of extracellular matrix components. The obtained extracellular
matrix closely resembles properties of native tissue but lacks immunogenic factors of cellular components leading to immune
responses in patients. In the surgical practice, sufficient availability
of decellularized heart valves of different sizes is required. Heart
valves that are stored in liquid solutions after decellularization
processing are prone to degradation reactions and bacterial contamination. Therefore, appropriate preservation methods are
needed to ensure off-the-shelf availability. Freeze-drying allows
room temperature storage and easy transport.
Freeze-drying increases the shelf stability of biological materials
by removing water and slowing down degradation reactions.
Freeze-drying can also be applied to store excised heart valves.
The efficacy of freeze-dried homografts has been investigated in
the 1960s and 1970s, but tests on in vivo performance yielded
unsatisfactory results [5, 6]. Curtil et al. [7] reported that freezedrying of allograft heart valves resulted in porous leaflets after
rehydration. Surprisingly, no lyoprotectants were used in these
studies to protect the tissues from the damaging effects of freezedrying. Freeze-drying damage is visible as pores in the tissue caused
by ice crystal formation during the freezing step.
We have established methods for freeze-drying of decellularized heart valves using sucrose as lyoprotectant [8–10]. Sucrose
treatment preserves the extracellular matrix (ECM) histoarchitecture. However, pretreatment with very high sucrose concentrations
is needed (!40% w/v) to avoid pore formation [10]. Just like other
disaccharides, sucrose protects biomolecules by replacing water
that is normally hydrogen bonded and by forming a stabilizing
glassy state in which degradation reactions are kinetically limited.
In order to estimate loading times needed to load tissues with
sufficient amounts of sucrose, permeation kinetics of protective
molecules into heart valve tissues first needs to be investigated.
We have recently demonstrated that sucrose-protected freezedried decellularized pulmonary heart valves show excellent clinical
performance and cell repopulation potential in juvenile sheep [11].
Heart valve scaffolds, which are meant to be used as implants,
can be produced using decellularization approaches, which typically
comprise of treatment of an excised heart valve tissue construct
with detergents followed by extensive washing. Freeze-drying
requires pretreatment of the decellularized valves with protectants
(e.g., sucrose), followed by controlled freezing and drying. The
drying process is divided in a primary and a secondary drying step.
During primary drying, the freezable water fraction is removed
from frozen material under reduced pressure by sublimation,
whereas during secondary drying, the unfrozen water is removed
through desorption, resulting in a product with a water content
typically less than 5% (w/w). This chapter outlines the decellularization process to remove cells from porcine heart valves, the
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Willem F. Wolkers and Andres Hilfiker
matrix closely resembles properties of native tissue but lacks immunogenic factors of cellular components leading to immune
responses in patients. In the surgical practice, sufficient availability
of decellularized heart valves of different sizes is required. Heart
valves that are stored in liquid solutions after decellularization
processing are prone to degradation reactions and bacterial contamination. Therefore, appropriate preservation methods are
needed to ensure off-the-shelf availability. Freeze-drying allows
room temperature storage and easy transport.
Freeze-drying increases the shelf stability of biological materials
by removing water and slowing down degradation reactions.
Freeze-drying can also be applied to store excised heart valves.
The efficacy of freeze-dried homografts has been investigated in
the 1960s and 1970s, but tests on in vivo performance yielded
unsatisfactory results [5, 6]. Curtil et al. [7] reported that freezedrying of allograft heart valves resulted in porous leaflets after
rehydration. Surprisingly, no lyoprotectants were used in these
studies to protect the tissues from the damaging effects of freezedrying. Freeze-drying damage is visible as pores in the tissue caused
by ice crystal formation during the freezing step.
We have established methods for freeze-drying of decellularized heart valves using sucrose as lyoprotectant [8–10]. Sucrose
treatment preserves the extracellular matrix (ECM) histoarchitecture. However, pretreatment with very high sucrose concentrations
is needed (!40% w/v) to avoid pore formation [10]. Just like other
disaccharides, sucrose protects biomolecules by replacing water
that is normally hydrogen bonded and by forming a stabilizing
glassy state in which degradation reactions are kinetically limited.
In order to estimate loading times needed to load tissues with
sufficient amounts of sucrose, permeation kinetics of protective
molecules into heart valve tissues first needs to be investigated.
We have recently demonstrated that sucrose-protected freezedried decellularized pulmonary heart valves show excellent clinical
performance and cell repopulation potential in juvenile sheep [11].
Heart valve scaffolds, which are meant to be used as implants,
can be produced using decellularization approaches, which typically
comprise of treatment of an excised heart valve tissue construct
with detergents followed by extensive washing. Freeze-drying
requires pretreatment of the decellularized valves with protectants
(e.g., sucrose), followed by controlled freezing and drying. The
drying process is divided in a primary and a secondary drying step.
During primary drying, the freezable water fraction is removed
from frozen material under reduced pressure by sublimation,
whereas during secondary drying, the unfrozen water is removed
through desorption, resulting in a product with a water content
typically less than 5% (w/w). This chapter outlines the decellularization process to remove cells from porcine heart valves, the
732
Willem F. Wolkers and Andres Hilfiker