CN116763990A - Jellyfish cell removing material and preparation method and application thereof - Google Patents
Jellyfish cell removing material and preparation method and application thereof Download PDFInfo
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- CN116763990A CN116763990A CN202211326288.2A CN202211326288A CN116763990A CN 116763990 A CN116763990 A CN 116763990A CN 202211326288 A CN202211326288 A CN 202211326288A CN 116763990 A CN116763990 A CN 116763990A
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- ADWNFGORSPBALY-UHFFFAOYSA-M sodium;2-[dodecyl(methyl)amino]acetate Chemical compound [Na+].CCCCCCCCCCCCN(C)CC([O-])=O ADWNFGORSPBALY-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
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- 239000000758 substrate Substances 0.000 description 1
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- 230000009897 systematic effect Effects 0.000 description 1
- GWIKYPMLNBTJHR-UHFFFAOYSA-M thiosulfonate group Chemical group S(=S)(=O)[O-] GWIKYPMLNBTJHR-UHFFFAOYSA-M 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses jellyfish cell removing material and a preparation method and application thereof. The method comprises the following steps: performing decellularization treatment on jellyfish tissues by adopting a decellularization agent; the decellularizing agent includes a surfactant. The method for preparing the decellularized jellyfish tissue material is simple, reduces the application of chemical reagents, can completely remove cell components, and minimizes the residual DNA in the decellularized jellyfish tissue material; meanwhile, the damage to the three-dimensional space structure of jellyfish extracellular matrix (ECM) protein and ECM fiber is reduced, and the natural molecular structure of collagen in jellyfish ECM can be completely reserved; the decellularized jellyfish material prepared by the method can be effectively used for tissue engineering applications such as cell three-dimensional culture, skin injury, cartilage and cornea repair and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to jellyfish cell removing material, and a preparation method and application thereof.
Background
Decellularized materials (Decellularized materials, DMs) are of increasing interest because of their inherent structure, higher bioactivity, lower immunogenicity and good biodegradability, which are difficult to mimic with synthetic materials. The cell-free biological scaffold material has good application potential in the aspects of cell and organ transplantation treatment, biomedical engineering, medical dressing delivery carrier, pharmaceutical preparation and the like.
The cell-removing materials (materials mainly used for wound repair and tissue regeneration) used in clinic at present mainly come from the tissues of mammal dermis, small intestine and the like. Typically, the decellularized material is prepared by chemically and physically removing animal cells from animal tissue to reduce the immunogenicity of the animal tissue. The technology still has some defects and hidden troubles at present: (1) The decellularized material mainly comes from mammal tissues (such as dermis and small intestine), and the tissue structure of the higher mammal is complex, and complicated depilation and blood and fat removal are required before decellularizing; (2) The cell-free material of the mammal may be provided with infectious viruses such as foot-and-mouth disease viruses, and the risk of infection to people exists; (3) The substrate materials such as the dermis of mammal have high crosslinking degree and limited liquid absorption capacity.
Extracellular matrix, as a very conserved structure evolved in various animal tissues, has low rejection even when implanted into different species. In the field of tissue engineering, the extracellular matrix (Extracellular matrix, ECM) material after cell removal has good biocompatibility and tissue regeneration performance, so that the complete ECM can be obtained by optimizing the cell removal process, and the extracellular matrix (Extracellular matrix, ECM) material has wide application prospect in repairing skin wound surfaces and bone defects. Related studies have shown that cell removal methods that maintain the native molecular structure of ECM and reduce ECM damage are highly desirable in favor of cell proliferation and migration. However, the cell removal method often has a certain influence on the biochemical composition, structure and biomechanical properties of the tissue, and may cause irreversible damage to the three-dimensional structure of ECM, thereby negatively affecting the function of the repaired tissue. Therefore, there is a need for systematic development and characterization of decellularization processes and methods to develop a method for preserving the integrity of ECM structures.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a preparation method of jellyfish cell removal material, which is simple, and can remove jellyfish cells without damaging collagen structure of jellyfish ECM.
The invention also provides the jellyfish cell removing material prepared by the method.
