CN104491927A - Decellularized tongue matrix material and preparation method thereof - Google Patents
Decellularized tongue matrix material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a decellularized tongue matrix material and a preparation method thereof. The decellularized tongue matrix material is characterized in that tongue tissues of healthy animals are selected for physical, chemical and enzyme comprehensive method processing; lower osmatic solution, high osmatic solution, Triton X-100 and nuclease low-temperature processing is carried out to prepare the decellularized tongue matrix material; and the decellularized tongue matrix material with the good biocompatibility and the appropriate biodegradation rate is obtained. The material can be used for tongue regeneration and repairing and tongue disease studying models. The material is wide in source, easy to obtain, low in price and easy to manufacture.
Description
Technical field
This belongs to and the manufacture field of bio-medical material, is specifically related to a kind of de-cell tongue host material and preparation method thereof.
Background technology
Extracellular matrix (extracellular matrix, ECM) is to the protein in the mesenchyme of extracellular and polysaccharide macromolecular material by emiocytosis.ECM, by highly organizing orderly extracellular microenvironment, forms complicated rack conjunctive tissue structure, has crucial regulating action to the cellular physiological events such as existence, differentiation of cell.Therefore, the ideal carrier that the support substrate of ECM and timbering material are Tissue Engineering Study and tissue repair can be simulated.The contact of cell and ECM is dynamic, and the change of ECM will cause the change of adjacent cells morphology and function.For organizational project ideal stent material require for plantation cell the microenvironment (comprise the composition of ECM, structure and biomechanical characterization etc.) identical or similar with ECM in body is provided.But, natural ECM is made up of (being mainly divided into collagen, glycoprotein, aminoglycan and Dan Baiduotang proteoglycan PG, the large class of elastin laminin 4) multiple proteins, and have complicated structure, not easily reconstruct and ECM in body form identical and that structure is consistent ECM material in vitro.Going cellular processes from the ECM organized and Organ procurement is natural by different chemical, physics or zymetology, providing new effective solution route for solving this difficult problem.Decellularization substrate has ECM protein and the micro structure of tissue or cell-specific, has good mechanical characteristic, cell compatibility and biological degradability simultaneously, is conducive to cell adhesion, growth, proliferation and growth.
The method preparing acellular matrix material has a lot, mainly by the method for physics, chemistry and enzyme integrated treatment.Chinese Patent Application No. be 201110134511.9 patent of invention relate to a kind of method preparing acellular dermal matrix material, although cell can be removed more up hill and dale and the processing time short, have employed the process of sodium lauryl sulphate (SDS) in preparation process.Due to the strong elute effect of SDS, destroy cytostromatic structural intergrity and biological activity to a great extent, the extracellular matrix using SDS to prepare in addition has potential bio-toxicity, is unfavorable for that this host material is rebuild the three dimensional structure of tissue.Chinese Patent Application No. be 201210237911.7 patent of invention relate to the method for removing cells preparing a kind of extracellular matrix support material.Although have employed pyranglucoside to reduce the potential source biomolecule toxicity that detergent may cause repopulating cell in cleaning process, but the tissue be mainly used in containing more collagen fiber and elastic fibers and organ, be applied to muscle fiber and enrich and to be not exclusively suitable in the tongue tissue of prosperity.
Summary of the invention
The object of the invention is the deficiency in order to overcome above technology, healthy animal tongue is adopted to be organized as raw material, the chemical substances such as applying composite enzyme inorganic agent and alkali, provide a kind of de-cell tongue host material with good biocompatibility and suitable biodegradation rate.
