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Cryopreservation of plants: Stress and antioxidants
Cryobiology 73 (2016) 399e443
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Abstracts e CRYO 2016 S001 CLINICAL TRIALS OF CELL BASED AND CELL BASED GENE ENHANCED THERAPIES: THE OTTAWA EXPERIENCE D. Courtman. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada The Cellular Biotherapeutics program at the Ottawa Hospital Research Institute is testing and producing novel translational cell and cell based gene therapies, culminating in early stage clinical trials for pulmonary hypertension, myocardial infarction, septic shock, and multiple sclerosis. The application of endothelial progenitor cells (EPCs) for the treatment of cardiovascular disease holds great promise, yet preclinical studies suggest that patient derived autologous cells are less effective. We have pioneered the development of gene enhanced autologous cell based treatments for pulmonary hypertension and myocardial ischemia. Our first-in-human dose escalation clinical trial in pulmonary hypertension (NCT00469027) utilized peripheral blood derived early outgrowth EPCs transiently transfected via electroporation with a human eNOS plasmid and delivered directly to the pulmonary circulation. We demonstrated that eNOStransfected EPCs to patients with stable, severe PAH was well tolerated, and resulted in a trend towards short-term hemodynamic improvement, especially in patients with high baseline pulmonary vascular resistance and concomitant therapy with phosphodiesterase type 5 inhibitors. Although, there was no long term sustained hemodynamic improvement, there were significant increases in 6 minute walk time seen at both 1 month, and persisting to 3 and 6 months post cell based gene therapy. To apply our therapy to cardiac patients, we developed a non-mobilized automated apheresis procedure combined with GMP manufacturing in environmentally controlled isolator units and in addition adopted a cationic polymer for eNOS transfection to limit cellular manipulations. We initiated a placebo controlled randomized trial (NCT00936819) testing the role of eNOS transfection in autologous EPCs in acute MI patients, with 24 treated to date. Our early results suggest that eNOS transfected autologous EPCs are safe and potentially effective treatments for cardiovascular disorders. To facilitate more advanced trials with broader distribution, including more repetitive dosing strategies, enhanced cryopreservation protocols designed to maintain cell potency will need to be developed. S002 BIOPRESERVATION CONSIDERATIONS IN THE EVOLVING FIELD OF CELLULAR THERAPIES
appropriate reagents, and Quality/Regulatory considerations to the transportation and storage of source material and final product. S003 ESTABLISHING CRYOPRESERVED CELL THERAPEUTIC SHELF LIFE: HISTORICAL PERSPECTIVE AND CLINICAL APPLICATION E. Woods. Ossium Health, Inc., San Francisco, California, United States Establishing a shelf life for a cryopreserved cellular therapeutic can be controversial. For more established systems such as hematopoietic stem cells (HSC), the consensus is typically 5 years with ongoing testing to demonstrate stability over time. However, once a cell is taken to cryogenic temperatures, biological time all but ceases as liquid water does not exist below 130 C. The only states that do are crystalline or glassy, and in both cases the viscosity is so high (>1013 as opposed to 10 4 poises at 20 C) that diffusion is not significant over less than geological time spans. Ordinary chemical reactions requiring molecular motion do not occur. The only types of reactions that can occur at cryogenic temperatures are photophysical resulting from background ionizing radiation. Ionization occurs when absorbed radiation has enough energy to eject one or more orbital electrons from an atom. This can cause direct damage when a target molecule itself (such as DNA) reacts with radiation. Indirectly, radiolysis of water through ionization can lead to generation of free radicals, which can then form radicals with other molecules causing damage. Background ionizing radiation has been estimated to be around 1.0e2.0 mSv/yr (or 0.1e0.2 rad/yr) depending on location. Given that this is the only potential for damage to a properly cryopreserved cell, analysis of its effects becomes relevant. Estimates determined from numerous studies indicate time required for damage to occur via such methods are on order of hundreds to tens of thousands of years. Some literature has indicated drops in survival after extended periods of cryostorage. The likely reason for this apparent phenomenon is inappropriate maintenance of storage. While cell cryopreservation is normally controlled, sample storage can be undefined and uncontrolled, with temperature fluctuations occurring during sample transfer to LN2 tanks, sample storage, sample sorting, and sample removal. S004 CRYOPRESERVATION OF PLANTS: STRESS AND ANTIOXIDANTS P. Saxena. University of Guelph, Guelph, Ontario, Canada
