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Protocols for thawing and cryoprotectant dilution of heart valves
Cryobiology 50 (2005) 17–20 www.elsevier.com/locate/ycryo
Protocols for thawing and cryoprotectant dilution of heart valvesq W. John Armitage*, Wayne Dale, Emily A Alexander Bristol Heart Valve Bank, Department of Clinical Science, University of Bristol, Bristol, BS1 2LX, United Kingdom Received 16 July 2004; accepted 20 September 2004 Available online 9 December 2004
Abstract Purpose: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. Methods: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1—Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2—Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37°C water bath. The DMSO was removed by 4-step dilution. Experiment 3— Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). Results: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. Conclusion: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Heart valve; Cryopreservation; DMSO; Warming rate
q
This work was funded by institutional sources. Corresponding author. Fax: +44 117 904 6624. E-mail address: [email protected] (W.J. Armitage).
*
Human heart valve allografts can be successfully stored by cryopreservation [2,3], which greatly improves the logistics of storage and distribution and minimizes wastage. Although the need for a viable population of valve leaflet fibroblasts in allografts has not been unequivocally demon-
0011-2240/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2004.09.005
18
W.J. Armitage et al. / Cryobiology 50 (2005) 17–20
strated, it is generally considered that the success of heart valve cryopreservation is at least in part due to the preservation of these cells. Since abrupt changes in cryoprotectant concentration can cause osmotic damage to cells [1], multi-step protocols for the removal of cryoprotectants are advocated by some heart valve banks. The time taken to prepare a valve for surgery may, however, be an important consideration since the thawing and cryoprotectant dilution is typically carried out in the operating theatre while the patient (often a child) is on cardio-pulmonary bypass. For valves frozen in 100 ml of cryoprotectant medium, such as 10% v/v dimethyl sulphoxide (DMSO), thawing takes approximately 10 min, and removal of the DMSO by 4-step dilution protocol adds a further 12–15 min, giving a total of at least 20 min before the valve can be implanted. Our aim was to reduce this time by increasing the warming rate and reducing the time taken to dilute out the cryoprotectant, but without compromising the integrity of the valve leaflet fibroblasts.
Methods Three sets of experiments were carried out. Pulmonary and aortic valves were dissected from porcine hearts obtained from an abattoir. The hearts were processed within just a few hours of retrieval, well within the time limits acceptable for the processing of human heart valves. For all three sets of experiments, valves were first exposed to 10% (v/v) DMSO using a 2-step protocol. Valves were initially exposed to 5% (v/v) DMSO in EagleÕs MEM for 10 min at ambient temperature (22°C) and then to 10% DMSO for a further 10 min. After each experimental treatment, the outgrowth of fibroblasts from valve leaflet explants in primary tissue culture was chosen as a measure cellular viability. Each group contained at least four valves.
Table 1 Stepwise dilution protocols for valves exposed to 10% (v/v) DMSO Step
Step duration (ml)
Volume added (ml)
Total volume (ml)
4-Step dilution 1 2 3 4
3 3 3 3
100 200 400 400
200 400 800 400a
2-Step dilution 1 2
3 9
100 400
200 400a
1-Step dilution 1
12
400
400a
The diluent was HartmannÕs solution and the starting volume, including the valve, was 100 ml in all cases. Temperature was maintained at 0–4°C and, in each case, the dilution protocol took 12 min to complete. a Valve transferred to 400-ml fresh HartmannÕs solution.
Experiment 2—Warming rate The only way to increase the warming rate without raising the temperature of the water bath used for thawing (37°C) was to reduce the volume of cryoprotectant solution from the standard 100 ml used for freezing the valves. Valves were cooled at 1°C/min to 100°C in 100, 50, 25, or 0 ml of 10% DMSO. After storage overnight in the vapour phase of a liquid nitrogen refrigerator, the valves were allowed to warm slowly to 80°C and then warmed in a 37°C water bath until all the ice had melted. After thawing the DMSO was removed by 4-step dilution (Table 1). Experiment 3—Standard vs. Modified protocol Finally, valves frozen and thawed by a modified protocol, based on the outcome of Experiments 1 and 2, were compared with valves frozen by the standard protocol used in Bristol Heart Valve Bank (Table 2).