The invention also provides application of the jellyfish cell removing material.
The invention also provides a biological stent material.
The invention also provides application of the biological stent material.
In one aspect of the present invention, a method for preparing jellyfish decellularized material is provided, the method comprising the steps of: and (3) performing cell removal treatment on jellyfish tissues by adopting a cell removing agent to obtain the jellyfish tissue.
In some embodiments of the invention, the decellularizing agent comprises a surfactant.
In some embodiments of the invention, the surfactant comprises an ionic surfactant and/or a nonionic surfactant.
In some embodiments of the invention, the ionic surfactant is at least one of SDS (Sodium dodecyl sulfate ), SNL (Sodium N-dodecanoylsalcinate, sodium N-dodecyl sarcosinate), SD (Sodium deoxycholate ), CHAPS (3- [3- (cholestamidopropyl) dimethylamino ] propane sulfonate), SB-10 (Sulfobetaine 10, thiobetaine 10), and CH (Cholesteryl hemisuccinate, cholesterol hemisuccinate).
In some embodiments of the invention, the nonionic surfactant is at least one of Triton X100 (Triton X100, polyethylene glycol tert-octylphenyl ether), IGEPAL CA-630 (polyoxyethylene octylphenol ether), TWEEN (TWEEN), DDM (n-Dodecyl β -D-maltoside ), nonidet P40 (ethylphenyl polyethylene glycol), and Digitin (digitonin).
In some embodiments of the invention, the ionic surfactant is at least one of 0.1% to 10% SDS, 0.1% to 10% SNL, 0.1% to 10% SD, 0.1% to 10% CHAPS, 0.1% to 10% SB-10, and 0.1% to 10% CH.
In some embodiments of the invention, the ionic surfactant is at least one of 0.1% to 10% sd, 0.1% to 10% snl, and 0.1% to 10% chaps.
In some embodiments of the invention, the ionic surfactant is at least one of 3% to 8% sd, 0.5% to 5% snl, and 1% to 10% chaps.
In some embodiments of the invention, the ionic surfactant is at least one of 3% to 6% sd, 0.5% to 2% snl, and 1% to 4% chaps.
In some embodiments of the invention, the nonionic surfactant is at least one of 0.1% to 10% Triton, 0.1% to 10% IGEPAL CA-630, 0.1% to 10% Tween, 0.1% to 10% DDM, 0.1% to 10% Nonidet P40, and 0.1% to 10% digitin.
In some embodiments of the invention, the nonionic surfactant is at least one of 0.1% to 10% triton, 0.1% to 10% igepal CA-630, and 0.1% to 10% tween.
In some embodiments of the invention, the nonionic surfactant is at least one of 3% to 8% triton, 3% to 8% igepal CA-630, and 3% to 8% tween.
In some embodiments of the invention, the tween comprises one of tween 20, tween 40, tween 60, tween 65 and tween 80.
In some embodiments of the invention, the decellularizing agent comprises 0.1% to 10% sd.
In some embodiments of the invention, the decellularizing agent comprises 1% to 10% sd.
In some embodiments of the invention, the decellularizing agent comprises 3% to 8% sd.
In some embodiments of the invention, the decellularization agent comprises 5% sd.
In some embodiments of the invention, the method further comprises the step of cross-linking the jellyfish tissue after the decellularization agent treatment.
In some embodiments of the invention, the crosslinking agent employed in the crosslinking treatment contains a chemically reactive group that is reactive with at least one of an amino group, a thiol group, a carboxyl group, an aldehyde group, and a hydroxyl group.
In some embodiments of the invention, the chemically reactive group of the crosslinker that reacts with the amino group is at least one of NHS ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine, and aldehyde group.
In some embodiments of the invention, the aldehyde-based chemically reactive groups include at least one of formaldehyde, paraformaldehyde, glutaraldehyde, glucose, and formalin.
In some embodiments of the invention, the chemically reactive group of the crosslinker that reacts with the thiol group is at least one of maleimide, haloacetyl, pyridyldimercapto, thiosulfonate, and vinyl sulfone.
