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1.
pH and excipient profiles during formulation of highly concentrated biotherapeutics using bufferless media.
Jabra, MG, Tao, Y, Moomaw, JF, Yu, Z, Hotovec, BJ, Geng, SB, Zydney, AL
Biotechnology and bioengineering. 2020;(11):3390-3399
Abstract
Several models have been developed to describe the shifts in pH and excipient concentrations seen during diafiltration of monoclonal antibody (mAb) products accounting for both Donnan equilibrium and electroneutrality constraints. However, these models have assumed that the mAb charge is either constant or only a function of pH, assumptions that will not be valid when formulating highly concentrated mAbs using bufferless or low-buffered media due to the change in local H+ concentration at the protein surface. The objective of this study was to incorporate the effects of both pH and ionic strength on the mAb charge, through the use of a charge regulation model based on the amino acid sequence of the mAb, into an appropriate mass balance model to describe the pH and excipient profiles during diafiltration. The model involves no adjustable parameters, with the protein charge evaluated directly from the protonation/deprotonation of the ionizable amino acids accounting for the electrostatic interactions between the charged mAb and the H+ ions. Model predictions are in excellent agreement with experimental data for the pH and ion concentrations during diafiltration of a mAb and fusion protein with different isoelectric points and different formulation conditions. Model simulations are then used to obtain fundamental insights into the factors controlling the diafiltration behavior as well as guidelines for development of diafiltration processes to achieve target bufferless formulation conditions.
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2.
A gnotobiotic growth assay for Arabidopsis root microbiota reconstitution under iron limitation.
Harbort, CJ, Hashimoto, M, Inoue, H, Schulze-Lefert, P
STAR protocols. 2020;(3):100226
Abstract
We present a gnotobiotic system for microbiota reconstitution on Arabidopsis thaliana under contrasting iron availability. This system induces iron starvation in plants by providing an unavailable form, mimicking conditions in alkaline soils. Inoculation of taxonomically diverse bacteria reconstitutes plants with a synthetic microbiota, allowing observation of nutrient-dependent interactions with commensals. Experimental optimization, including media composition and preparation of seedlings and bacteria, is discussed. This system provides a framework that can be adapted to study plant-microbiota interactions in further nutritional contexts. For complete details on the use and execution of this protocol, please refer to Harbort et al. (2020).
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3.
The Effects of Prolonged Storage on ARPE-19 Cells Stored at Three Different Storage Temperatures.
Islam, R, Corraya, RM, Pasovic, L, Khan, AZ, Aass, HCD, Eidet, JR, Utheim, TP
Molecules (Basel, Switzerland). 2020;(24)
Abstract
This study aimed to investigate how prolonged storage of adult retinal pigment epithelial (ARPE-19) cell sheets affects cell metabolism, morphology, viability, and phenotype. ARPE-19 cell sheets were stored at three temperatures (4 °C, 16 °C, and 37 °C) for three weeks. Metabolic status and morphology of the cells were monitored by sampling medium and examining cells by phase-contrast microscopy, respectively, throughout the storage period. Cell viability was analyzed by flow cytometry, and phenotype was determined by epifluorescence microscopy after the storage. Lactate production and glucose consumption increased heavily, while pH dropped considerably, through storage at 37 °C compared to 4 °C and 16 °C. During storage, morphology started to deteriorate first at 4 °C, then at 37 °C, and was maintained the longest at 16 °C. Viability of the cells after three weeks of storage was best preserved at 16 °C, while cells stored at 4 °C and 37 °C had reduced viability. Dedifferentiation indicated by reduced expression of retinal pigment epithelium-specific protein 65 (RPE65), zonula occludens protein 1 (ZO-1), and occludin after three weeks of storage was noticed in all experimental groups compared to control. We conclude that storage temperature affects the metabolic status of ARPE-19 cells and that 16 °C reduces metabolic activity while protecting viability and morphology.
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4.
Clinical phase II and III studies of an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform.
