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Model-Guided Design and Optimization of CPA Perfusion Protocols for Whole Organ Cryopreservation

Han, Zonghu, Rao, Joseph Sushil, Ramesh, Srivasupradha, Hergesell, Jan, Namsrai, Bat-Erdene, Etheridge, Michael L., Finger, Erik B., Bischof, John C.·2023·Annals of Biomedical Engineering
DOI 10.1007/s10439-023-03255-5PMC PMC10518287
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Abstract

Vitrification could enable long-term organ preservation, but only after loading high-concentration, potentially toxic cryoprotective agents (CPAs) by perfusion. In this paper, we combine a two-compartment Krogh cylinder model with a toxicity cost function to theoretically optimize the loading of CPA (VMP) in rat kidneys as a model system. First, based on kidney perfusion experiments, we systematically derived the parameters for a CPA transport loading model, including the following: V b = 86.0% ( r a = 3.86 μm), L p = 1.5 × 10 –14 m 3 /(N·s), ω = 7.0 × 10 –13 mol/(N·s), σ = 0.10. Next, we measured the toxicity cost function model parameters as α = 3.12 and β = 9.39 × 10 –6 . Combining these models, we developed an improved kidney-loading protocol predicted to achieve vitrification while minimizing toxicity. The optimized protocol resulted in shorter exposure (25 min or 18.5% less) than the gold standard kidney-loading protocol for VMP, which had been developed based on decades of empirical practice. After testing both protocols on rat kidneys, we found comparable physical and biological outcomes. While we did not dramatically reduce toxicity, we did reduce the time. As our approach is now validated, it can be used on other organs lacking defined toxicity data to reduce CPA exposure time and provide a rapid path toward developing CPA perfusion protocols for other organs and CPAs.

