CN106172369A - The antioxidation of a kind of optimization takes off Cell protective solutions - Google Patents
The antioxidation of a kind of optimization takes off Cell protective solutions Download PDFInfo
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- CN106172369A CN106172369A CN201610511580.XA CN201610511580A CN106172369A CN 106172369 A CN106172369 A CN 106172369A CN 201610511580 A CN201610511580 A CN 201610511580A CN 106172369 A CN106172369 A CN 106172369A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The antioxidation that the present invention relates to a kind of optimization takes off Cell protective solutions, can whole process be used for taking off cell processes, and structural intergrity and the biological nature of cell tissue de-to protection play a key effect.Its composition is: DMEM cell culture medium, L histidine hydrochloride, allopurinol, chondroitin sulfate, low molecular dextran, hydroxypropyl methyl cellulose, HEPES buffer, hydrochloric acid dexamethasone, reduced glutathione, levofloxacin；It it is the one pink liquid that possesses certain ph, particular crystal and colloid osmotic pressure.Needed raw material of the present invention is easy to get, price economy, is that a kind of suitability is strong, and applied range, antioxygenic property is superior, the optimization Cell protective solutions that can shield in biological tissue takes off cell processes.
Description
Technical field
The invention belongs to Chemical composition that technical field, the antioxidation of a kind of optimization takes off Cell protective solutions and system thereof
Preparation Method and application.
Background technology
21 century is that bioscience completely newly develops century with rapid changepl. never-ending changes and improvements, slowly rises as past 10 years biomedical sector
A Chaoyang TERM (Tissue engineering & Regenerative Medicine organizational project and the regeneration risen
Medical science) enjoy the world to attract attention.In the research and application of TERM, relevant cell free technology occupies very important status.
De-cell refers to that the biomedical engineering field process by extracellular mesenchyme Yu cell separation, separating obtained cell outer room are filled
Matter support can be used for artificial organ and tissue regeneration.The mixed method using physics, chemistry, ferment treatment and three is the current world
In the range of the method for removing cells that commonly used, its purpose is all integrity and the life of the cell mechanism for ensureing de-cell tissue
Thing characteristic is not damaged, and makes immunogenicity low, possesses the ideal biomaterial of good biocompatibility.But de-cell is adopted
Method the longest, condition is violent, unavoidably taken off cell tissue is caused relatively havoc in de-cell processes.Therefore
A kind of protective measure of exploitation (such as protection liquid) protects linked groups's organizational structure integrity in de-cell processes and will fill up
The blank of this respect.
The macromolecular material applying synthetic is repaired more by tissue transplantation the most clinically, often there is self degradation
Can be low, the problem of histocompatibility difference, biological engineering material such as de-cell eye conjunctiva, blood vessel and tendon/ligament etc. is in clinic
Application aspect is the most urgent.Therefore organizational structure is complete, biological nature is not damaged biological engineering material is obtained for clinic
Application also have very important significance.
Summary of the invention
The antioxidation that the invention provides a kind of optimization takes off the preparation method and application of Cell protective solutions.
Organization protection of the present invention its composition of liquid is:
1, DMEM cell culture medium powder 5-10g/L, is configured to maintain the mother solution of cytotrophy composition；
2, L-Histidine hydrochlorate 2.87-3.83g/L, makes crystalloid osmotic pressure maintain between 330-380mOsm/kgH2O；
3, chondroitin sulfate 25-30g/L, hydroxypropyl methyl cellulose 2-8g/L, Dextran-20-35g/L, come
The colloid osmotic pressure maintaining its liquid is 310-350mOsm/kg H2Between O.Due to chondroitin sulfate, hydroxypropyl methyl fiber
Element and the synergism of low molecular dextran, it is possible to reduce the negative charge in protection liquid, keep certain colloid osmotic simultaneously
Pressure, makes in de-cell processes, the inside and outside pressure balanced of de-cell tissue；
4, allopurinol 0.5-0.8g/L, it is possible to reduce the de-cell tissue anti-oxidation function when anaerobic condition；
5, HEPES buffer 20-25ml/L, is 7.2-8.0 in order to regulate pH value；
6, hydrochloric acid dexamethasone 1-2mg/L, reduced glutathione 1.5-3g/L, with the lysosome of stable cell tissue
Film and biomembrane, strengthen the defensive ability/resistance ability of cell induced by endotoxin, preferably keep histiocytic form and function；
7, levofloxacin 0.1-0.2g/L, as the fluoroquinolone of a new generation, can kill the leather that donor carries simultaneously
The pathogen that Lan Shi is positive and negative.
