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Corneal Preservation and Storage: Importance for Penetrating Keratoplasty Success

Learn about the history, types, and methods of corneal storage systems. Understand the functions and contents of storage media such as K-Sol, Dexsol, and Eusol-C. Discover the benefits of organ culture and cryopreservation for long-term corneal viability.

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Corneal Preservation and Storage: Importance for Penetrating Keratoplasty Success

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  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 • To maintain the endothelial viability & integrity. • Prevent corneal autolysis during storage • Long term corneal storage.

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

  5. 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)

  6. 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.

  7. 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

  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. • 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

  10. 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.

  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 timeENRICHED 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 • 7.00 7.25 7.50 contimanation

  18. 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)

  19. 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%

  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 • 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.

  24. 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

  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 • 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

  27. 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.

  28. Thank you

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