The present invention is realized by following technical scheme:
(1) choose healthy animal tongue tissue, be cut into the fritter of 1 centimeter square, wash away bloodstain with PBS buffer for subsequent use;
(2) tongue is organized multigelation 3 times, smudge cells;
(3) tongue piece of tissue is placed in 75% ethanol to sterilize;
(4) tongue piece of tissue is suspended from aseptic tertiary effluent, 4 DEG C of moderate-speed mixer process 12 hours;
(5) tongue chunk is knitted be suspended from 1M NaCl sterile solution, 4 DEG C of moderate-speed mixer process 24 hours;
(6) tongue piece of tissue is suspended from the sterile buffer of 2%TritonX-100 molten in, 4 DEG C of moderate-speed mixer process 48 hours;
(7) tongue piece of tissue is suspended from 5mM CaCl
2with 5mM MgCl
2in sterile solution, 4 DEG C of moderate-speed mixer process 24 hours;
(8) tongue piece of tissue transferred in the EP pipe of the aseptic D-Hanks buffer containing DNase I, 37 DEG C process 24 hours;
(9) tongue piece of tissue be suspended from aseptic PBS buffer solution, 4 DEG C of moderate-speed mixer process 24 hours, obtain described de-cell tongue host material.
Further, choose in described step (1) be organized as fresh virus-free infection, without the animals tongue of medical history.
Further, in described step (2), the method for smudge cells is taken out for piece of tissue being put into-80 DEG C after freezing 30 minutes, thaws 37 DEG C of water-baths, multigelation like this 3 times.
Further, in described step (6), sterile buffer can select the Tris-HCl buffer of PBS buffer or pH=7.4.
Further, described de-cell tongue host material can be positioned in the PBS containing 90 μ g/ml ammonia benzyls, preserves more than 2 months for 4 DEG C.
The present invention also comprises the de-cell tongue host material prepared according to the method for above-mentioned any one.
The present invention takes off all operations in the preparation method of cell tongue host material and all carries out in aseptic superclean bench.
Adopt the de-cell tongue host material prepared by this method, the requirement of following medical application and medical research can be met:
1, as the succedaneum of tongue, for the reparation that Post operation tongue damages and lacks.
2, for the reparation of tongue ulcer, the treatment because of ill fitting prosthesis and the tongue ulcer caused of biting is particularly useful for.
3, may be used for department of stomatology anaplasty, as the reparation etc. of cleft palate.
4, can be used as cell culture and organizational project external model material, as the research etc. of growing research for tongue regeneration and tongue, carcinoma of tongue occurs and infiltrates.
The present invention has the following advantages:
1, under maintenance animals tongue three-dimensional tissue constructs constant prerequisite, thoroughly remove the cell component in animal tongue, obtain the tongue substrate with complete three-dimensional tissue's structure.
2, have good mechanical property, can plastic property, good cell differentiation propagation environment and good adhesion property;
3, method is easy, easy, economical, reasonable, safe, reliable.
4, whole technical process belongs to and cleans preparation, environmentally safe.
Accompanying drawing explanation
Fig. 1 a is the HE dyeing microphotograph of the fresh mice tongue tissue containing cell, and Fig. 1 b is the HE dyeing microphotograph of the mice tongue cell epimatrix material after de-cell process.
Fig. 2 a is the electron scanning micrograph of the fresh mice tongue tissue containing cell, and Fig. 2 b is the electron scanning micrograph of the mice tongue cell epimatrix material after de-cell process.
Fig. 3 is that after fresh mice tongue tissue DNA content and de-cell process, mice tongue organizes the DNA content block diagram in ECM.
Fig. 4 a is Collagen I ImmunohistochemistryResults Results of the fresh mice tongue tissue containing cell, and Fig. 4 b is Collagen I ImmunohistochemistryResults Results of the mice tongue cell epimatrix material after de-cell process.
Fig. 5 a is Collagen IV ImmunohistochemistryResults Results of the fresh mice tongue tissue containing cell, and Fig. 5 b is Collagen IV ImmunohistochemistryResults Results of the mice tongue cell epimatrix material after de-cell process.
Fig. 6 a is the Fibronectin ImmunohistochemistryResults Results of the fresh mice tongue tissue containing cell, and Fig. 6 b is the Fibronectin ImmunohistochemistryResults Results of the mice tongue cell epimatrix material after de-cell process.
Fig. 7 a is the Laminin ImmunohistochemistryResults Results of the fresh mice tongue tissue containing cell, and Fig. 7 b is the Laminin ImmunohistochemistryResults Results of the mice tongue cell epimatrix material after de-cell process.
