta, washing with 0.9% physiological saline for several times in biological safety cabinet, stripping with sterile medical forceps to remove residual chorion substance, and removing blood components. The amnion is laid on a sterile square one-way acellular device (10x10x3cm) (with a downward matrix layer), the edge of the amnion is wrapped on a groove of the device by a sterile elastic medical rubber band for fixation, and the unfixed amnion is cut off along the groove by sterile medical scissors. Placing the fixed amnion into TrypLETMIn a mixed solution of Select Enzyme and Triton X-100, the mixture was shaken at 37 ℃ for 1 hour. After shaking and cleaning with sterile normal saline for 5 times, the fixed amnion is put into a mixed solution of DNA enzyme and RNA enzyme, and shaking treatment is carried out for 24 hours at 37 ℃. And finally, shaking and cleaning the obtained product for 5 times by using sterile physiological saline to obtain the human amniotic extracellular matrix scaffold material.
Detection and evaluation: HE staining shows that the amnion treated by the method has no cell residue, the appearance is semitransparent, and the results of DNA content, collagen content and tensile strength are as follows:
the amnion nucleic acid treated by the method has low residue, and the mechanical property and the main structural components of the amnion matrix are well reserved.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (4)
1. A unidirectional amnion decellularization method is characterized by comprising the following steps:
a) stripping and cleaning fresh amnion;
b) flatly laying and fixing the amnion obtained in the step a) on a support body with a plane;
c) placing the amnion treated in the step b) into a decellularization solution for treatment;
d) washing the amnion treated in the step c), and then putting the amnion into a nuclease solution for treatment;
e) washing the amnion treated in the step d) to obtain a human acellular amnion;
flatly paving the amnion obtained in the step a) on a support body with a plane, and flatly paving the matrix layer of the amnion downwards on the surface of the support body;
when the amnion is treated in the steps c), d) and e), the amnion is fixed on a support body with a plane;
the support body ensures that the matrix layer of the amnion is not directly contacted with a decellularization solution or a nuclease solution, and the decellularization solution or the nuclease solution only permeates into the matrix layer of the amnion through the epithelial layer of the amnion;
the cell removing solution comprises a main component and a preparation solution, wherein the main component is TrypLETMA combination of Select Enzyme, trypsin, neutral protease, non-ionic detergent, zwitterionic detergent; the preparation solution is sterile 0.9% sodium chloride injection, phosphate buffer solution, D-Hanks solution or HEPES solution;
TrypLETMthe concentration of the Select Enzyme is 0.001-5% v/v, the concentration of trypsin is 0.001-5% v/v, the concentration of neutral protease is 0.001-5% v/v, the concentration of nonionic detergent is 0.001-5% v/v, and the concentration of zwitterionic detergent is 0.001-5% v/v;
the support body comprises a plane as a support surface and a groove positioned on the side surface of the support surface, the support body is a rigid object, and the method for flatly fixing the amnion obtained in the step a) on the support body with the plane comprises the following steps: the amnion is laid on the plane of the support body, and the edge of the laid amnion is fixed in the groove on the side surface of the support body by the elastic ring.
2. The method of claim 1, wherein the amnion treated in step b) is placed in a decellularization solution under the following conditions: shaking at 4-37 deg.C for 1min-48 h.
3. The unidirectional amnion decellularization method of claim 1, wherein the nuclease solution is a totipotent nuclease solution or a broad-spectrum nuclease solution, or a mixed solution of DNase and RNAse,
the totipotent Nuclease solution or the broad-spectrum Nuclease solution is selected from Super Nuclease, Benzonase Nuclease or Biolonase Nuclease; washing the amnion treated in the step c), and then putting the amnion into a nuclease solution for treatment under the following conditions: shaking at 37 deg.C for 1-48 hr.
4. The method of claim 1, wherein the washing conditions in step d) and step e) are as follows: and (3) shaking and cleaning the treated amnion by using sterile 0.9% sodium chloride injection at the temperature of 4-37 ℃.
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