cardiac anomalies. Cryopreserved allografts have especially benefited children with congenital heart disease since the use of alternative mechanical and xenogeneic tissue valves has historically been
limited in this patient population. They are also used in women of
childbearing age and older patients with memory problems who
may not be relied upon to keep up with the medications required
for mechanical valves.
In the last century, cryopreserved, control rate frozen DMSO
protected, human heart valves were used in approximately 20% of
the tissue heart valve procedures performed annually [1–4]. They
are currently less utilized because of new xenogeneic valves, which
are impacting aortic valve utilization, and because of failures in
young patients [5], particularly infants [6, 7]. The pathophysiology
of allograft heart valve failure is not fully understood [8–10]. Immunology definitely played a significant role in failure in young
patients, which led to the development of decellularization methods to minimize the recipient immune response upon implantation.
We, however, hypothesized that the rapid deterioration seen in
some allograft heart valve recipients might be due, at least in part,
to disruptive interstitial ice damage that occurred during cryopreservation leading subsequently to accelerated valve degeneration
upon implantation. About 75% of the area of conventional
DMSO cryopreserved heart valve leaflets, the most important functional component of a heart valve, is occupied by ice in cryosubstituted samples [11, 12]. It was hard to imagine that such
extensive ice formation was not damaging the tissue. Both light
and electron microscopy demonstrated that the ice was extracellular. Upon thawing and rehydration, the leaflets looked histologically normal because the ice domains within the extracellular matrix
closed up as the ice melted.
Our first heart valve vitrification studies utilized pieces of porcine aortic valves and intact rat aortic valves in small volumes of a
vitrification solution (VS) called VS55 to reflect its 55% (w/v) of
cryoprotectant solutes that was originally designated VS41A by its
originators [13]. VS55 consists of an 8.4 M mixture of
1,2-propanediol, formamide, and dimethyl sulfoxide in EuroCollins (EC) solution. EC is an extracellular-type hypothermic organ
preservation solution that was developed in the 1970s by Collins
and subsequently modified in Europe, where it was the gold standard for hypothermic storage of human kidneys for several years.
The protocol is described in US Patent #6,740,484 [14, 15] and in
Protocol 1 below. In vitro studies with animal tissues have repeatedly demonstrated that Protocol 1 maintained ~80% heart valve
leaflet cell viability immediately after rewarming, returning to control values over a few days in tissue culture. This protocol was
excellent for small volume tissue samples where relatively rapid
cooling and warming rates that avoided ice formation could be
achieved. However, upon scale up to full-sized heart valves,
594
Kelvin G. M. Brockbank et al.
limited in this patient population. They are also used in women of
childbearing age and older patients with memory problems who
may not be relied upon to keep up with the medications required
for mechanical valves.
In the last century, cryopreserved, control rate frozen DMSO
protected, human heart valves were used in approximately 20% of
the tissue heart valve procedures performed annually [1–4]. They
are currently less utilized because of new xenogeneic valves, which
are impacting aortic valve utilization, and because of failures in
young patients [5], particularly infants [6, 7]. The pathophysiology
of allograft heart valve failure is not fully understood [8–10]. Immunology definitely played a significant role in failure in young
patients, which led to the development of decellularization methods to minimize the recipient immune response upon implantation.
We, however, hypothesized that the rapid deterioration seen in
some allograft heart valve recipients might be due, at least in part,
to disruptive interstitial ice damage that occurred during cryopreservation leading subsequently to accelerated valve degeneration
upon implantation. About 75% of the area of conventional
DMSO cryopreserved heart valve leaflets, the most important functional component of a heart valve, is occupied by ice in cryosubstituted samples [11, 12]. It was hard to imagine that such
extensive ice formation was not damaging the tissue. Both light
and electron microscopy demonstrated that the ice was extracellular. Upon thawing and rehydration, the leaflets looked histologically normal because the ice domains within the extracellular matrix
closed up as the ice melted.
Our first heart valve vitrification studies utilized pieces of porcine aortic valves and intact rat aortic valves in small volumes of a
vitrification solution (VS) called VS55 to reflect its 55% (w/v) of
cryoprotectant solutes that was originally designated VS41A by its
originators [13]. VS55 consists of an 8.4 M mixture of
1,2-propanediol, formamide, and dimethyl sulfoxide in EuroCollins (EC) solution. EC is an extracellular-type hypothermic organ
preservation solution that was developed in the 1970s by Collins
and subsequently modified in Europe, where it was the gold standard for hypothermic storage of human kidneys for several years.
The protocol is described in US Patent #6,740,484 [14, 15] and in
Protocol 1 below. In vitro studies with animal tissues have repeatedly demonstrated that Protocol 1 maintained ~80% heart valve
leaflet cell viability immediately after rewarming, returning to control values over a few days in tissue culture. This protocol was
excellent for small volume tissue samples where relatively rapid
cooling and warming rates that avoided ice formation could be
achieved. However, upon scale up to full-sized heart valves,
594
Kelvin G. M. Brockbank et al.