Peak Cell Density Research Articles

Effects of seasonal variations and environmental factors on phytoplankton community structure and abundance in Beibu Gulf, China

The seasonal variation in phyla within the phytoplankton community is evident in the Beibu Gulf region, with Bacillariophyta being the most diverse group, accounting for 55% of genera in spring and slightly increasing to 57.9% in autumn. In contrast, Pyrrophyta exhibits a higher number of genera and taxa in spring at 43.3%, but this percentage declines to 40% in autumn. During both seasons, the phylum Cyanophyta had a limited occurrence, making up only 1.7% and 2.1% of the total species counts, but in the spring season, it emerged as the dominant phylum with a high cell density of 95.4%, surpassing other phyla such as Aphanocapsa elachista, Guinardia flaccida, Chaetoceros laciniosus, Trichodesmium thiebautii, and Thalassiosira subtilis. Conversely, in the autumn season, cyanophyta constituted merely 4% of the overall cell density. The relative abundance of Bacillariophyta species (Bacteriastrum sp. >Chaetoceros affinis > Chaetoceros lorenzianus > Thalassionema nitzschioides > Chaetoceros curvisetus) displayed a distinct trend, reaching its peak cell density of 94% during autumn. The significance of this study lies in its findings that different types of phytoplankton, such as dinoflagellates and diatoms, have distinct growth patterns in the northern Beibu Gulf. It also highlights the importance of nutrient levels, particularly nitrate, nitrite, silicate, and soluble reactive phosphorus, in influencing the composition of phytoplankton communities during the spring season. Additionally, the study reveals a negative relationship between ammonium concentration and water temperature during both spring and autumn. These findings suggest that changes in nutrient availability during seasonal shifts play a critical role in shaping phytoplankton dynamics in marine ecosystems. The observed negative relationship between ammonium concentration and water temperature raises questions about the potential impacts of climate change on phytoplankton dynamics in the Beibu Gulf. As temperatures continue to rise, it is possible that the nutrient-rich conditions in the spring season may be altered, leading to changes in the composition and abundance of phytoplankton populations. This could potentially have cascading effects on the overall ecosystem. Therefore, studying the interplay between temperature and nutrient availability is essential for accurately assessing the future health and productivity of the Beibu Gulf's phytoplankton communities.

Local hydrodynamic in coastal system affects the coccolithophore community at a short spatial scale

Coccolithophores are an ecologically and biogeochemically important group of calcifying marine plankton, contributing globally to both the organic carbon pump and the carbonate counter pump. Understanding their distribution in today's ecosystems is crucial for both paleoenvironmental reconstruction and for predicting their fate under climate change and environmental pollution. Traditionally regarded as open ocean dwellers, more recent studies have highlighted their importance in coastal systems.Here we describe the coccolithophore community in the Catalan Sea (Northwestern Mediterranean), off the Ebro River Delta, based on an oceanographic survey conducted in autumn 2019. We identify three environmentally distinct regimes: mixed (mostly shallow), transition (upper photic layers offshore), and stratified deep waters (below 75 m). Total coccolithophore density is driven by E. huxleyi and peaks in the mixed zone where nutrient concentrations are low, but nitrite concentrations are high. We propose that peak cell densities are partly explained by a switch from nitrate to nitrite usage similarly to diatoms behavior. Species diversity peaks in deeper offshore waters due to vertical stratification. Helicosphaera carteri and Algirosphaera robusta benefit from Emiliania huxleyi decrease both in coastal and offshore environment.

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers.

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

Boosting Productivity for Advanced Biomanufacturing by Re-Using Viable Cells.

Monoclonal antibodies (mAb) have gained enormous therapeutic application during the last decade as highly efficient and flexible tools for the treatment of various diseases. Despite this success, there remain opportunities to drive down the manufacturing costs of antibody-based therapies through cost efficiency measures. To reduce production costs, novel process intensification methods based on state-of-the-art fed-batch and perfusion have been implemented during the last few years. Building on process intensification, we demonstrate the feasibility and benefits of a novel, innovative hybrid process that combines the robustness of a fed-batch operation with the benefits of a complete media exchange enabled through a fluidized bed centrifuge (FBC). In an initial small-scale FBC-mimic screening, we investigated multiple process parameters, resulting in increased cell proliferation and an elongated viability profile. Consecutively, the most productive process scenario was transferred to the 5-L scale, further optimized and compared to a standard fed-batch process. Our data show that the novel hybrid process enables significantly higher peak cell densities (163%) and an impressive increase in mAb amount of approximately 254% while utilizing the same reactor size and process duration of the standard fed-batch operation. Furthermore, our data show comparable critical quality attributes (CQAs) between the processes and reveal scale-up possibilities and no need for extensive additional process monitoring. Therefore, this novel process intensification strategy yields strong potential for transfer into future industrial manufacturing processes.