A. Abazari. BioLife Solutions Inc., Bothell, Washington, United States Cellular therapies are cell and tissue products sourced from biological material that are employed as “living drugs.“ .” As such, they require specialized biological support, namely biopreservation, to protect structural integrity and viability during ex vivo processing and handling, and to ensure consistency for eventual in-patient delivery. Successful biopreservation entails optimal recovery, viability, and a return to function of cells post-preservation. However, to deliver clinical and commercial efficacy, certain limitations and restriction are applied to ensure compliance and compatibility with Good Manufacturing Practices (GMPs). This presentation will address biopreservation methods as practiced in commercial manufacturing of cell therapy products and some of the inherent deficiencies and challenges from evaluation, selection, and qualification of
http://dx.doi.org/10.1016/j.cryobiol.2016.09.009
In cryopreservation processes, cells and tissues are exposed to extreme stresses caused by freezing temperatures, dehydration, and toxic effect of cryoprotection chemicals. The generation of reactive oxygen species in response to multiple stresses of cryopreservation has been recognized as one of the most detrimental factors associated with poor recovery and growth of cryopreserved tissues. Antioxidants such as ascorbic acid, reduced glutathione, and glycine betaine have been shown to alleviate stress in cryopreservation. The indoleamines, melatonin and serotonin, which are thought to have evolved to combat the oxidative stress of photosynthesis and cellular respiration, also exhibit strong antioxidant properties. Recent research has found that melatonin and serotonin can directly reduce and neutralize the damaging effects of reactive oxygen species. In addition, the capacity of these compounds to enhance the
400
Abstracts / Cryobiology 73 (2016) 399e443
activity of other antioxidant enzymes such as catalase and superoxide dismutase has made them useful in combatting extreme stresses such as dehydration and freezing experienced by plant tissues during cryopreservation. Furthermore, melatonin and serotonin have a dual effect in their abilities to modulate plant growth, although this has been indirectly linked to their antioxidant capacity, as reactive oxygen species have been found to modulate some plant signaling pathways. However, melatonin and serotonin are emerging independently as their own class of plant growth regulators due to the metabolic complexes with which they are able to mediate plant responses. This dual effect of melatonin and serotonin makes them a unique class of cryoprotectant as they not only enhance explant survival during cryopreservation, they also promote growth and regeneration after recovery. This presentation will provide an overview of recent research on the role of melatonin and serotonin in cryopreservation of a range of plant species. S005 CRYOPRESERVATION AND THE CHALLENGE ENDANGERED EXCEPTIONAL SPECIES
OF
vitro. Generally, pre-cryopreservation light quality mainly affected the early recovery (survival) whereas post-cryopreservation illumination had significant effect on the late recovery (regeneration). Specifically, blue light applied before cryopreservation significantly increased survival whereas a combination of red and blue LEDs during recovery phase increased regeneration into whole new plants. Therefore, the modification of light spectral quality is a promising tool for increasing the cryopreservation success of potato shoot tips in vitro. Source of funding: This work was funded by national scholarship program Kristjan Jaak (to JE), which is funded and managed by Archimedes Foundation in collaboration with the Estonian Ministry of Education and € nning Foundation Research. Additional funding was provided by Tauno To and Niemi Foundation (to JE). S007 OPTIMIZATION OF A MULTISTAGE-DIALYSIS MICRODEVICE EXTRACTION OF CRYOPROTECTANTS FROM A CELL SUSPENSION
FOR
PRESERVING
V. Pence. Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio, United States Cryopreservation is a critical tool for the ex situ conservation of endangered plant species. For those species that cannot be preserved by traditional seed-banking, cryopreservation techniques offer approaches for preserving non-seed plant tissues, including zygotic embryos, pollen, gametophytes, somatic embryos, and shoot tips. These methods have been developed primarily for economically important species, but when they are applied to endangered species, there are additional challenges. Lack of previous research and information on the targeted species, small amounts of available material, unusual species adaptations, and limited institutional resources are the rule rather than the exception. Furthermore, there is the need to maintain genetic diversity and the imperative to preserve species that otherwise might be lost. These challenges apply not only to the development of cryopreservation protocols, but also to the in vitro protocols that are often required for supplying appropriate tissues for freezing. Work on growing and cryopreserving shoot tips of several species threatened or endangered in the United States from several locations and habitats will illustrate these challenges and will highlight further work that is needed to make cryopreservation more efficient and effective for conservation. Source of funding: Institute of Museum of Library Services and the Cincinnati Zoo and Botanical Garden S006 DOES LIGHT MATTER? THE EFFECT OF LIGHT SPECTRAL QUALITY ON CRYOPRESERVATION SUCCESS OF THE POTATO (SOLANUM TUBEROSUM L.) €, H. H€ aggman. University of Oulu, Oulu, Finland J. Edesi*, A. Pirttila * Corresponding author.
