Project

Biomimetic approaches to cell preservation

Goal: To improve cell preservation techniques by applying protection strategies observed in naturally occurring states of low-water tolerance.

Methods: Calorimetry, Cell Viability Assay, High Resolution Respirometry, Confocal Raman Microscopy

Updates
0 new
1
Recommendations
0 new
0
Followers
0 new
14
Reads
0 new
387

Project log

Michael A Menze
added a research item
To deal with increasingly severe periods of dehydration related to global climate change, it becomes increasingly important to understand the complex strategies many organisms have developed to cope with dehydration and desiccation. While it is undisputed that late embryogenesis abundant (LEA) proteins play a key role in the tolerance of plants and many anhydrobiotic organisms to water limitation, the molecular mechanisms are not well understood. In this review we recap the current knowledge of the physiological roles of LEA proteins and discuss their potential molecular functions. As these are ultimately linked to conformational changes in the presence of binding partners, posttranslational modifications or water deprivation, we give a detailed summary of the current knowledge on the structure-function relationship of LEA proteins, including their disordered state in solution, coil-to-helix transitions, self-assembly and their recently discovered ability to undergo liquid-liquid phase separation (LLPS). We point out the promising potential of LEA proteins in biotechnological and agronomic applications and summarize recent advances. We identify the most relevant open questions and discuss major challenges in establishing a solid understanding of how these intriguing molecules accomplish their tasks as cellular sentinels at the limits of surviving water scarcity.
Michael A Menze
added a research item
Cell-based therapies have garnered significant interest to treat cancer and other diseases. Acoustofluidic technologies are in development to improve cell therapy manufacturing by facilitating rapid molecular delivery across the plasma membrane via ultrasound and microbubbles (MBs). In this study, a three-dimensional (3D) printed acoustofluidic device was used to deliver a fluorescent molecule, calcein, to human T cells. Intracellular delivery of calcein was assessed after varying parameters such as MB face charge, MB concentration, flow channel geometry, ultrasound pressure, and delivery time point after ultrasound treatment. MBs with a cationic surface charge caused statistically significant increases in calcein delivery during acoustofluidic treatment compared to MBs with a neutral surface charge (p < 0.001). Calcein delivery was significantly higher with a concentric spiral channel geometry compared to a rectilinear channel geometry (p < 0.001). Additionally, calcein delivery was significantly enhanced at increased ultrasound pressures of 5.1 MPa compared to lower ultrasound pressures between 0–3.8 MPa (p < 0.001). These results demonstrate that a 3D-printed acoustofluidic device can significantly enhance intracellular delivery of biomolecules to T cells, which may be a viable approach to advance cell-based therapies.
Michael A Menze
added a research item
No PDF available ABSTRACT T-cell therapies are rapidly emerging for treatment of cancer and other diseases but are limited by inefficient non-viral delivery methods. Acoustofluidic devices are in development to enhance non-viral delivery to cells. The effect of acoustofluidic parameters, such as channel geometry, on molecular loading in human T cells was assessed using 3D-printed acoustofluidic devices. Devices with rectilinear channels (1- and 2-mm diameters) were compared directly with concentric spiral channel geometries. Intracellular delivery of a fluorescent dye (calcein, 100 μg/ml) was evaluated in Jurkat T cells using flow cytometry after ultrasound treatment with cationic microbubbles (2.5% v/v). B-mode ultrasound pulses (2.5 MHz, 3.8 MPa output pressure) were generated by a P4-1 transducer on a Verasonics Vantage ultrasound system. Cell viability was assessed using propidum iodine staining (10 μg/ml). Intracellular molecular delivery was significantly enhanced with acoustofluidic treatment in each channel geometry, but treatment with the 1-mm concentric spiral geometry further enhanced delivery after acoustofluidic treatment compared to both 1- and 2-mm rectilinear channels (ANOVA p < 0.001, n = 6/group). These results indicate that 3D-printed acoustofluidic devices enhance molecular delivery to T cells, and channel geometry modulates intracellular loading efficiency. This approach may offer advantages to improve manufacturing of T cell therapies.
Michael A Menze
added a research item
Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS- based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.
Michael A Menze
added a research item
Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at -1 ˚C/min to -80 ˚C, then either storing the cells at -80 ˚C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.