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Fig. 1 Krogh model geometry. a Krogh cylinder stacks and each unit with description of relevant parameters. b Cross section view of the Krogh cylinder, rk is the apothem of the hexagonal prism (Krogh unit), ra is the apothem of the hexagonal prism of the active volume, and rc is the radius of the capillary. The gray area refers to the non-solvent (or solid) inactive volume
Fig. 1 Krogh model geometry. a Krogh cylinder stacks and each unit with description of relevant parameters. b Cross section view of the Krogh cylinder, rk is the apothem of the hexagonal prism (Krogh unit), ra is the apothem of the hexagonal prism of the active volume, and rc is the radius of the capillary. The gray area refers to the non-solvent (or solid) inactive volumeCC BY · PubMed Central
Fig. 2 Schematic diagram of the a hypothermic perfusion circuit and b non-survival acute transplant model
Fig. 2 Schematic diagram of the a hypothermic perfusion circuit and b non-survival acute transplant modelCC BY · PubMed Central
Fig. 3 Parameters measured by kidney perfusion. a For ra, the kidney was perfused with LM5, LM5 + 150 mM lactose, and LM5 + 300 mM lactose solutions, and ra was fit based on Eq. (12). b For Lp, the kidney was perfused with LM5 and LM5 + 300 mM lactose solutions, and Lp was fit based on Eq. (1). c For ω and σ, the kidney was perfused with LM5 and 25% VMP solutions, and ω and σ were fit based on Eq. (2). d Summary table of the measured parameter values (mean ± standard deviation). Each parameter was determined from n = 3 separate kidney perfusion experiments
Fig. 3 Parameters measured by kidney perfusion. a For ra, the kidney was perfused with LM5, LM5 + 150 mM lactose, and LM5 + 300 mM lactose solutions, and ra was fit based on Eq. (12). b For Lp, the kidney was perfused with LM5 and LM5 + 300 mM lactose solutions, and Lp was fit based on Eq. (1). c For ω and σ, the kidney was perfused with LM5 and 25% VMP solutions, and ω and σ were fit based on Eq. (2). d Summary table of the measured parameter values (mean ± standard deviation). Each parameter was determined from n = 3 separate kidney perfusion experimentsCC BY · PubMed Central
Fig. 4 Toxicity cost function measurements using kidney slices and the results of kidney loading protocol optimization. a The loading and unloading protocol for kidney slices is shown. This was used to measure the toxicity rate at the testing concentration, e.g., 100% VMP. The time interval is 4 min for each step to reach the final testing concentration (e.g., 25 to 100%), and the exposure time at the testing concentration ranges from 15 to 120 min. b Viability of the kidney slices after 15–120-min exposure at different testing concentrations is shown (note: assumption is that toxicity is mainly from final testing concentration). n = 4 for each concentration at each exposure time. c) The toxicity rate as a function of the concentration of VMP was fit based on experimental data shown in b using Eq. (3). d Model-predicted minimum toxicity cost function values at each ramp rate were calculated. Solid and hollow stars represent the toxicity of the existing and optimized protocols, respectively. For ramp rates above ~ 64 mM/min, the ramp duration is limited due to increased osmotic stress, which subsequently requires a substantially longer “plateau” region at higher CPA concentrations, which produces a rapid increase in the minimum predicted toxicity (see Figures S2–4 in Supplementary material)
Fig. 4 Toxicity cost function measurements using kidney slices and the results of kidney loading protocol optimization. a The loading and unloading protocol for kidney slices is shown. This was used to measure the toxicity rate at the testing concentration, e.g., 100% VMP. The time interval is 4 min for each step to reach the final testing concentration (e.g., 25 to 100%), and the exposure time at the testing concentration ranges from 15 to 120 min. b Viability of the kidney slices after 15–120-min exposure at different testing concentrations is shown (note: assumption is that toxicity is mainly from final testing concentration). n = 4 for each concentration at each exposure time. c) The toxicity rate as a function of the concentration of VMP was fit based on experimental data shown in b using Eq. (3). d Model-predicted minimum toxicity cost function values at each ramp rate were calculated. Solid and hollow stars represent the toxicity of the existing and optimized protocols, respectively. For ramp rates above ~ 64 mM/min, the ramp duration is limited due to increased osmotic stress, which subsequently requires a substantially longer “plateau” region at higher CPA concentrations, which produces a rapid increase in the minimum predicted toxicity (see Figures S2–4 in Supplementary material)CC BY · PubMed Central
Fig. 5 Comparison between the existing and the optimized VMP loading protocols. a, e The arterial concentration and pressure during perfusion loading. b, f The model-predicted and experimental perfusion resistances for the representative cases. R2 values equal 0.987 and 0.983 for b and f, respectively. c, g The model-predicted CPA concentration inside the kidney tissue for the representative cases. d, h The model-predicted tissue volume change for the representative cases. The dashed line represents the volume limit leading to irreversible osmotic damage, 45%. The hatched area in e, f, g, h represents time saved in the optimized protocol over the existing protocol. Data are representative cases from n = 3.
Fig. 5 Comparison between the existing and the optimized VMP loading protocols. a, e The arterial concentration and pressure during perfusion loading. b, f The model-predicted and experimental perfusion resistances for the representative cases. R2 values equal 0.987 and 0.983 for b and f, respectively. c, g The model-predicted CPA concentration inside the kidney tissue for the representative cases. d, h The model-predicted tissue volume change for the representative cases. The dashed line represents the volume limit leading to irreversible osmotic damage, 45%. The hatched area in e, f, g, h represents time saved in the optimized protocol over the existing protocol. Data are representative cases from n = 3.CC BY · PubMed Central
Fig. 6 Physical (vitrification) and biological assessments of rat kidneys loaded by the two protocols. a, b Representative photo and micro-CT image of vitrified kidney perfused with the existing loading protocol. d, e Representative photo and micro-CT image of vitrified kidney perfused with the optimized loading protocol. Uniform Hounsfield units (HU) ~500 (yellow-orange) in the micro-CT images indicate vitrification. Some ice is visible on resected connective tissue (blue), but does not impact kidney viability for transplant. c, f Representative intra-operative pictures of orthotopic transplanted rat kidneys after CPA perfusion using existing and optimized protocols. Data are representative cases from n = 3
Fig. 6 Physical (vitrification) and biological assessments of rat kidneys loaded by the two protocols. a, b Representative photo and micro-CT image of vitrified kidney perfused with the existing loading protocol. d, e Representative photo and micro-CT image of vitrified kidney perfused with the optimized loading protocol. Uniform Hounsfield units (HU) ~500 (yellow-orange) in the micro-CT images indicate vitrification. Some ice is visible on resected connective tissue (blue), but does not impact kidney viability for transplant. c, f Representative intra-operative pictures of orthotopic transplanted rat kidneys after CPA perfusion using existing and optimized protocols. Data are representative cases from n = 3CC BY · PubMed Central

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Metadata

Authors
Han, Z., Rao, J. S., Ramesh, S., Hergesell, J., Namsrai, B.-E., Etheridge, M. L., Finger, E. B., & Bischof, J. C.
ORCID
Bischof, John C.
Venue
Annals of Biomedical Engineering
Publisher
Springer Science and Business Media LLC
Published
2023-06-23
Volume
51
Issue
10
Pages
2216-2228
ISSN
  • 0090-6964
  • 1573-9686
DOI
10.1007/s10439-023-03255-5
PMC
PMC10518287
Cited by
21
References
1
Language
en
Licence
Funders
  • National Institutes of Health (NIH R01DK117425, NIH R01DK132211, NIH R01HL135046)
  • National Science Foundation (NSF EEC 1941543)
  • Biostasis Research Institute

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