Transcript 8-CORNEAL-PRESERVATION
CORNEAL PRESERVATION & STORAGE
DR.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 MEDIA
K-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
PROCELL
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)
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.