1. CORNEAL PRESERVATION & STORAGE DR.REKHA. Lions Eye hospital BANGALORE

2. IMPORTANCE OF STORAGE SUCCESS OF PENENTRATING KERATOPLASTY DEPEND UPON CORNEAL ENDOTHELIAL INTEGRITY.

3. CORNEAL STORAGE SYSTEM AIM w To maintain the endothelial viability & integrity. w Prevent corneal autolysis during storage w Long term corneal storage.

4. HISTORY w First corneal preservation. w 1911 Mgitot in J.A.M.A. w Stored cornea in Hemolyzed blood serum at 5-7deg.centigrade. w Cornea viable for2days

5. STORAGE TYPES SHORT-4deg.C(days) w Moist chamber(1day),M.K.Media(2-4days) INTERMEDIATE-14days- 4deg.C(wks) w K-Sol, Dexsol,OptisolGS,Procell, Eusol-C LONG(months) w EuropeanOrgan culture-120days(37deg.C) w Eurosol-31deg.C-28days(New) w Cryoprev.-Unlimited-(-80degC)

6. MOIST CHAMBER METHOD w Filatov &Castroviejo. w Whole globe is stored in a sterile jar. w Jar has saline Humidification. w Temp of 4degrees C. w Popular untill 1970. w Simple & in- expensive. w Grafting on emergency basis.

7. McCAREY&KUFMAN STORAGE Corneo-scleral excision is necessary& immersed with endothelial side up. w 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. w Scheduling of surgery

8. 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..

9. OPTISOL.G.S. w Introduced in 1991 w Contents w MEM, 1.35%Chondrotin Sulphate, 1mMSodiumpyruvate,1mM non-essential aminoacids, Antioxidants, 1%dextran40,ATP,Iron,cholestrol,L- hydroxyproline,Vitamine,2antibiotics- Genticyn,Stepomycin. Expensive

10. EUSOL-C w Store at 4deg.C w Storage time 14days, SIMILAR TO DEXOL w CONTENTS w Dextran,Sodium Piruvate,Glucose,Essential &non-essential aminoacids, mineral salts, Vitamins, Gentamin, hepesbuffer, Bicarbonate, Phenol Red. w

11. 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

12. 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

13. 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.

14. 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.

15. 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.

16. 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

17. Ph COLOUR REFERENCE CHART w 7.00 7.25 7.50 contimanation

18. 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

19. ORGAN CULTURE w 1936,by Archer & Trevor-Roper. w Being used since1974. w Refrigeration not required. w 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%

20. 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.

21. TISSUE CULTURE MEDIA EUROSOL New Duration of storage 28days. Temp 31deg.C. Better then the European culture media. Maintains intact endothelium, epithelium & Keratocyte.

22. 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.

23. CRYOPRESERVATION w Kaufman &Capella.1954 w Cornea is stored in Liquid Nitrogen with DimethylSulfoxide(DMSO). w DMSO,Prevents intracellular damage by ice crystals. w Not a procedure used.Holds a lot of research interest.

24. CRYOPRESERVATION ADVANTAGES:Long-term storage at- 80deg.C. DISADVANTAGES:Technically difficult,Limited tissue tolerance to freez- thaw. Rapid thaw at 37 deg.C. w 1964 w 18months w Tissue strored in Liquid nitrogen at --197deg C w At the time of thawing the cial containing cornea is placed in water bath at temp of 60dec C at 1min. w Trained techiques for Freezing & thawing

25. GLYCERINE PRESERVATION Patch graft or Lamellar Keraoplasty. Corneo-scleral button. 100%glycerine. Enothelium is nonviable Use with in 1 year. Stored at room temp.

26. NOTE w HYPOTHERMIC (CS+Hyperkalaemic) w Storage 2wks,epithelial not well preserved,not used w ORGAN CULTURE w At one time predominating in Europe w Organ culture storage-4wks w CRYOPRESERVATION w Not used, tissure strored for emergency use

27. FUTURE w Improve endothelial cell layer viability &compact stroma. w Maintain Keratocyte viability. w Reduce corneal autolysis during storage. w Thin cornea in spite of long time storage. w No contamenation w Easy to use & transport w Inexpensive.

28. Thank you