Endo, M, Tanishima, M, Ibaragi, K, Hayashida, K, Fukuda, T, Tanabe, T, Naruse, T, Kino, Y, Ueda, K
Influenza and other respiratory viruses. 2020;(5):551-563
Abstract
BACKGROUND We have developed an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform (KD-295). OBJECTIVES In accordance with Japanese guidelines for development of pandemic prototype vaccines, the phase II study was conducted in a double-blind, randomized, parallel-group comparison study and the phase III study was conducted in an open-label, non-randomized, uncontrolled study. METHODS Healthy adult volunteers aged 20 - 64 years enrolled in the phase II and III studies (N = 248 and N = 369) received KD-295 intramuscularly twice with a 21-day interval. After administration, immune response and adverse events were evaluated. In the phase II study, four different vaccine formulations were compared: MA (3.75 μg hemagglutinin [HA] antigen + AS03 adjuvant system), MB (3.75 μg HA + 1/2AS03), HA (7.5 μg HA + AS03), and HB (7.5 μg HA + 1/2AS03). In the phase III study, the MA formulation was further evaluated. RESULTS In the phase II study, all four vaccine formulations were well-tolerated and no SAE related to vaccination were observed. The MA formulation was slightly more immunogenic and less reactogenic among the vaccine formulations. Therefore, the MA formulation was selected for the phase III study, and it was well-tolerated and no serious adverse drug reactions were observed. The vaccine fulfilled the three immunogenicity criteria described in the Japanese guidelines. CONCLUSIONS These data indicate that the MA formulation of KD-295 was well-tolerated and highly immunogenic and it can be considered a useful pandemic and pre-pandemic influenza vaccine.
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5.
Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix.
Weiss, OE, Hendler, RM, Baranes, D
Journal of visualized experiments : JoVE. 2020;(160)
Abstract
Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture. Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices. This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.
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6.
Regenerative effects of spring water-derived bacterial lysates on human skin fibroblast in in vitro culture: preliminary results.
Nicoletti, G, Saler, M, Tresoldi, MM, Faga, A, Benedet, M, Cristofolini, M
The Journal of international medical research. 2019;(11):5777-5786
Abstract
OBJECTIVE Previous studies have shown regenerative power of the skin with Comano (Trento, Italy) spring water and resident non-pathogenic microflora. This study investigated the action of bacterial lysates that were isolated from Comano spring water on in vitro culture of human skin fibroblasts. METHODS For this study, we selected the following four bacterial lysates: L1 (closest relative: Rudaea cellulosilytica), L2 (closest relative: Mesorhizobium erdmanii), L3 (closest relative: Herbiconiux ginsengi), and L4 (closest relative: Fictibacillus phosphorivorans). Human fibroblasts were cultured under Dulbecco’s modified Eagle’s medium (DMEM) with bacterial lysates added or DMEM (controls). Cell proliferation was evaluated by spectrophotometric absorbance analysis after the XTT-Microculture Tetrazolium Assay. RESULTS At 24 hours, cultures with L2, L3, and L4 showed a higher absorbance compared with controls. At 48 hours, cultures with L1, L2, and L3 showed slightly lower absorbance compared with controls, and culture with L4 showed a higher absorbance than in the other experimental conditions. At 72 hours, absorbance was lower in cultures with L1, L2, and L3 than in controls, and absorbance was higher in culture with L4 than in the other experimental conditions. CONCLUSIONS Our study indicates a favorable action of Comano spring water microbiota on proliferation of human skin fibroblasts.
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7.
Generation of human induced pluripotent stem cell lines from patients with selective IgA deficiency.
Arias-Fuenzalida, J, Yu, J, Du, L, Custodio, J, Notarangelo, LD, Hammarström, L, Pan-Hammarström, Q
Stem cell research. 2019;:101613
Abstract
Selective immunoglobulin-A deficiency (IgAD) is the most common primary immunodeficiency (PID) in the Western world and results in higher susceptibility to infections, autoimmune disorders and malignancies. We generated human induced pluripotent stem cell lines from two patients with selective IgAD, PHAi001 and PHAi002. Patient samples were reprogrammed using non-integrative based methods. Pluripotency of the PHAi001 and PHAi002 cell lines was confirmed by their expression of stem cell markers and capacity to differentiate into cells of the three germ layers. The PHAi001 and PHAi002 lines are a unique resource for experimental modeling of selective IgAD and associated disorders.