On the retinae of Glis and Graphiurus: photoreceptor and ganglion cell populations, an absence of shortwave-sensitive cones, and some other features (Rodentia: Gliridae)

The retina of the fat dormouse Glis glis was studied histologically. Opsin immunolabeling identified an unusually dense population of rod photoreceptors (ca. 600,000–780,000/mm²) and a low-density population of L cone photoreceptors containing the longwave-sensitive (LWS) cone opsin, with a shallow maximum of ca. 3,300/mm² in temporal retina and ca. 2,000–3,000/mm² in the remaining retina. Hence the cones comprise only 0.25–0.50% of the photoreceptors. There are no S cones expressing the shortwave-sensitive (SWS1) opsin that is the second cone opsin in most mammals, conveying dichromatic colour vision. We conclude that the fat dormouse is a cone monochromat. Sequencing of the tuning-relevant exon 1 of the SWS1 opsin gene revealed that the gene would have coded for a UV-sensitive visual pigment, but that it contains mutational changes making it nonfunctional. Retinal interneurons (rod bipolar cells, horizontal cells, several amacrine cell types) have rodent-typical features. NeuN-labeled presumed retinal ganglion cells have densities between ca. 4000/mm² in temporal central retina and 850–1300/mm² in peripheral retina. The peak ganglion cell density would result in an estimated visual acuity maximum of ca. 1.8 cycles/degree or ca. 33 minutes of arc. Assessment of a further Gliridae species, the African dormouse Graphiurus sp., also revealed a high rod density, low L cone density, and an absence of SWS1 opsin.

Retinal Ganglion Cell Topography and Spatial Resolution in Three Indian Pteropodid Bats

Pteropodidae is the only phytophagous bat family that predominantly depends on visual and olfactory cues for orientation and foraging. During daytime, pteropodids of different species roost in sites with varying light exposure. Pteropodids have larger eyes relative to body size than insectivorous bats. Retinal topography has been studied in less than 10% of the approximately 200 pteropodid species, a behavioural estimation of spatial resolution is available only for Pteropus giganteus, and little is known about the relationship between their roost site preference and visual ecology. We present retinal ganglion cell topographic maps and anatomical estimates of spatial resolution in three southern Indian pteropodid species with different roosting preferences. Ganglion cell densities are between 1,000 and 2,000 cells/mm² in the central retina and lower in the dorsal and ventral periphery. All three species have a temporal area in the retina with peak ganglion cell densities of 4,600–6,600 cells/mm². As a result, the foliage-roosting Cynopterus sphinx and the cave-roosting Rousettus leschenaultii have similar anatomical resolution (2.7 and 2.8 cycles/degree, respectively). The anatomical estimate for the larger tree-roosting P. giganteus (4.0 cycles/degree) is higher than the spatial resolution determined earlier in behavioural tests. Like other pteropodids and unlike other vertebrates, all three species have choroidal papillae. Based on 15 pteropodid species studied to date, we find no relationship between roost type and eye size or visual acuity. For a general understanding of the sensory ecology of pteropodids that perform key ecosystem services in the tropics, it will be essential to study additional species.

Real-time online monitoring of insect cell proliferation and baculovirus infection using digital differential holographic microscopy and machine learning.

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

Scaling‐up of an Insect Cell‐based Virus Production Process in a Novel Single‐use Bioreactor with Flexible Agitation

AbstractA novel single‐use bioreactor was recently introduced to the market that is agitated by impellers suspended on flexible ropes rather than a rigid shaft. Computational fluid dynamics (CFD) models were created and validated by particle image velocimetry (PIV) to predict the bioreactor's fluid flow and mixing. The data were then used to scale‐up a Spodoptera frugiperda, subclone 9 (Sf9) insect cell‐based production of recombinant adeno‐associated virus (AAV) from a benchtop glass bioreactor to the single‐use system with 30 L working volume. This viral vector is one of the most commonly used in gene therapies. The volumetric power input was kept constant while maintaining reasonable mixing times and shear stresses between the scales. Peak cell densities of up to 7.2 × 106 cells mL−1 and maximum virus titers of 1.7 × 1011 vg mL−1 were achieved. Similar cell growth and metabolite profiles further proved the successful process transfer between the two geometrically non‐similar bioreactor systems. The pilot bioreactor yielded between 3.3 and 4.8 × 1015 vg that, depending on the therapy, can be sufficient for the treatment of a single patient.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

The Growth of Chlorella sp. With Varying Nutrient Concentration

The aim of this study was to determine the effect of differences addition of culture medium concentrations on the growth of microalgae type Chlorella sp. Samples were obtained from the Center for Brackish Water Cultivation Fisheries (BBPBAP), Jepara. This research was conducted at the Laboratory of Nutrition and Natural Feed Sorong Marine and Fisheries Polytechnic with an experimental method using four treatments with addition of walne medium, there were A (adding only at the beginning of cultivation); treatment B (adding every day); C (adding every two days), and treatment; D (adding every three days). The cell growth performance was significantly found in treatment C with the addition of nutrients every two days with a peak cell density of Chlorella sp was 2,628,450 cells / ml. There is a need for mass culture assay to determine the level of effectiveness and efficiency.