In some embodiments of the invention, the chemically reactive group of the crosslinker that reacts with the carboxyl group is a carbodiimide.
In some embodiments of the invention, the crosslinking agent reacts with aldehyde groups to form at least one of the chemically reactive groups hydrazide, alkoxyamine, and NHS ester.
In some embodiments of the invention, the chemically reactive group of the crosslinker that reacts with the hydroxyl group is an isocyanate.
In some embodiments of the invention, the crosslinking agent is NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide).
In some embodiments of the invention, the NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) are added in a mass ratio of 10:1 to 1:10.
in some embodiments of the invention, the NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) are added in a mass ratio of 1:4.
in some embodiments of the invention, the time of the crosslinking treatment is from 4 to 20 hours.
In some embodiments of the invention, the time of the crosslinking treatment is 8 to 16 hours.
In some embodiments of the invention, the crosslinking is followed by a step of drying the resulting jellyfish tissue.
In some embodiments of the invention, the drying is freeze-drying.
In some embodiments of the invention, the jellyfish is at least one of jellyfish, cubic jellyfish, coronal jellyfish, cross jellyfish, flag jellyfish, and root jellyfish.
In some embodiments of the invention, the jellyfish is at least one of jellyfish, bamboo hat jellyfish, purple sea thorn jellyfish, mediterranean omelanuginose jellyfish, black warrior jellyfish, blue jellyfish, silver coin jellyfish, alert jellyfish, dilemma jellyfish, starfish jellyfish, white spot jellyfish, brindy river jellyfish, lighthouse jellyfish, nepheline jellyfish, and sea month jellyfish.
In some embodiments of the invention, the jellyfish tissue is prepared by subjecting jellyfish to salt displacement with pure water and then cutting.
In some embodiments of the invention, the jellyfish tissue is jellyfish umbrella tissue.
In some embodiments of the invention, the jellyfish tissue is jellyfish antenna tissue.
In some embodiments of the invention, the decellularization reagent is treated for a time of 0.02 to 10 days.
In some embodiments of the invention, the decellularization reagent is treated for a time of 0.5 to 8 days.
In some embodiments of the invention, the step of washing the jellyfish tissue with a washing solution is further included after the decellularizing treatment of the jellyfish tissue with the decellularizing agent.
In some embodiments of the invention, the wash solution is PBS or water.
In some embodiments of the invention, the washing time is from 0.5 to 14 hours.
In a second aspect of the present invention, jellyfish decellularized material prepared by the above method is provided.
In a third aspect of the invention, there is provided the use of the jellyfish decellularized material described above for the preparation of scaffold biomaterials.
In some embodiments of the invention, the use is in the preparation of a product that supports growth of animal cells.
In some embodiments of the invention, the use is in the manufacture of a product for promoting wound repair.
In some embodiments of the invention, the wound repair comprises wound repair of related wounds such as burns, scalds, incised wounds, chronic infectious wounds and the like.
In some embodiments of the invention, the wound repair further comprises wound repair of skin, bone, spinal cord, heart, muscle, nerve, blood vessel, oral cavity, cornea, cartilage or other associated wound surface of damaged or malformed tissue.
In some embodiments of the invention, the wound repair further comprises repair of a damaged heart.
In some embodiments of the invention, the use is in the preparation of a product that promotes tissue regeneration.
In a fourth aspect of the present invention, a biological scaffold material is presented, comprising jellyfish decellularized material prepared by the above method.
In a fifth aspect of the invention, there is provided the use of a biological scaffold material as described above in the preparation of a product for supporting growth of animal cells.
In some embodiments of the invention, the use is in the manufacture of a product for promoting wound repair.
In some embodiments of the invention, the wound repair comprises wound repair of related wounds such as burns, scalds, incised wounds, chronic infectious wounds and the like.
In some embodiments of the invention, the wound repair further comprises wound repair of the skin, bone, spinal cord, muscle, nerve, blood vessel, oral cavity, cornea, cartilage or other associated wound surface of damaged or malformed tissue.
In some embodiments of the invention, the wound repair further comprises repair of a damaged heart.
