Effect Of Cell Density Research Articles

Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

Immune quorum sensing dictates IFN-I response dynamics in human plasmacytoid dendritic cells.

Type I interferons (IFN-Is) are key in fighting viral infections, but also serve major roles beyond antiviral immunity. Crucial is the tight regulation of IFN-I responses, while excessive levels are harmful to the cells. In essence, immune responses are generated by single cells making their own decisions, which are based on the signals they perceive. Additionally, immune cells must anticipate the future state of their environment, thereby weighing the costs and benefits of each possible outcome, in the presence of other potentially competitive decision makers (i.e., IFN-I producing cells). A rather new cellular communication mechanism called quorum sensing describes the effect of cell density on cellular secretory behaviors, which fits well with matching the right amount of IFN-Is produced to fight an infection. More competitive decision makers must contribute relatively less and vice versa. Intrigued by this concept, we assessed the effects of immune quorum sensing in pDCs, specialized immune cells known for their ability to mass produce IFN-Is. Using conventional microwell assays and droplet-based microfluidics assays, we were able the characterize the effect of quorum sensing in human primary immune cells in vitro. These insights open new avenues to manipulate IFN-I response dynamics in pathological conditions affected by aberrant IFN-I signaling.

Amelioration of L-methionine production by Alcaligenes faecalis ATCXT3624: Empirical optimization of culture conditions

Background: L-methionine production at the industrial scale suffers from various drawbacks, including the production and separation of the L-enantiomer of the amino acid methionine by chemical production methods. This strain exhibiting resilience to feedback inhibition by L-methionine provided a promising avenue for enhanced production. The present study has tried to uplift the production rate of the amino acid by a developed L-methionine-resistant strain Alcaligenes faecalis ATCXT 3624 through surpassing feedback inhibition. Methods: Empirical optimization of all fermentation conditions was studied by observing the effects of initial pH, temperature, inoculum age, medium volume, cell density, and different micronutrients, which enhanced L-methionine production. In addition, efforts were made to find suitable nitrogen and carbon sources. Results: Eventually, 23.8±0.22 mg/mL of L-methionine was obtained under optimized fermentation conditions, which is significantly (p<0.05) higher than that produced before optimized fermentation conditions. Conclusion: Eventually, it can be assumed that the strain effectively produced the amino acid at an enhanced rate under optimized conditions.

Effects of process intensification on homogeneity of an IgG1:κ monoclonal antibody during perfusion culture

The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products’ efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs).Key points• Molecular integrity may suffer with increasing process intensity.• Galactosylated and sialylated N-glycans may decrease.• Perfusion culture appears to maintain protein charge structure.

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compounds in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV-depletion from the extracellular medium restored transfection efficiency in conditions suffering from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UGCG enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV-depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12x106 cells/mL. These results suggest that the interaction between polyplexes and extracelluar and intracellular vesicles plays a crutial role in the CDE, providing insights for the development of strategies to mitigate its impact.

Direct measurement of dynamic attractant gradients reveals breakdown of the Patlak-Keller-Segel chemotaxis model.

Chemotactic bacteria not only navigate chemical gradients, but also shape their environments by consuming and secreting attractants. Investigating how these processes influence the dynamics of bacterial populations has been challenging because of a lack of experimental methods for measuring spatial profiles of chemoattractants in real time. Here, we use a fluorescent sensor for aspartate to directly measure bacterially generated chemoattractant gradients during collective migration. Our measurements show that the standard Patlak-Keller-Segel model for collective chemotactic bacterial migration breaks down at high cell densities. To address this, we propose modifications to the model that consider the impact of cell density on bacterial chemotaxis and attractant consumption. With these changes, the model explains our experimental data across all cell densities, offering insight into chemotactic dynamics. Our findings highlight the significance of considering cell density effects on bacterial behavior, and the potential for fluorescent metabolite sensors to shed light on the complex emergent dynamics of bacterial communities.