Michael A Menze
added a research item
Preservation of erythrocytes in a desiccated state for storage at ambient temperature could simplify blood transfusions in austere environments, such as rural clinics, far-forward military operations, and during space travel. Currently, storage of erythrocytes is limited by a short shelf-life of 42 days at 4 °C, and long-term preservation requires a complex process that involves the addition and removal of glycerol from erythrocytes before and after storage at −80 °C, respectively. Natural compounds, such as trehalose, can protect cells in a desiccated state if they are present at sufficient levels inside the cell, but mammalian cell membranes lack transporters for this compound. To facilitate compound loading across the plasma membrane via ultrasound and microbubbles (sonoporation), a polydimethylsiloxane-based microfluidic device was developed. Delivery of fluorescein into erythrocytes was tested at various conditions to assess the effects of parameters such as ultrasound pressure, ultrasound pulse interval, microbubble dose, and flow rate. Changes in ultrasound pressure and mean flow rate caused statistically significant increases in fluorescein delivery of up to 73 ± 37% (p < 0.05) and 44 ± 33% (p < 0.01), respectively, compared to control groups, but no statistically significant differences were detected with changes in ultrasound pulse intervals. Following freeze-drying and rehydration, recovery of viable erythrocytes increased by up to 128 ± 32% after ultrasound-mediated loading of trehalose compared to control groups (p < 0.05). These results suggest that ultrasound-mediated molecular delivery in microfluidic channels may be a viable approach to process erythrocytes for long-term storage in a desiccated state at ambient temperatures.
Michael A Menze
added a research item
Late embryogenesis abundant (LEA) proteins are found in desiccation-tolerant species from all domains of life. Despite several decades of investigation, the molecular mechanisms by which LEA proteins confer desiccation tolerance are still unclear. In this study, dielectrophoresis (DEP) was used to determine the electrical properties of Drosophila melanogaster (Kc167) cells ectopically expressing LEA proteins from the anhydrobiotic brine shrimp, Artemia franciscana. Dielectrophoresis-based characterization data demonstrate that the expression of two different LEA proteins, AfrLEA3m and AfrLEA6, increases cytoplasmic conductivity of Kc167 cells to a similar extent above control values. The impact on cytoplasmic conductivity was surprising, given that the concentration of cytoplasmic ions is much higher than the concentrations of ectopically expressed proteins. The DEP data also supported previously reported data suggesting that AfrLEA3m can interact directly with membranes during water stress. This hypothesis was strengthened using scanning electron microscopy, where cells expressing AfrLEA3m were found to retain more circular morphology during desiccation, while control cells exhibited a larger variety of shapes in the desiccated state. These data demonstrate that DEP can be a powerful tool to investigate the role of LEA proteins in desiccation tolerance and may allow to characterize protein-membrane interactions in vivo, when direct observations are challenging.
Michael A Menze
added a research item
Update of our Blood Project on WHAS11-TV https://www.whas11.com/article/news/health/method-developed-by-louisville-researchers-could-preserve-blood-for-decades/417-5d221bfc-16fe-4966-bf70-5f4eb467e262
Michael A Menze
added a research item
The shelf-life of donated red blood cells (RBCs) for transfusions is currently limited to six weeks when stored under refrigeration. This causes supply shortages worldwide and prevents transfusions in locations that lack access to cold-chain storage. Recently, a new approach to store RBCs as a dried powder at ambient temperature was developed. This method utilizes an ultrasound-integrated microfluidic platform to induce intracellular delivery of compounds that protect cells during desiccation and rehydration. The objective of this study was to detect cavitation emissions in order to optimize parameters for molecular delivery to RBCs in this system. Ultrasound was continuously generated in the microfluidic channels using an 8-MHz PZT plate and acoustic emissions were passively detected with an identical PZT plate aligned coaxially. Fluorescein and lipid-coated microbubbles were added to RBC solutions in order to nucleate cavitation and enhance intracellular molecular uptake as measured by flow cytometry. Increased levels of broadband emissions were detected at microfluidic flow rates associated with higher fluorescein delivery to RBCs. These results suggest that inertial cavitation plays an important role in enhancing molecular delivery to RBCs in the microfluidic channels. Optimization of this system may enhance delivery of protective compounds for long-term preservation of blood.