In some embodiments of the invention, the use is in the preparation of a product that promotes tissue regeneration.
In some embodiments of the invention, the tissue comprises skin, bone, spinal cord, muscle, nerve, blood vessel, oral cavity, cornea, cartilage, or other damaged or malformed tissue.
In some embodiments of the invention, the tissue regeneration further comprises regeneration of damaged cardiac tissue.
In some embodiments of the invention, the use is in the preparation of a product that promotes angiogenesis.
In some embodiments of the invention, the use is in the preparation of a tissue replacement.
In some embodiments of the invention, the tissue comprises skin, bone, spinal cord, muscle, nerve, blood vessel, oral cavity, cornea, cartilage, or other damaged or malformed tissue.
In some embodiments of the invention, the tissue further comprises tissue of a damaged heart.
In some embodiments of the invention, the use is in the preparation of a structural implant for cosmetic surgery.
According to some embodiments of the invention, at least the following benefits are provided: the method for preparing the decellularized jellyfish tissue material is simple, reduces the application of chemical reagents, can completely remove cell components, and minimizes the residual DNA in the decellularized jellyfish tissue material; meanwhile, the damage to the three-dimensional space structure of jellyfish extracellular matrix (ECM) protein and ECM fiber is reduced, and the natural molecular structure of collagen in jellyfish ECM can be completely reserved; the decellularized jellyfish material prepared by the method can be effectively used for tissue engineering applications such as cell three-dimensional culture, skin injury, cartilage and cornea repair and the like.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing the results of a fluorescent quantitative determination of jellyfish cell DNA residues in a test example of the present invention;
FIG. 2 is a graph of imaging results of HE stained tissue section test to remove jellyfish cells in a test case of the present invention;
FIG. 3 is a graph showing SEM imaging results of the ECM structure of jellyfish after decellularization by various methods in the test examples of the present invention;
FIG. 4 is a graph showing the results of imaging after staining of Collagen Hybrid Peptide (CHP) for testing the structural integrity of collagen molecules in tissues according to the test examples of the present invention;
FIG. 5 is a graph showing the digital quantification of fluorescent signals in an image scan after CHP staining in a test example of the present invention;
FIG. 6 is a graph showing the results of evaluation of cell activity of a three-dimensional mammalian cell cultured from a decellularized jellyfish tissue material in a test example of the present invention;
FIG. 7 is a graph showing the results of experiments for repairing a full-thickness injury of rat skin using a decellularized jellyfish tissue material in a test example of the present invention;
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
EXAMPLE 1 preparation of jellyfish cell-free Material
The embodiment prepares the jellyfish cell removal material, which comprises the following specific processes:
1. jellyfish pretreatment
Fresh jellyfish (edible jellyfish, rhopilema esculentum, from the pearl sea aquatic market) feeler was taken for the experiment. After removing impurities on the surface of jellyfish, the jellyfish is washed and subjected to salt replacement by ultrapure water. The liquid change is carried out once per hour, the shaking table is at 4 ℃ and the speed is 100rpm, and the liquid change is repeated for 5 times. Jellyfish tissue was then cut into 2cm×2cm pieces for subsequent decellularization.
2. Jellyfish tissues were subjected to decellularization treatment.
After washing the jellyfish tissue slices 3 times with PBS, the slices were continuously shaken with 0.1% -10% SD of decellularizing agent at room temperature for 5 days, shaking table, 100rpm, and changing the solution every 12 hours for 120 hours. Subsequently, after washing the decellularized jellyfish with PBS, the first three times for 10 minutes was changed to one time, followed by changing to one time every half hour (repeating 3 times), followed by washing overnight (12 hours), and the resulting jellyfish decellularized material was prepared.
3. The jellyfish cell-removed material is subjected to a crosslinking treatment.
The resulting jellyfish decellularized material was washed with ultrapure water, and 50mL of MES buffer solution (0.1M, pH=4.7) containing NHS (N-hydroxysuccinimide, 2.5 mg/mL) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 10 mg/mL) was reacted with the washed jellyfish decellularized tissue material at room temperature for 12 hours to carry out chemical crosslinking. Subsequently, the crosslinked jellyfish material was washed with ultrapure water, washed in an ultrasonic cleaner for 30 minutes, and washed 3 times to remove the crosslinking agent. And then dehydrating with 30%, 50%, 70% and 100% tertiary butanol for 30 minutes, and freeze-drying to obtain the cross-linked decellularized jellyfish tissue material.
Example 2
This example prepared a jellyfish decellularized material, differing from example 1 in that 0.1% -10% sd of the decellularizing agent was replaced with 0.1% -10% SDS.
Example 3
This example produced a jellyfish decellularized material that differed from example 1 in that 0.1% -10% sd of the decellularizing agent was replaced with 0.1% -10% snl.
Example 4
This example produced a jellyfish decellularized material that differed from example 1 in that 0.1% -10% sd of the decellularizing agent was replaced with 0.1% -10% chaps.
Example 5
This example produced a jellyfish decellularized material that differed from example 1 in that 0.1% -10% SD of the decellularizing agent was replaced with 0.1% -10% IGEPAL CA-630.
Example 6
This example prepared a jellyfish decellularized material, differing from example 1 in that 0.1% -10% sd of the decellularizing agent was replaced with 0.1% -10% tween-20.
Test examples
This test example tests the performance of jellyfish decellularized material prepared in examples 1-6 and crosslinked decellularized jellyfish tissue material prepared in examples 1-6 (5% SD, 1% SDS, 1% SNL, 2% CHAPS, 5% IGEPAL CA-630 and 5% Tween-20 were used as the decellularizing agent in examples 1-6, respectively).
1. DNA detection
To evaluate whether DNA remains in the jellyfish after decellularization, the total DNA content of the jellyfish decellularized material and the natural jellyfish prepared in examples 1 to 6 was measured. Briefly, the jellyfish cell-removed material samples prepared in examples 1 to 6 were first freeze-dried, and the samples were weighed (dry weight 5 mg) after freeze-drying. The DNA in the sample was then extracted using a DNA extraction kit (purchased from Shanghai Biotechnology Co., ltd.) and the residual DNA content was determined using a DNA micro-fluorescence detection kit.
The results of the fluorescent quantitative detection of the residual DNA in the decellularization process are shown in FIG. 1, and it can be seen from the figure that 5% SD, 1% SNL and 1% SDS are preferable for decellularization.
2. HE-stained tissue sections of decellularized post-treated jellyfish
The natural jellyfish and the jellyfish cell removal materials prepared in examples 1-6 are placed into a dehydration frame to be marked, and the tissue is fully washed by running water to remove the paraformaldehyde fixing solution. And dehydrating the tissue by using alcohol from low concentration to high concentration, and carrying out transparency, wax dipping, tissue embedding and slicing on the dehydrated tissue. Paraffin sections were taken for conventional dewaxing to water and tissues were completely immersed in hematoxylin for 3-5min until nuclei were stained evident. And fully washing the slices in PBS buffer solution, performing eosin counterstaining, observing the dyeing degree under a mirror, and sealing the slices with neutral resin after the slices are dried.
HE stained tissue sections of decellularized post-treated jellyfish were scanned using an EVOS fluorescence microscope (40 Xobjective, light cube: BF for HE staining imaging). As a result, as shown in FIG. 2, it was revealed that jellyfish cells from 5% SD, 2% CHAPS, 1% SDS, and 5% IGEPAL groups were cellular, and that no nuclei were found in jellyfish treated with the above-mentioned single detergent, and a cell-free porous structure was exhibited, as compared with the natural tissue.
3. SEM imaging
The cross-linked decellularized jellyfish tissue material samples and natural jellyfish cell samples prepared in examples 1 to 6 were dehydrated using gradient t-butanol (30%, 50%, 70%, 100%) and then lyophilized. A small piece of jellyfish ECM was cut and fixed with conductive adhesive. And (5) performing metal spraying on the sample by using a vacuum sputtering device. And (3) scanning and observing the surface morphology and the pore size of the prepared jellyfish ECM by using a scanning electron microscope, and comparing the morphology change and the surface morphology of the jellyfish collagen fiber after the natural jellyfish tissue and various reagents are decellularized.
SEM imaging results of the surface, cross section and interior of jellyfish after cell removal treatment in different examples are shown in fig. 3, and it can be seen from the graph that after cell removal, collagen fibers of 1% SNL group and 1% SDS group are thickened and entangled, fiber structure arrangement is disordered, and collagen fiber shape is distorted. Jellyfish ECM collagen fiber after 5% SD, 2% CHAPS, 5% Tween and 5% IGEPAL are similar to natural jellyfish in shape and structure, dense in collagen fiber, regular in shape and cellular and net-shaped pore structure.
4. CHP staining
Collagen is used as the protein with the highest content in ECM and has a special triple helix structure, and once collagen molecules are destroyed in the cell removal process, the original triple helix structure is lost to be in a single-chain state, and collagen hybrid peptide (Collagen hybridization peptide, CHP, the sequence of which is Cy5-Ahx- (GPO) is designed and synthesized in the early stage 9 Cy5 is the fluorescent dye sulfocyanine 5, brand: lumiprobe number: 63320, ahx is aminocaproic acid, G is glycine, P is proline, O is hydroxyproline) which is able to specifically target the binding of these structurally disrupted collagen molecules, thus allowing the alteration of ECM structure from direct examination of decellularization.
After washing the natural jellyfish cell-removed material (jellyfish cell-removed material prepared in examples 1 to 6) with PBS, dehydration was performed in 30% sucrose for 12 hours at 4 ℃, shaking at 100rpm,30% sucrose: OCT was replaced at 1:1 ratio for 12h, then embedded with OCT and frozen, cut into 8 μm thick sections, and adhered to a slide. The OCT complex was removed by three rounds of washing with 1 x PBS for 5 minutes each before CHP staining. Since CHP can self-assemble into trimers in solution when stored at 4 ℃, and cannot hybridize with the unwound collagen chains, CHP needs to be thermally dissociated into monomers by heating immediately prior to use in order to stain denatured collagen in tissue sections. A15. Mu.M solution of Cy5 fluorescent-labeled CHP in 1 XPBS was heated in a 80℃water bath for 10min and immediately cooled to room temperature in an ice/water bath. 100 μL of quenched Cy5-Ahx- (GPO) 9 The solution was added rapidly to each tissue section. After overnight incubation at 4 ℃, three washes with PBS (1×) were followed by blocking with anti-fluorescence quenchers.
Frozen tissue sections after decellularization and CHP staining were scanned using an EVOS fluorescence microscope (20-fold objective, cy5 light cube for CHP fluorescence imaging and scanning).
The experimental results are shown in fig. 4-5, where it can be seen that 1% SDs has the greatest effect on the unwinding of the collagen structure of the descaled jellyfish, and that 5% IGEPAL, 2% chaps and 5% SD have much less effect on the destruction of the collagen structure of the descaled jellyfish than 1% SDs, with 5% SD having no detectable damage to the molecular structure of the descaled jellyfish collagen.
5. Construction of jellyfish scaffold/cell culture System
The cross-linked decellularized jellyfish tissue material (1.5 mg) of the system mass (the cross-linked decellularized jellyfish tissue material prepared in example 1 and example 2, respectively) was weighed into a 96-well plate, and ultraviolet sterilization was performed on a cell table for 60min. mu.L of the cell suspension to be cultured (human dermal fibroblast H103, human cornea cell HCF and mouse knee joint chondrocyte) were all purchased from scientific cell reagent (KWH) 1×10 by a pipette 4 Cells/scaffolds) were directly inoculated onto the decellularized jellyfish scaffold prepared in example 1. The cell-loaded jellyfish scaffold was then placed in a cell incubator (37 ℃,5% CO) 2 ) The cells were allowed to adhere to the scaffolds by incubation for 2h, then 200 μl fresh medium was added for 3 days. The medium was subjected to liquid exchange every two days. Equivalent amounts of mammalian cell suspension directly cultured in plastic 96-well plates without jellyfish cell-free scaffolds were used as blank controls. The system was operated at 37℃with 5% CO 2 Hatching in the humidified atmosphere of the jellyfish stent/cell culture system is constructed.