Experiment 1—Dilution of DMSO Primary tissue culture Valves were subjected to 4-, 2-, or single-step dilution with HartmannÕs solution at 0–4°C to remove the DMSO (see Table 1 for details). Untreated controls were not exposed to DMSO.
After experimental treatment, the leaflets were dissected from the valves. Each leaflet was divided into four pieces and each piece placed in a separate
W.J. Armitage et al. / Cryobiology 50 (2005) 17–20
19
Table 2 Details of the Standard and Modified protocols used for Experiment 3 Protocol
Standard
Modified
Addition of 10% DMSO Volume of 10% DMSO Cooling rate
2-Step 100 ml 1°C/min to 100°C 140°C 4-Step
2-Step 50 ml 1°C/min to 100°C 140°C 1-Step
Storage temperature Dilution to remove DMSOa a
See Table 1 for details.
well of a 12-well tissue culture plate. The growth medium was EagleÕs MEM supplemented with 10% fetal bovine serum. The explants were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed every 2–3 days. The tissue explants were monitored daily for cell growth, which, after 2 weeks, was graded as follows: 0—no cell outgrowth evident 1—cell outgrowth, but sparse distribution around explant 2—good growth of cells surrounding explant and spreading out 3—confluent cell cultures.
Data analysis
Fig. 1. Effect of dilution protocol on growth of cells from valve leaflet explants after two weeks in culture. Controls were not exposed to DMSO. The numbers of explants are given in each group showing little or no growth of cells (i.e., growth =1, solid bars) vs. good growth/confluence (i.e., growth >1, hatched bars).
graded 61), v2 analysis was not possible. However, the results strongly suggest that 1-step dilution was not detrimental to the cells. Warming rate Fig. 2 shows that warming rate also had little effect on the number of explants showing good outgrowth of cells at 2 weeks (v2 = 2.3, df = 3, p = 0.52). Volumes less than 50 ml were insufficient to immerse valves completely, but a reduction from 100 to 50 ml halved the time taken to
v2 tests or FisherÕs exact test were used to compare the numbers of explants in the different groups showing cell growth graded P2 after two weeks in culture. The level of significance was set at 5%.
Results Dilution of DMSO The rate of dilution of DMSO had little effect on the number of explants showing outgrowth of cells at 2 weeks, although the 4-step dilution appeared to be less favourable (Fig. 1). Since all of the explants in the 1-step dilution group showed good outgrowth of cells (i.e., none were
Fig. 2. Effect of warming rate (controlled by volume of cryoprotectant medium) on growth of cells from valve leaflet explants after two weeks in culture. The numbers of explants are given in each group showing little or no growth of cells (i.e., growth =1, solid bars) vs. good growth/confluence (i.e., growth >1, hatched bars).
20
W.J. Armitage et al. / Cryobiology 50 (2005) 17–20
Fig. 3. Effect of the standard vs. the modified cryopreservation protocol on growth of cells from valve leaflet explants after two weeks in culture. Controls were not cryopreserved. The numbers of explants are given in each group showing little or no growth of cells (i.e., growth =1, solid bars) vs. good growth/ confluence (i.e., growth >1, hatched bars).
warm a valve. The time taken to warm from 79 to 0°C was 8 min (SD = 1.1 min) for 100 ml and 4 min (SD = 0.6 min) for 50 ml. These times were equivalent to linearized warming rates of, respectively, 10 and 20°C/min. Standard vs. Modified protocol Valves were cryopreserved according either to the standard protocol used at the time by Bristol Heart Valve Bank or to a Modified protocol that reduced the warming time and applied a singlestep dilution protocol for removal of the cryoprotectant. Fig. 3 suggests that the outgrowth of cells from the two cryopreserved groups was slightly less than the untreated control (v2 = 7.8, df = 2, p = 0.02). A direct comparison between just the standard and modified protocols using FisherÕs exact test showed no difference between the cryopreservation protocols (p = 0.23).
