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