Assessing the Viability of Commercial Media for the Mass Culture of Chaetoceros muelleri

The microalgae Chaetoceros muelleri is considered a highly nutritious feed for the cultured larvae of the tropical sea cucumber Holothuria scabra. Due to the cost of analytical grade culture media used in the production of C. muelleri, there is a need to evaluate cheap alternative commercial media to decrease the cost of producing quality live microalgal food. In this study, two different indoor batch culture systems (1 L glass bottles and 10 L plastic carboys) were used to evaluate the effectiveness of two conventional (modified F/2 and Walne’s) and one commercial (Epizyme AGP complete) microalgal culture media. Results of the 1 L glass bottle experiment showed that the peak cell density of C. muelleri in AGP (1,241 ± 116 x 104 cells ml-1) was not significantly different from the modified F/2 (1,584 ± 41 x 104 cells ml-1) and Walne’s medium (1,319 ± 162 x 104 cells ml-1) (Kruskal-Wallis test, p=0.78). Likewise, in the plastic carboy experiment, the maximum cell density of C. muelleri in Walne’s medium (750 ± 144 x 104 cells ml-1) and F/2 medium (653 ± 79 x 104 cells ml-1) were higher, but not significantly different from AGP (496 ± 184 x 104 cells ml-1) (Kruskal-Wallis test, p=0.43). The highest growth rate in the glass bottle cultures was the modified F/2 (0.38 div day-1), while AGP was the lowest (0.34 div. day-1). On the other hand, in carboy culture, AGP was higher (0.17 div.day-1) compared to modified F/2 (0.15 div. day-1) and Walne’s medium (0.13 div. day-1). The exponential growth phase was similar in the glass bottles, while in the carboy, the exponential phase was reached at a shorter time in the AGP treatment than those in the modified F/2 and Walne’s media. The findings showed that AGP medium is an adequate alternative to replace the conventional media (modified F/2 and Walne’s) during the secondary stock culture for C. muelleri. The viability of using cheaper and more readily available commercial AGP media for the indoor culture production of C. muelleri can contribute to cost-effective scaling-up of the hatchery production of quality H. scabra larvae and early juveniles.

Visual Corticocortical Inputs to Ferret Area 18.

Visual cortical areas in the adult mammalian brain are linked by a network of interareal feedforward and feedback circuits. We investigated the topography of feedback projections to ferret (Mustela putorius furo) area 18 from extrastriate areas 19, 21, and Ssy. Our objective was to characterize the anatomical organization of the extrastriate feedback pool to area 18. We also wished to determine if feedback projections to area 18 share similar features as feedback projections to area 17. We injected the tracer cholera toxin B subunit (CTb) into area 18 of adult ferrets to visualize the distribution and pattern of retrogradely labeled cells in extrastriate cortex. We find several similarities to the feedback projection to area 17: (i) Multiple visual cortical areas provide feedback to area 18: areas 19, 21, Ssy, and weaker inputs from posterior parietal and lateral temporal visual areas. Within each area a greater proportion of feedback projections arises from the infragranular than from the supragranular layers. (ii) The cortical area immediately rostral to area 18 provides the greatest proportion of total cortical feedback, and has the greatest peak density of cells providing feedback to area 18. (iii) The spacing (peak cell density and nearest neighbor distances) of cells in extrastriate cortex providing feedback to areas 17 and 18 are similar. However, peak density of feedback cells to area 18 is comparable in the supra- and infragranular layers, whereas peak density of feedback cells to area 17 is higher in the infragranular layers. Another prominent difference is that dorsal area 18 receives a cortical input that area 17 does not: from ventral cortex representing the upper visual field; this appears to be roughly 25% of the feedback input to area 18. Lastly, area 17 receives a greater proportion of cortical feedback from area 21 than from Ssy, whereas area 18 receives more feedback from Ssy than from area 21. While the organization of feedback projections from extrastriate cortex to areas 17 and 18 is broadly similar, the main difference in input topography might arise due to differences in visual field representations of the two areas.

Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation.

Monoclonal antibodies are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute that has ramifications for patient safety and physiological efficacy-one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-F-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from Days 4 to 7 in a batch process used for IgG1 production from Chinese hamster ovary cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by ∼30%. The supplements caused significant changes in gene expression by Day 4 of the cultures where 2FP addition significantly reduced fucosyltransferase 8 and nucleotide sugar transporter gene expression (by ∼2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1-6 genes (by 1.5-3-fold). These gene expression data alongside glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes.

