Concentrated Cell Suspensions Research Articles

3D structured capillary cell suspensions aided by aqueous two-phase systems.

We report a facile technique for 3D structuring of living cells by forming capillary cell suspensions based on an aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and dextran (DEX) solutions. We demonstrate the formation of water-in-water (DEX-in-PEG) capillary bridges using concentrated suspensions of yeast cells which show enhanced rheological properties and distinctive 3D patterns. Capillary structured cell suspensions can potentially find applications in novel ways of 3D cell culturing, instant tissue engineering and many biomedical investigations.

Investigation on absorption cross-section of photosynthetic pigment molecules based on a mechanistic model of the photosynthetic electron flow-light response in C3, C4 species and cyanobacteria grown under various conditions.

Investigation on intrinsic properties of photosynthetic pigment molecules participating in solar energy absorption and excitation, especially their eigen-absorption cross-section (σ ik) and effective absorption cross-section (σ ' ik), is important to understand photosynthesis. Here, we present the development and application of a new method to determine these parameters, based on a mechanistic model of the photosynthetic electron flow-light response. The analysis with our method of a series of previously collected chlorophyll a fluorescence data shows that the absorption cross-section of photosynthetic pigment molecules has different values of approximately 10-21 m2, for several photosynthetic organisms grown under various conditions: (1) the conifer Abies alba Mill., grown under high light or low light; (2) Taxus baccata L., grown under fertilization or non-fertilization conditions; (3) Glycine max L. (Merr.), grown under a CO2 concentration of 400 or 600 μmol CO2 mol-1 in a leaf chamber under shaded conditions; (4) Zea mays L., at temperatures of 30°C or 35°C in a leaf chamber; (5) Osmanthus fragrans Loureiro, with shaded-leaf or sun-leaf; and (6) the cyanobacterium Microcystis aeruginosa FACHB905, grown under two different nitrogen supplies. Our results show that σ ik has the same order of magnitude (approximately 10-21 m2), and σ ' ik for these species decreases with increasing light intensity, demonstrating the operation of a key regulatory mechanism to reduce solar absorption and avoid high light damage. Moreover, compared with other approaches, both σ ik and σ ' ik can be more easily estimated by our method, even under various growth conditions (e.g., different light environment; different CO2, NO2, O2, and O3 concentrations; air temperatures; or water stress), regardless of the type of the sample (e.g., dilute or concentrated cell suspensions or leaves). Our results also show that CO2 concentration and temperature have little effect on σ ik values for G. max and Z. mays. Consequently, our approach provides a powerful tool to investigate light energy absorption of photosynthetic pigment molecules and gives us new information on how plants and cyanobacteria modify their light-harvesting properties under different stress conditions.

In Ukrainian

Low-temperature 1H NMR spectroscopy and DSC methods were used to study the hydration process of Lactobacillus, the influence of a weakly polar organic environment on it, and the encapsulation of cells with silica and the possibility of penetration of such an active substance as trifluoroacetic acid (TFAA) into them. It is shown that the spectral parameters of water in concentrated cell suspensions of Lactobacillus significantly depend on the concentration of the suspensions, which is probably related to the possibility of forming a stable cell gel, which can be encapsulated by silica particles both in the air environment and in the environment without its destruction chloroform with the addition of trifluoroacetic acid. There are two maxima corresponding to R = 2 and 20-100 nm on the distribution curves of the radii of clusters of unfreezing water. The contribution to the distribution of the second maximum increases with increasing water concentration. On the DSC-thermograms of lactobacilli, the value of the thermal effect related to the amount of bound water is much smaller than the thermal effect of ice melting, which is due to the presence of a significant amount of non-freezing water.