Michael A Menze
added 3 research items
Late Embryogenesis Abundant (LEA) proteins are a remarkable group of intrinsically disordered proteins (IDPs) that confer desiccation tolerance to plants and animals that can enter a cryptobiotic state during their life cycle. AfrLEA6 contains seed maturation domains (SMD) and is expressed in the anhydrobiotic cysts of the brine shrimp Artemia franciscana. in vitro analyses of AfrLEA6 reveal a series of protein phase transitions during desiccation. As ionic strength or molecular crowding with Ficoll-400 increases, AfrLEA6 undergoes a liquid-liquid phase separation (LLPS), forming protein droplets. AfrLEA6 droplets are also inducible by reducing the sample pH from 8.0 to 6.5 and cooling protein solutions from 25˚C to 4˚C. These conditions are notable in the context of the cysts of A. franciscana, which can naturally undergo a cytoplasmic pH shift from 7.9 to 6.5 in response to severe hypoxia. In the hydrated state, AfrLEA6 droplets exclude green fluorescent protein demonstrating that the protein droplet may be selective for inclusions of specific targets. SEM and AFM reveal that AfrLEA6 may also undergoes a phase shift to a hydrogel structure, as ionic strength and crowding increase, which is reversible upon rehydration. However, early during dehydration formed hydrogels dry into a reversible glassy state during complete desiccation. The LLPS of AfrLEA6 may confer desiccation tolerance by selectively incorporating sensitive protein targets and shielding them from desiccation induced denaturation during early drying. Any incorporated proteins may then be stabilized within a glassy compartment in the fully desiccated state and released upon rehydration (supported by NSF IOS-1659970).
Our understanding of protein liquid-liquid phase separation (LLPS; ‘membraneless organelles’) and its importance in a wide range of biological phenomena is rapidly growing. Unexpectedly, protein LLPS may also play a role in the desiccation- and osmotic-stress tolerance of encysted Artemia franciscana (brine shrimp) embryos. AfrLEA6 is an intrinsically disordered protein in Artemia that shares homology with seed maturation proteins (SMPs) found in some plant seeds. SMPs have been linked to the duration in which a seed remains viable in the dried state. Therefore, it was hypothesized that AfrLEA6 may play a role in sustained tolerance to water stress. This hypothesis was tested by ectopically expressing AfrLEA6 in desiccation-sensitive Drosophila melanogaster (Kc167) cells and exposing these cells to water stress. AfrLEA6 was found to increase both desiccation and osmotic-stress tolerance of Kc167 cells. Furthermore, confocal microscopy was used to image LLPS of AfrLEA6 in vivo. Staining cells with Nile Red, a lipophilic dye, suggested that AfrLEA6 causes the cytosol to interact with Nile Red like an aqueous-organic cosolvent mixture. Altered solvent properties may decrease the thermodynamic stability of unfolded proteins and reduce native protein conformational mobility yielding cytosolic wide stabilization of native proteins. Altogether, these data support the hypothesis that AfrLEA6 plays a role during water loss and indicates that AfrLEA6 significantly impacts the physicochemical properties of the cytosol. (Supported by NSF IOS-1659970.)
Late Embryogenesis Abundant (LEA) proteins are a class of highly hydrophilic intrinsically disordered polypeptides (IDP) that are found in many plants and some anhydrobiotic animals. Over 15 distinct LEA proteins, belonging to three different classification groups (1,3 and 6), have been found in Artemia franciscana and several of these proteins have been shown to be involved in the anhydrobiotic life history stage of these Branchiopods. The exact mechanisms by which specific LEA proteins protect brine shrimp embryos during desiccation is largely unknown. To gain understanding into the possible mechanisms of protection conferred by group 1 and 6 LEA proteins, enzyme assays were utilized to investigate the effect of AfLEA1.1 and AfrLEA6 on lactate dehydrogenase (LDH) activity in lysate of Drosophila melanogaster Kc167 cells after desiccation and rehydration. Cell lysates were utilized to probe for specific interactions between LDH and LEA proteins during water-stress in a proteome system. This may closer resemble potential interaction in the cytoplasm than observed in a binary protein study with purified enzymes and a specific LEA protein. Results show that AfLEA1.1 added to purified LDH protected the enzyme during desiccation and rehydration, however, when added to cell lysate, no protection of enzymatic activity was observed after rehydration compared to LEA-free control lysates. Similarly, no protection of LDH activity by AfrLEA6 was observed when the protein was added to cell lysates before desiccation compared to LEA-free controls. It appears likely that the protection of enzymatic activity observed by AfLEA1.1 in the binary protein system might be an overestimate and LDH is not a specific target of AfLEA1.1 under physiological conditions (supported by NSF IOS-1659970).
