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CORNEAL PRESERVATION & STORAGEDR.REKHA.
Lions Eye hospital
BANGALORE
IMPORTANCE OF STORAGE
SUCCESS OF PENENTRATING
KERATOPLASTY
DEPEND UPON CORNEAL
ENDOTHELIAL INTEGRITY.
CORNEAL STORAGE SYSTEM
AIM To maintain the endothelial viability &
integrity. Prevent corneal autolysis during storage Long term corneal storage.
HISTORY
First corneal preservation.
1911 Mgitot in J.A.M.A.
Stored cornea in Hemolyzed blood serum at 5-7deg.centigrade.
Cornea viable for2days
STORAGE TYPES SHORT-4deg.C(days) Moist chamber(1day),M.K.Media(2-4days)
INTERMEDIATE-14days- 4deg.C(wks) K-Sol, Dexsol,OptisolGS,Procell, Eusol-C
LONG(months) EuropeanOrgan culture-120days(37deg.C) Eurosol-31deg.C-28days(New) Cryoprev.-Unlimited-(-80degC)
MOIST CHAMBER METHOD Filatov &Castroviejo. Whole globe is stored in a sterile jar. Jar has saline Humidification. Temp of 4degrees C. Popular untill 1970. Simple & in- expensive. Grafting on emergency basis.
McCAREY&KUFMAN STORAGE Corneo-scleral excision is necessary&
immersed with endothelial side up. Contains
Tc199,dextran-40 1%, PH7.0-7.5,Osmolality
295=355 & gentamycin sulphate75-150
micro gm/ml ,HEPES as buffer.
Phenol red as indicator. Scheduling of surgery
STORAGE MEDIAK-SOL: Tc 199,MEM&Earles media,
HEPES,Gentamicin,Chondroitin
sulphate2.5% .
1988 proprionibacteria contamination
DEXOL: MEM, 1.35%Chondrotin
Sulphate, 1mM Sodium pyruvate,
1mM non- essential aminoacids,
Antioxidants, 1%dextran40.
.
OPTISOL.G.S. Introduced in 1991 Contents MEM, 1.35%Chondrotin
Sulphate, 1mMSodiumpyruvate,1mM
non-essential aminoacids, Antioxidants,
1%dextran40,ATP,Iron,cholestrol,L-
hydroxyproline,Vitamine,2antibiotics-
Genticyn,Stepomycin. Expensive
EUSOL-C
Store at 4deg.C Storage time 14days, SIMILAR TO DEXOL CONTENTS Dextran,Sodium Piruvate,Glucose,Essential
&non-essential aminoacids, mineral salts, Vitamins, Gentamin, hepesbuffer, Bicarbonate, Phenol Red.
FUNCTIONS OF CONTENTS
BASE MEDIA
a)Tissue-culture199-aminoacids,salts,buffer&energy to support life.
b)Minimum Essential Media(MEM) with Earles salt- matches natural salts &buffers of human tissue,electrolites similar to aqueous
FUNCTIONS OF CONTENTS
BUFFER
HEPES(N-2-hydroxyethyl-piperazine-N-2ethane-sulfonic acid)
-used for cultivation of sensitive mammalian cells
-provides optimal physiological Ph in the range7-7.2
COMMON FEATURES OF MEDIA ANTIBIOTIC
Gentamicin only /&Streptomycin
DEXTRAN
Polysaccharide,negatively charged.
Prevents tissue swelling.
Used alone does not provide sufficient tissue stability & viability for extended storage.
COMMON FEATURES OF MEDIA CHONDROTIN SULFATE
Polysaccharide,negatively charged
Endothelial integrity&acts as a osmotic
agent.Superior to dextran.Longer storage
time ENRICHED
Various salts,aminoacids essential &non-
essential,vit.,phosphates &antioxidents to enhance cell & tissue viability & health during storage.
COMMON MEDIA FEATURES ATP precursors--> Provide energy for
pumping function.
Anti-Oxidents--->Neutralize metabolic waste,maintain DNS synthesis
Vitamins-->Provides additional nutritional cell supplements.
Sodium Piruvate&Glucose--> Energy supply
Colour Phenol Red-->Visual aid for pH
indication.
CHECK MEDIA The cornea which is stored in
MKmedia,Dexol,optisolGS.Thefollowing parameters to be checked before using
Intact seal,Expiry date,Turbity, Colour (rose red) ,precipitates & FB.
COLOUR CHANGE INDICATES
Yellow-- Bacterial contamination.
Red--- Unacceptable Ph.
Cloudy-- Contamination
Ph COLOUR REFERENCE CHART 7.00
7.25
7.50
contimanation
MEM,1.35%chondrotin sulphate,1mM
sodium pyruvate1mM,Non-essential aminoacids,Antioxidants,Dextran40,
Humen insulin10mic.g/ml & Human epidermal growth factor(hEGF10ng/ml) to improve long term endothelial survival after PKP.(experiments have not proved it, opth1994 feb101(2)252-9 LassJH et all)
PROCELL
ORGAN CULTURE 1936,by Archer & Trevor-Roper. Being used since1974. Refrigeration not required. Complicated,expensive & well trained
microbiologist.
EUROPEAN ORGAN CULTURE
31-37deg.C, EMEM, MEM, IMDM ,
HEPES, NaHCO3,Peni,Strep,Ampo-B,
BOVINE SERUM 2-10ML,Dextran4-10%
ORGAN CULTURE
contents
Eagles media,Earles salt with out L-glutamine,L-glutamine1%final conc.,Decomlemented calf serum10%final conc.Penicilllin 100units/ml,, Gentamicin100microgram/ml. AmphotericianB 0.25% microgram/ml.
TISSUE CULTURE MEDIA EUROSOL New
Duration of storage 28days.
Temp 31deg.C.
Better then the European culture media.
Maintains intact endothelium, epithelium
& Keratocyte.
ORGAN CULTURE
ADVANTAGES:Endothelial integrity 120days,Epithelial integrity35days,Culture report prior to transplantation,stored at 4deg,centi.
DISADVANTAGES:Cornea thickens with time,Microbial contamination, Complicated expensive techniques, Greater loss of endothelial afterPK.
CRYOPRESERVATION
Kaufman &Capella.1954 Cornea is stored in Liquid Nitrogen with
DimethylSulfoxide(DMSO). DMSO,Prevents intracellular damage by ice
crystals. Not a procedure used.Holds a lot of
research interest.
CRYOPRESERVATION
ADVANTAGES:Long-term storage at- 80deg.C.DISADVANTAGES:Technically difficult,Limited tissue tolerance to freez- thaw. Rapid thaw at 37 deg.C. 1964 18months Tissue strored in Liquid nitrogen at --197deg C At the time of thawing the cial containing cornea is placed
in water bath at temp of 60dec C at 1min. Trained techiques for Freezing & thawing
GLYCERINE PRESERVATION
Patch graft or Lamellar Keraoplasty.
Corneo-scleral button.
100%glycerine.
Enothelium is nonviable
Use with in 1 year.
Stored at room temp.
NOTE
HYPOTHERMIC (CS+Hyperkalaemic) Storage 2wks,epithelial not well preserved,not
used
ORGAN CULTURE At one time predominating in Europe Organ culture storage-4wks
CRYOPRESERVATION Not used, tissure strored for emergency use
FUTURE
Improve endothelial cell layer viability
&compact stroma. Maintain Keratocyte viability. Reduce corneal autolysis during storage. Thin cornea in spite of long time storage. No contamenation Easy to use & transport Inexpensive.
Thank you