Fig. 8 a is that the HE of fresh pig tongue tissue containing cell dyes microphotograph (× 100), and Fig. 8 b is that the HE of pig tongue cell epimatrix material after de-cell process dyes microphotograph (× 100).
Fig. 9 is the electron scanning micrograph of the pig tongue extracellular matrix after de-cell process.
Figure 10 is that after fresh pig tongue tissue DNA content and de-cell process, pig tongue organizes the DNA content block diagram in ECM.
Figure 11 a is the HE dyeing microphotograph of the fresh rat tongue tissue containing cell, and Figure 11 b is HE dyeing microphotograph (× 100) of the Rat Tongue extracellular matrix after de-cell process.
Figure 12 is the electron scanning micrograph of the Rat Tongue cell epimatrix material after de-cell process.
Figure 13 is that after fresh rat tongue tissue DNA content and de-cell process, Rat Tongue organizes the DNA content block diagram in ECM.
Figure 14 a and Figure 14 b is the HE dyeing microphotograph of mice tongue ECM material after injection tongue cancer cells Cal27 cultivates 20 days.Wherein Figure 14 b (400 ×) partial enlarged drawing that is Figure 14 a (100 ×).
Figure 15 a and Figure 15 b is the HE dyeing microphotograph of skin model after inoculation tongue cancer cells Cal27 cultivates 14 days utilizing type i collagen to make.Wherein Figure 15 b (400 ×) partial enlarged drawing that is Figure 15 a (100 ×).
Detailed description of the invention
Below by embodiment, the present invention is specifically described.
The making of embodiment 1 mice tongue ECM
(1) draw materials: the tongue tissue choosing healthy mice, clean both sides with PBS and remove bloodstain.For subsequent use as-80 DEG C of refrigerators in EP pipe.
(2) smudge cells: piece of tissue is taken out from-80 DEG C of refrigerators and puts back to-80 DEG C of refrigerators more freezing 30 minutes after 37 DEG C of water-baths thaw, multigelation like this 3 times.
(3) sterilization and preparation: piece of tissue as in the ethanol of 75%, with stitching thread, tongue piece of tissue is sling, hang in the wide mouthed bottle of 250ml, and place the magnetic stirring bar of one piece of 1cm at the bottom of bottle.
(4) prerinse and fragmentation: the aseptic tertiary effluent adding 250ml, as (gear 2) on magnetic stirring apparatus 4 DEG C process 12 hours.
(5) cell debris is cleaned: changed to by tongue tissue in the small-sized agitation cycle device that 250ml 1M NaCl sterile solution is housed.4 DEG C process 24 hours.
(6) broken and clean: tongue tissue to be changed in the small-sized agitation cycle device of the aseptic PBS buffer solution that 250ml 2%TritonX-100 is housed.4 DEG C process 48 hours.
(7) clean and strengthen enzyme work: tongue tissue being changed to 250ml 5mM CaCl is housed
2with 5mM MgCl
2in the small-sized agitation cycle device of sterile solution.4 DEG C process 24 hours.
(8) remove residual nucleic acid: transferred to by tongue tissue in the EP pipe of the aseptic D-Hanks buffer of 1ml of the DNase I (sigma) containing 300 units, 37 DEG C process 24 hours.
(9) clean: changed to by tongue tissue in the small-sized agitation cycle device that 250ml aseptic PBS buffer solution is housed, 4 DEG C process 24 hours.
Evaluation of result
1, ECM constructed observation: ECM 10% neutral formalin is fixed, is dewatered, paraffin embedding, is cut into 0.5 μm of thick thin slice, through dewaxing dehydration, Hematoxylin-eosin (HE) dyes, observe ECM structure, its HE dyeing microphotograph is shown in Fig. 1 b, make the HE dyeing of fresh mice tongue tissue, its microphotograph is shown in Fig. 1 a simultaneously.Mice ECM structure prepared by visible the inventive method, confirms that no cell structure remains, and extra-cellular matrix structure keeps complete.
2, ECM Ultrastructural observation: ECM tissue is dry with critical point drying instrument, gold-plated, sem observation, Fig. 2 b is shown in by shooting electron micrograph, takes fresh mice tongue organizing electronic micro mirror photo simultaneously and sees Fig. 2 a.Contrast two figure as seen by mice ECM structure prepared by the inventive method, intactly can slough cellularity, and remain the fibre structure of extra-cellular matrix structure more in good condition.
3, in ECM, DNA content detects: after being ground by ECM tissue, DNA is extracted with DNeasy Blood & Tissue Kit (QIAGEN) cracking digestion, and measure DNA content with Nano-drop (Thermo), and contrast with fresh mice tongue tissue DNA content, the results are shown in Figure 3.In the mice ECM that visible the inventive method is obtained, DNA content decreases 90.98%, shows that successfully eliminating most nucleic acid remains.
4, in ECM, the SABC of Collagen I albumen detects: ECM 10% neutral formalin is fixed, dewater, paraffin embedding, be cut into 0.5 μm of thick thin slice, through dewaxing dehydration, remove peroxidase, antigen retrieval, lowlenthal serum is closed, and hatches CollagenI antibody (dilution ratio 1:500) (abcam) 4 DEG C and spends the night, hatch two anti-room temperatures 1 hour, DAB develops the color, and mounting is observed, and shooting microphotograph as shown in Figure 4 b; Take the Collagen I SABC microphotograph 4a of fresh mice tongue tissue in contrast simultaneously.
5, in ECM, the SABC of Collagen IV albumen detects: ECM 10% neutral formalin is fixed, dewater, paraffin embedding, be cut into 0.5 μm of thick thin slice, through dewaxing dehydration, remove peroxidase, antigen retrieval, lowlenthal serum is closed, and hatches Collagen IV antibody (dilution ratio 1:300) (abcam) 4 DEG C and spends the night, two anti-incubated at room 1 hour, DAB develops the color, and mounting observes shooting microphotograph as shown in Figure 5 b; Take the Collagen IV SABC microphotograph 5a of fresh mice tongue tissue in contrast simultaneously.
6, in ECM, the SABC of laminin albumen detects: ECM 10% neutral formalin is fixed, dewater, paraffin embedding, be cut into 0.5 μm of thick thin slice, through dewaxing dehydration, remove peroxidase, antigen retrieval, lowlenthal serum is closed, and hatches laminin antibody (dilution ratio 1:200) (abcam) 4 DEG C and spends the night, two anti-incubated at room 1 hour, DAB develops the color, and mounting is observed, and shooting microphotograph as shown in Figure 6 b; Take the Laminin SABC microphotograph 6a of fresh mice tongue tissue in contrast simultaneously.
7, in ECM, the SABC of fibronectin albumen detects: ECM 10% neutral formalin is fixed, dewater, paraffin embedding, be cut into 0.5 μm of thick thin slice, through dewaxing dehydration, remove peroxidase, antigen retrieval, lowlenthal serum is closed, and hatches fibronectin antibody (dilution ratio 1:250) (abcam) 4 DEG C and spends the night, two anti-incubated at room 1 hour, DAB develops the color, and mounting is observed, and shooting microphotograph as shown in Figure 7b; Take the fibronectin SABC microphotograph 7a of fresh mice tongue tissue in contrast simultaneously.
Embodiment 2, the making of pig tongue ECM
(1) draw materials: the tongue tissue choosing healthy pig, is decomposed into the fritter of a centimeter square, clean both sides with PBS and remove bloodstain.For subsequent use as-80 DEG C of refrigerators in EP pipe.
(2) smudge cells: piece of tissue is taken out from-80 DEG C of refrigerators and puts back to-80 DEG C of refrigerators more freezing 30 minutes after 37 DEG C of water-baths thaw, multigelation like this 3 times.
(3) sterilization and preparation: piece of tissue as in the ethanol of 75%, with stitching thread, tongue piece of tissue is sling, hang in the wide mouthed bottle of 250ml, and place the magnetic stirring bar of one piece of 1cm at the bottom of bottle.
