Nilay Chakraborty

Late embryogenesis abundant proteins protect human hepatoma cells during acute desiccation

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporter was used for intracellular loading of this disaccharide. Cells were rapidly and uniformly desiccated to low water content (<0.12 g H2O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, compared with 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.

A Role for Microwave Processing in the Dry Preservation of Mammalian Cells

Dry preservation involves removing water from samples so that degradative biochemical processes are slowed and extended storage is possible. Recently this approach has been explored as a method for preserving living mammalian cells. The current work explores the use of microwave processing to enhance evaporation rates and to improve drying uniformity, thereby overcoming some of the challenges in this field. Mouse macrophage cells (J774) were pre‐incubated in full complement media containing 50 mM trehalose, for 18‐h, to allow for endocytosis of trehalose. Droplets of experimental and control (no intracellular trehalose) cell suspensions were placed on coverslips in a microwave cavity. Water was evaporated using intermittent microwave heating (600 W, 30 s intervals). Samples were dried to various moisture levels, rehydrated, and then survival was assessed after a 45‐min recovery period using Calcein‐AM/PI fluorescence and Trypan Blue exclusion assays. The metabolic activity of dried cells (4.3 gH2O/gdw) was assessed after rehydration using a resazurin reduction assay. Apoptosis levels were also measured. Post‐ rehydration survival correlated with the final moisture content achieved, consistent with other drying methods. Intracellular trehalose provided protection against injury associated with moisture loss. Metabolic assays revealed normal growth in surviving cells, and these survival levels were consistent with results from apoptosis assays (Pu2009>u20090.05). Brightfield and fluorescence images of microwave‐dried samples revealed a uniform distribution of cells within the dried matrix and profilometry analysis demonstrated that solids were uniformly distributed throughout the sample. Microwave‐processing successfully facilitated rapid and uniform dehydration of cell‐based samples. Biotechnol. Bioeng. 2008;100: 782–796.

Trehalose Transporter from African Chironomid Larvae Improves Desiccation Tolerance of Chinese Hamster Ovary Cells

Dry preservation has been explored as an energy-efficient alternative to cryopreservation, but the high sensitivity of mammalian cells to desiccation stress has been one of the major hurdles in storing cells in the desiccated state. An important strategy to reduce desiccation sensitivity involves use of the disaccharide trehalose. Trehalose is known to improve desiccation tolerance in mammalian cells when present on both sides of the cell membrane. Because trehalose is membrane impermeant the development of desiccation strategies involving this promising sugar is hindered. We explored the potential of using a high-capacity trehalose transporter (TRET1) from the African chironomid Polypedilum vanderplanki[21] to introduce trehalose into the cytoplasm of mammalian cells and thereby increase desiccation tolerance. When Chinese hamster ovary cells (CHO) were stably transfected with TRET1 (CHO-TRET1 cells) and incubated with 0.4M trehalose for 4h at 37°C, a sevenfold increase in trehalose uptake was observed compared to the wild-type CHO cells. Following trehalose loading, desiccation tolerance was investigated by evaporative drying of cells at 14% relative humidity. After desiccation to 2.60g of water per gram dry weight, a 170% increase in viability and a 400% increase in growth (after 7days) was observed for CHO-TRET1 relative to control CHO cells. Our results demonstrate the beneficial effect of intracellular trehalose for imparting tolerance to partial desiccation.

A spin-drying technique for lyopreservation of mammalian cells.

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 0.2–0.6xa0M trehalose solutions at a variety of spinning speeds ranging from 1000 to 4000xa0rpm. The model developed here predicts the formation of sugar films with thicknesses of 200–1000xa0nm, which was in good agreement with experimental results. Preliminary data suggest that after spin drying cells to about 0.159xa0±xa00.09xa0gH2O/gdw (nxa0=xa011, ±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 0.4xa0gH2O/gdw (Acker et al. Cell Preserv. Technol. 1(2):129–140, 2002; Elliott et al. Biopreserv. Biobank. 6(4):253–260, 2009).

Metabolic preconditioning of cells with AICAR-riboside : improved cryopreservation and cell-type specific impacts on energetics and proliferation.

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.

Anhydrous Preservation of Mammalian Cells: Cumulative Osmotic Stress Analysis

Cumulative osmotic stress models have been previously used to successfully describe the dehydration kinetics of recalcitrant seeds. For example, Liang and Sun demonstrated that a cumulative stress model effectively described the dehydration rate-dependence of recalcitrant seed viability under various drying conditions. In contrast, most studies describing the functionality of mammalian cells following drying conditions have been end-point oriented, describing cell viability as a function of the final moisture content reached or the duration of drying. This study applies a thermodynamics-based water metric similar to the Liang and Sun model to describe the viability of J774 mouse macrophage cells as a function of both moisture content and time of drying. Cells were incubated in full-complement DMEM media containing 50 mM trehalose, to enable trehalose loading by endocytosis. Treated cells and untreated controls that were not previously incubated in trehalose were dried in hypertonic (508 mOsm) and isotonic (308 mOsm) solutions of trehalose (200 mM) in phosphate-buffered saline (PBS). Various levels of dehydration were achieved by placing droplets of cell suspension in a desiccator for time periods up to 2 h. Cells were then immediately rehydrated and viability was assessed 45-min after rehydration. Cell viability was evaluated using a combination of Trypan Blue staining for membrane integrity of detached cells and Calcein AM - ethidium bromide fluorescence as a live-dead assay for attached cells. The cellular response was then evaluated as a function of cumulative osmotic stress, defined as the integral of the deviation in osmolality from isotonic conditions as a function of time. The results of this modeling suggested that significant cell injury was occurring in a moderate osmolality range, and that modulation of osmotic stresses in this range could lead to improved processing outcomes.

