new attempt to freeze-drying red blood cells using …...5th annual international conference on...
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5th Annual International Conference on Lyophilization and Freeze Drying
Bologna, Italy, March 2012
New attempt to freeze-drying red blood cells using stevioside as an alternative of lyoprotectant
Virgilio Tattini Jr., Mariana M. Vilhena, Ludmila Moroz, Denise T. Fantoni, Ronaldo N.M. Pitombo
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Dr. Virgilio Tattini JuniorPhD Lyophilization Technology
Professor Ronaldo Pitombo
Thomas Jennings
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Introduction
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• Several attempts were made to improve RBC storage conditions:
Cryo/lyoptortectants;
Process engineering: freeze conditions, temperature profiles;
• Something is missing ?
• More important than lyoprotectants itself !
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Aim
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Material and Methods
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Loading RBC
ElectroporationImmersion
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Thermal Analisys
(RBC, Stevioside, RBC + Stevioside )
DSC Lyostat
Tg´ Tcryst.Tmelt.
Tcol. Tcryst.Tmelt.
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FreezingSF (shelves): +25 to -55ºC (± 1,5ºC/min.);FF (N2): +25 to – 150ºC (± 35ºC/min.);
Primary DryingTsh: -35ºCPch: 130 mTorrTco: -80 ºC
Secondary DryingTsh: 25ºCPch: 130 mTorrTco: -80 ºC
Freeze-drying
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Hematology analysis and hemolysis measurement
Percent hemolysis (Drabkin’s reagent);
% Hemolysis = 100 x (OD540 of the free Hb)(OD540 of the total Hb)
Radiometer blood gas analyzer
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Morphology
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Results
Fig.1.Comparison between immersion and electroporation methodologies on the pH values of RBC suspensionincorporated with stevioside at different concentrations.
Optimal pH
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Fig.2. Comparison between immersion and Electroporation methodologies on the gasometry of RBCsuspension incorporated with stevioside at different concentrations.
Optimal CO2Optimal O2
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Fig.3. Comparison between immersion and electroporation methodologies on the degree of hemolysis ofRBC suspension incorporated with stevioside at different concentrations.
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Fig.4. Morphological characteristics of RBCs, incorporated with stevioside by immersion, obtained byoptical microscopy of 500 x magnitude. (A) stevioside 1%; (B) stevioside 3%; (C) stevioside 5% and (D)stevioside 10%.
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Fig.5. Morphological characteristics of RBCs, incorporated with stevioside by electroporation, obtained byoptical microscopy of 500 x magnitude. (A) stevioside 1%; (B) stevioside 3%; (C) stevioside 5% and (D)stevioside 10%.
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Fig.6. DSC curve showing the glass transition temperature of the maximally freeze-concentratedregion (Tg´) of RBC´s incorporated with stevioside 3% by electroporation.
Tg´ midpoint
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Fig.7. Freeze-drying microscopy images of RBC´s with stevioside 3% incorporated by electroporation. (A)Crystallization of the sample at – 14.3ºC; (B) Freeze-drying front formed at – 44ºC; (C) Progress ofsublimation front at – 37ºC; (D) Collapse of the sample at – 34.3ºC.
RBC suspensionRBC suspension CrystallizationCrystallization
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Determining the optimum freezing rate during the freeze-drying process of RBC
Fig.8. Comparison between slow freezing and fastfreezing on the pH values of freeze-dried RBC´sincorporated with stevioside 3% by electroporation.
Fig.9. Comparison between slow freezing and fastfreezing on the gasometry results of freeze-dried RBC´sincorporated with stevioside 3% by electroporation.
Optimal pH
Optimal CO2
Optimal O2
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Fig.10. Comparison between slow freezing (A) and fastfreezing (B) on the SEM of freeze-dried RBC´s incorporatedwith stevioside 3% by electroporation. Magnitude of 2000and 5000 X respectively.
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Conclusion
Stevioside (1 and 3%) – OK
Freeze-drying – not OK
Future
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THANK YOU
Dr. Virgilio Tattini JuniorPhD Lyophilization Technology