(4) prerinse and fragmentation: the aseptic tertiary effluent adding 250ml, as (gear 2) on magnetic stirring apparatus 4 DEG C process 12 hours.
(5) cell debris is cleaned: changed to by tongue tissue in the small-sized agitation cycle device that 250ml 1M NaCl sterile solution is housed.4 DEG C process 24 hours.
(6) broken and clean: tongue tissue to be changed in the small-sized agitation cycle device of the aseptic PBS buffer solution that 250ml 2%TritonX-100 is housed.4 DEG C process 48 hours.
(7) clean and strengthen enzyme work: tongue tissue being changed to 250ml 5mM CaCl is housed
2with 5mM MgCl
2in the small-sized agitation cycle device of sterile solution.4 DEG C process 24 hours.
(8) remove residual nucleic acid: transferred to by tongue tissue in the EP pipe of the aseptic D-Hanks buffer of 1ml of the DNase I (sigma) containing 300 units, 37 DEG C process 24 hours.
(9) clean: tongue tissue is changed in the small-sized agitation cycle device that 250ml aseptic PBS buffer solution is housed.4 DEG C process 48 hours.
Evaluation of result
1, ECM constructed observation: ECM 10% neutral formalin is fixed, is dewatered, paraffin embedding, is cut into 0.5 μm of thick thin slice, through dewaxing dehydration, HE dyes, and its HE dyeing microphotograph is shown in Fig. 8 b, make the HE dyeing of fresh pig tongue tissue, its microphotograph is shown in Fig. 8 a simultaneously.In pig ECM structure prepared by visible the inventive method, no cell structure remains, and extra-cellular matrix structure keeps complete.
2, ECM Ultrastructural observation: ECM tissue is dry with critical point drying instrument, gold-plated, sem observation, Fig. 9 is shown in by shooting electron micrograph.Pig ECM structure prepared by visible the inventive method, intactly can slough cellularity, and remain the fibre structure of extra-cellular matrix structure more in good condition.
3, in ECM, DNA content detects: after being ground by ECM tissue, DNA is extracted with DNeasy Blood & Tissue Kit (QIAGEN) cracking digestion, and measure DNA content with Nano-adrop (Thermo), and contrast with fresh mice tongue tissue DNA content, the results are shown in Figure 10.In pig ECM prepared by visible the inventive method, DNA content reduces 97.17%, shows that successfully eliminating most nucleic acid remains.
Embodiment 3, the making of Rat Tongue ECM
(1) draw materials: the tongue tissue choosing healthy rat, is decomposed into the fritter of a centimeter square, clean both sides with PBS and remove bloodstain.For subsequent use as-80 DEG C of refrigerators in EP pipe.
(2) smudge cells: piece of tissue is taken out from-80 DEG C of refrigerators and puts back to-80 DEG C of refrigerators more freezing 30 minutes after 37 DEG C of water-baths thaw, multigelation like this 3 times.
(3) sterilization and preparation: piece of tissue as in the ethanol of 70%, with stitching thread, tongue piece of tissue is sling, hang in the wide mouthed bottle of 250ml, and place the magnetic stirring bar of one piece of 1cm at the bottom of bottle.
(4) prerinse and fragmentation: the aseptic tertiary effluent adding 250ml, as (gear 2) on magnetic stirring apparatus 4 DEG C process 12 hours.
(5) cell debris is cleaned: changed to by tongue tissue in the small-sized agitation cycle device that 250ml 1M NaCl sterile solution is housed.4 DEG C process 24 hours.
(6) broken and clean: tongue tissue to be changed in the small-sized agitation cycle device of the aseptic PBS buffer solution that 250ml 2%TritonX-100 is housed.4 DEG C process 24 hours.
(7) clean and strengthen enzyme work: tongue tissue being changed to 250ml 5mM CaCl is housed
2with 5mM MgCl
2in the small-sized agitation cycle device of sterile solution.4 DEG C process 24 hours.
(8) remove residual nucleic acid: transferred to by tongue tissue in the EP pipe of the aseptic D-Hanks buffer of 1ml of the DNase I (sigma) containing 300 units, 37 DEG C process 24 hours.