6. Cell activity assay
After human dermal fibroblasts H103 (purchased from Punuocele, cat. No. CP-H103), human keratocytes HCF (purchased from Start, cat. No. CD 5024) and mouse knee chondrocytes chondryte (purchased from Punuocele, cat. No. CP-M087) were respectively implanted into the jellyfish scaffold/cell culture system prepared in step 5, cell viability and proliferation on the jellyfish scaffold were evaluated using a CCK8 detection kit. Briefly, fresh medium containing 10% CCK8 reagent was first prepared, then after the original medium was aspirated, 150. Mu.L of fresh medium containing 10% CCK-8 solution was added to each well, incubated for 3 hours at 37℃in an incubator with 5% carbon dioxide, and the Optical Density (OD) value of the scaffolds in solution was measured at 450nm using an enzyme-labeled instrument to evaluate cell activity.
The experimental results of CCK8 activity evaluation of three cells in jellyfish ECM scaffolds after different decellularization methods are shown in fig. 6, from which it can be seen that the activity of cells cultured with jellyfish matrix with 5% SD removal of cells is significantly higher than that of a cell blank control group (No martrix) without providing jellyfish matrix directly cultured in an orifice plate.
7. Animal experiment
Male SPF-grade pathogen-free SD rats were purchased from the animal laboratory center in Guangdong province for 12, 6-8 weeks, 200-250 g. The rats were divided into 2 groups, 5% SD group (cross-linked cell-free jellyfish tissue material group prepared in example 1) and control group, respectively, and were individually placed under standard temperature and humidity conditions with an illumination/darkness cycle of 12 hours, and were free to obtain food and water. The scaffolds were cut to a size of 1cm by 1cm prior to implantation. Gas anesthesia was first performed with 3% isoflurane, and 12 SD rats (male, 200-250 g) were each with 4 1cm diameter long incisions made in the backs of the rats. After sterilization by iodophor shaving, 4 circular full-thickness skin defects 10mm in diameter were cut into the dorsal side of each rat using a punch (10 mm. Times.10 mm). The wound was covered with decellularized jellyfish ECM material and rats without any treatment of the wound served as a blank. All wounds were then wrapped with a clear film and gauze. Wound observation photographing is carried out on the 3 rd, 7 th and 14 th days after operation.
The experimental results are shown in fig. 7, and it can be seen from the graph that the healing observations of the 5% SD group and the control group on days 0,3,7, and 14 in the full-thickness injury of the rats show that the healing process of the 5% SD group rat skin is faster than that of the control group. On the third day, when the control group had an inflammatory phase in which interstitial fluid oozes, the 5% SD group had begun to crusted up, and the result shows that the decellularized jellyfish ECM material prepared in this example can accelerate wound healing.
The intact collagen structure is very important for the normal function of the ECM and cells can easily recognize the denatured triple helix of collagen. Many cellular receptors, such as DDR1, DDR2 and integrins α2β1, α1β1, α10β1 and α11β1, bind only to sites on the triple helical collagen chain and not to the denatured collagen chain. These receptors regulate basic cellular processes including proliferation, adhesion and migration; on the other hand, denatured collagen may expose hidden cell binding sites that are inactive in the triple helix conformation, such as multiple RGD polypeptides that can be recognized by specialized subgroups of integrins. Thus, the unwinding of the collagen triple helix will result in a change in cell behavior in the ECM.
The jellyfish cell removing material has the advantages of wide sources, processed and safe properties, high bioactivity and compatibility and wide application range.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A method of preparing jellyfish decellularized material, comprising the steps of: performing decellularization treatment on jellyfish tissues by adopting a decellularization agent; the decellularizing agent includes a surfactant.
2. The method of preparation according to claim 1, wherein the surfactant comprises an ionic surfactant and/or a nonionic surfactant; preferably, the ionic surfactant is at least one of SDS, SNL, SD, CHAPS, SB-10 and CH; preferably, the nonionic surfactant is at least one of Triton, IGEPAL CA-630, tween, DDM, nonidet P40 and Digitin.