Michael A Menze
added 2 research items
Background Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Promising new avenues in cell stabilization include biomimetic approaches based on intracellular conditions found in animals with the ability to maintain viable cells and tissues in a frozen or desiccated state (cryptobiosis). Our approach addresses storage limitations by lyophilizing human RBCs into a powder that could theoretically be stored for several years and be rehydrated as needed for transfusion. This process involves the non-reducing sugar trehalose, a compound with well-established cytoprotective properties in cryptobiotic animals. However, trehalose is impermeable to mammalian cells which hampered progress in using this promising biomolecule in RBC preservation. Our approach utilizes sonoporation for sugar loading of RBCs. Sonoporation is a process in which transient pores are induced in the cell membrane by the ultrasound-mediated oscillation of gas microbubbles. The objective of this study is to verify the efficacy of trehalose loading and long-term stability and functionality of preserved blood units. Methods Human red blood cells were obtained with informed consent from donors. RBCs were resuspended and diluted in loading buffer containing trehalose immediately prior to treatment. Lipid-coated microbubbles were added to samples and B-mode ultrasound pulses were applied using an ultrasound imaging system. Trehalose uptake into RBC was confirmed enzymatically. RBCs were cooled to -80 °C at a rate of -1 °C/min followed by freeze-drying. Dried RBCs were stored at ambient temperature and resuspended in deionized water. Cell recovery was measured using automated cell counting and cell viability was assessed by staining with calcein-AM. Results Sonoporation-mediated trehalose loading has minimal toxicity as 95-100% of RBCs were recovered after treatment. Trehalose-loaded RBCs showed >95% recovery and viability after storage at -80 °C, whereas a significantly reduced recovery of 20-40% was observed without treatment (n=12; p<0.05). Recovery of lyophilized RBCs after rehydration was 16-30% and cell viability in this cell population was 70-80% (n=12). Without trehalose loading, no viable RBCs were recovered after freeze-drying and rehydration. Conclusions Our results demonstrate that sonoporation enhances delivery of trehalose into RBCs and dramatically increases recovery of intact RBCs following freezing/thawing or drying/rehydration. Although further testing is needed to evaluate RBC function in vivo after dry preservation, our approach offers significant potential to help stabilize the clinical blood supply and to increase the accessibility of blood transfusions in the future.
Michael A Menze
added a research item
This review compares the molecular strategies employed by anhydrobiotic invertebrates to survive extreme water stress. Intrinsically disordered proteins (IDPs) play a central role in desiccation tolerance in all species investigated. Various hypotheses about the functions of anhydrobiosis‐related intrinsically disordered (ARID) proteins, including late embryogenesis abundant (LEA) and tardigrade‐specific intrinsically disordered proteins, were evaluated by broad sequence characterization. A surprisingly wide range in sequence characteristics including hydropathy and the frequency and distribution of charges was discovered. Interestingly, two clusters of similar proteins were found that potentially correlate with distinct functions. This may indicate two broad groups of ARID proteins, composed of one group that folds into functional conformations during desiccation and a second group that potentially displays functions in the hydrated state. A broad range of physiochemical properties suggest that folding may be induced by factors such as hydration level, molecular crowding, and interactions with binding partners. This plasticity may be required to fine tune the ARID‐proteome response at different hydration levels during desiccation. Furthermore, the sequence properties of some LEA proteins share qualities with IDPs known to undergo liquid‐liquid phase separations during environmental challenges. This article is protected by copyright. All rights reserved
Michael A Menze
added a research item
Red blood cells (RBCs) must be continuously maintained at 4 °C, which poses a significant barrier to blood transfusions in far-forward locations where uninterrupted cooling is challenging. Furthermore, the maximal shelf-life of RBC transfusion units is only 42 days. The short shelf-life of transfusable units also creates a significant barrier to developing strategic blood reserves for national emergency situations. Therefore, a method to preserve RBCs in a dried state for long-term storage at ambient temperatures with rapid rehydration would offer significant benefits for civilian and military transfusion needs. We have developed a novel approach to load RBCs with a naturally-occurring sugar that acts as cell protectant, trehalose, to preserve RBCs in a dried state. Trehalose is a non-reducing carbohydrate found in plants and lower animals that enables these organisms to survive water-limited states such as dehydration or freezing, but the protectant cannot penetrate RBC membranes. We have developed a novel loading method that utilizes “sonoporation”, a phenomenon caused by ultrasound-mediated oscillation and collapse of microbubbles (MB) near cell membranes which induces transient pore formation and enables uptake of cell impermeant compounds such as trehalose. The combination of our unique loading technique with parallel microfluidic devices allows for high throughput without compromising loading efficiency or cell recovery. This project was funded by NIH grant U01HL127518 and the NSF I-Corps program (Patent pending).
Michael A Menze
added a research item
Blood transfusions are one of the most common medical procedures in hospitals, but shortages of erythrocytes often occur due to their limited shelf life (6 weeks) when refrigerated. Preservation of erythrocytes in a dried state offers a potential solution to challenges faced with blood storage, and preservative compounds such as trehalose have been identified in organisms that survive desiccation in nature. However, these compounds do not readily cross mammalian cell membranes. Therefore, we are exploring the use of sonoporation to facilitate their delivery into erythrocytes ex vivo. In this study, we assessed the effect of cavitation activity on delivery of a fluorescent compound similar in size to trehalose (fluorescein) to human erythrocytes. Microbubbles were added to erythrocyte solutions and sonicated (2.5 MHz, 4 cycles) at various pressures and durations. Fluorescence was quantified with flow cytometry. The amplitude of broadband emissions in the first 8 seconds of sonication did not correlate with delivery (r² = 0.23), whereas after 8 seconds the broadband emissions amplitude was associated with increased delivery to erythrocytes (r² = 0.97). These results suggest that the timing of cavitation activity, rather than the amplitude alone, may be an important factor in ultrasound-mediated delivery of compounds into erythrocytes.
Michael A Menze
added a research item
In order to preserve and store cells and cellular products, especially those with a short shelf-life as seen with red blood cells, cryopreservation is utilized. This method relies on dissolving the sample a cryoprotective solution, cooling the sample and storing in the vapor phase of liquid nitrogen. Researchers at U of L have developed a sonoporation method to deliver tetralose to cells, which can be frozen and/or dried using convective, or freeze-drying for long-term preservation. Using this technology, researchers can successfully preserve cells and cellular products long-term without the use of cytotoxic DMSO.
Michael A Menze
added a research item
UofL’s Mark Hebert features our project to preserve red blood cells in a desiccated state. To access the youtube video please copy and paste the above url into your browser.
Michael A Menze
added an update
Michael A Menze
added a research item
INTRODUCTION Blood transfusions are critical life-saving medical procedures required for survival following trauma or surgery. However, red blood cells (RBCs) can only be stored for a maximum of 42 days at 4 °C, which poses a significant barrier to blood transfusions on long-term space missions. RBCs can be frozen for long-term storage, but a complex glycerolization process is required and careful maintenance of temperatures are needed. Furthermore, upon thawing the glycerol must be removed using a sensitive process. Therefore, a method to preserve RBCs in a dried state would offer significant benefits for long-term space missions in addition to many terrestrial applications, including military needs. We have developed a novel approach to load RBCs with a naturally-occurring cell protectant, trehalose, to preserve RBCs in a dried state. Trehalose is a non-reducing sugar found in plants and lower animals that enables these organisms to survive water-limited states such as dehydration or freezing. However, mammalian cell membranes lack trehalose transporters and are impermeable to this sugar, which requires active loading into these cells. To overcome this permeability challenge, we have developed a novel approach utilizing ultrasound and microbubbles to mediate trehalose delivery into RBCs. This method utilizes "sonoporation", a phenomenon caused by ultrasound-mediated oscillation and collapse of microbubble near cell membranes which induces transient pore formation and enables uptake of compounds such as trehalose. The objective of this study was to evaluate whether ultrasound-mediated trehalose delivery is a viable stratregy for dry preservation of RBCs.