De-cell tissue of the present invention protection liquid is pink liquid.
Accompanying drawing explanation
Fig. 1 whole process adds the de-cell eye biopsy of conjunctiva DAPI dyeing preserving protection liquid
Fig. 2 whole process adds the de-cell eye biopsy of conjunctiva DAPI dyeing of sterilized water for injection
Detailed description of the invention
Being specifically described the present invention below by embodiment, these embodiments are served only for being described in further detail explanation originally
Invention, it is impossible to be interpreted as limiting the scope of the present invention, making nonessential adjustment within scope needs
Side of the present invention authorizes.
Embodiments of the invention 1
Antioxidation takes off the preparation of Cell protective solutions:
1, take DMEM powder 10g and add deionized water 500ml, autoclaving after dissolving；
2, take chondroitin sulfate 25g and add deionized water 275ml heating for dissolving autoclaving, make chondroitin sulfate molten
Liquid；Low molecular dextran 20g adds water for injection 100ml and dissolves autoclaving, makes low molecular dextran solution；L-group
Propylhomoserin hydrochlorate 2.87g, adds water for injection 100ml and dissolves autoclaving, make L-Histidine HCI solution；
3, take sterile chamber and add DMEM culture fluid 500ml, autoclaving chondroitin sulfate 275ml, low molecule dextrose
Anhydride solution 100ml, L-Histidine HCI solution 100ml；Add allopurinol 0.5g, hydroxypropyl methyl cellulose 5g, reduction
Type glutathione 2g, dexamethasone injection 2mg, levofloxacin 0.1g mix；
4, Hepes buffer regulation pH value is to 7.4；
5, regulation crystalloid osmotic pressure is 340mOsm/kgH2O；
6, regulation colloid osmotic pressure is 350mOsm/kgH2O。
Embodiments of the invention 2
Use de-Cell protective solutions effect cell free to pig tendon tissue and Observation of Histological Structure and detection
Fresh pig takes out tendon tissue, aseptic process in killing latter 3 hours, and tendon aponeurotomy is made the group of 1cm × 2cm
Knit sheet, take 5 be sealed in fill 10ml embodiment 1 joined preserve liquid plastic bag in；Another matched group is that 5 pieces of tissue are sealed in
Fill in the plastic bag of 10ml BSS.Under 600MP height hydrostatic pressure condition, processing 8 times, each time is 3 minutes；Again ligament is taken
After going out, being placed in the protection liquid containing 0.2% sodium lauryl sulphate+1000U/ml DNA enzymatic, temperature is 25 DEG C, sets shaking table speed
Degree is 100 revs/min, processes 2 hours；Take out de-cell tendon and be placed in rinsing 2 hours in protection liquid.The enzyme of matched group and de-sludging
Agent and last rinsing process and all carry out in BSS.
Textural bands row dyeing to the de-cell tendon/ligament of preparation.Take the two groups of specimen of tendon after de-cell to
Giving paraffin embedding, section (5 μm), HE dye observation.Observation of cell nuclear composition (haematoxylin dyeing), collagen arrangement form (Yihong
Dyeing), make comparisons with normal tendon.Quick freezing is cut into slices, routine hematoxylin-Yihong (HE) dyeing and DAPI
(DAPI) (Invitrogen, the U.S.) fluorescence staining, the de-cell degree of detection.Result: nothing in the tissue slice of de-cell tendon
DAPI positive staining, shows to remain without DNA.The tendon tissue structure using this de-Cell protective solutions to protect is substantially complete, cell
Removing is clean.And matched group, after de-cell, tissue edema is obvious, tissue fibers arrangement disorder.
