CN106172369B - A kind of anti-oxidant de- Cell protective solutions of optimization - Google Patents
A kind of anti-oxidant de- Cell protective solutions of optimization Download PDFInfo
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- CN106172369B CN106172369B CN201610511580.XA CN201610511580A CN106172369B CN 106172369 B CN106172369 B CN 106172369B CN 201610511580 A CN201610511580 A CN 201610511580A CN 106172369 B CN106172369 B CN 106172369B
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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 present invention relates to a kind of anti-oxidant de- Cell protective solutions of optimization, whole can be used to take off cell processes, the structural intergrity and biological nature for protecting de- cell tissue are played a key effect.Its composition is：DMEM cell culture mediums, L histidine hydrochlorides, allopurinol, chondroitin sulfate, low molecular dextran, hydroxypropyl methyl cellulose, HEPES buffer solution, hydrochloric acid dexamethasone, reduced glutathione, lavo-ofloxacin；It is the pink liquid that one kind possesses certain ph, particular crystal and colloid osmotic pressure.Raw material is easy to get needed for the present invention, price economy, is a kind of strong applicability, has a wide range of application, and antioxygenic property is superior, the optimization Cell protective solutions that can be shielded in biological tissue takes off cell processes.
Description
Technical field
The invention belongs to Chemical composition that technical field, the anti-oxidant de- Cell protective solutions and its system of specially a kind of optimization
Preparation Method and application.
Background technology
21 century is the bioscience century that completely newly development is maked rapid progress, and is slowly risen as past 10 years biomedical sector
A Chaoyang --- TERM (Tissue engineering ＆ Regenerative Medicine organizational projects and the regeneration risen
Medical science) enjoy the world to attract attention.In TERM research and application, related cell free technology occupies very important status.
De- cell refers to the process of that biomedical engineering field separates extracellular mesenchyma with cell, and separating obtained cell outer room fills
Matter support can be used for man-made organ and regeneration.It is the current world using physics, chemistry, ferment treatment and the mixed method of three
In the range of the method for removing cells that generally uses, its purpose is all to ensure the integrality of the cell mechanism of de- cell tissue and life
Thing characteristic is not damaged, and makes the ideal biomaterial that immunogenicity is low, possesses good biocompatibility.But de- cell is adopted
Often time-consuming for method, and condition is violent, and taken off cell tissue is caused compared with havoc unavoidably in de- cell processes.Therefore
Developing a kind of safeguard measure and protecting institutional framework integrality of the linked groups in de- cell processes (such as protection liquid) to fill up
The blank of this respect.
Tissue transplantation clinically at present is repaired applies artificial synthesized high polymer material more, self degradation often be present
Can be low, the problem of histocompatbility difference, biological engineering material such as takes off cell eye conjunctiva, blood vessel and tendon/ligament in clinic
Application aspect is very urgent.Therefore the biological engineering material that acquirement institutional framework is complete, biological nature is not damaged is for clinic
Application also have very important significance.
The content of the invention
The invention provides a kind of preparation method and application of the anti-oxidant de- Cell protective solutions of optimization.
Its composition of organization protection's liquid of the present invention is：
1st, DMEM cell culture based powders 5-10g/L, it is configured to maintain the mother liquor of cytotrophy composition；
2nd, L-Histidine hydrochloride 2.87-3.83g/L, crystal osmotic pressure is made to maintain between 330-380mOsm/kgH2O；
3rd, chondroitin sulfate 25-30g/L, hydroxypropyl methyl cellulose 2-8g/L, Dextran-20-35g/L, come
The colloid osmotic pressure for maintaining its liquid is 310-350mOsm/kg H2Between O.Due to chondroitin sulfate, hydroxypropyl methyl fiber
The synergy of element and low molecular dextran, it is possible to reduce the negative electrical charge in protection liquid, while keep certain colloid osmotic
Pressure, make in de- cell processes, the inside and outside pressing of the de- cell tissue of institute is weighed；
4th, allopurinol 0.5-0.8g/L, anti-oxidation function of the de- cell tissue in anaerobic condition can be reduced；
5th, HEPES buffer solution 20-25ml/L, it is 7.2-8.0 to adjust pH value；
6th, 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；
7th, lavo-ofloxacin 0.1-0.2g/L, as the fluoquinolone of a new generation, the leather that donor carries can be killed simultaneously
Pathogen positive and negative Lan Shi.