Light is one of the most important factors affecting growth and morphogenesis of plants. Light intensity, photoperiod, and spectral composition greatly affect morphogenetic responses of in vitro plants and specific wavelengths drive different responses during different developmental stages. Therefore, light intensity as well as spectral composition during pre- and post-cryopreservation culture of in vitro plants significantly affect the cryopreservation result. In addition, there is an increasing trend to replace the widely used fluorescent tubes with light emitting diode (LEDs) based illumination sources in tissue culture chambers which may affect the cryopreservation result of plant germplasm. Therefore, we studied the effect of six different light spectral qualities provided by cool white fluorescent tubes (CW), warm white (HQI), white LEDs (W), blue LEDs (B), red LEDs (R) and a combination of 90% red 10% blue (LEDs) applied either before or after cryopreservation, with the emphasis on shoot tip recovery of five different potato cultivars. Light spectral quality had a significant effect on growth characteristics of potato plants in vitro and some light qualities clearly affected cryopreservation success of potato shoot tips in
L. Zou*, W. Ding. University of Science and Technology of China, Hefei, Anhui, China * Corresponding author.
The cell samples for lab-on-a-chip use are often a trace of cryopreserved frozen-thawed cells, and the cryoprotectants (CPAs) inside these cells may affect the results of the study. Therefore, the device for cryoprotectant removal from these samples is of great significance. In our previous study, a multistage-dialysis microdevice (MDM) was proposed to remove cryoprotectants from CPA-laden cell suspension. However, the performance (clearance efficiency, survival, and recovery rate of cells) and the size of the device were not satisfactory and needs to be further optimized. In this study, the volume ratio of cell suspension and wash solutions, a three-layer structure, a wide range of operating conditions and a large area of the dialysis will be considered in the optimized device. Subsequently, the fluorescence experiments and deglycerolized experiments under different conditions (flow rate, glycerin concentration, hematocrit, and the membrane pore size) will be conducted to confirm the functions and performance of the optimized device. The goal of our research is to minimize size and improve the performance of the device. Our further work may provide a tool to avoid the side effects of cryoprotectants on lab-on-a-chip applications of cryopreserved cells. Source of funding: This work was partly supported by the Fundamental Research Funds for the Central Universities, National Natural Science Foundation of China (Grant No. 81571768), the Natural Science Foundation of Anhui Province (Grant No. 1408085ME96), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20133402120033), and the Fundamental Research Funds for the Central Universities of China (Grant Nos. WK3490000001 and WK2100000001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. S008 A NOVEL CRYOPROTECTANT HAEMATOPOIETIC STEM CELLS
FOR
PRESERVATION
OF
HUMAN
N. Dolezalova 1, *, N. Georgakopoulos 1, E. Calderbank 2, E. Laurenti 2, N. Slater 3, K. Mahbubani 3, K. Saeb-Parsy 1. 1 Department of Surgery, University of Cambridge, and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom; 2 Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom; 3 Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom * Corresponding author.