Michael A Menze
added 17 research items
Hematopoietic stem and progenitor cells (HPCs) are a heterogenic population of cells used to treat a number of human diseases. Multilineage differentiation is a required function in successful hematopoietic reconstitution af- ter transplantation of cryopreserved grafts. Conventional use of the cryoprotectant dimethyl sulfoxide (DMSO) has resulted in some reports of infusion related toxicity attributed to DMSO and/or damage to cells during freeze- thawing procedures. The purpose of this study was to explore the use of trehalose, a nontoxic disaccharide of glu- cose, as an alternative cryoprotectant. Trehalose was introduced into HPCs using the P2Z receptor, known to form nonselective pores in the presence of extracellular adenosine 5� -triphosphate (ATP4� ). Cells loaded with trehalose were frozen and stored at � 80°C for 4 months. After storage, cells were thawed and evaluated for differentiation capacity and clonogenic output. Results obtained with this technique were compared to traditional freezing pro- tocols using 10% (v/v) DMSO. Clonogenic output of cells frozen with trehalose was approximately 90% of that of unfrozen control cells. Furthermore, there were no significant alterations in phenotypic markers of differentia- tion, activation, and proliferation. These data demonstrate that preservation of HPC function with trehalose is su- perior to that obtained with DMSO and this method could be widely adapted to any cell or tissue type express- ing the P2Z receptor. Furthermore, cells loaded with trehalose can potentially be freeze-dried for storage at ambient temperatures.
In species whose evolutionary history has provided natural tolerance to dehydration and freezing, metabolic depression is often a pre-requisite for survival. We tested the hypothesis that preconditioning of mammalian cells with 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) to achieve metabolic depression will promote greater survivorship during cryopreservation. AICAR is used extensively to stimulate AMP-activated protein kinase (AMPK), which can result in downregulation of biosynthetic processes. We showed that the metabolic interconversion of AICAR was cell-type dependent. Accumulation of 5-aminoimidazole-4-carboxamide-1b-D-ribofuranosyl-5'-monophosphate (ZMP), as well as other metabolites that possess multiple phosphates (i.e., ZDP, ZTP), varied approximately 3.5-fold across the cell lines tested. AICAR treatment also significantly influenced the concentrations of cellular adenylates (ATP, ADP, and AMP). Depression of cell metabolism and proliferation with AICAR treatment differed among cell lines. Proliferation for a given cell line was negatively correlated with the fold-increase achieved in the 'effective adenylate ratio' ([AMP]+[ZMP])/[ATP]) after AICAR treatment. Metabolic preconditioning with AICAR promoted a significant increase in viability post-freezing in J774.A1 macrophages, HepG2/C3A cells and primary hepatocytes but not in NIH/3T3 fibroblasts or OMK cells. The effect of AICAR on viability after freezing was positively correlated (r(2)=0.94) with the fold-increase in the 'effective adenylate ratio'. Thus for each cell line, the greater the depression of metabolism and proliferation due to preconditioning with AICAR, the greater was the survivorship post-freezing.
Stabilization of cellular material in the presence of glass-forming sugars at ambient temperatures is a viable approach that has many potential advantages over current cryogenic strategies. Experimental evidence indicates the possibility to preserve biomolecules in glassy matrices of low-molecular mobility using "glass-forming" sugars like trehalose at ambient temperatures. However, when cells are desiccated in trehalose solution using passive drying techniques, a glassy skin is formed at the liquid/vapor interface of the sample. This glassy skin prevents desiccation of the sample beyond a certain level of dryness and induces non-uniformities in the final water content. Cells trapped underneath this glassy skin may degrade due to a relatively high molecular mobility in the sample. This undesirable result underscores the need for development of a uniform, fast drying technique. In the present study, we report a new technique based on the principles of "spin drying" that can effectively address these problems. Forced convective evaporation of water along with the loss of solution due to centrifugal force leads to rapid vitrification of a thin layer of trehalose containing medium that remains on top of cells attached to the spinning glass substrate. The glassy layer produced has a consistent thickness and a small "surface-area-to-volume" ratio that minimizes any non-homogeneity. Thus, the chance of entrapping cells in a high-mobility environment decreases substantially. We compared numerical predictions to experimental observations of the drying time of 0.2-0.6 M trehalose solutions at a variety of spinning speeds ranging from 1000 to 4000 rpm. The model developed here predicts the formation of sugar films with thicknesses of 200-1000 nm, which was in good agreement with experimental results. Preliminary data suggest that after spin drying cells to about 0.159 ± 0.09 gH₂O/gdw (n = 11, ±SE), more than 95% of cells were able to preserve their membrane integrity. Membrane integrity after spin drying is therefore considerably higher than what is achieved by conventional drying methods; where about 90% of cells lose membrane integrity at 0.4 gH₂O/gdw (Acker et al. Cell Preserv. Technol. 1(2):129-140, 2002; Elliott et al. Biopreserv. Biobank. 6(4):253-260, 2009).
Michael A Menze
added a project goal
To improve cell preservation techniques by applying protection strategies observed in naturally occurring states of low-water tolerance.