Embodiments of the invention 3
Use de-Cell protective solutions effect cell free to eye conjunctival tissue and Observation of Histological Structure and detection
Fresh pig takes out eye conjunctival tissue in killing latter 2 hours, is eliminated as much as subconjunctival tissue, and aseptic process, by eye
Conjunctival tissue is cut into the tissue of 0.5cm × 1cm, and wherein eye conjunctival tissue 5 is sealed in and fills the joined preservation of 10ml embodiment 1
In the plastic bag of liquid；Another matched group is that a conjunctival tissue 5 is sealed in the plastic bag filling 10ml BSS.In 300MP Gao Jing
Under the conditions of pressure, processing 3 times, each time is 1 minute；After again eye conjunctival tissue being taken out, it is placed in containing 0.2% lauryl sulphate acid
In the protection liquid of sodium+1000U/ml DNA enzymatic, temperature is 25 DEG C, sets shaking table speed as 100 revs/min, processes 1 hour；Take
Go out de-cell eye conjunctiva and be placed in rinsing 1 hour in protection liquid.The enzyme of matched group processes all at BSS with detergent and last rinsing
In carry out.
Textural bands row dyeing to the de-cell eye conjunctiva of preparation.Take the two groups of specimen of eye conjunctival tissue after de-cell
Giving paraffin embedding, section (5 μm), HE dye observation.Observation of cell nuclear composition (haematoxylin dyeing), collagen arrangement form (she
Red colouring), make comparisons with normal eyes conjunctival tissue.Quick freezing is cut into slices, routine hematoxylin-Yihong (HE) dyeing and diamidino benzene
Base indole (DAPI) (Invitrogen, the U.S.) fluorescence staining, the de-cell degree of detection.Result: the tissue slice of de-eye conjunctiva
Middle positive staining without DAPI, shows to remain without DNA.Use tendon that this de-Cell protective solutions protects and ligament tissue structure basic
Completely, cell removing is clean.And matched group, after de-cell, eye conjunctival epithelium comes off, tissue fibers arrangement disorder.
Embodiments of the invention 4
Use de-Cell protective solutions effect cell free to vascular tissue and constructed observation and detection
Fresh pig is killed and is taken out heart arter vascular tissue in latter 4 hours, and vascular tissue is cut by aseptic process, pave into
The tissue of 1X1.5cm, vascular tissue 5 is sealed in and fills in the plastic bag that the embodiment 1 of 10ml is joined protection liquid；Another comparison
Group is sealed in the plastic bag filling 10ml BSS for vascular tissue 5.Under 500MP height hydrostatic pressure condition, process 5 times, every time
Time is 5 minutes；After vascular tissue being taken out again, it is placed in the protection containing 0.2% sodium lauryl sulphate+1000U/ml DNA enzymatic
In liquid, temperature is 25 DEG C, sets shaking table speed as 100 revs/min, processes 2 hours；Take out vascular tissue and be placed in drift in protection liquid
Wash 2 hours.The enzyme of matched group processes with detergent and last rinsing and all carries out in BSS.
Textural bands row dyeing to the de-cellular vascular of preparation.Taking the vascular tissue after de-cell, two groups of specimen are given
Giving paraffin embedding, section (5 μm), HE dye observation.Observation of cell nuclear composition (haematoxylin dyeing), collagen arrangement form (Yihong
Dyeing), make comparisons with normal vascular tissues.Quick freezing is cut into slices, routine hematoxylin-Yihong (HE) dyeing and diamidino phenyl Yin
Diindyl (DAPI) (Invitrogen, the U.S.) fluorescence staining, the de-cell degree of detection.Result: in the tissue slice of de-cellular vascular
Positive staining without DAPI, shows to remain without DNA.The vascular tissue's structure using this Cell protective solutions to protect is substantially complete, cell
Removing is clean.And matched group, original vascular tissue structural damage after de-cell, fibrous fracture, edema is obvious.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (5)
1. the antioxidation optimized takes off a Cell protective solutions, and its composition is:
(1) DMEM cell culture medium powder 5-10g/L
(2) L-Histidine hydrochlorate 2.87-3.83g/L；
(3) chondroitin sulfate 25-30g/L；
(4) Dextran-20-35g/L；
(5) hydroxypropyl methyl cellulose 2-8g/L；
(6) allopurinol 0.5-0.8g/L；
(7) HEPES buffer 20-25ml/L；
(8) hydrochloric acid dexamethasone 1-2mg/L；
(9) reduced glutathione 1.5-3g/L；
(10) levofloxacin 0.1-0.2g/L.