Clinical transplantation of haematopoietic stem cells (HSCs) usually includes a period of cryopreservation before infusion into patients. Dimethyl sulphoxide (Me2SO) is the most commonly used cryoprotectant for this purpose, despite its known cellular and systemic toxicity. Trehalose, a disaccharide, is a promising alternative, but is unable to cross the plasma membrane and can only provide extracellular protection. The amphipathic biopolymer PP-50, made of a poly (L-lysine iso-phthalamide) backbone
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Abstracts e CRYO 2016 S001 CLINICAL TRIALS OF CELL BASED AND CELL BASED GENE ENHANCED THERAPIES: THE OTTAWA EXPERIENCE D. Courtman. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada The Cellular Biotherapeutics program at the Ottawa Hospital Research Institute is testing and producing novel translational cell and cell based gene therapies, culminating in early stage clinical trials for pulmonary hypertension, myocardial infarction, septic shock, and multiple sclerosis. The application of endothelial progenitor cells (EPCs) for the treatment of cardiovascular disease holds great promise, yet preclinical studies suggest that patient derived autologous cells are less effective. We have pioneered the development of gene enhanced autologous cell based treatments for pulmonary hypertension and myocardial ischemia. Our first-in-human dose escalation clinical trial in pulmonary hypertension (NCT00469027) utilized peripheral blood derived early outgrowth EPCs transiently transfected via electroporation with a human eNOS plasmid and delivered directly to the pulmonary circulation. We demonstrated that eNOStransfected EPCs to patients with stable, severe PAH was well tolerated, and resulted in a trend towards short-term hemodynamic improvement, especially in patients with high baseline pulmonary vascular resistance and concomitant therapy with phosphodiesterase type 5 inhibitors. Although, there was no long term sustained hemodynamic improvement, there were significant increases in 6 minute walk time seen at both 1 month, and persisting to 3 and 6 months post cell based gene therapy. To apply our therapy to cardiac patients, we developed a non-mobilized automated apheresis procedure combined with GMP manufacturing in environmentally controlled isolator units and in addition adopted a cationic polymer for eNOS transfection to limit cellular manipulations. We initiated a placebo controlled randomized trial (NCT00936819) testing the role of eNOS transfection in autologous EPCs in acute MI patients, with 24 treated to date. Our early results suggest that eNOS transfected autologous EPCs are safe and potentially effective treatments for cardiovascular disorders. To facilitate more advanced trials with broader distribution, including more repetitive dosing strategies, enhanced cryopreservation protocols designed to maintain cell potency will need to be developed. S002 BIOPRESERVATION CONSIDERATIONS IN THE EVOLVING FIELD OF CELLULAR THERAPIES
appropriate reagents, and Quality/Regulatory considerations to the transportation and storage of source material and final product. S003 ESTABLISHING CRYOPRESERVED CELL THERAPEUTIC SHELF LIFE: HISTORICAL PERSPECTIVE AND CLINICAL APPLICATION E. Woods. Ossium Health, Inc., San Francisco, California, United States Establishing a shelf life for a cryopreserved cellular therapeutic can be controversial. For more established systems such as hematopoietic stem cells (HSC), the consensus is typically 5 years with ongoing testing to demonstrate stability over time. However, once a cell is taken to cryogenic temperatures, biological time all but ceases as liquid water does not exist below 130 C. The only states that do are crystalline or glassy, and in both cases the viscosity is so high (>1013 as opposed to 10 4 poises at 20 C) that diffusion is not significant over less than geological time spans. Ordinary chemical reactions requiring molecular motion do not occur. The only types of reactions that can occur at cryogenic temperatures are photophysical resulting from background ionizing radiation. Ionization occurs when absorbed radiation has enough energy to eject one or more orbital electrons from an atom. This can cause direct damage when a target molecule itself (such as DNA) reacts with radiation. Indirectly, radiolysis of water through ionization can lead to generation of free radicals, which can then form radicals with other molecules causing damage. Background ionizing radiation has been estimated to be around 1.0e2.0 mSv/yr (or 0.1e0.2 rad/yr) depending on location. Given that this is the only potential for damage to a properly cryopreserved cell, analysis of its effects becomes relevant. Estimates determined from numerous studies indicate time required for damage to occur via such methods are on order of hundreds to tens of thousands of years. Some literature has indicated drops in survival after extended periods of cryostorage. The likely reason for this apparent phenomenon is inappropriate maintenance of storage. While cell cryopreservation is normally controlled, sample storage can be undefined and uncontrolled, with temperature fluctuations occurring during sample transfer to LN2 tanks, sample storage, sample sorting, and sample removal. S004 CRYOPRESERVATION OF PLANTS: STRESS AND ANTIOXIDANTS P. Saxena. University of Guelph, Guelph, Ontario, Canada