(9) clean: tongue tissue is changed in the small-sized agitation cycle device that 250ml aseptic PBS buffer solution is housed.4 DEG C process 24 hours.
Evaluation of result
1, ECM constructed observation: ECM 10% neutral formalin is fixed, is dewatered, paraffin embedding, is cut into 0.5 μm of thick thin slice, through dewaxing dehydration, HE dyes, observe its HE of ECM structure dyeing microphotograph and see Figure 11 b, make the HE dyeing of fresh rat tongue tissue, its microphotograph is shown in Figure 11 a simultaneously.In rat ECM structure prepared by visible the inventive method, no cell structure remains, and extra-cellular matrix structure keeps complete.
2, ECM Ultrastructural observation: ECM tissue is dry with critical point drying instrument, and gold-plated, sem observation, is shown in Figure 12.Rat ECM structure prepared by visible the inventive method, intactly can slough cellularity, and remain the fibre structure of extra-cellular matrix structure more in good condition.
3, in ECM, DNA content detects: after being ground by ECM tissue, DNA is extracted with DNeasy Blood & Tissue Kit (QIAGEN) cracking digestion, and measure DNA content with Nano-adrop (Thermo), and contrast with fresh rat tongue tissue DNA content, the results are shown in Figure 13.In the rat ECM that visible the inventive method is obtained, DNA content reduces 98.93%, shows that successfully eliminating most nucleic acid remains.
Embodiment 4, mice tongue ECM is in Human Tongue Carcinoma Lines propagation and the application infiltrating research
(1) cell is injected: prepare 10
6cal27 cancer cell of oral cavity.With aseptic pin, tongue ECM tissue obtained for embodiment 1 is fixed on brand-new stencil plate in superclean bench.With alcohol burner, Pasteur's pipe is pulled the glass needle that bore is approximately about 100 μm, draw Cal27 cancer cell of oral cavity and inject ECM tissue.
(2) cultivate: the tongue ECM injecting Cal27 cancer cell of oral cavity is organized the Tissue Culture Dish put as the 3.5cm being added with 3mlDF+10%FBS culture fluid, culture dish is put in the CO of 37 DEG C
2incubator, regularly changes liquid, cultivates 20 days.
(3) tissue is fixed: organize the tongue ECM after cultivating as in 10% neutral formalin, fix 48 hours.
(4) film-making: fixing ECM tissue PBS buffer is washed 5 minutes, through automatic dehydrator dehydration, paraffin embedding, paraffin section, is cut into the thin slice of 5 μm, dries sheet and spends the night, 4 DEG C of preservations.
(5) HE dyeing: dewaxed by tissue slice dimethylbenzene, use haematoxylin dyeing 5 minutes after ethanol gradient rehydration, eosin stains 1 minute, ethanol serial dehydration, dimethylbenzene is changed thoroughly, and resinene mounting is observed.Figure 14 a and Figure 14 b is the HE dyeing microphotograph of mice tongue ECM material after injection Human Tongue Carcinoma Lines Cal27 cultivates 20 days.As can be seen from the figure Cal27 cell has very high multiplication capacity and tissue infiltration's property in mice tongue ECM material of the present invention.
Supplementing at this utilizes skin model to cultivate the method for tongue cancer cells, for tongue ECM materials control.
(1) foundation of skin model: will containing 1 × 10
5the RD culture fluid of the 10%FBS of cancer nests fibroblast (Cancer associated fibroblast, CAF) and acid collagen (collagen I) are got 2ml and are joined in a hole of 24 orifice plates after mixing homogeneously rapidly on ice.Continuous culture 1 week, allows CAF cellular contraction skin model.
(2) cultivate: Oxford cup is vertically positioned over above skin model, adds 1 × 10 wherein
6individual Cal27 cell.24 orifice plates are put in the CO of 37 DEG C
2incubator, cultivates with DF+10%FBS culture fluid, regularly changes liquid, cultivate 14 days.
(3) tissue is fixed: by the skin model after cultivation as in 10% neutral formalin, fix 48 hours.