De- cell tissue protection liquid of the present invention is pink liquid.
Brief description of the drawings
The whole additions of Fig. 1 preserve the de- cell eye conjunctiva section DAPI dyeing of protection liquid
The de- cell eye conjunctiva section DAPI dyeing of the whole addition sterilizeds water for injection of Fig. 2
Embodiment
The present invention is specifically described 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, nonessential adjustment is made within the scope of the present invention to be needed
Present invention side authorizes.
Embodiments of the invention 1
The preparation of anti-oxidant de- Cell protective solutions：
1st, DMEM powder 10g is taken to add deionized water 500ml, autoclaving after dissolving；
2nd, take chondroitin sulfate 25g to add deionized water 275ml and dissolve by heating autoclaving, it is molten that chondroitin sulfate is made
Liquid；Low molecular dextran 20g adds water for injection 100ml dissolving autoclavings, and low molecular dextran solution is made；L- groups
Propylhomoserin hydrochloride 2.87g, water for injection 100ml dissolving autoclavings are added, L-Histidine HCI solution is made；
3rd, sterile chamber is taken to add DMEM nutrient solution 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 are mixed；
4th, Hepes buffer solutions adjust pH value to 7.4；
5th, it is 340mOsm/kgH to adjust crystal osmotic pressure2O；
6th, it is 350mOsm/kgH to adjust colloid osmotic pressure2O。
Embodiments of the invention 2
Using de- Cell protective solutions to the cell free effect of pig tendon tissue and Observation of Histological Structure and detection
Fresh pig takes out tendon tissue after killing in 3 hours, aseptic process, and tendon aponeurotomy is made to 1cm × 2cm group
Piece is knitted, takes 5 to be sealed in and fills in the polybag that 10ml embodiments 1 match somebody with somebody preservation liquid；Another control group is that 5 pieces of tissues are sealed in
In the polybag for filling 10ml BSS.Under the conditions of 600MP high static pressures, handle 8 times, each time is 3 minutes；Ligament is taken again
After going out, it is placed in the protection liquid containing 0.2% lauryl sodium sulfate+1000U/ml DNA enzymatics, temperature is 25 DEG C, setting shaking table speed
Spend for 100 revs/min, handle 2 hours；The de- cell tendon of taking-up, which is placed in protection liquid, to be rinsed 2 hours.The enzyme of control group and de-sludging
Agent and last rinsing processing are carried out in BSS.
The textural bands row of de- cell tendon/ligament of preparation is dyed.Take two groups of samples of tendon after de- cell to
Give FFPE, section (5 μm), HE dyeing observations.Observe nuclear fraction (haematoxylin dyeing), collagen arrangement form (Yihong
Dyeing), made comparisons with normal tendon.Snap frozen is cut into slices, routine hematoxylin-Yihong (HE) dyeing and diamidino phenyl indole
(DAPI) (Invitrogen, the U.S.) fluorescent staining, de- cell degree is detected.As a result：Nothing in the histotomy of de- cell tendon
DAPI positive stainings, show no DNA residuals.It is substantially complete that the tendon tissue structure of Cell protective solutions protection is taken off using this, cell
Removing is clean.And control group, tissue edema is obvious after de- cell, tissue fibers arrangement disorder.
Embodiments of the invention 3
Using de- Cell protective solutions to the cell free effect of eye conjunctiva tissue and Observation of Histological Structure and detection
Fresh pig takes out eye conjunctiva tissue after killing in 2 hours, be eliminated as much as subconjunctival tissue, aseptic process, by eye
Conjunctival tissue is cut into 0.5cm × 1cm tissue, and wherein 5, eye conjunctiva tissue, which is sealed in, fills 10ml embodiments 1 and match somebody with somebody preservation
In the polybag of liquid；Another control group is that 5, eye conjunctiva tissue is sealed in the polybag for filling 10ml BSS.In 300MP Gao Jing
Under the conditions of pressure, handle 3 times, each time is 1 minute；After eye conjunctiva tissue is taken out again, it is placed in containing 0.2% dodecyl sulphate
In the protection liquid of sodium+1000U/ml DNA enzymatics, temperature is 25 DEG C, sets shaking table speed as 100 revs/min, is handled 1 hour；Take
Go out de- cell eye conjunctiva be placed in protection liquid in rinse 1 hour.The enzyme of control group is handled in BSS with detergent and last rinsing
Middle progress.
