CN106135197B - A kind of cornea mid-term preservation liquid of serum-free composition - Google Patents
A kind of cornea mid-term preservation liquid of serum-free composition Download PDFInfo
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- CN106135197B CN106135197B CN201610511681.7A CN201610511681A CN106135197B CN 106135197 B CN106135197 B CN 106135197B CN 201610511681 A CN201610511681 A CN 201610511681A CN 106135197 B CN106135197 B CN 106135197B
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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 cornea mid-term preservation liquid of serum-free composition, its composition is：Compound electrolyte intra-ocular flushing liquor, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, chondroitin sulfate, sodium hyaluronate, HEPES buffer solution, hydrochloric acid dexamethasone, reduced glutathione, lavo-ofloxacin；Be it is a kind of there is particular electrolyte ion concentration, pH value, blush, the transparency liquid of crystal osmotic pressure and colloid osmotic pressure.Raw material needed for the corneal storage medium is easy to get, and without serum composition, avoids and infects some special viral risks, it carrys out the osmotic pressure of the inside and outside liquid of statocyte with various ions, and preservation effect is notable.
Description
Technical field
The invention belongs to Chemical composition that technical field, the cornea mid-term preservation liquid of specially a kind of serum-free composition and
Its preparation method and application.
Background technology
The store method of cornea is mostly vital preservation in terms of clinical practice.Vital preservation (the eye bank of common cornea
One of important technology) short-term, mid-term, long-term 3 kinds can be divided into according to the active holding time of endothelial cell.It is short-term to protect
The method of depositing is that the holding time is within 24 hours by the preservation of 4 DEG C of wet room after cornea tissue aseptic process.Mid-term preservation typically uses
Preserve liquid to preserve, using cornea activity preservative fluid is most commonly used that in current world wide, such as Optisol can be in cornea preservation
Chrotoplast activity was up to 14 days.It is long-term to preserve, such as organ culture method, can cornea preservation tissue activity 4 weeks, for clinic.
In view of the holding time of short-term preservation is excessively of short duration, the method for long-term preservation method is complicated, and required equipment is expensive, is not easy
Popularization and application, so it is the current method most approved and the focus developed to carry out Mid-term preservation with corneal storage medium.But very
The balance of the nutrition of tissue and fluid of inside and outside cell is maintained with serum composition in more mid-term preservation liquids, though there is certain effectiveness, is deposited
Infecting some special viral risks.Thus it is inside and outside to carry out statocyte for various ions of the application without biotic component (serum)
The osmotic pressure of liquid is the direction for developing new corneal storage medium.
Nowadays China's cornea activity preservative fluid still relies on import, such as most-often used Optisol preserves liquid, although protecting
It is notable but expensive to hold endothelial cell activity aspect function, and the selling without this product due to the country, can not promote should
With.Therefore, China at present more applies short-range process cornea preservation, and this causes originally just serious deficient corneal donor material source
Because preservation problem more adds crisis.As can be seen here, develop preferably, and the new cornea middle term preserving fluid to tally with the national condition is with most
Limits are urgent problems to be solved using limited donor material.
The research and development of corneal storage medium, the construction to eye bank of China will play an important role, and be beneficial to the guarantor of corneal donor
Deposit, and it is between unit, interzone Rational flow, and for adequately and reasonably utilizing precious corneal donor resource, treatment
More eye illness sufferers have very important significance.
The content of the invention
The invention provides one kind external pressure in keratocyte is maintained without serum composition, with particular electrolyte ion concentration
The preparation scheme of the cornea mid-term preservation liquid of balance.
Cornea of the present invention preserves liquid its composition：
1st, compound electrolyte intra-ocular flushing liquor is as preservation liquid mother liquor；
2nd, sodium chloride 2.34-2.92g/L, magnesium sulfate 0.6-0.72g/L, potassium dihydrogen phosphate 2.72-4.08g/L, make crystal
Osmotic pressure is maintained between 330-380mOsm/kgH2O；
3rd, chondroitin sulfate 25-30g/L, sodium hyaluronate 5-10g/L, it is 310- to maintain the colloid osmotic pressure of its liquid
350mOsm/kg H2Between O.Due to the synergy of chondroitin sulfate, sodium hyaluronate, it is possible to reduce negative in corneal storage medium
Electric charge, while certain colloid osmotic pressure is kept, in storage life oedema does not occur for the cornea for making to be saved；
4th, hydrochloric acid dexamethasone 1-2mg/L, reduced glutathione 1.5-3g/L, with stablize keratocyte lysosome membrane and
Biomembrane, strengthen the defensive ability/resistance ability of cell induced by endotoxin, preferably keep the form and function of keratocyte；
5th, HEPES buffer solution 20-25ml/L, it is 7.2-7.5 to adjust pH value；
6th, lavo-ofloxacin 0.1-0.2g/L, to kill the positive and negative pathogens of the leather Lan Shi of donor carrying.