In English

A promising direction for long-term storage of cells at relatively high temperatures may be their encapsulation with nanoscale biologically inert materials capable of creating a shell around microdroplets of a cell suspension, which, on the one hand, provides the possibility of gas exchange between the suspension and the external environment, and on the other hand, inhibits the processes of cell life, so transferring them to a state close to suspended animation. The method of low-temperature 1H NMR spectroscopy was used to study the process of hydration of lactobacilli, the effect of a weakly polar organic medium on it and the encapsulation of cells with silica. The aim of this work was to study the hydration of cell suspensions and the viability of lactic acid bacteria cells encapsulated with silica and the penetration possibility of such an active substance as trifluoroacetic acid into them. As a result of the studies carried out, it has been shown that the spectral parameters of water in concentrated cell suspensions of lactic acid bacteria strongly depend on the concentration of the suspension, which is probably associated with the possibility of the formation of a stable cell gel, which can be encapsulated by silica particles without its destruction in both air and a chloroform medium with addition of trifluoroacetic acid. The radial distribution curves of non-freezing water clusters have two maxima corresponding to R = 2 and 20–100 nm. The contribution to the distribution of the second maximum increases with increasing water concentration.

4418 Optimization and Validation of a Silk Scaffold-Based Neural Tissue Construct

OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

Formate Is Required for Growth of the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui Lacking Hydrogen-Dependent Carbon Dioxide Reductase (HDCR).

The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.

Capacity of the medullary cavity of tibia and femur for intra-bone marrow transplantation in mice.

Intra-bone marrow transplantation (IBMT) has been adapted for mouse models to improve the seeding efficiency of transplanted hematopoietic stem and progenitor cells. Commonly used injection volumes for IBMT into the tibia differ between 10 and 40 μL even though considerable amounts of injected cells leak into the blood circulation immediately after injection. Injection of 3 μL trypan blue into the tibia of dead BALB/c mice showed staining in large vessels of hind limbs, even without supporting circulation. We therefore tested the effective capacity of the medullary cavity of dissected tibiae and femora of different mouse strains by bioluminescence imaging after injection of luciferase expressing cells. Cell leakage was already observed at 3 μL of injection volume and the measured emission rate increased significantly when 5 and 10 μL of volume with the same cell concentration were injected. Surprisingly, increasing injection volumes containing constant cell amounts resulted in comparable emission rates, suggesting a similar amount of leaked and absorbed cells independent of the injection volume. However, the absorption of a specific amount of injected cells could not be confirmed, as the ratio of leaked to absorbed cells was similar between IBMT that were performed with a constant injection volume containing either low or high cell amounts. In summary, for optimal cell transplantation via IBMT in mice we suggest to inject a high concentrated cell suspension with a maximum injection volume of 3 μL.

Method and Apparatus for the Automated Delivery of Continuous Neural Stem Cell Trails Into the Spinal Cord of Small and Large Animals.

Immature neurons can extend processes after transplantation in adult animals. Neuronal relays can form between injected neural stem cells (NSCs) and surviving neurons, possibly improving recovery after spinal cord injury (SCI). Cell delivery methods of single or multiple bolus injections of concentrated cell suspensions thus far tested in preclinical and clinical experiments are suboptimal for new tract formation. Nonuniform injectate dispersal is often seen due to gravitational cell settling and clumping. Multiple injections have additive risks of hemorrhage, parenchymal damage, and cellular reflux and require additional surgical exposure. The deposition of multiply delivered cells boluses may be uneven and discontinuous. To develop an injection apparatus and methodology to deliver continuous cellular trails bridging spinal cord lesions. We improved the uniformity of cellular trails by formulating NSCs in hyaluronic acid. The TrailmakerTM stereotaxic injection device was automatized to extend a shape memory needle from a single-entry point in the spinal cord longitudinal axis to "pioneer" a new trail space and then retract while depositing an hyaluronic acid-NSC suspension. We conducted testing in a collagen spinal models, and animal testing using human NSCs (hNSCs) in rats and minipigs. Continuous surviving trails of hNSCs within rat and minipig naive spinal cords were 12 and 40 mm in length. hNSC trails were delivered across semi-acute contusion injuries in rats. Transplanted hNSCs survived and were able to differentiate into neural lineage cells and astrocytes. The TrailmakerTM creates longitudinal cellular trails spanning multiple levels from a single-entry point. This may enhance the ability of therapeutics to promote functional relays after SCI.

