CN103436489A - Reagent for acellular processing of animal skin tissue, and processing method thereof - Google Patents
Reagent for acellular processing of animal skin tissue, and processing method thereof Download PDFInfo
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- CN103436489A CN103436489A CN2013103894694A CN201310389469A CN103436489A CN 103436489 A CN103436489 A CN 103436489A CN 2013103894694 A CN2013103894694 A CN 2013103894694A CN 201310389469 A CN201310389469 A CN 201310389469A CN 103436489 A CN103436489 A CN 103436489A
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Abstract
The invention relates to the technical field of acellular processing of an animal skin tissue in biomedical engineering, and particularly relates to a reagent for acellular processing of a animal skin tissue, and a processing method thereof. The reagent is a solution prepared from SDS (sodium dodecyl sulfonate), NaOH and water. The processing method comprises the following steps of: A, acellular processing of SDS.NaOH; B, glutaraldehyde cross-linking; and C, sterilization. In the technical scheme disclosed by the invention, the animal skin tissue is directly processed by adopting the solution containing the SDS and the NaOH to obtain an acellular dermal matrix. Compared with other methods, the processing method provided by the invention has the characteristics of being simple and convenient, efficient, cheap, suitable for popularization and the like. The dermal matrix processed by the method is thorough in cell removal, a basement membrane complex is completely reserved, and the contents of collagen IV, fibronectin, elastin, HLA-DR (human leukocyte antigen DR) and the like in derma are very low.
Description
Technical field
The present invention relates to the cell free processing technology field of animal skin tissue in biomedical engineering, is a kind of for the treatment of the cell free solution of animal skin tissue and treatment process thereof specifically.
Background technology
In recent years, acellular dermal matrix (acellular dermalma-trix, ADM), as a kind of emerging corium sub, is subject to people's attention gradually.Desirable ADM should possess: cellular constituent and I, II type cell compatibility antigen are completely removed, retain complete basilar membrane complex body (basement membrane complex.BMC), immunocompetence is very low, can not bring out the specific cell immunoreaction (being rejection) produced for allograft, also can not bring out non-specific foreign body reaction, good biocompatibility.Research shows, the immunogenicity of allogeneic dermis is mainly the immune response that the cellular constituents such as epidermic cell due to donor skin, the inoblast in corium, endotheliocyte excite, the acellular composition extracellular matrix protein of corium and collagen (being ADM) is relative immunologic incompetence, can for good and all be present in host's body.Livesey etc. observe ADM and have complete basilar membrane complex body.Retain the basilar membrane complex body, can form two faces of basilar membrane and corium, the corium face is conducive to the quick vascularization of ADM, the substrate face can be epithelial dividing a word with a hyphen at the end of a line and provides a natural plane with field planting, the epithelization that is conducive to ADM, as a template, make the host cells such as patient's self inoblast and endotheliocyte again grow into, form new vessel.
At present the acellular dermal preparation method has: 1. Dispase11 and Triton method: skin sample is first through Dispase1I processing (2.5U/ml solution, act on 48h under 4 ℃), process (0.5% solution through TritonX 1 again, act on 48h under room temperature), cell in skin can be removed, but still residual cell debris arranging, basement membrane structure destroys greatlyr simultaneously, relatively is unfavorable for the epithelial cell tactophily.2. high salt and the SDS method of oozing: skin sample first oozes salt through height to be processed (NaC1 solution 1mol/L, 37 ℃ of effect 24h) and intactly removes epidermis; Then processing (0.5%SDS solution acts on 1h under room temperature) with the rupture of membranes agent removes the cell in skin sample.This method cell removes also more thorough, and it is complete that basilar membrane retains, but type Ⅳ collagen in corium, Zeta protein, elastin, HLA-DR equal size are more, therefore, have relatively high immunogenicity.
Summary of the invention
The objective of the invention is the problem existed for above-mentioned technology, provide a kind of for the treatment of the cell free solution of animal skin tissue and by this cell free method of solution-treated animal skin tissue.
For achieving the above object, the technical solution used in the present invention is: a kind of for the treatment of the cell free reagent of animal skin tissue, this reagent is by SDS, NaOH and the formulated solution of water.