A. Abazari. BioLife Solutions Inc., Bothell, Washington, United States Cellular therapies are cell and tissue products sourced from biological material that are employed as “living drugs.“ .” As such, they require specialized biological support, namely biopreservation, to protect structural integrity and viability during ex vivo processing and handling, and to ensure consistency for eventual in-patient delivery. Successful biopreservation entails optimal recovery, viability, and a return to function of cells post-preservation. However, to deliver clinical and commercial efficacy, certain limitations and restriction are applied to ensure compliance and compatibility with Good Manufacturing Practices (GMPs). This presentation will address biopreservation methods as practiced in commercial manufacturing of cell therapy products and some of the inherent deficiencies and challenges from evaluation, selection, and qualification of
http://dx.doi.org/10.1016/j.cryobiol.2016.09.009
In cryopreservation processes, cells and tissues are exposed to extreme stresses caused by freezing temperatures, dehydration, and toxic effect of cryoprotection chemicals. The generation of reactive oxygen species in response to multiple stresses of cryopreservation has been recognized as one of the most detrimental factors associated with poor recovery and growth of cryopreserved tissues. Antioxidants such as ascorbic acid, reduced glutathione, and glycine betaine have been shown to alleviate stress in cryopreservation. The indoleamines, melatonin and serotonin, which are thought to have evolved to combat the oxidative stress of photosynthesis and cellular respiration, also exhibit strong antioxidant properties. Recent research has found that melatonin and serotonin can directly reduce and neutralize the damaging effects of reactive oxygen species. In addition, the capacity of these compounds to enhance the
400
Abstracts / Cryobiology 73 (2016) 399e443
activity of other antioxidant enzymes such as catalase and superoxide dismutase has made them useful in combatting extreme stresses such as dehydration and freezing experienced by plant tissues during cryopreservation. Furthermore, melatonin and serotonin have a dual effect in their abilities to modulate plant growth, although this has been indirectly linked to their antioxidant capacity, as reactive oxygen species have been found to modulate some plant signaling pathways. However, melatonin and serotonin are emerging independently as their own class of plant growth regulators due to the metabolic complexes with which they are able to mediate plant responses. This dual effect of melatonin and serotonin makes them a unique class of cryoprotectant as they not only enhance explant survival during cryopreservation, they also promote growth and regeneration after recovery. This presentation will provide an overview of recent research on the role of melatonin and serotonin in cryopreservation of a range of plant species. S005 CRYOPRESERVATION AND THE CHALLENGE ENDANGERED EXCEPTIONAL SPECIES
OF
vitro. Generally, pre-cryopreservation light quality mainly affected the early recovery (survival) whereas post-cryopreservation illumination had significant effect on the late recovery (regeneration). Specifically, blue light applied before cryopreservation significantly increased survival whereas a combination of red and blue LEDs during recovery phase increased regeneration into whole new plants. Therefore, the modification of light spectral quality is a promising tool for increasing the cryopreservation success of potato shoot tips in vitro. Source of funding: This work was funded by national scholarship program Kristjan Jaak (to JE), which is funded and managed by Archimedes Foundation in collaboration with the Estonian Ministry of Education and € nning Foundation Research. Additional funding was provided by Tauno To and Niemi Foundation (to JE). S007 OPTIMIZATION OF A MULTISTAGE-DIALYSIS MICRODEVICE EXTRACTION OF CRYOPROTECTANTS FROM A CELL SUSPENSION
FOR
PRESERVING
V. Pence. Cincinnati Zoo and Botanical Garden, Cincinnati, Ohio, United States Cryopreservation is a critical tool for the ex situ conservation of endangered plant species. For those species that cannot be preserved by traditional seed-banking, cryopreservation techniques offer approaches for preserving non-seed plant tissues, including zygotic embryos, pollen, gametophytes, somatic embryos, and shoot tips. These methods have been developed primarily for economically important species, but when they are applied to endangered species, there are additional challenges. Lack of previous research and information on the targeted species, small amounts of available material, unusual species adaptations, and limited institutional resources are the rule rather than the exception. Furthermore, there is the need to maintain genetic diversity and the imperative to preserve species that otherwise might be lost. These challenges apply not only to the development of cryopreservation protocols, but also to the in vitro protocols that are often required for supplying appropriate tissues for freezing. Work on growing and cryopreserving shoot tips of several species threatened or endangered in the United States from several locations and habitats will illustrate these challenges and will highlight further work that is needed to make cryopreservation more efficient and effective for conservation. Source of funding: Institute of Museum of Library Services and the Cincinnati Zoo and Botanical Garden S006 DOES LIGHT MATTER? THE EFFECT OF LIGHT SPECTRAL QUALITY ON CRYOPRESERVATION SUCCESS OF THE POTATO (SOLANUM TUBEROSUM L.) €, H. H€ aggman. University of Oulu, Oulu, Finland J. Edesi*, A. Pirttila * Corresponding author.