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8.
Cellular effects of paclitaxel-loaded iron oxide nanoparticles on breast cancer using different 2D and 3D cell culture models.
Lugert, S, Unterweger, H, Mühlberger, M, Janko, C, Draack, S, Ludwig, F, Eberbeck, D, Alexiou, C, Friedrich, RP
International journal of nanomedicine. 2019;:161-180
Abstract
BACKGROUND Magnetic drug targeting (MDT) is an effective alternative for common drug applications, which reduces the systemic drug load and maximizes the effect of, eg, chemotherapeutics at the site of interest. After the conjugation of a magnetic carrier to a chemotherapeutic agent, the intra-arterial injection into a tumor-afferent artery in the presence of an external magnetic field ensures the accumulation of the drug within the tumor tissue. MATERIALS AND METHODS In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) coated with lauric acid and human serum albumin as carriers for paclitaxel (SPIONLA-HSA-Ptx). To investigate whether this particle system is suitable for a potential treatment of cancer, we investigated its physicochemical properties by dynamic light scattering, ζ potential measurements, isoelectric point titration, infrared spectroscopy, drug release quantification, and magnetic susceptibility measurements. The cytotoxic effects were evaluated using extensive toxicological methods using flow cytometry, IncuCyte® live-cell imaging, and growth experiments on different human breast cancer cell lines in two- and three-dimensional cell cultures. CONCLUSION The data showed that next to their high magnetization capability, SPIONLA-HSA-Ptx have similar cytostatic effects on human breast cancer cells as pure paclitaxel, suggesting their usage for future MDT-based cancer therapy.
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9.
Low-cost cultivation and sporulation of alkaliphilic Bacillus sp. strain AK13 for self-healing concrete.
Hong, M, Kim, W, Park, W
Journal of microbiology and biotechnology. 2019;(12):1982-1992
Abstract
The alkaliphilic, calcium carbonate precipitating Bacillus sp. strain AK13 can be utilized in concrete for self-repairing. A statistical experimental design was used to develop an economical medium for its mass cultivation and sporulation. Two types of screening experiment were first conducted to identify substrates that promote the growth of the AK13 strain: the first followed a one-factor-at-a-time factorial design and the second a two-level full factorial design. Based on these screening experiments, barley malt powder and mixed grain powder were identified as the substrates that most effectively promoted the growth of the AK13 strain from a range of 21 agricultural products and by-products. A quadratic statistical model was then constructed using a central composite design and the concentration of the two substrates was optimized. The estimated growth and sporulation of Bacillus sp. strain AK13 in the proposed medium were 3.08 ± 0.38 × 108 and 1.25 ± 0.12 × 108 CFU/ml, respectively, which meant that the proposed low-cost medium was approximately 45 times more effective than the commercial medium in terms of the number of cultivatable bacteria per unit price. The spores were then powdered via a spray-drying process to produce a spore powder with a spore count of 2.0 ± 0.7 × 109 CFU/g. The AK13 spore powder was mixed with cement paste, yeast extract, calcium lactate, and water. The yeast extract and calcium lactate generated the highest CFU/ml for AK13 at a 0.4:0.4 ratio compared to 0.4:0.25 (the original ratio of the B4 medium) and 0.4:0.8. Twenty-eight days after the spores were mixed into the mortar, the number of vegetative cells and spores of the AK13 strain had reached 106 CFU/g within the mortar. Cracks in the mortar under 0.29 mm were healed in 14 days. Calcium carbonate precipitation was observed on the crack surface. The mortar containing the spore powder was thus concluded to be effective in terms of healing micro-cracks.
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10.
Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells.
Costantini, D, Overi, D, Casadei, L, Cardinale, V, Nevi, L, Carpino, G, Di Matteo, S, Safarikia, S, Valerio, M, Melandro, F, et al
Scientific reports. 2019;(1):5559
Abstract
Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota's Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs.