loading/dehydration step with sucrose, as well as the freeze-drying
and rehydration procedure. Furthermore, a standard histological
procedure is described to assess the tissue structure after
rehydration.
2 Materials
2.1 Decellularization
and Freeze-Drying
of Heart Valves
1. Braunol disinfection solution (7.5% povidone-iodine).
2. Decellularization solutions: (a) 0.5% (v/v) Triton X-100 and
(b) 0.5% (w/v) sodium dodecyl sulfate (SDS) in filter-sterilized
water.
3. Phosphate-buffered saline solution (PBS): 137 mM NaCl,
27 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , and
pH 7.4.
4. PBS+: PBS supplemented with 1% (w/v) penicillin/streptomycin, 1% (w/v) partricin, and 1% (w/v) gentamycin.
5. Shaking incubator for continuously shaking during decellularization and washing.
6. Lyoprotectant incubation and freeze-drying medium.
(a) PBS/sucrose: 5% (w/v) sucrose in PBS. Prepare 100 mL
(for processing of one heart valve), filter-sterilize using
0.22 μm filters, and store at 4
C until use.
(b) PBS/sucrose: 80% (w/v) sucrose in PBS. Prepare 300 mL
(for processing of one heart valve), filter-sterilize using
0.22 μm filters, and store at 4
C until use (see Note 1).
7. Water bath or incubator for loading the scaffolds with protectants at elevated temperatures (37
C).
8. Freeze-dryer (e.g., Virtis Advantage Plus Benchtop freezedryer from SP scientific, Warminster, PA, USA) (see Note 2).
9. Vacuum sealer and vacuum sealing bags to store freeze-dried
samples.
10. Refrigerator.
2.2 Histological
Evaluation of Tissue
Structure
1. Formalin (3.5%, v/v) solution for fixation of tissue specimens.
2. Ethanol (70, 75, 80, 85, 90, 95, and 100% v/v solutions in
distilled water), isopropanol (100%), and Roticlear (Carl Roth
GmbH, Karlsruhe, Germany) for dehydration of tissue specimens, and liquid paraffin for embedding.
3. Leica TP 1020 automatic tissue processor (Leica, Wetzlar,
Germany) for dehydration of tissue samples with increasing
alcohol concentrations and final embedding in liquid paraffin.
4. Microtome (e.g., manual rotary Reichert-Jung 2040 autocut
microtome, Leica, Wetzlar, Germany) for preparing tissue
sections.
Freeze-Dried Heart Valves
733
and rehydration procedure. Furthermore, a standard histological
procedure is described to assess the tissue structure after
rehydration.
2 Materials
2.1 Decellularization
and Freeze-Drying
of Heart Valves
1. Braunol disinfection solution (7.5% povidone-iodine).
2. Decellularization solutions: (a) 0.5% (v/v) Triton X-100 and
(b) 0.5% (w/v) sodium dodecyl sulfate (SDS) in filter-sterilized
water.
3. Phosphate-buffered saline solution (PBS): 137 mM NaCl,
27 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , and
pH 7.4.
4. PBS+: PBS supplemented with 1% (w/v) penicillin/streptomycin, 1% (w/v) partricin, and 1% (w/v) gentamycin.
5. Shaking incubator for continuously shaking during decellularization and washing.
6. Lyoprotectant incubation and freeze-drying medium.
(a) PBS/sucrose: 5% (w/v) sucrose in PBS. Prepare 100 mL
(for processing of one heart valve), filter-sterilize using
0.22 μm filters, and store at 4
C until use.
(b) PBS/sucrose: 80% (w/v) sucrose in PBS. Prepare 300 mL
(for processing of one heart valve), filter-sterilize using
0.22 μm filters, and store at 4
C until use (see Note 1).
7. Water bath or incubator for loading the scaffolds with protectants at elevated temperatures (37
C).
8. Freeze-dryer (e.g., Virtis Advantage Plus Benchtop freezedryer from SP scientific, Warminster, PA, USA) (see Note 2).
9. Vacuum sealer and vacuum sealing bags to store freeze-dried
samples.
10. Refrigerator.
2.2 Histological
Evaluation of Tissue
Structure
1. Formalin (3.5%, v/v) solution for fixation of tissue specimens.
2. Ethanol (70, 75, 80, 85, 90, 95, and 100% v/v solutions in
distilled water), isopropanol (100%), and Roticlear (Carl Roth
GmbH, Karlsruhe, Germany) for dehydration of tissue specimens, and liquid paraffin for embedding.
3. Leica TP 1020 automatic tissue processor (Leica, Wetzlar,
Germany) for dehydration of tissue samples with increasing
alcohol concentrations and final embedding in liquid paraffin.
4. Microtome (e.g., manual rotary Reichert-Jung 2040 autocut
microtome, Leica, Wetzlar, Germany) for preparing tissue
sections.
Freeze-Dried Heart Valves
733