The textural bands row of the de- cell eye conjunctiva of preparation is dyed.Take two groups of samples of eye conjunctiva tissue after de- cell
Give FFPE, section (5 μm), HE dyeing observations.Observe nuclear fraction (haematoxylin dyeing), collagen arrangement form (she
Red colouring), made comparisons with normal eyes conjunctival tissue.Snap frozen is cut into slices, routine hematoxylin-Yihong (HE) dyeing and diamidino benzene
Base indoles (DAPI) (Invitrogen, the U.S.) fluorescent staining, detect de- cell degree.As a result：The histotomy of de- eye conjunctiva
Middle no DAPI positive stainings, show no DNA residuals.Using this take off Cell protective solutions protect tendon and ligament tissue structure it is basic
Completely, cell removing is clean.And control group, the eye conjunctiva exuviation after de- cell, tissue fibers arrangement disorder.
Embodiments of the invention 4
Using de- Cell protective solutions to the cell free effect of vascular tissue and structure observation and detection
Fresh pig takes out heart arter vascular tissue after killing in 4 hours, aseptic process, vascular tissue is cut, pave for
1X1.5cm tissue, vascular tissue 5 be sealed in fill 10ml embodiment 1 match somebody with somebody protection liquid polybag in；Another control
Group is sealed in the polybag for filling 10ml BSS for vascular tissue 5.Under the conditions of 500MP high static pressures, processing 5 times, every time
Time is 5 minutes；After vascular tissue is taken out again, the protection containing 0.2% lauryl sodium sulfate+1000U/ml DNA enzymatics is placed in
In liquid, temperature is 25 DEG C, sets shaking table speed as 100 revs/min, is handled 2 hours；Take out vascular tissue and be placed in protection liquid and float
Wash 2 hours.The enzyme of control group is carried out with detergent and last rinsing processing in BSS.
The textural bands row of the de- cellular vascular of preparation is dyed.The vascular tissue after de- cell is taken, two groups of samples are given
Give FFPE, section (5 μm), HE dyeing observations.Observe nuclear fraction (haematoxylin dyeing), collagen arrangement form (Yihong
Dyeing), made comparisons with normal vascular tissues.Snap frozen is cut into slices, routine hematoxylin-Yihong (HE) dyeing and diamidino phenyl Yin
Diindyl (DAPI) (Invitrogen, the U.S.) fluorescent staining, detect de- cell degree.As a result：In the histotomy of de- cellular vascular
Without DAPI positive stainings, show no DNA residuals.The vascular tissue's structure protected using this Cell protective solutions is substantially complete, cell
Removing is clean.And control group, original vascular tissue's structural damage after de- cell, fibrous fracture, oedema are obvious.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (5)
1. a kind of anti-oxidant de- Cell protective solutions of optimization, its composition are：
(1) DMEM cell culture based powders 5-10g/L
(2) L-Histidine hydrochloride 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 solution 20-25ml/L；
(8) hydrochloric acid dexamethasone 1-2mg/L；
(9) reduced glutathione 1.5-3g/L；
(10) lavo-ofloxacin 0.1-0.2g/L.
2. de- Cell protective solutions as claimed in claim 1, make crystal osmotic pressure maintain 330- with L-Histidine hydrochloride
380mOsm/kgH2Between O.
3. de- Cell protective solutions as claimed in claim 1, right using chondroitin sulfate, hydroxypropyl methyl cellulose and low molecule
Revolve the synergy of glucosides, it is possible to reduce the negative electrical charge in protection liquid, while keep colloid osmotic to be pressed in 310-350mOsm/
kgH2Between O, make in de- cell processes, the inside and outside pressing of the de- cell tissue of institute is weighed.
4. de- Cell protective solutions as claimed in claim 1, cell is maintained using hydrochloric acid dexamethasone and reduced glutathione
The stability of film.
5. de- Cell protective solutions as claimed in claim 1, by adding allopurinol to reduce antioxygen when being organized in anaerobic condition
Change function, play a part of protective tissue structural integrity and biological nature in de- cell processes.
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| Application Number | Priority Date | Filing Date | Title |
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| 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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| 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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| CN106172369B true CN106172369B (en) | 2018-01-12 |
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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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2016
- 2016-07-04 CN CN201610511580.XA patent/CN106172369B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Dextran Preserves Native Corneal Structure During Decellularization;Amy P. Lynch 等;《TISSUE ENGINEERING》;20160512;第22卷(第6期);561-572 * |
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