Effect of High Cell Density to Lipid Content Microalgae Chlorella vulgaris on Photoautotrophic Cultivation

Chlorella vulgaris is one of microalgae types that has essential ingredients beneficial tohumans, such as being a source of good lipid. Chlorella sp. have high lipid content, up to30%, under autotrophic conditions considered as a promising candidate for commercial lipidproduction due to its fast growth and easy cultivation. High cell density sedimentation can beinvestigated as a starving method in cultivation to make microalgae conditions less favorablein obtaining nutrient to lipid accumulation. Starving is one of the stress conditions carried outwith the aim of reducing nutrients in the microalgae cultivation process. The aim of this studywas to determine the effect of high cell density on increasing lipid content in Chlorellavulgaris. The results of this study showed that high cell density method affected theproductivity of Chlorella vulgaris biomass. The data obtained were analyzed using ANOVAα=0.05. The highest productivity and lipid content was obtained from the control sample witha biomass value of 0.36±0.03 g/l and a lipid content of 56.2±4.6% (P<0.05). The conclusionof this study is high cell density may not increase the production of lipid content fromChlorella vulgaris

Effect of Cell Density of a Methacrylic Acid-Based Hydrogel Implant on Embedded Islet Function and Viability.

Subcutaneous delivery of islets in a methacrylic acid-based hydrogel may offer a functional cure for type 1 diabetes. Here we show in mice that the hydrogel is able to provide sufficient vasculature to support islet function and viability, when islets are used at a low islet volume fraction (i.e., cell density). The Krogh cylinder model was used to mathematically estimate the effect of implant volume, for a fixed islet dose (600 islet equivalents [IEQ]), on the minimum vessel density required to maintain sufficient pO2 within the graft. Modeling suggested that 200 μL implants would have low enough islet densities and enough vessels to have islets remain viable, but that 50 μL implants would not; this was confirmed experimentally through measurement of glucose level in streptozotocin-induced diabetic severe combined immunodeficiency disease (SCID/bg) mice, comparing 200 and 50 μL implants, both with 600 IEQ. Vessel densities were ∼20-30 vessels/mm2 independent of implant volume and vessels were sufficient to increase subcutaneous oxygen tension, as measured with microcapsules containing oxygen sensitive material (a platinum [Pt] porphyrin); both these results were determined without cells. These results are useful in thinking about the scale-up of this system to humans: to maintain a low islet density (∼0.5%), many more islets will require attention to the subcutaneous implant configuration to satisfy the oxygen needs of the cells.

Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations.

Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.

Production of adeno-associated viral vector serotype 6 by triple transfection of suspension HEK293 cells at higher cell densities.

In recent years, the use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has increased, leading to a rise in the amount of AAV vectors required during pre-clinical and clinical trials. AAV serotype 6 (AAV6) has been found to be efficient in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG), making large-scale production of AAV6 necessary. Suspension cell-based platforms are currently limited to low cell density productions due to the widely reported cell density effect (CDE), which results in diminished production at high cell densities and decreased cell-specific productivity. This limitation hinders the potential of the suspension cell-based production process to increase yields. In this study, we investigated the improvement of the production of AAV6 at higher cell densities by transiently transfecting HEK293SF cells. The results showed that when the plasmid DNA was provided on a cell basis, the production could be carried out at medium cell density (MCD, 4 × 106 cells/mL) resulting in titers above 1010 VG/mL. No detrimental effects on cell-specific virus yield or cell-specific functional titer were observed at MCD production. Furthermore, while medium supplementation alleviated the CDE in terms of VG/cell at high cell density (HCD, 10 × 106 cells/mL) productions, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations for AAV production in HCD processes. The MCD production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing. This article is protected by copyright. All rights reserved.

Relationship between oxygen partial pressure and inhibition of cell aggregation of human adipose tissue-derived mesenchymal stem cells stored in cell preservation solutions

IntroductionThis study investigated the storage conditions under which cell aggregation occurs and the conditions that inhibit cell aggregation when human adipose tissue-derived mesenchymal stem cells (hADSCs) are stored in lactated Ringer’s solution (LR) supplemented with 3% trehalose and 5% dextran 40 (LR-3T-5D). MethodsWe first examined the effects of storage temperature and time on the aggregation and viability of hADSCs stored in LR and LR-3T-5D. The cells were stored at 5 °C or 25 °C for various times up to 24 h. We then evaluated the effects of storage volume (250-2,000 μL), cell density (2.5-20 × 105 cells/mL), and nitrogen gas replacement on aggregation, oxygen partial pressure (pO2), and viability of hADSCs stored for 24 h at 25 °C in LR-3T-5D. ResultsWhen stored in LR-3T-5D, viability did not change under either condition compared with pre-storage, but the cell aggregation rate increased significantly with storage at 25 °C for 24 h (p<0.001). In LR, the aggregation rate did not change under either condition, but cell viability decreased significantly after 24 h at both 5 °C and 25 °C (p < 0.05). The cell aggregation rates and pO2 tended to decrease with increasing solution volume and cell density. Nitrogen gas replacement significantly decreased the cell aggregation rate and pO2 (p < 0.05). However, there were no differences in viability among cells stored under conditions of different storage volumes, densities, and nitrogen gas replacement. ConclusionsAggregation of cells after storage at 25 °C in LR-3T-5D may be suppressed by increasing the storage volume and cell density as well as by incorporating nitrogen replacement, which lowers the pO2 in the solution.

