Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc.

BACKGROUND: Bacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo. RESULTS: The attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50-95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals. CONCLUSIONS: In summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

In silico-designed vitrification protocols: an approach to improve survival of in vitro produced-bovine embryos.

In brief: In silico predictions validated in this study demonstrate the potential for designing shorter equilibration protocols that improve post-warming re-expansion and hatching rates of D7 and D8 in vitro-produced bovine embryos. Our results benefit the livestock industry by providing a refined and reproducible approach to cryopreserving bovine embryos, which, in addition, could be useful for other mammalian species. Abstract: The cryopreservation of in vitro-produced (IVP) embryos is vital in the cattle industry for genetic selection and crossbreeding programs. Despite its importance, there is no standardized protocol yielding pregnancy rates comparable to fresh embryos. Current approaches often neglect the osmotic tolerance responses to cryoprotectants based on temperature and time. Hereby, we propose improved vitrification methods using shorter dehydration-based protocols. Blastocysts cultured for 7 (D7) or 8 days (D8) were exposed to standard equilibration solution (ES) at 25ºC and 38.5ºC. Optimized exposure times for each temperature and their impact on post-warming re-expansion, hatching rates, cell counts, and apoptosis rate were determined. In silico predictions aligned with in vitro observations, showing original volume recovery within 8 min 30 s at 25ºC or 3 min 40 s at 38.5ºC (D7 blastocysts) and 4 min 25 s at 25ºC and 3 min 15 s at 38.5ºC (D8 blastocysts) after exposure to ES. Vitrification at 38.5ºC resulted in D7 blastocysts re-expansion and hatching rates (93.1% and 38.1%, respectively) comparable to fresh embryos (100.0% and 32.4%, respectively), outperforming the 25ºC protocol (86.2% and 24.4%, respectively; P < 0.05). No differences were observed between D7 and D8 blastocysts using the 38.5ºC protocol. Total cell number was maintained for D7 and D8 blastocysts vitrified at 38.5ºC but decreased at 25ºC (P < 0.05). Apoptosis rates increased post-warming (P < 0.05), except for D8 blastocysts vitrified at 38.5ºC, resembling fresh controls. In conclusion, based on biophysical permeability data, new ES incubation times of 3 min 40 s for D7 blastocysts and 3 min 15 s for D8 blastocysts at 38.5ºC were validated for optimizing vitrification/warming methods for bovine IVP blastocysts.

Conditional in vivo deletion of LYN kinase has little effect on a BRCA1 loss-of-function-associated mammary tumour model.

LYN kinase is expressed in BRCA1 loss-of-function-dependent mouse mammary tumours, in the cells of origin of such tumours, and in human breast cancer. Suppressing LYN kinase activity in BRCA1-defective cell lines as well as in in vitro cultures of Brca1-null mouse mammary tumours is deleterious to their growth. Here, we examined the interaction between LYN kinase and BRCA1 loss-of-function in an in vivo mouse mammary tumour model, using conditional knockout Brca1 and Lyn alleles. Comparison of Brca1 tumour cohorts showed little difference in mammary tumour formation between animals that were wild type, heterozygous or homozygous for the conditional Lyn allele, although this was confounded by factors including incomplete Lyn recombination in some tumours. RNA-sequencing analysis demonstrated that tumours with high levels of Lyn gene expression had a slower doubling time, but this was not correlated with levels of LYN staining in tumour cells themselves. Rather, high Lyn expression and slower tumour growth were likely a result of B-cell infiltration. The multifaceted role of LYN indicates that it is likely to present difficulties as a therapeutic target in breast cancer.

Osmotic response during kidney perfusion with cryoprotectant in isotonic or hypotonic vehicle solution.