Light is one of the most important factors affecting growth and morphogenesis of plants. Light intensity, photoperiod, and spectral composition greatly affect morphogenetic responses of in vitro plants and specific wavelengths drive different responses during different developmental stages. Therefore, light intensity as well as spectral composition during pre- and post-cryopreservation culture of in vitro plants significantly affect the cryopreservation result. In addition, there is an increasing trend to replace the widely used fluorescent tubes with light emitting diode (LEDs) based illumination sources in tissue culture chambers which may affect the cryopreservation result of plant germplasm. Therefore, we studied the effect of six different light spectral qualities provided by cool white fluorescent tubes (CW), warm white (HQI), white LEDs (W), blue LEDs (B), red LEDs (R) and a combination of 90% red 10% blue (LEDs) applied either before or after cryopreservation, with the emphasis on shoot tip recovery of five different potato cultivars. Light spectral quality had a significant effect on growth characteristics of potato plants in vitro and some light qualities clearly affected cryopreservation success of potato shoot tips in
L. Zou*, W. Ding. University of Science and Technology of China, Hefei, Anhui, China * Corresponding author.
The cell samples for lab-on-a-chip use are often a trace of cryopreserved frozen-thawed cells, and the cryoprotectants (CPAs) inside these cells may affect the results of the study. Therefore, the device for cryoprotectant removal from these samples is of great significance. In our previous study, a multistage-dialysis microdevice (MDM) was proposed to remove cryoprotectants from CPA-laden cell suspension. However, the performance (clearance efficiency, survival, and recovery rate of cells) and the size of the device were not satisfactory and needs to be further optimized. In this study, the volume ratio of cell suspension and wash solutions, a three-layer structure, a wide range of operating conditions and a large area of the dialysis will be considered in the optimized device. Subsequently, the fluorescence experiments and deglycerolized experiments under different conditions (flow rate, glycerin concentration, hematocrit, and the membrane pore size) will be conducted to confirm the functions and performance of the optimized device. The goal of our research is to minimize size and improve the performance of the device. Our further work may provide a tool to avoid the side effects of cryoprotectants on lab-on-a-chip applications of cryopreserved cells. Source of funding: This work was partly supported by the Fundamental Research Funds for the Central Universities, National Natural Science Foundation of China (Grant No. 81571768), the Natural Science Foundation of Anhui Province (Grant No. 1408085ME96), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20133402120033), and the Fundamental Research Funds for the Central Universities of China (Grant Nos. WK3490000001 and WK2100000001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. S008 A NOVEL CRYOPROTECTANT HAEMATOPOIETIC STEM CELLS
FOR
PRESERVATION
OF
HUMAN
N. Dolezalova 1, *, N. Georgakopoulos 1, E. Calderbank 2, E. Laurenti 2, N. Slater 3, K. Mahbubani 3, K. Saeb-Parsy 1. 1 Department of Surgery, University of Cambridge, and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom; 2 Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom; 3 Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom * Corresponding author.
Clinical transplantation of haematopoietic stem cells (HSCs) usually includes a period of cryopreservation before infusion into patients. Dimethyl sulphoxide (Me2SO) is the most commonly used cryoprotectant for this purpose, despite its known cellular and systemic toxicity. Trehalose, a disaccharide, is a promising alternative, but is unable to cross the plasma membrane and can only provide extracellular protection. The amphipathic biopolymer PP-50, made of a poly (L-lysine iso-phthalamide) backbone