Imaging of a high concentration of iron labeled cells with positive contrast in a rat knee.

The sweep imaging with Fourier transformation (SWIFT) imaging technique has been shown to provide positive contrast from diluted cell suspensions labeled with super-paramagnetic iron oxide (SPIO) in a tissue, as an alternative to T2*-weighted imaging. Here we demonstrate a variation of the SWIFT technique that yields a hyperintense signal from a concentrated cell suspension. The proposed technique provides minimal background signal from host tissue and facilitates visualization of injected cells. The proton resonance frequency and linewidth were determined for SPIO solutions of different concentrations. The original SWIFT sequence was modified and a dual saturation Gaussian shape RF pulse with ~200 Hz bandwidth was incorporated into the acquisition protocol to suppress host tissue and fat signals. This modification of the original acquisition protocol permits the detection of a hyperintense signal from grafted cells with minimal background signal from the host tissue. SPIO particles not only induce broadening of NMR line-width but also an initiate proton resonance frequency shift. This shift is linearly proportional to the concentration of the iron oxide particles and induced by the bulk magnetic susceptibility of SPIOs. The shift of the resonance frequency of iron labeled cells allowed us effectively suppress the host tissues with saturation RF pulse to improve MRI detection of grafted cells. Iron oxide particles increase the resonance frequency of water proton signal. This shift permitted us to add the tissue/fat saturation RF pulse into the original SWIFT acquisition protocol and detect distinct hyperintense signals from grafted cells with minimal background signal from the host tissue.

Investigating the Anticancer Activity of 1,2,3-Triazole Analogues of Alpha Amino Acids on Prostate Cancer Cells

Event Abstract Back to Event Investigating the Anticancer Activity of 1,2,3-Triazole Analogues of Alpha Amino Acids on Prostate Cancer Cells Raven Richardson1*, Brandon Sellers1, Karelle Aiken1 and Ria Ramoutar1 1 Georgia Southern University, United States Prostatic carcinoma is the most common malignancy amongst males and the second leading cause of cancer death in American men (ACS, 2017). In recent years, the number of studies of anticancer compounds have increased with a shift to focus on the effects of various triazole derivatives. The cynosure of the Aiken lab group has been the synthesis of various 1,2,3-triazole analogues of alpha-amino acids as potential drugs with anticancer activity. The present study is extended research into the efficacy of various derivatives of 1,2,3-triazoles on prostatic carcinoma cells, PC-3, over five days. The Aiken lab synthesized two analogues, BTS and BTSI, wher BTS contains an added hydroxyl group and BTSI is a sulfur based compound. PC-3 cells were plated on a 96-well microtiter cell culture plate with 100 μL of cells per well from a concentrated cell suspension to ensure at least 1000 cells per well for testing. After a 24-hour incubation period, 2-fold serial dilutions of the triazole derivatives with concentration ranges of 1.953 μM to 2000 μM were used to treat each well. MTT assays and SoftMax Pro software were used to analyze and quantify PC-3 cell viability after five days of analogue treatment. The first analogue from the Aiken lab group tested, BTS, showed low potential of anticancer activity due to elevated concentration levels as efficacy improved most after at least 62.5 μM of BTS was used to treat PC-3 cells. The second analogue, BTSI, is currently under investigation to determine the efficacy of anticancer activity. Acknowledgements The McNair Program (COSM) & The Department of Chemistry and Biochemistry of Georgia Southern University, Briana Burney, Dr. Timothy Tolentino, Dr. Amanda Stewart, Dominique Rivers, and Dr. Jonathan Arambula. References 1 Fu, D.-J.; Liu, J.-F.; Zhao, R.-H.; Li, J.-H.; Zhang, S.-Y.; Zhang, Y.-B. Molecules 2017, 22 (9), 1470. 2 Aiken, K.; Bunn, J.; Sutton, S.; Christianson, M.; Winder, D.; Freeman, C.; Padgett, C.; Mcmillen, C.; Ghosh, D.; Landge, S. Magnetochemistry 20 Keywords: PC-3, 1,2,3 - triazole, Anticancer properties, MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Carcinoma Conference: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference , Orlando, Florida, United States, 17 Sep - 20 Sep, 2018. Presentation Type: Poster Presentation Topic: Biochemistry Citation: Richardson R, Sellers B, Aiken K and Ramoutar R (2019). Investigating the Anticancer Activity of 1,2,3-Triazole Analogues of Alpha Amino Acids on Prostate Cancer Cells. Front. Chem. Conference Abstract: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference . doi: 10.3389/conf.fchem.2018.01.00050 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Ms. Raven Richardson, Georgia Southern University, Statesboro, United States, Rr03898@georgiasouthern.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Raven Richardson Brandon Sellers Karelle Aiken Ria Ramoutar Google Raven Richardson Brandon Sellers Karelle Aiken Ria Ramoutar Google Scholar Raven Richardson Brandon Sellers Karelle Aiken Ria Ramoutar PubMed Raven Richardson Brandon Sellers Karelle Aiken Ria Ramoutar Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Radial spreading in filter paper: A simple micromethod for the measurement of flow properties of concentrated red cell suspensions

Radial spreading in filter paper: A simple micromethod for the measurement of flow properties of concentrated red cell suspensions

Lactic acid production using free cells of bacteria and filamentous fungi and cells immobilized in polyvinyl alcohol cryogel: A comparative analysis of the characteristics of biocatalysts and processes

The results from studies and a comparative analysis of process characteristics are presented for lactic acid (LA) production from glucose, performed under batch conditions of the long-duration use of different biocatalyst samples comprising cells of Lactobacillus сasei bacteria and Rhizopus oryzae filamentous fungi immobilized in polyvinyl alcohol (PVA) cryogel or applied as concentrated suspensions. It is established that for LA production, the bacteria and fungi must be used in the form of PVA-cryogel-immobilized cells because their half-life in this form is considerably longer than that of concentrated cell suspensions. After 200 h of the batch use of the same immobilized cell samples, the amounts of accumulated LA were similar for both fungal (920 ± 5 g) and bacterial (895 ± 5 g) biocatalysts. The fungal biocatalyst, however, was characterized by a twice higher rate of substrate conversion to product (0.92 g LA per 1 g glucose) than the bacterial biocatalyst. The half-life of the immobilized fungal biocatalyst was 80 days (96 working cycles), ten times longer than that of the bacterial biocatalyst. A comparison of our data and the literature data demonstrated the promise of using fungal cells immobilized in PVA cryogel to produce LA: the process based on their use is superior to all known processes in its main indicators, i.e., the rate of LA conversion to glucose and the maximum accumulated concentration of the product.

Feasibility and Safety of Intra-arterial Pericyte Progenitor Cell Delivery Following Mannitol-Induced Transient Blood-Brain Barrier Opening in a Canine Model.

Stem cell therapy is currently being studied with a view to rescuing various neurological diseases. Such studies require not only the discovery of potent candidate cells but also the development of methods that allow optimal delivery of those candidates to the brain tissues. Given that the blood-brain barrier (BBB) precludes cells from entering the brain, the present study was designed to test whether hyperosmolar mannitol securely opens the BBB and enhances intra-arterial cell delivery. A noninjured normal canine model in which the BBB was presumed to be closed was used to evaluate the feasibility and safety of the tested protocol. Autologous adipose tissue-derived pericytes with platelet-derived growth factor receptor β positivity were utilized. Cells were administered 5 min after mannitol pretreatment using one of following techniques: (1) bolus injection of a concentrated suspension, (2) continuous infusion of a diluted suspension, or (3) bolus injection of a concentrated suspension that had been shaken by repeated syringe pumping. Animals administered a concentrated cell suspension without mannitol pretreatment served as a control group. Vital signs, blood parameters, neurologic status, and major artery patency were kept stable throughout the experiment and the 1-month posttreatment period. Although ischemic lesions were noted on magnetic resonance imaging in several mongrel dogs with concentrated cell suspension, the injection technique using repeated syringe shaking could avert this complication. The cells were detected in both ipsilateral and contralateral cortices and were more frequent at the ipsilateral and frontal locations, whereas very few cells were observed anywhere in the brain when mannitol was not preinjected. These data suggest that intra-arterial cell infusion with mannitol pretreatment is a feasible and safe therapeutic approach in stable brain diseases such as chronic stroke.

A Simple and Efficient Method for Preparing Cell Slides and Staining without Using Cytocentrifuge and Cytoclips.

Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

Genomic, proteomic, and biochemical analysis of the organohalide respiratory pathway in Desulfitobacterium dehalogenans.

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1(T) consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.

Effects of synthetic biomacromolecule addition on the flow behavior of concentrated mesenchymal cell suspensions.

In the fields of tissue engineering and regenerative medicine, many researchers and companies alike are investigating the utility of concentrated mesenchymal stem cell suspensions as therapeutic injectables, with the hope of regenerating the damaged tissue site. These cells are seldom used alone, being instead combined with synthetic biomacromolecules, such as branched poly(ethylene glycol) (PEG) polymers, in order to form cross-linked hydrogels postinjection. In this article, we present the results of a detailed experimental and analytical investigation into the impacts of a range of eight-arm PEG polymers, each presenting functional end groups, on the rheological properties of concentrated living cells of mesenchymal origin. Using two-photon confocal microscopy, we confirmed that the aggregates formed by the cells are fractal structures, the dimension of which changed with PEG polymer type addition. From these results and the observed substantial variation in rheological footprint with increasing volume fraction and different PEG polymer type, we propose a number of mechanisms driving such structural changes. Lastly, we derived a modified Krieger-Dougherty model to produce a master curve for the relative viscosity as a function of volume fraction over the range of conditions investigated (including shear stress and PEG polymer type), from which we extract the adhesion force between individual cells within these concentrated suspensions. The outcomes of this study provide new insights into the complex interactions occurring in concentrated mesenchymal cell suspensions when combined with synthetic biomacromolecules commonly used as precursors in tissue engineering hydrogels, highlighting their substantial impacts on the resultant rheological footprint.

Freeze-drying of lactic acid bacteria.

Lactic acid bacteria are of great importance for the food and biotechnology industry. They are widely used as starters for manufacturing food (e.g., yogurt, cheese, fermented meats, and vegetables) and probiotic products, as well as for green chemistry applications. Freeze-drying or lyophilization is a convenient method for preservation of bacteria. By reducing water activity to values below 0.2, it allows long-term storage and low-cost distribution at suprazero temperatures, while minimizing losses in viability and functionality. Stabilization of bacteria via freeze-drying starts with the addition of a protectant solution to the bacterial suspension. Freeze-drying includes three steps, namely, (1) freezing of the concentrated and protected cell suspension, (2) primary drying to remove ice by sublimation, and (3) secondary drying to remove unfrozen water by desorption. In this chapter we describe a method for freeze-drying of lactic acid bacteria at a pilot scale, thus allowing control of the process parameters for maximal survival and functionality recovery.

Production of free and glycosylated isoflavones in in vitro soybean (Glycine max L.) hypocotyl cell suspensions and comparison with industrial seed extracts

Glycine max is one of the major sources of phytochemicals, in particular of isoflavones, a class of phytoestrogens with ascertained beneficial effects on human health. In the present study, in vitro callus production from soybean hypocotyl seedling explants and cell suspensions were optimized. Time-courses having 20, 40 and 60 g/L of initial cell inoculum were performed to determine the concentration most suitable for isoflavone production. The amount of total polyphenols and total flavonoids as well as the antioxidant capacity of both cell and culture media fractions were measured by means of spectrophotometric methods. The levels of aglycone and glycosylated isoflavones (didzein, genistein, glycitein, didzin, genistin, glycitin), as well as of ferulic acid, vanillic acid and vanillin, were determined by HPLC–DAD. On average, 93.5 % of the total (cells plus media) isoflavones in soybean cell suspensions were detected as aglycones. Concentrated cell cultures as well as industrial soybean seed extracts were enzymatically hydrolyzed to release the aglycones and their metabolic profiles were analysed by HPLC–DAD. In contrast to cell suspensions, in undigested seed extract the aglycon form represented only 16.8 % of the total isoflavones amount. After enzymatic treatment, the antioxidant capacity increased by 30 and 33 %, respectively, in concentrated cell and seed extracts, demonstrating the presence of a larger amount of bioactive metabolites after digestion. At the present extraction conditions, soybean concentrated cell suspensions yielded 5.8-fold more total isoflavones (mostly in the free form) than seed extracts, leading to hypothesise their possible use as ingredients for cosmetic and nutraceutical applications.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

017 Cryomicroscopic analysis of damage mechanisms during freezing and thawing of a human diploid fibroblast cell line

Human diploid cell strains are useful for commercial production of viral vaccines. For example, the MRC-5 cell line (lung fibroblast) is used to produce vaccines against chickenpox and other childhood diseases. To support the safe mass production of such vaccines, it is necessary to cryopreserve standardized batches of concentrated cell suspension, thus ensuring a consistent supply of cells for manufacturing. However, the cryopreservation process can result in deleterious intracellular ice formation (IIF), or trigger other mechanisms that also lead to cell damage. Therefore, towards improving the efficiency of MRC-5 cryopreservation techniques, the IIF phenomenon was investigated in cell suspensions using a high-speed video cryomicroscopy system. Previous high-speed imaging studies have visualized the IIF process in adherent cells, but not in suspended cells. In the present study, the effect of cooling rate on IIF in MRC-5 cells in suspension was determined. High-speed videos were captured at recording rates of 540–1200 frames/s during freezing to −60 °C at various rates of cooling (1–120 °C/min). In the suspended cells, intracellular ice formation manifested as an advancing solidification front, with minimal change in cell transparency; the median duration of such IIF events was 1 ms during rapid cooling (120 °C/min), demonstrating the importance of high-speed imaging techniques to detect intracellular freezing. The cumulative incidence of IIF increased with increasing cooling rate, from 0% at 1 °C/min to 96% at 120 °C/min. The mean temperature of IIF was −21.7 °C ( n = 75) and −33.0 °C ( n = 853) during cooling at 15 °C/min and 120 °C/min, respectively. For MRC-5 cells cooled at 120 °C/min in the presence of 5% Me 2 SO, the mean IIF temperature was depressed to −46.1 °C. Whereas the majority of cells did not darken significantly during cooling, prominent cell darkening was often observed upon warming of the frozen cells. Video recordings were acquired during warming from −60 °C to 4 °C at a warming rate of 120 °C/min. In cells with intracellular ice, the probability of darkening during warming was greater than 50%, and darkening during warming appeared to be more likely for cells that had been frozen at a higher cooling rate. In slowly cooled samples, another phenomenon was observed during rapid warming: prior to melting of extracellular ice, a transient swelling was seen in many cells. Because the magnitude of these volume excursions was often large (e.g., doubling the original cell volume), it is likely that such events are damaging, and may represent a new form of slow-cooling injury. Source of funding: Funded in part by a gift from Merck & Co., Inc. to Villanova University. Conflict of interest: None declared. jens.karlsson@villanova.edu