Modulation of cellular stress response via the erythropoietin/CD131 heteroreceptor complex in mouse mesenchymal-derived cells

Tissue-protective properties of erythropoietin (EPO) have let to the discovery of an alternative EPO signaling via an EPO-R/CD131 receptor complex which can now be specifically targeted through pharmaceutically designed short sequence peptides such as ARA290. However, little is still known about specific functions of alternative EPO signaling in defined cell populations. In this study, we investigated effects of signaling through EPO-R/CD131 complex on cellular stress responses and pro-inflammatory activation in different mesenchymal-derived phenotypes. We show that anti-apoptotic, anti-inflammatory effects of ARA290 and EPO coincide with the externalization of CD131 receptor component as an immediate response to cellular stress. In addition, alternative EPO signaling strongly modulated transcriptional, translational, or metabolic responses after stressor removal. Specifically, we saw that ARA290 was able to overcome a TNFα-mediated inhibition of transcription factor activation related to cell stress responses, most notably of serum response factor (SRF), heat shock transcription factor protein 1 (HSF1), and activator protein 1 (AP1). We conclude that alternative EPO signaling acts as a modulator of pro-inflammatory signaling pathways and likely plays a role in restoring tissue homeostasis.Key message• Erythropoietin (EPO) triggers an alternative pathway via heteroreceptor EPO/CD131.• ARA290 peptide specifically binds EPO/CD131 but not the canonical EPO/EPO receptor.• Oxidative stress and inflammation promote cell surface expression of CD131.• ARA290 prevents tumor necrosis factor-mediated inhibition of stress-related genes.• Alternative EPO signaling modulates inflammation and promotes tissue homeostasis.

High-resolution intracellular viscosity measurement using time-dependent fluorescence anisotropy

A low-cost pulsed laser is used in conjunction with a homebuilt laser confocal-scanning epifluorescence microscope having submicron lateral and axial spatial resolution to determine cytoplasmic viscosity at specific intracytoplasmic locations in J774 mouse macrophage cells. Time-dependent fluorescence anisotropy measurements are made at each location and global deconvolution techniques are used to determine rotational correlation times. These rotational correlation times are related to the hydrated volume of 8-hydroxyperene-1,3,6-trisulfonic acid (HPTS) to calculate viscosity at specific points inside the cell. In the cytoplasmic areas measured, rotational correlation times of HPTS ranged from 0.186 ns to 0.411 ns, corresponding to viscosities ranging from 1.00 +/- 0.03 cP to 2.21+/- 0.05 cP.

Cryopreservation of Spin-Dried Mammalian Cells

This study reports an alternative approach to achieve vitrification where cells are pre-desiccated prior to cooling to cryogenic temperatures for storage. Chinese Hamster Ovary (CHO) cells suspended in a trehalose solution were rapidly and uniformly desiccated to a low moisture content (<0.12 g of water per g of dry weight) using a spin-drying technique. Trehalose was also introduced into the cells using a high-capacity trehalose transporter (TRET1). Fourier Transform Infrared Spectroscopy (FTIR) was used to examine the uniformity of water concentration distribution in the spin-dried samples. 62% of the cells were shown to survive spin-drying in the presence of trehalose following immediate rehydration. The spin-dried samples were stored in liquid nitrogen (LN2) at a vitrified state. It was shown that following re-warming to room temperature and re-hydration with a fully complemented cell culture medium, 51% of the spin-dried and vitrified cells survived and demonstrated normal growth characteristics. Spin-drying is a novel strategy that can be used to improve cryopreservation outcome by promoting rapid vitrification.

Confocal and force probe imaging system for simultaneous three-dimensional optical and mechanical spectroscopic evaluation of biological samples

We present the design and operation of a novel instrument for the simultaneous three-dimensional measurements of localized properties using optical and mechanical probes. In this instrument the mechanical and optical probes are stationary relative to the instrument frame while the specimen can be navigated in three-dimensional space in the probing field, translating over a range of 64.5 microm by 49.7 microm by 31.5 microm in each axis, respectively, at closed loop speeds of 10 Hz. A large aperture is provided in the center of the moving platform so that an optical lens can image the specimen from below. An additional z-direction translator has been integrated with this instrument to independently move a force probe that contacts the specimen from above with a translation range of 16 microm. Furthermore, there is an additional seven degrees of freedom providing adjustments to independently position and/or align the scanner and force probe relative to the optical imaging lens. Initial results of both optical and mechanical scans demonstrate 6 nm localization from single molecule fluorescence measurements, as well as single pair fluorescence energy transfer measurements indicating molecular separations of about 2 nm.