Corneal storage medium of the present invention is blush, transparency liquid；Using 20ml as a bottled unit, for monolithic
The preservation of cornea.
Brief description of the drawings
Fig. 1 people's fresh cornea is put into corneal storage medium 0 day, and eye bank is detected with Corneal Specular, and endothelial cell number 2300/
mm2
Fig. 2 people's fresh cornea is put into corneal storage medium 14 days, and eye bank is detected with Corneal Specular, endothelial cell number 2100
Individual/mm2
Fig. 3 people's fresh cornea is put into corneal storage medium 0 day, and madder Soviet Union red colouring, 50% endothelial cell is more typical
Hexagon, 2350/mm of cell number2
Fig. 4 people's fresh cornea is put into corneal storage medium 14 days, and alizarin Su Hong and tire expect blue combined staining, and 45% corneal endothelium is thin
Born of the same parents are more typical hexagon, the coloring of about 8-10% endothelial cells core, illustrate that cell has lost activity, 2150/mm of cell number2
The fresh cornea of Fig. 5 cats is put into corneal storage medium 14 days, and madder Soviet Union red colouring, 65% endothelial cell is more typical five
Side shape, 4150/mm of cell number2
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 corneal storage medium：
1st, compound electrolyte intra-ocular flushing liquor 500ml is stand-by as mother liquor；
2nd, take chondroitin sulfate 25g to add deionized water 275ml and dissolve by heating autoclaving, it is molten that chondroitin sulfate is made
Liquid；Sodium chloride 2.93g, magnesium sulfate 0.6g, potassium dihydrogen phosphate 2.72g add water for injection 100ml dissolving autoclavings, sodium are made
Magnesium potassium salt soln；Sodium hyaluronate 5g, water for injection 100ml dissolving autoclavings are added, sodium hyaluronate solution is made；
3rd, sterile chamber is taken to add compound electrolyte intra-ocular flushing liquor 500ml, autoclaving chondroitin sulfate 275ml, sodium
Magnesium potassium salt soln 100ml, sodium hyaluronate solution 100ml；Reduced glutathione 3g, Dexamethasone Injection 1.5mg are added, it is left
Ofloxacin injection 0.2g is mixed；
4th, Hepes buffer solutions adjust pH value to 7.2
5th, it is 340mOsm/kgH to adjust crystal osmotic pressure2O；
6th, it is 350mOsm/kgH to adjust colloid osmotic pressure2O；
7th, it is packed as in every 20ml preservation bottle, 4 degree of refrigerations are standby.
Embodiments of the invention 2
Corneal preserves the effect detection of liquid：
1st, fresh (being obtained after government official in 2 hours) pig eyeball, aseptic process, and total corneal of the clip with 1-2mm limbus of sclera are taken,
It is respectively placed in the Optisol cornea activity preservative fluids of the corneal storage medium and 20ml of the preparation of 20ml embodiments 1 as control；
2nd, preserved 3 days, 7 days and 14 days under the conditions of 4 DEG C, in case being detected to preservation effect.
3rd, in preserving 3 days taking-up corneal films, row bores blue dyeing, the corneal endothelium that as a result discovery cornea activity preservative fluid preserves
There is no difference between the quantity of cell, the transparency of hexagon state and cornea and the Optisol cornea activity preservative fluids compareed；
4th, corneal film is taken out when preserving 1 week, cone of going is blue to be dyed, in the cornea for as a result finding cornea activity preservative fluid preservation
There is no difference between the quantity of chrotoplast, the transparency of hexagon state and cornea and the Optisol cornea activity preservative fluids compareed；
5th, corneal film is taken out when preserving to 2 weeks, the blue dyeing of row cone, as a result finds the cornea that cornea activity preservative fluid preserves
The transparency of the quantity of endothelial cell, hexagon state and cornea does not have between the Optisol cornea activity preservative fluids compareed
Difference, the quantity of cornea activity preservative fluid group endothelial cell declines, but corneal film endothelial cell number is in 1800/mm2More than, angle
Film transparency is good.
Embodiments of the invention 3
Cornea middle term preserving fluid preserves cat cornea penetrating keratoplasty：
1st, fresh (being obtained after government official in 2 hours) cat corneal graft is taken to match somebody with somebody in embodiment 1 in corneal storage medium, 4 DEG C of refrigerations
Preserve 2 weeks, wait to transplant；
2nd, healthy domestic cat 6, it is conventional to apply diameter 7.0mm trepans, drilling central cornea after row anesthesia；After cutting cornea,
The cat corneal graft deposited 2 weeks of going bail for is placed on cutting pillow, is drilled through plant piece from interior surface with the trepan of 7.25mm diameters, is covered in cat
Cornea is planted on hole, 10/0 nylon suture interrupted suture, and watertight forms anterior chamber；
3rd, the slit lamp observation corneal graft transparency of postoperative 1 day, 3 days, 5 days, 7 days and 14 days, uses corneal endothelium in 14 days
Microscopy surveys corneal graft endothelium number；As a result find, postoperative 1-3 days, plant piece is transparent, Mild edema, 5 days after operation, plants piece transparency
It is always maintained at well, postoperative 14 days, corneal endothelium spectroscopy endothelial cell number was in 1800/mm2More than.