3. The method of claim 2, wherein the ionic surfactant is at least one of 0.1% to 10% sds, 0.1% to 10% snl, 0.1% to 10% sd, 0.1% to 10% chaps, 0.1% to 10% sb-10, and 0.1% to 10% ch; the nonionic surfactant is at least one of 0.1 to 10 percent of Triton, 0.1 to 10 percent of IGEPAL CA-630, 0.1 to 10 percent of Tween, 0.1 to 10 percent of DDM, 0.1 to 10 percent of Nonidet P40 and 0.1 to 10 percent of digitin; preferably, the ionic surfactant is at least one of 0.1% to 10% sd, 0.1% to 10% snl, and 0.1% to 10% chaps; preferably, the nonionic surfactant is at least one of 0.1% to 10% triton, 0.1% to 10% igepal CA-630 and 0.1% to 10% tween.
4. The method according to claim 1, wherein the treatment time of the decellularizing agent is 0.02 to 10d; preferably, the treatment time of the decellularization reagent is 0.5-8 d.
5. The method according to claim 1, further comprising a step of crosslinking the jellyfish tissue after the decellularization agent treatment; preferably, the crosslinking agent used in the crosslinking treatment contains a chemically reactive group that can react with at least one of an amino group, a mercapto group, a carboxyl group, an aldehyde group, and a hydroxyl group; more preferably, the chemically reactive group of the crosslinking agent that reacts with the amino group is at least one of NHS ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine, and aldehyde group; the chemical reaction group of the crosslinking agent and the mercapto reaction is at least one of maleimide, haloacetyl, pyridine dimercapto, thiosulfate and vinyl sulfone; the chemical reaction group of the crosslinking agent and the carboxyl is carbodiimide; at least one of a chemically reactive group of a hydrazide, an alkoxyamine, and an NHS ester, in which the crosslinking agent reacts with an aldehyde group; the chemical reaction group of the crosslinking agent and the hydroxyl is isocyanate; more preferably, the time of the crosslinking treatment is 4 to 20 hours.
6. The method of claim 1, wherein the jellyfish is at least one of jellyfish, cubic jellyfish, crown jellyfish, cross jellyfish, flag jellyfish, and root jellyfish, preferably wherein the jellyfish is at least one of jellyfish, bamboo hat jellyfish, purple sea thorn jellyfish, mediterranean jellyfish, black warrior jellyfish, blue jellyfish, silver coin jellyfish, alert jellyfish, dilemma jellyfish, s jellyfish, white spot jellyfish, bried river jellyfish, lighthouse jellyfish, nephelia fimbriae, and sea month jellyfish.
7. Jellyfish decellularized material prepared by the preparation method according to any one of claims 1 to 6.
8. Use of the jellyfish decellularized material of claim 7 in any one of the following (1) - (4):
(1) Preparing a scaffold biological material;
(2) A product that supports growth of animal cells;
(3) Preparing a product for promoting wound repair;
(4) A product is prepared that promotes tissue regeneration.
9. A biological scaffold material comprising the jellyfish decellularized material of claim 7.
10. Use of the biological scaffold material of claim 9 in any one of the following (1) - (6):
(1) Preparing a product supporting the growth of animal cells;
(2) Preparing a product for promoting wound repair;
(3) Preparing a structural implant for cosmetic surgery;
(4) Preparing a product for promoting angiogenesis;
(5) Preparing a tissue replacement;
(6) Preparing a product for promoting tissue regeneration; preferably, the tissue comprises skin, bone, cartilage, spinal cord, muscle, nerve, blood vessel, oral cavity, cornea or other damaged or malformed tissue.
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| CN202211326288.2A CN116763990A (en) | 2022-10-25 | 2022-10-25 | Jellyfish cell removing material and preparation method and application thereof |
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| CN202211326288.2A CN116763990A (en) | 2022-10-25 | 2022-10-25 | Jellyfish cell removing material and preparation method and application thereof |
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