Organ cryopreservation would revolutionize transplantation by overcoming the shelf-life limitations of conventional organ storage. To prepare an organ for cryopreservation, it is first perfused with cryoprotectants (CPAs). These chemicals can enable vitrification during cooling, preventing ice damage. However, CPAs can also cause toxicity and osmotic damage. It is a major challenge to find the optimal balance between protecting the cells from ice and avoiding CPA-induced damage. In this study, we examined the organ perfusion process to shed light on phenomena relevant to cryopreservation protocol design, including changes in organ size and vascular resistance. In particular, we compared perfusion of kidneys (porcine and human) with CPA in either hypotonic or isotonic vehicle solution. Our results demonstrate that CPA perfusion causes kidney mass changes consistent with the shrink-swell response observed in cells. This response was observed when the kidneys were relatively fresh, but disappeared after prolonged warm and/or cold ischemia. Perfusion with CPA in a hypotonic vehicle solution led to a significant increase in vascular resistance, suggesting reduced capillary diameter due to cell swelling. This could be reversed by switching to perfusion with CPA in isotonic vehicle solution. Hypotonic vehicle solution did not cause notable osmotic damage, as evidenced by low levels of lactate dehydrogenase (LDH) in the effluent, and it did not have a statistically significant effect on the delivery of CPA into the kidney, as assessed by computed tomography (CT). Overall, our results show that CPA vehicle solution tonicity affects organ size and vascular resistance, which may have important implications for cryopreservation protocol design.

Accelerating cryoprotectant delivery using vacuum infiltration.

The ability to cryopreserve bone marrow within the vertebral body (VB) would offer significant clinical and research benefits. However, cryopreservation of large structures, such as VBs, is challenging due to mass transport limitations that prevent the effective delivery of cryoprotectants into the tissue. To overcome this challenge, we examined the potential of vacuum infiltration, along with carbonation, to increase the penetration of cryoprotectants. In particular, we hypothesized that initial exposure to high-pressure carbon dioxide gas would introduce bubbles into the tissue and that subsequent vacuum cycling would cause expansion and contraction of the bubbles, thus enhancing the transport of cryoprotectant into the tissue. Experiments were carried out using colored dye and agarose gel as a model revealing that carbonation and vacuum cycling result in a 14% increase in dye penetration compared to the atmospheric controls. Experiments were also carried out by exposing VBs isolated from human vertebrae to 40% (v/v) DMSO solution. CT imaging showed the presence of gas bubbles within the tissue pores for carbonated VBs as well as control VBs. Vacuum cycling reduced the bubble volume by more than 50%, most likely resulting in replacement of this volume with DMSO solution. However, we were unable to detect a statistically significant increase in DMSO concentration within the VBs using CT imaging. This research suggests that there may be a modest benefit to carbonation and vacuum cycling for introduction of cryoprotectants into larger structures, like VBs.

Permeation of individual cryoprotectants and their different combinations into mouse liver tissue.

Development of successful tissue cryopreservation methods requires specific knowledge regarding tissue permeation of individual cryoprotective agents (CPAs) and their combinations. The present study assessed the permeation of dimethyl sulfoxide, ethylene glycol, and propylene glycol into liver tissue, and addressed whether the diffusion coefficient of individual CPAs changes when combining CPAs. To do this, mouse liver slices were exposed at room temperature to 3.5 mol/L concentrations of CPAs individually or in combination for 15, 30, 45, and 60 min. Subsequently, tissue CPA concentrations were determined using a gas chromatography/mass spectrometry (GC/MS) method. Our results show that (1) the GC/MS method allows measurement of multiple CPA concentrations in a single small tissue sample, (2) dimethyl sulfoxide has a higher diffusion coefficient than ethylene glycol and propylene glycol, and (3) the CPA diffusivity appears to decrease in mixtures with multiple CPAs. These findings may help the development of effective tissue cryopreservation methods.

Multiple cryoprotectant toxicity model for vitrification solution optimization.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells.

SCOPE: Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs. METHODS AND RESULTS: Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel. CONCLUSION: BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

A single-site pilot feasibility randomized trial of a supportive care mobile application intervention for patients with advanced cancer and caregivers.