Clay-bacteria interaction: Effect of bacterial cell density on sedimentation behavior and fabric map of kaolinite clay

Clay-bacteria interactions are ubiquitous and the presence of bacteria has a pronounced effect on the mechanical, chemical, and electrical behaviors of clays. However, the role of microscale clay-bacteria interactions to the macro-scale engineering behaviors and properties of clays remains poorly understood. This study presents changes in the microstructure of kaolinite in association with bacteria and the resulting sedimentation behavior of bacterium-associated kaolin suspensions. Sedimentation tests were performed using suspensions composed of kaolin and model bacteria Shewanella oneidensis MR-1 while varying the fluid pH and cell density. The results reveal that kaolinite presents face-to-face aggregated microstructures with the presence of bacteria, attributable to sharing and/or exchange of cations between kaolinite and bacterial cells. Meanwhile, at a pH value lower than the isoelectric point of the kaolinite edge surfaces, the clay-bacteria association appears less pronounced comparing to the higher pH, owing to the competition between negative (kaolinite faces and bacteria surfaces) over positive (kaolinite edges) charges. The fabric change caused by kaolinite-bacteria association accelerates kaolin settling and reduces the final void ratios. A fabric map for bacterium-asssociated kaolinite is suggested based on the sedimentation behaviors and scanning electron microscopy observations. This study advances the understanding of particle-level clay-bacteria interactions and the sedimentation behavior of clays in suspensions with vigorous microbial activities.

Evaluation of the Relationship between Bacteria Concentration and the Strength and Durability of Self-compacting Concrete Incorporating Sporosarcina pasteurii

This research was carried out to evaluate the relationship between the incorporation of calcite precipitation bacteria, sporosarcina pasteurii using calcium lactate as nutrient source and the properties of calcined clay and limestone powder blended self-compacting concrete. Ten mixes were designed and designated S0 to S9 with S0 the control (without bacteria and nutrient) and S1 to S9 at varying bacteria and calcium lactate concentrations and the effect of the bacteria cell density and calcium lactate concentration on the compressive strength, sorptivity and tensile strength with age evaluated using experimental program and statistical packages (ANOVA and post hoc tests). The result of both the experimental program and statistical evaluation shows that the incorporation of sporosarcina pasteurii and calcium lactate as nutrient had a positive impact on the properties of the ternary blended self-compacting concrete.

Vibrio cholerae Invasion Dynamics of the Chironomid Host Are Strongly Influenced by Aquatic Cell Density and Can Vary by Strain.

Cholera has been a human scourge since the early 1800s and remains a global public health challenge, caused by the toxigenic strains of the bacterium Vibrio cholerae. In its aquatic reservoirs, V. cholerae has been shown to live in association with various arthropod hosts, including the chironomids, a diverse insect family commonly found in wet and semiwet habitats. The association between V. cholerae and chironomids may shield the bacterium from environmental stressors and amplify its dissemination. However, the interaction dynamics between V. cholerae and chironomids remain largely unknown. In this study, we developed freshwater microcosms with chironomid larvae to test the effects of cell density and strain on V. cholerae-chironomid interactions. Our results show that chironomid larvae can be exposed to V. cholerae up to a high inoculation dose (109 cells/mL) without observable detrimental effects. Meanwhile, interstrain variability in host invasion, including prevalence, bacterial load, and effects on host survival, was highly cell density-dependent. Microbiome analysis of the chironomid samples by 16S rRNA gene amplicon sequencing revealed a general effect of V. cholerae exposure on microbiome species evenness. Taken together, our results provide novel insights into V. cholerae invasion dynamics of the chironomid larvae with respect to various doses and strains. The findings suggest that aquatic cell density is a crucial driver of V. cholerae invasion success in chironomid larvae and pave the way for future work examining the effects of a broader dose range and environmental variables (e.g., temperature) on V. cholerae-chironomid interactions. IMPORTANCE Vibrio cholerae is the causative agent of cholera, a significant diarrheal disease affecting millions of people worldwide. Increasing evidence suggests that the environmental facets of the V. cholerae life cycle involve symbiotic associations with aquatic arthropods, which may facilitate its environmental persistence and dissemination. However, the dynamics of interactions between V. cholerae and aquatic arthropods remain unexplored. This study capitalized on using freshwater microcosms with chironomid larvae to investigate the effects of bacterial cell density and strain on V. cholerae-chironomid interactions. Our results suggest that aquatic cell density is the primary determinant of V. cholerae invasion success in chironomid larvae, while interstrain variability in invasion outcomes can be observed under specific cell density conditions. We also determined that V. cholerae exposure generally reduces species evenness of the chironomid-associated microbiome. Collectively, these findings provide novel insights into V. cholerae-arthropod interactions using a newly developed experimental host system.