Discussion The rationale for decreasing the rate of dilution of cryoprotectants after thawing by increasing the number of steps in a dilution protocol is to lessen the risk of cellular damage from osmotic stress [1]. Heart valves, however, are typically exposed to only 10% v/v DMSO and, given that DMSO tends to cross cell membranes readily, a multi-step
protocol for the removal of this concentration of DMSO after thawing is perhaps not warranted. Our results supported this contention, showing that a single-step dilution was as well-tolerated by the valve leaflet fibroblasts as a 4-step protocol. Similarly, reducing the volume of cryoprotectant medium as a means of increasing the warming rate, also had little effect on the outgrowth of cells from the explants. For the final experiment, valves were frozen in 50-ml of cryoprotectant medium. This volume was chosen because it was just sufficient to be able to freeze a valve completely immersed in medium, which we felt might better protect the valve from physical trauma during subsequent handling. Freezing in smaller volumes, which would have resulted in more rapid warming, was just as effective at preserving cellular viability. This could be an option should a further reduction in the time taken to prepare a valve for surgery be considered desirable. In the final experiment, cryopreservation resulted in slightly fewer explants achieving good outgrowth of cells compared with unfrozen control tissue; but the effect was small. Moreover, the modified protocol using more rapid warming and single-step dilution of the cryoprotectant was just as effective at preserving the cells within the tissue matrix after cryopreservation as the longer and more complicated standard protocol. Overall, the time taken for thawing a valve and removing the cryoprotectant was reduced from >20 to <10 min. This result should have a positive impact on valve replacement surgery, shortening the time that patients spend on cardiopulmonary bypass.
References [1] W.J. Armitage, P. Mazur, Toxic and osmotic effects of glycerol on human granulocytes, Am. J. Physiol. 247 (1984) C382–C389. [2] M.F. OÕBrien, E.G. Stafford, M.A. Gardner, P.G. Pohlner, D.C. McGiffin, A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies, J. Thorac. Cardiovasc. Surg. 94 (1987) 812–823. [3] M.F. OÕBrien, S. Harrocks, E.G. Stafford, M.A. Gardner, P.G. Pohlner, P.J. Tesar, F. Stephens, The homograft aortic valve: a 29-year, 99.3% follow up of 1,022 valve replacements, J. Heart Valve Dis. 10 (2001) 334–344.
Protocols for thawing and cryoprotectant dilution of heart valvesq W. John Armitage*, Wayne Dale, Emily A Alexander Bristol Heart Valve Bank, Department of Clinical Science, University of Bristol, Bristol, BS1 2LX, United Kingdom Received 16 July 2004; accepted 20 September 2004 Available online 9 December 2004
Abstract Purpose: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. Methods: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1—Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2—Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37°C water bath. The DMSO was removed by 4-step dilution. Experiment 3— Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). Results: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. Conclusion: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Heart valve; Cryopreservation; DMSO; Warming rate
q
This work was funded by institutional sources. Corresponding author. Fax: +44 117 904 6624. E-mail address: [email protected] (W.J. Armitage).
*
Human heart valve allografts can be successfully stored by cryopreservation [2,3], which greatly improves the logistics of storage and distribution and minimizes wastage. Although the need for a viable population of valve leaflet fibroblasts in allografts has not been unequivocally demon-
0011-2240/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2004.09.005
18
W.J. Armitage et al. / Cryobiology 50 (2005) 17–20
strated, it is generally considered that the success of heart valve cryopreservation is at least in part due to the preservation of these cells. Since abrupt changes in cryoprotectant concentration can cause osmotic damage to cells [1], multi-step protocols for the removal of cryoprotectants are advocated by some heart valve banks. The time taken to prepare a valve for surgery may, however, be an important consideration since the thawing and cryoprotectant dilution is typically carried out in the operating theatre while the patient (often a child) is on cardio-pulmonary bypass. For valves frozen in 100 ml of cryoprotectant medium, such as 10% v/v dimethyl sulphoxide (DMSO), thawing takes approximately 10 min, and removal of the DMSO by 4-step dilution protocol adds a further 12–15 min, giving a total of at least 20 min before the valve can be implanted. Our aim was to reduce this time by increasing the warming rate and reducing the time taken to dilute out the cryoprotectant, but without compromising the integrity of the valve leaflet fibroblasts.
Methods Three sets of experiments were carried out. Pulmonary and aortic valves were dissected from porcine hearts obtained from an abattoir. The hearts were processed within just a few hours of retrieval, well within the time limits acceptable for the processing of human heart valves. For all three sets of experiments, valves were first exposed to 10% (v/v) DMSO using a 2-step protocol. Valves were initially exposed to 5% (v/v) DMSO in EagleÕs MEM for 10 min at ambient temperature (22°C) and then to 10% DMSO for a further 10 min. After each experimental treatment, the outgrowth of fibroblasts from valve leaflet explants in primary tissue culture was chosen as a measure cellular viability. Each group contained at least four valves.
Table 1 Stepwise dilution protocols for valves exposed to 10% (v/v) DMSO Step
Step duration (ml)
Volume added (ml)
Total volume (ml)
4-Step dilution 1 2 3 4
3 3 3 3
100 200 400 400
200 400 800 400a
2-Step dilution 1 2
3 9
100 400
200 400a
1-Step dilution 1
12
400
400a
The diluent was HartmannÕs solution and the starting volume, including the valve, was 100 ml in all cases. Temperature was maintained at 0–4°C and, in each case, the dilution protocol took 12 min to complete. a Valve transferred to 400-ml fresh HartmannÕs solution.
Experiment 2—Warming rate The only way to increase the warming rate without raising the temperature of the water bath used for thawing (37°C) was to reduce the volume of cryoprotectant solution from the standard 100 ml used for freezing the valves. Valves were cooled at 1°C/min to 100°C in 100, 50, 25, or 0 ml of 10% DMSO. After storage overnight in the vapour phase of a liquid nitrogen refrigerator, the valves were allowed to warm slowly to 80°C and then warmed in a 37°C water bath until all the ice had melted. After thawing the DMSO was removed by 4-step dilution (Table 1). Experiment 3—Standard vs. Modified protocol Finally, valves frozen and thawed by a modified protocol, based on the outcome of Experiments 1 and 2, were compared with valves frozen by the standard protocol used in Bristol Heart Valve Bank (Table 2).
Experiment 1—Dilution of DMSO Primary tissue culture Valves were subjected to 4-, 2-, or single-step dilution with HartmannÕs solution at 0–4°C to remove the DMSO (see Table 1 for details). Untreated controls were not exposed to DMSO.
After experimental treatment, the leaflets were dissected from the valves. Each leaflet was divided into four pieces and each piece placed in a separate
W.J. Armitage et al. / Cryobiology 50 (2005) 17–20
19
Table 2 Details of the Standard and Modified protocols used for Experiment 3 Protocol
Standard
Modified
Addition of 10% DMSO Volume of 10% DMSO Cooling rate
2-Step 100 ml 1°C/min to 100°C 140°C 4-Step
2-Step 50 ml 1°C/min to 100°C 140°C 1-Step
Storage temperature Dilution to remove DMSOa a
See Table 1 for details.
well of a 12-well tissue culture plate. The growth medium was EagleÕs MEM supplemented with 10% fetal bovine serum. The explants were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was changed every 2–3 days. The tissue explants were monitored daily for cell growth, which, after 2 weeks, was graded as follows: 0—no cell outgrowth evident 1—cell outgrowth, but sparse distribution around explant 2—good growth of cells surrounding explant and spreading out 3—confluent cell cultures.
Data analysis
Fig. 1. Effect of dilution protocol on growth of cells from valve leaflet explants after two weeks in culture. Controls were not exposed to DMSO. The numbers of explants are given in each group showing little or no growth of cells (i.e., growth =1, solid bars) vs. good growth/confluence (i.e., growth >1, hatched bars).
graded 61), v2 analysis was not possible. However, the results strongly suggest that 1-step dilution was not detrimental to the cells. Warming rate Fig. 2 shows that warming rate also had little effect on the number of explants showing good outgrowth of cells at 2 weeks (v2 = 2.3, df = 3, p = 0.52). Volumes less than 50 ml were insufficient to immerse valves completely, but a reduction from 100 to 50 ml halved the time taken to
v2 tests or FisherÕs exact test were used to compare the numbers of explants in the different groups showing cell growth graded P2 after two weeks in culture. The level of significance was set at 5%.
Results Dilution of DMSO The rate of dilution of DMSO had little effect on the number of explants showing outgrowth of cells at 2 weeks, although the 4-step dilution appeared to be less favourable (Fig. 1). Since all of the explants in the 1-step dilution group showed good outgrowth of cells (i.e., none were
Fig. 2. Effect of warming rate (controlled by volume of cryoprotectant medium) on growth of cells from valve leaflet explants after two weeks in culture. The numbers of explants are given in each group showing little or no growth of cells (i.e., growth =1, solid bars) vs. good growth/confluence (i.e., growth >1, hatched bars).
20
W.J. Armitage et al. / Cryobiology 50 (2005) 17–20
Fig. 3. Effect of the standard vs. the modified cryopreservation protocol on growth of cells from valve leaflet explants after two weeks in culture. Controls were not cryopreserved. The numbers of explants are given in each group showing little or no growth of cells (i.e., growth =1, solid bars) vs. good growth/ confluence (i.e., growth >1, hatched bars).
warm a valve. The time taken to warm from 79 to 0°C was 8 min (SD = 1.1 min) for 100 ml and 4 min (SD = 0.6 min) for 50 ml. These times were equivalent to linearized warming rates of, respectively, 10 and 20°C/min. Standard vs. Modified protocol Valves were cryopreserved according either to the standard protocol used at the time by Bristol Heart Valve Bank or to a Modified protocol that reduced the warming time and applied a singlestep dilution protocol for removal of the cryoprotectant. Fig. 3 suggests that the outgrowth of cells from the two cryopreserved groups was slightly less than the untreated control (v2 = 7.8, df = 2, p = 0.02). A direct comparison between just the standard and modified protocols using FisherÕs exact test showed no difference between the cryopreservation protocols (p = 0.23).
Discussion The rationale for decreasing the rate of dilution of cryoprotectants after thawing by increasing the number of steps in a dilution protocol is to lessen the risk of cellular damage from osmotic stress [1]. Heart valves, however, are typically exposed to only 10% v/v DMSO and, given that DMSO tends to cross cell membranes readily, a multi-step
protocol for the removal of this concentration of DMSO after thawing is perhaps not warranted. Our results supported this contention, showing that a single-step dilution was as well-tolerated by the valve leaflet fibroblasts as a 4-step protocol. Similarly, reducing the volume of cryoprotectant medium as a means of increasing the warming rate, also had little effect on the outgrowth of cells from the explants. For the final experiment, valves were frozen in 50-ml of cryoprotectant medium. This volume was chosen because it was just sufficient to be able to freeze a valve completely immersed in medium, which we felt might better protect the valve from physical trauma during subsequent handling. Freezing in smaller volumes, which would have resulted in more rapid warming, was just as effective at preserving cellular viability. This could be an option should a further reduction in the time taken to prepare a valve for surgery be considered desirable. In the final experiment, cryopreservation resulted in slightly fewer explants achieving good outgrowth of cells compared with unfrozen control tissue; but the effect was small. Moreover, the modified protocol using more rapid warming and single-step dilution of the cryoprotectant was just as effective at preserving the cells within the tissue matrix after cryopreservation as the longer and more complicated standard protocol. Overall, the time taken for thawing a valve and removing the cryoprotectant was reduced from >20 to <10 min. This result should have a positive impact on valve replacement surgery, shortening the time that patients spend on cardiopulmonary bypass.
References [1] W.J. Armitage, P. Mazur, Toxic and osmotic effects of glycerol on human granulocytes, Am. J. Physiol. 247 (1984) C382–C389. [2] M.F. OÕBrien, E.G. Stafford, M.A. Gardner, P.G. Pohlner, D.C. McGiffin, A comparison of aortic valve replacement with viable cryopreserved and fresh allograft valves, with a note on chromosomal studies, J. Thorac. Cardiovasc. Surg. 94 (1987) 812–823. [3] M.F. OÕBrien, S. Harrocks, E.G. Stafford, M.A. Gardner, P.G. Pohlner, P.J. Tesar, F. Stephens, The homograft aortic valve: a 29-year, 99.3% follow up of 1,022 valve replacements, J. Heart Valve Dis. 10 (2001) 334–344.