PURPOSE: Mobile health interventions can improve patient care. We developed the Digital Supportive Care Awareness and Navigation (D-SCAN) application (app) to facilitate symptom monitoring and use/awareness of cancer supportive care resources. This study tested feasibility, usability/satisfaction, and preliminary efficacy of D-SCAN. METHODS: We randomized 50 patients with advanced cancer to receive the D-SCAN intervention or usual care; 10 caregivers also received D-SCAN. The primary feasibility outcome was determined by weekly symptom survey completion and end of study procedures. We assessed secondary outcomes including usability/satisfaction, awareness/use of supportive care resources, patient activation, and quality of life via various questionnaires including the Net Promoter Score (NPS), Patient Activation Measure (PAM-13), Functional Assessment of Cancer Therapy-General (FACT-G), and Caregiver Oncology Quality of Life (CarGOQOL) questionnaire. RESULTS: Seventy-six percent of intervention patients met feasibility criteria, exceeding our pre-determined threshold of 75%. Usability/satisfaction by NPS was high, at 14.3% and 12.5% for patients and caregivers, respectively. Intervention patient and caregiver resource awareness increased by a mean of 3.7 (p = 0.27) and 4.1 items, respectively. Supportive care resource utilization increased by a mean of 0.8 items for intervention patients (p = 0.70) and 0.6 for caregivers. PAM-13 increased by a mean of 1.6 for intervention patients (p = 0.65). FACT-G increased by a mean of 1.1 for intervention patients (p = 0.91), and CarGOQoL increased by a mean of 2.2 (p = 0.41). CONCLUSION: D-SCAN is a feasible, usable, and satisfactory intervention for augmenting patient and caregiver supportive care. Further testing is necessary to formally assess D-SCAN's efficacy and impact on patients and caregivers. CLINICAL TRIAL REGISTRATION NUMBER: NCT03628794. Registered on August 14th, 2018.

Impact of equilibration duration combined with temperature on the outcome of bovine oocyte vitrification.

The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality.

The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes.

Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic Me2SO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of Me2SO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to Me2SO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to Me2SO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions.

Microfluidic photoreactor to treat neonatal jaundice.

While in most cases, jaundice can be effectively treated using phototherapy, severe cases require exchange transfusion, a relatively risky procedure in which the neonate's bilirubin-rich blood is replaced with donor blood. Here, we examine extracorporeal blood treatment in a microfluidic photoreactor as an alternative to exchange transfusion. This new treatment approach relies on the same principle as phototherapy but leverages microfluidics to speed up bilirubin removal. Our results demonstrate that high-intensity light at 470 nm can be used to rapidly reduce bilirubin levels without causing appreciable damage to DNA in blood cells. Light at 470 nm was more effective than light at 505 nm. Studies in Gunn rats show that photoreactor treatment for 4 h significantly reduces bilirubin levels, similar to the bilirubin reduction observed for exchange transfusion and on a similar time scale. Predictions for human neonates demonstrate that this new treatment approach is expected to exceed the performance of exchange transfusion using a low blood flow rate and priming volume, which will facilitate vascular access and improve safety.

General tissue mass transfer model for cryopreservation applications.

Successful cryopreservation of complex specimens, such as tissues and organs, would greatly benefit both the medical and scientific research fields. Vitrification is one of the most promising techniques for complex specimen cryopreservation, but toxicity remains a major challenge because of the high concentration of cryoprotectants (CPAs) needed to vitrify. Our group has approached this problem using mathematical optimization to design less toxic CPA equilibration methods for cells. To extend this approach to tissues, an appropriate mass transfer model is required. Fick's law is commonly used, but this simple modeling framework does not account for the complexity of mass transfer in tissues, such as the effects of fixed charges, tissue size changes, and the interplay between cell membrane transport and transport through the extracellular fluid. Here, we propose a general model for mass transfer in tissues that accounts for all of these phenomena. To create this model, we augmented a previously published acellular model of mass transfer in articular cartilage to account for the effects of cells. We show that the model can accurately predict changes in CPA concentration and tissue size for both articular cartilage and pancreatic islets, tissue types with vastly different properties.

Human induced pluripotent stem cell derived kidney organoids as a model system for studying cryopreservation.