Strain, cell density, and nutrient condition affect patterns of diurnal vertical migration and superoxide production in a red-tide alga.

A red tide occurs when cell densities of autotrophic microalgae and some heterotrophic protists increase dramatically and thereby change the color of the sea. Red tides sometimes have negative impacts on human activities, such as fisheries and tourism. Most red-tide flagellates display diurnal vertical migration (DVM) in which cells normally migrate upward during the day and downward at night. This behavior promotes active growth, due to the effective acquisition of nutrients and light, as well as population density increase and cell aggregation. However, the factors and their interactions influencing DVM remain to be clarified, such that no algorithm exists that can precisely simulate the DVM pattern and the development of a red tide in the field. Chattonella marina complex (hereafter Chattonella) is a representative microalga of harmful red tides and some previous studies has suggested that Chattonella's DVM plays important roles in development of a red tide. Chattonella can produce a large amount of superoxide (•O2 -), which is responsible for the regulation of various physiological processes as well as its toxicity against microorganisms and animals. In the present study, we examined the effects of strain, growth phase, cell density, and nutrient deficiency on the pattern of DVM. In addition, we also measured the •O2 - level in most experiments to assess the relationship between DVM and •O2 - production. Some strains displayed clear DVM, whereas others aggregated at the surface all day in a fixed condition. Strains' DVM patterns did not show a relationship with •O2 - production. Moreover, the DVM became less clear at high cell density and in nitrogen- or phosphorus-depleted conditions. Although a previous study reported that the •O2 - production rate increased during the light period and decreased during the dark period, regardless of cell density, the diurnal pattern of •O2 - became less clear at a higher cell density in a Chattonella strain used in the present study. Our findings indicate that DVM and •O2 - production by a Chattonella population composed of various strains can change across developmental phases and environmental conditions. This characteristic may produce adaptability in species and increase the chances of a massive population increase.

Optimization of precision nanofiber micelleplexes for DNA delivery.

As nucleic acid (NA) technologies continue to revolutionize medicine, new delivery vehicles are needed to effectively transport NA cargoes into cells. Uniform and length-tunable nanofiber micelleplexes have recently shown promise as versatile polymeric delivery vehicles for plasmid DNA, however the effects of several key parameters on micelleplex transfection and stability remain unknown. In this work, we compare poly(fluorenetrimethylenecarbonate)-b-poly(2-(dimethylamino)ethyl methacrylate) (PFTMC-b-PDMAEMA) nanofiber micelleplexes to nanosphere micelleplexes and PDMAEMA polyplexes, examining the effects of complexation buffer, the temporal and serum stability of nanofiber micelleplexes, as well as the effects of cell density, cell type, and polymer DPn upon transfection efficiency and cell viability. These studies are vital for understanding the formation and biological activity of micelleplexes in more detail and should inform the future design of more advanced polymeric NA delivery systems.