Further, SDS concentration 0.0087M/L～1.0000M/L in described solution, NaOH concentration 0.01M/L～1.00M/L.
Further, SDS concentration 0.0087M/L in described solution, NaOH concentration 0.01M/L.
Another object of the present invention is to provide the cell free treatment process of a kind of animal skin tissue, and step is as follows:
The de-cell of A, SDSNaOH is processed;
B, glutaraldehyde cross-linking;
C, sterilizing.
Further, in described steps A, the de-cell processing of SDSNaOH comprises the steps:
1., skin graft is put into to the container of suitable size, the more de-cell damping fluid of the SDSNaOH prepared is poured in container;
2., container is put in the isothermal vibration incubator, 25～42 ℃, shake 1～48 hour;
3., by purified water, clean 1～48 hour, within every 2～3 hours, change liquid once.
Further, described step 1. the ratio of the de-cell damping fluid of skin graft and SDSNaOH be 10cm
2～200cm
2: 50ml～300ml.
Further, described step 2. middle culture temperature is 37 ℃, and the time is 24 hours.
Further, in described step B, glutaraldehyde cross-linking comprises the steps:
1., skin graft is put into to the container of suitable size; The glutaraldehyde cross-linking liquid prepared is poured in container;
2., container is put in the isothermal vibration incubator, 25～42 ℃, shake 1～120 minute;
3., adding concentration is that 0.01M/L～1M/L glycine stops crosslinking reaction;
4., by purified water, clean 1～48 hour, within every 2～3 hours, change liquid once.
Further, described step 1. in the ratio of skin graft and glutaraldehyde cross-linking liquid be 10cm
2～200cm
2: 50ml～300ml.
Further, described step 2. middle culture temperature is 37 ℃, and the time is 24 hours.
Sodium lauryl sulphate (SDS), have another name called sodium laurylsulfate (sodium laurylsulfate, SLS), belongs to sulfuric acid, and molecular weight is 288.38, and this product is white or faint yellow crystallization, very easily water-soluble.The modal purposes of SDS is as the denaturing agent of protein and hydrotropy reagent (as SDS-PAGE), and it can rupture in molecule and intermolecular hydrogen bond, makes the molecule unfolding, destroy protein two, tertiary structure.And nearly all protein all can be dissolved in SDS, even comprise hydrophobic and protein sex change.Therefore, SDS can be used as biomembranous effective solvating agent, sloughs the cellular constituent in corium.
Compared with prior art, useful technique effect of the present invention is:
(1), in technical scheme of the present invention, adopt and to contain SDS and NaOH solution and directly process animal skin tissue and obtain acellular dermal matrix, present method and additive method comparison, have easy, effective, inexpensive, the characteristics such as should promote.
(2) present method is processed the dermal matrix of gained, and cell is removed thoroughly, the basilar membrane complex body retains type Ⅳ collagen in complete, corium, Zeta protein, elastin, HLA-DR equal size are few.
(3) ADM that adopts present method to process, immunocompetence is low, can not bring out the specific cell immunoreaction (being rejection) produced for allograft, also can not bring out non-specific foreign body reaction, good biocompatibility.
Embodiment
The invention will be further described for following specific embodiment.
Embodiment 1
Take SDS 2.51g, NaOH 0.4g is dissolved in 1L distilled water, shakes that it is fully dissolved is standby.
The de-cell of A, SDSNaOH is processed
1., the animal skin graft is pressed to 10cm
2join in the container of the SDSNaOH that contains 50ml;
2., container is put in the isothermal vibration incubator, concussion 1 hour under 25 ℃ of conditions;
3., by purified water, clean 1 hour, within every 2 hours, change liquid once.
B, glutaraldehyde cross-linking
1., the skin graft in steps A is added in the glutaraldehyde cross-linking liquid container that the concentration of 50ml is 0.01%;
2., container is put in the isothermal vibration incubator, concussion 1 minute under 25 ℃ of conditions;
3., add the glycine that concentration is 0.01M/L to stop crosslinking reaction;
4., by purified water, clean 1 hour, within every 2 hours, change liquid once.
C, employing irradiation sterilization, dosage 1Kgy, obtain ADM.
Embodiment 2
Take SDS 28.838g, NaOH 4.0g is dissolved in 1L distilled water, shakes that it is fully dissolved is standby.
The de-cell of A, SDSNaOH is processed
1., the animal skin graft is pressed to 100cm
2join in the container of the SDSNaOH that contains 200ml;
2., container is put in the isothermal vibration incubator, concussion 24 hours under 37 ℃ of conditions;
3., by purified water, clean 24 hours, within every 2.5 hours, change liquid once.
B, glutaraldehyde cross-linking
1., the skin graft in steps A is added in the glutaraldehyde cross-linking liquid container that the concentration of 200ml is 0.1%;
2., container is put in the isothermal vibration incubator, concussion 60 minutes under 37 ℃ of conditions;
3., add the glycine that concentration is 0.1M/L and stop crosslinking reaction;
4., by purified water, clean 1 hour, within every 2 hours, change liquid once.
C, employing irradiation sterilization, dosage 20Kgy, obtain ADM.
Embodiment 3
Take SDS 288.38g, NaOH 40g is dissolved in 1L distilled water, shakes that it is fully dissolved is standby.
The de-cell of A, SDSNaOH is processed
1., skin graft is pressed to 200cm
2join in the container of the SDSNaOH that contains 300ml;
2., container is put in the isothermal vibration incubator, concussion 48 hours under 42 ℃ of conditions;
3., by purified water, clean 48 hours, within every 3 hours, change liquid once.
B, glutaraldehyde cross-linking
1., the skin graft in steps A is added in the glutaraldehyde cross-linking liquid container that the concentration of 300ml is 1.0%;
2., container is put in the isothermal vibration incubator, concussion 120 minutes under 42 ℃ of conditions;
3., add the glycine that concentration is 1M/L and stop crosslinking reaction;
4., by purified water, clean 48 hours, within every 3 hours, change liquid once.
C, employing irradiation sterilization, dosage 35Kgy, obtain ADM.
Adopting the ADM that above-mentioned embodiment makes is off-white color or faint yellow, without epidermis, be shaped as sheet or mesh, more soft, flexible, edge is more neat, immunocompetence is low, can not bring out the specific cell immunoreaction (being rejection) produced for allograft, also can not bring out non-specific foreign body reaction, good biocompatibility.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. for the treatment of the cell free reagent of animal skin tissue, it is characterized in that, this reagent is by SDS, NaOH and the formulated solution of water.
2. the reagent of processing for the treatment of the de-cell of animal skin tissue according to claim 1, is characterized in that SDS concentration 0.0087M/L～1.0000M/L in described solution, NaOH concentration 0.01M/L～1.00M/L.
3. the reagent of processing for the treatment of the de-cell of animal skin tissue according to claim 1, is characterized in that SDS concentration 0.0087M/L in described solution, NaOH concentration 0.01M/L.
4. according to the cell free treatment process of the described animal skin tissue of claim 1～3 any one, it is characterized in that, step is as follows:
The de-cell of A, SDSNaOH is processed;
B, glutaraldehyde cross-linking;
C, sterilizing.
5. the cell free treatment process of animal skin tissue according to claim 4, is characterized in that, in described steps A, the de-cell of SDSNaOH is processed and comprised the steps:
1., the animal skin graft is put into to container, then by the de-cell damping fluid of the SDSNaOH prepared pour in container;
2., container is put in the isothermal vibration incubator, under 25～42 ℃ of conditions, shake 1～48 hour;
3., by purified water, clean 1～48 hour, within every 2～3 hours, change liquid once.
6. the cell free treatment process of animal skin tissue according to claim 5, is characterized in that, the described step 1. ratio of animal skin graft and the de-cell damping fluid of SDSNaOH is 10cm
2～200cm
2: 50ml～300ml.
7. the cell free treatment process of animal skin tissue according to claim 5, is characterized in that, described step 2. in culture temperature be 37 ℃, the time is 24 hours.