The ability to cryopreserve organs would have an enormous impact in transplantation medicine. To investigate organ cryopreservation strategies, experiments are typically done on whole organs, or on cells in 2D culture. Whole organs are not amenable to high throughput investigation, while conventional 2D culture is limited to a single cell type and lacks the complexity of the whole organ. In this study, we examine kidney organoids as a model system for studying cryopreservation. Consistent with previous studies, we show that kidney organoids comprised of multiple cell types can be generated in 96-well plates, with an average of about 8 organoids per well. We present a live/dead staining and image analysis method for quantifying organoid viability and show that this method can be used for assessing cryoprotectant toxicity. Our results highlight the potential for using organoids for high throughput investigation of cryopreservation approaches.

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches.

The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.

Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy.

Fibroblast growth factor homologous factors (FHFs) are intracellular proteins which regulate voltage-gated sodium (Nav) channels in the brain and other tissues. FHF dysfunction has been linked to neurological disorders including epilepsy. Here, we describe two sibling pairs and three unrelated males who presented in infancy with intractable focal seizures and severe developmental delay. Whole-exome sequencing identified hemi- and heterozygous variants in the N-terminal domain of the A isoform of FHF2 (FHF2A). The X-linked FHF2 gene (also known as FGF13) has alternative first exons which produce multiple protein isoforms that differ in their N-terminal sequence. The variants were located at highly conserved residues in the FHF2A inactivation particle that competes with the intrinsic fast inactivation mechanism of Nav channels. Functional characterization of mutant FHF2A co-expressed with wild-type Nav1.6 (SCN8A) revealed that mutant FHF2A proteins lost the ability to induce rapid-onset, long-term blockade of the channel while retaining pro-excitatory properties. These gain-of-function effects are likely to increase neuronal excitability consistent with the epileptic potential of FHF2 variants. Our findings demonstrate that FHF2 variants are a cause of infantile-onset developmental and epileptic encephalopathy and underline the critical role of the FHF2A isoform in regulating Nav channel function.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

Mathematical Modeling of Protectant Transport in Tissues.

Mass transfer of protectant chemicals is a fundamental aspect of cryopreservation and freeze-drying protocols. As such, mass transfer modeling is useful for design of preservation methods. Cell membrane transport modeling has been successfully used to guide design of preservation methods for isolated cells. For tissues, though, there are several mass transfer modeling challenges that arise from phenomena associated with cells being embedded in a tissue matrix. Both cells and the tissue matrix form a barrier to the free diffusion of water and protective chemicals. Notably, the extracellular space becomes important to model. The response of cells embedded in the tissue is dependent on the state of the extracellular space which varies both spatially and temporally. Transport in the extracellular space can also lead to changes in tissue size. In this chapter, we describe various mass transfer models that can be used to describe transport phenomena occurring during loading of tissues with protective molecules for cryopreservation applications. Assumptions and simplifications that limit the applicability of each of these models are discussed.

Implications of variability in cell membrane permeability for design of methods to remove glycerol from frozen-thawed erythrocytes.

In North America, red blood cells (RBCs) are currently cryopreserved in a solution of 40% glycerol. While glycerol is not inherently toxic to humans, it must be removed prior to transfusion to prevent intravascular osmotic hemolysis. The current deglycerolization procedure requires about 45 min per RBC unit. We previously presented predictions suggesting that glycerol could be safely removed from RBCs in less than 1 min. However, experimental evaluation of these methods resulted in much higher hemolysis than expected. Here we extend our previous study by considering both concentration-dependence of permeability and variability in permeability values in the mathematical optimization algorithm. To establish a model for the concentration dependence of glycerol permeability, we combined literature data with new measurements of permeability in the presence of 40% glycerol. To account for cell-dependent variability we scaled the concentration-dependent permeability model to define a permeability range for optimization. Methods designed using a range extending to 50% of the model-predicted glycerol permeability had a duration of less than 3 min and resulted in hemolysis ranging from 34% to 83%; hemolysis values were highly dependent on the blood donor. Extending the permeability range to 5% of the model-predicted value yielded a 30 min method that resulted in an average hemolysis of 12%. Our results suggest high variability in the glycerol permeability between donors and within a population of cells from the same donor. Such variability has broad implications for design of methods for equilibration of cells with cryoprotectants.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.