Collective organization from cellular disorder

In biology, form follows function. Spatial organization of cells within tissues is a critical aspect of this form. Injuries, drugs, and genetic mutations that perturb cellular organization disrupt tissue and organ functions. Indeed, the high degree of order and organization in tissues like heart muscle and bone is one impressive aspect of histological images. The influence of entropic crowding in cell monolayersIppolito et al.Biophysical JournalAugust 23, 2022In BriefCell-cell interaction dictates cell morphology and organization, which play a crucial role in the micro-architecture of tissues that guides their biological and mechanical functioning. Here, we investigate the effect of cell density on the responses of cells seeded on flat substrates using a novel statistical thermodynamics framework. The framework recognizes the existence of nonthermal fluctuations in cellular response and thereby naturally captures entropic interactions between cells in monolayers. Full-Text PDF Open Access

Effect of varying cell densities on the rheological properties of the bioink

The rheological characterization of any biopolymer solution is crucial for evaluating the overall printability or injectability of the hydrogel. However, the effect of cells in the cell-laden hydrogel's rheological profile is often ignored. As a result, there is a significant difference in the predicted and experimental outcome in the structural stability of the construct as well as on the cell viability, proliferation, and differentiation potential of the embedded cells. Our present study has addressed the effect of different cell densities (0.1 million cells/ml, 0.5 million cells/ml, 1 million cells/ml and 2 million cells/ml) of TVA-BMSCs on the flow property, modulus behaviour, gelation kinetics and printability of our proprietary silk fibroin-gelatin (5SF-6G) bioink. The cell-laden hydrogels demonstrated a characteristic shear thinning behaviour (low initial viscosity), low storage modulus and increased gelation time when compared to the acellular 5SF-6G hydrogel. The printability analysis also portrayed a square pore geometry with low spreading ratio in 1 million cells/ml encapsulated 5SF-6G hydrogel comparable to the acellular hydrogel. We postulated that incorporation of cells in the bioink interfered with the gelation mechanism of the mushroom tyrosinase in the 5SF-6G bioink by masking the active sites. Additionally, the mechanistic crosstalk between the cell-surface integrins with the cell-attachment motifs of the biomaterial alters the cellular biomechanics of the cell that in-turn profoundly impacts the rheological properties of the polymer blend. Therefore, cell density of 1 million cells/ml was considered the best fit for extrusion-based 3D bioprinting owing to its optimum rheological traits and printability index akin the acellular hydrogel.

The influence of entropic crowding in cell monolayers

Cell-cell interaction dictates cell morphology and organization, which play a crucial role in the micro-architecture of tissues that guides their biological and mechanical functioning. Here, we investigate the effect of cell density on the responses of cells seeded on flat substrates using a novel statistical thermodynamics framework. The framework recognizes the existence of nonthermal fluctuations in cellular response and thereby naturally captures entropic interactions between cells in monolayers. In line with observations, the model predicts that cell area and elongation decrease with increasing cell seeding density—both are a direct outcome of the fluctuating nature of the cellular response that gives rise to enhanced cell-cell interactions with increasing cell crowding. The modeling framework also predicts the increase in cell alignment with increasing cell density: this cellular ordering is also due to enhanced entropic interactions and is akin to nematic ordering in liquid crystals. Our simulations provide physical insights that suggest that entropic cell-cell interactions play a crucial role in governing the responses of cell monolayers.

Effect of cell density on decrease in hydraulic conductivity by microbial calcite precipitation

The effect of number of cells deposited on decrease in hydraulic conductivity of porous media using CaCO3 precipitation induced by Sporosarcina pasteurii (ATCC 11,859) was examined in columns packed with glass beads in the range of 0.25 mm and 3 mm in diameter. After resting Sporosarcina pasteurii cells were introduced into the columns, a precipitation solution, which consisted of 500 mM CaCl2 and 500 mM urea, was introduced under continuous flow conditions. It was shown that hydraulic conductivity was decreased by formation of microbially induced CaCO3 precipitation from between 8.37 * 10−1 and 6.73 * 10−2 cm/s to between 3.69 * 10−1 and 1.01 * 10−2 cm/s. The lowest hydraulic conductivity was achieved in porous medium consisting of the smallest glass beads (0.25 mm in diameter) using the highest density of cell suspension (OD600 2.25). The number of the deposited cells differed depending on the glass bead size of the columns. According to the experiments, 7 * 10−9 g CaCO3 was produced by a single resting cell. The urease activity, which led CaCO3 precipitation, depended on presence of high number of cells deposited in the column because the nutrients were not included in the precipitation solution and consequently, the amount of CaCO3 precipitated was proportional with the cell number in the column. A mathematical model was also developed to investigate the experimental results, and statistical analysis was also performed.