Embodiments of the invention 4
The inspection of corneal endothelial cell densities and endothelial cell form after the preservation of people's fresh cornea：
1st, people's cornea tissue (the dead donor of a variety of causes, drawn materials in 6 hours) totally 40, is positioned over eye bank's cornea
Preserve in bottle, each corneal material preserves liquid using 20ml, matches somebody with somebody wherein 20 corneas are put into embodiment 1 in preservation liquid, separately
20 Optisol cornea middle term preserving fluids for being put into import, sealed membrane seal bottleneck, are put in 4 DEG C of refrigerators and preserve.
2nd, it is monitored using eye bank's diagonal membrane material of special specular microscope, carries out endothelial cell count and body point
Analysis, detection time point is respectively 0 day, 1 day, 3 days, 7 days and 14 days.
3rd, result is found, 7 days holding times, corneal endothelial cell densities and 0 day no difference of science of statistics.Preserving 14 days
Afterwards, Ultrasonic pachymetry number is 2252 ± 32 (individual/mm2)；The side row ratio of endothelial cell six was from the 45% of 0 day, by 14 days
32%；The endothelial cell death rate is (9.10 ± 4.4) %.The preservation liquid and Optisol cornea middle term preserving fluids of the present invention
Effectiveness there is no difference.During matching somebody with somebody cornea middle term preserving fluid storage cornea using the embodiment 1, endothelial cell activity is steady
Fixed, change is smaller.
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 cornea mid-term preservation liquid of serum-free composition, its composition and content are as follows：
(1) compound electrolyte intra-ocular flushing liquor；
(2) sodium chloride 2.34-2.92g/L；
(3) magnesium sulfate 0.6-0.72g/L；
(4) potassium dihydrogen phosphate 2.72-4.08g/L；
(5) chondroitin sulfate 25-30g/L；
(6) sodium hyaluronate 5-10g/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；
Wherein, the compound electrolyte intra-ocular flushing liquor is as mother liquor；
The crystalline percolation of the cornea mid-term preservation liquid is pressed in 330-380mOsm/kg H2Between O；
The colloid osmotic pressure 310-350mOsm/kg H of the cornea mid-term preservation liquid2Between O.
2. cornea mid-term preservation liquid as claimed in claim 1, its crystal osmotic pressure pass through reasonable combination sodium chloride, magnesium sulfate
Maintained with the content of potassium dihydrogen phosphate.
3. cornea mid-term preservation liquid as claimed in claim 1, its colloid osmotic pressure by select and match chondroitin sulfate,
The content of sodium hyaluronate maintains.
4. cornea mid-term preservation liquid as claimed in claim 1, maintained using hydrochloric acid dexamethasone and reduced glutathione
The stability of cell membrane, can cornea preservation endothelial cell activity up to 2 weeks.
5. cornea mid-term preservation liquid as claimed in claim 1, using 20ml as a bottled unit, for monolithic cornea group
The preservation knitted.
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| CN108849858A (en) * | 2018-07-26 | 2018-11-23 | 姚晓明 | A kind of cornea middle term preserving fluid |
| CN109329274A (en) * | 2018-11-29 | 2019-02-15 | 镇江雷音再生医学科技有限公司 | A kind of cornea middle term preserving fluid and preparation method thereof containing recombination human serum albumin |
| CN115197913A (en) * | 2021-04-13 | 2022-10-18 | 江苏齐氏生物科技有限公司 | Primary corneal endothelial cell culture solution and application thereof |
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| IT1287227B1 (en) * | 1996-04-04 | 1998-08-04 | Fidia Spa In Amministrazione S | HYALURONIC ACID AS A COMPONENT OF THE CORNEA PRESERVATION LIQUID |
| CN1175822C (en) * | 2002-05-10 | 2004-11-17 | 刘继东 | Compound electrolyte intra-ocular flushing liquor |
| CN1270605C (en) * | 2004-11-15 | 2006-08-23 | 山东省眼科研究所 | Cornea middle term preserving fluid |
| CN104094925B (en) * | 2014-07-18 | 2015-11-18 | 广州优得清生物科技有限公司 | A kind of lamellar cornea conserving liquid |
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Effective date of registration: 20211109 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: BAIO DIESEL (BEIJING) BIOLOGICAL SCIENCE AND TECHNOLOGY CO.,LTD. |