2. de-Cell protective solutions as claimed in claim 1, makes crystalloid osmotic pressure maintain 330-with L-Histidine hydrochlorate
380mOsm/kgH2Between O.
3. de-Cell protective solutions as claimed in claim 1, uses chondroitin sulfate, hydroxypropyl methyl cellulose and low molecule right
The synergism of rotation glucosides, it is possible to reduce the negative charge in protection liquid, keeps colloid osmotic to be pressed in 310-350mOsm/ simultaneously
kgH2Between O, make in de-cell processes, the inside and outside pressure balanced of de-cell tissue.
4. de-Cell protective solutions as claimed in claim 1, application hydrochloric acid dexamethasone and reduced glutathione maintain cell
The stability of film.
5. de-Cell protective solutions as claimed in claim 1, reduces antioxygen when being organized in anaerobic condition by adding allopurinol
Change function, play protective tissue structural integrity and the effect of biological nature in de-cell processes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610511580.XA CN106172369B (en) | 2016-07-04 | 2016-07-04 | A kind of anti-oxidant de- Cell protective solutions of optimization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610511580.XA CN106172369B (en) | 2016-07-04 | 2016-07-04 | A kind of anti-oxidant de- Cell protective solutions of optimization |
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| CN106172369A true CN106172369A (en) | 2016-12-07 |
| CN106172369B CN106172369B (en) | 2018-01-12 |
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| CN201610511580.XA Active CN106172369B (en) | 2016-07-04 | 2016-07-04 | A kind of anti-oxidant de- Cell protective solutions of optimization |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110278940A (en) * | 2019-07-11 | 2019-09-27 | 河南省农业科学院畜牧兽医研究所 | A kind of Boar spermatozoa preservative agent and its preparation method and application |
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| CN1262866A (en) * | 1999-02-12 | 2000-08-16 | 山东省医学科学院眼科研究所 | Cornea activity preservative fluid |
| CN101590292A (en) * | 2009-06-11 | 2009-12-02 | 山东省眼科研究所 | The preparation method of acellular conjunctiva matrix and application |
| WO2009152384A1 (en) * | 2008-06-11 | 2009-12-17 | The Children's Mercy Hospital | Solutions for tissue engineering and methods of use |
| US8735054B1 (en) * | 2008-01-04 | 2014-05-27 | Lifecell Corporation | Acellular tissue matrix preservation solution |
| CN104511053A (en) * | 2015-03-06 | 2015-04-15 | 青岛中皓生物工程有限公司 | Decellularized porcine cornea tissue and preparation method and application thereof |
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2016
- 2016-07-04 CN CN201610511580.XA patent/CN106172369B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1262866A (en) * | 1999-02-12 | 2000-08-16 | 山东省医学科学院眼科研究所 | Cornea activity preservative fluid |
| US8735054B1 (en) * | 2008-01-04 | 2014-05-27 | Lifecell Corporation | Acellular tissue matrix preservation solution |
| WO2009152384A1 (en) * | 2008-06-11 | 2009-12-17 | The Children's Mercy Hospital | Solutions for tissue engineering and methods of use |
| CN101590292A (en) * | 2009-06-11 | 2009-12-02 | 山东省眼科研究所 | The preparation method of acellular conjunctiva matrix and application |
| CN104511053A (en) * | 2015-03-06 | 2015-04-15 | 青岛中皓生物工程有限公司 | Decellularized porcine cornea tissue and preparation method and application thereof |
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Cited By (1)
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| CN110278940A (en) * | 2019-07-11 | 2019-09-27 | 河南省农业科学院畜牧兽医研究所 | A kind of Boar spermatozoa preservative agent and its preparation method and application |
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| CN106172369B (en) | 2018-01-12 |
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Effective date of registration: 20211111 Address after: 611139 floors 1-4, building 15, No. 1919, Shuangyan Road, cross strait science and Technology Industrial Development Park, Wenjiang District, Chengdu, Sichuan Patentee after: Baiodisel (Chengdu) Biotechnology Co., Ltd Address before: 100176 601a, 6 / F, building 13, yard 8, Liangshuihe Second Street, Beijing Economic and Technological Development Zone, Beijing Patentee before: Baiodisel (Beijing) Biotechnology Co., Ltd |
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