(4) film-making: fixing skin model PBS buffer is washed 5 minutes, through automatic dehydrator dehydration, paraffin embedding, paraffin section, is cut into the thin slice of 5 μm, dries sheet and spends the night, 4 DEG C of preservations.
(5) HE dyeing: dewaxed by tissue slice dimethylbenzene, use haematoxylin dyeing 5 minutes after ethanol gradient rehydration, eosin stains 1 minute, ethanol serial dehydration, dimethylbenzene is changed thoroughly, and resinene mounting is observed.Figure 15 a and Figure 15 b is the HE dyeing microphotograph of skin model after inoculation Human Tongue Carcinoma Lines Cal27 cultivates 14 days.As can be seen from the figure Cal27 cell is mainly distributed in the surface of skin model, and cell is monolayer growth, does not show significantly propagation and infiltration phenomenon.
Above embodiment is only used to further illustrate the present invention, and can not be interpreted as limiting the scope of the invention, and the person skilled in the art in this field can make some nonessential improvement and adjustment according to foregoing invention content.
Claims (6)
1. a preparation method for de-cell tongue host material, comprises the following steps:
(1) choose healthy animal tongue tissue, be cut into the fritter of 1 centimeter square, wash away bloodstain with PBS buffer for subsequent use;
(2) by tongue piece of tissue multigelation 3 times, smudge cells;
(3) tongue piece of tissue is placed in 75% ethanol to sterilize;
(4) tongue piece of tissue is suspended from aseptic tertiary effluent, 4 DEG C of moderate-speed mixer process 12 hours;
(5) tongue chunk is knitted be suspended from 1M NaCl sterile solution, 4 DEG C of moderate-speed mixer process 24 hours;
(6) tongue piece of tissue is suspended from the sterile buffer of 2%TritonX-100 molten in, 4 DEG C of moderate-speed mixer process 48 hours;
(7) tongue piece of tissue is suspended from 5mM CaCl
2with 5mM MgCl
2in sterile solution, 4 DEG C of moderate-speed mixer process 24 hours;
(8) tongue piece of tissue transferred in the EP pipe of the aseptic D-Hanks buffer containing DNase I, 37 DEG C process 24 hours;
(9) tongue piece of tissue be suspended from aseptic PBS buffer solution, 4 DEG C of moderate-speed mixer process 24 hours, obtain described de-cell tongue host material.
2. the preparation method of de-cell tongue host material according to claim 1, is characterized in that: choose in described step (1) tongue be organized as fresh virus-free infection, without the animals tongue of medical history.
3. the preparation method of de-cell tongue host material according to claim 1, it is characterized in that: in described step (2), the method of smudge cells is taken out for piece of tissue being put into-80 DEG C after freezing 30 minutes, thaws 37 DEG C of water-baths, multigelation like this 3 times.
4. the preparation method of de-cell tongue host material according to claim 1, it is characterized in that: in described step (6), sterile buffer can select the Tris-HCl buffer of PBS buffer or pH=7.4.
5. the preparation method of de-cell tongue host material according to claim 1, is characterized in that: described de-cell tongue host material can be positioned in the PBS containing 90 μ g/ml ammonia benzyls, preserves more than 2 months for 4 DEG C.
6. according to the de-cell tongue host material that the method for Claims 1 to 5 any one prepares.
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| CN108992709A (en) * | 2018-07-27 | 2018-12-14 | 山东省眼科研究所 | A kind of acellular nerve host material and its preparation method and application |
| WO2021223674A1 (en) * | 2020-05-05 | 2021-11-11 | International Business Machines Corporation | Bioprinted living tissue with therapy capability |
| US11559389B2 (en) | 2020-05-05 | 2023-01-24 | International Business Machines Corporation | Bioprinted living tissue with therapy capability |
| GB2611437A (en) * | 2020-05-05 | 2023-04-05 | Ibm | Bioprinted living tissue with therapy capability |
| CN117563048A (en) * | 2023-11-13 | 2024-02-20 | 重庆医科大学附属口腔医院 | Preparation method and application of decellularized extracellular matrix tissue block |
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