8. the cell free treatment process of animal skin tissue according to claim 4, is characterized in that, in described step B, glutaraldehyde cross-linking comprises the steps:
1., skin graft is put into to the container of suitable size; The glutaraldehyde cross-linking liquid prepared is poured in container;
2., container is put in the isothermal vibration incubator, under 25～42 ℃ of conditions, shake 1～120 minute;
3., adding concentration is that 0.01M/L～1M/L glycine stops crosslinking reaction;
4., by purified water, clean 1～48 hour, within every 2～3 hours, change liquid once.
9. the cell free treatment process of animal skin tissue according to claim 8, is characterized in that, described step 1. in the ratio of skin graft and glutaraldehyde cross-linking liquid be 10cm
2～200cm
2: 50ml～300ml.
10. the cell free treatment process of animal skin tissue according to claim 8, is characterized in that, described step 2. incubator temperature is 37 ℃, and the time is 24 hours.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2013103894694A CN103436489A (en) | 2013-08-30 | 2013-08-30 | Reagent for acellular processing of animal skin tissue, and processing method thereof |
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|---|---|---|---|
| CN2013103894694A CN103436489A (en) | 2013-08-30 | 2013-08-30 | Reagent for acellular processing of animal skin tissue, and processing method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108030914A (en) * | 2017-12-19 | 2018-05-15 | 广州昕生医学材料有限公司 | A kind of Matrigel raw material and preparation method and application |
| CN108079363A (en) * | 2017-12-19 | 2018-05-29 | 广州昕生医学材料有限公司 | A kind of kit and its application that cell processing is taken off for animal tissue |
| CN111569151A (en) * | 2020-05-12 | 2020-08-25 | 上海亚朋生物技术有限公司 | Acellular dermal matrix tissue engineering scaffold and preparation method thereof |
| CN112190764A (en) * | 2020-09-17 | 2021-01-08 | 上海亚朋生物技术有限公司 | Allogeneic skin acellular method |
| CN114395525A (en) * | 2021-12-23 | 2022-04-26 | 北京鑫康辰医学科技发展有限公司 | Preparation method of acellular and antigen-removed xenogenic tendon |
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| CN102218162A (en) * | 2011-05-24 | 2011-10-19 | 山西奥瑞生物材料有限公司 | Preparation method of homologous acellular dermal matrix |
| CN102580141A (en) * | 2012-03-09 | 2012-07-18 | 潘银根 | Preparation method of acellular dermal matrix dressing |
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| CN102218162A (en) * | 2011-05-24 | 2011-10-19 | 山西奥瑞生物材料有限公司 | Preparation method of homologous acellular dermal matrix |
| CN102580141A (en) * | 2012-03-09 | 2012-07-18 | 潘银根 | Preparation method of acellular dermal matrix dressing |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108030914A (en) * | 2017-12-19 | 2018-05-15 | 广州昕生医学材料有限公司 | A kind of Matrigel raw material and preparation method and application |
| CN108079363A (en) * | 2017-12-19 | 2018-05-29 | 广州昕生医学材料有限公司 | A kind of kit and its application that cell processing is taken off for animal tissue |
| CN111569151A (en) * | 2020-05-12 | 2020-08-25 | 上海亚朋生物技术有限公司 | Acellular dermal matrix tissue engineering scaffold and preparation method thereof |
| CN111569151B (en) * | 2020-05-12 | 2021-12-17 | 上海亚朋生物技术有限公司 | Acellular dermal matrix tissue engineering scaffold and preparation method thereof |
| CN112190764A (en) * | 2020-09-17 | 2021-01-08 | 上海亚朋生物技术有限公司 | Allogeneic skin acellular method |
| CN114395525A (en) * | 2021-12-23 | 2022-04-26 | 北京鑫康辰医学科技发展有限公司 | Preparation method of acellular and antigen-removed xenogenic tendon |
| CN114395525B (en) * | 2021-12-23 | 2024-02-13 | 北京鑫康辰医学科技发展有限公司 | Preparation method of decellularized antigen-removed heterogeneous tendon |
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Application publication date: 20131211 |