Temperature Down-Shift Modifies Expression of UPR-/ERAD-Related Genes and Enhances Production of a Chimeric Fusion Protein in CHO Cells.

Low culture temperature enhances the cell-specific productivity of Chinese hamster ovary (CHO) cells expressing varied recombinant (r-) proteins, but the mechanisms remain unclear. Regulation of unfolded protein response (UPR) pathway genes, such as transcriptional regulatory factors and endoplasmic reticulum (ER)-resident proteins, appear to be involved in the improvements of r-protein production under low temperature conditions. The transcriptional regulation of UPR-specific targets is studied in response to decreased culture temperature in relation to production of a difficult-to-express protein. A clonally-derived CHO cell line expressing a chimeric fusion protein (human erythropoietin [hEPO] linked to a murine Fc region, hEPO-Fc) is evaluated in terms of growth, metabolism, r-protein production and UPR-/ER associated degradation (ERAD)-specific gene expression at standard (37°C) and low (32°C) temperature in batch and fed-batch systems. Low temperature decreased peak cell density, improved viability, generated cell cycle arrest in the G1 phase and enhanced hEPO-Fc expression in both batch and fed-batch cultures. A low culture temperature significantly upregulated genes encoding UPR-specific transcriptional activators (xbp1s, ddit3, and atf5) and ER-resident proteins (grp78, grp94, trib3, and ero1α), that are associated with folding and processing of proteins within the ER. Further, low culture temperature decreased expression of genes involved in ERAD (edem3, sels, herpud1, and syvn1) indicating a decreased potential for protein degradation.

Fifteen years of Pseudo-nitzschia in an Australian estuary, including the first potentially toxic P. delicatissima bloom in the southern hemisphere

In March 2018, an unprecedented bloom of a species of Pseudo-nitzschia occurred in Berowra Creek, a major tributary of the Hawkesbury River estuary, New South Wales, Australia (max cell abundance 5.7 × 106 cells L−1). Morphological and phylogenetic analysis showed the bloom was dominated by the species P. delicatissima, with toxin characterization by LC-MS confirming one of the seven strains isolated produced domoic acid (DA), at a concentration of 0.34 pg DA per cell.In response to this unprecedented bloom, we used a fifteen year series of phytoplankton and physico-chemical data to relate environmental factors to spatial and temporal variability of Pseudo-nitzschia species throughout the estuary. Species in the Pseudo-nitzschia delicatissima group consistently dominated all locations monitored throughout the estuary, with peak cell densities observed in the austral autumn (lowest in winter). Overall, Generalised Additive Models (GAMs) suggested that high cell concentrations of Pseudo-nitzschia were linked to an increase in soluble reactive phosphorus and a decrease in total nitrogen at all sites, with up to 55% of the deviance explained. Twenty species of Pseudo-nitzschia have now been reported in Australian coastal waters, four of which are confirmed DA producers. We hypothesise that increased urbanization, nutrient input and warmer waters could combine to increase Pseudo-nitzschia blooms in south-eastern Australia.

Spatial and Temporal Resolution of the Visual System of the Anna's Hummingbird (Calypte anna) Relative to Other Birds.

Hummingbirds are an emerging model for studies of the visual guidance of flight. However, basic properties of their visual systems, such as spatial and temporal visual resolution, have not been characterized. We measured both the spatial and temporal visual resolution of Anna's hummingbirds using behavioral experiments and anatomical estimates. Spatial visual resolution was determined behaviorally using the optocollic reflex and anatomically using peak retinal ganglion cell densities from retinal whole mounts and eye size. Anna's hummingbirds have a spatial visual resolution of 5-6 cycles per degree when measured behaviorally, which matches anatomical estimates (fovea: cycles per degree; area temporalis: cycles per degree; and whole eye average: ). To determine temporal visual resolution, we used an operant conditioning paradigm wherein hummingbirds were trained to use a flickering light to find a food reward. The limits of temporal visual resolution were estimated as 70-80 Hz. To compare Anna's hummingbirds with other bird species, we used a phylogenetically controlled analysis of previously published data on avian visual resolutions and body size. Our measurements for Anna's hummingbird vision fall close to and below predictions based on body size for spatial visual resolution and temporal visual resolution, respectively. These results indicate that the enhanced flight performance and foraging behaviors of hummingbirds do not require enhanced spatial or temporal visual resolution. This finding is important for interpreting flight control studies and contributes to a growing understanding of avian vision.

Systematic evaluation of high-throughput scale-down models for single-use bioreactors (SUB) using volumetric gas flow rate as the criterion

Developing representative bioreactor scale-down models is a critical part in process development and characterization. Here we present a systematic analysis of high-throughput scale-down models (ambr®250, 250 mL) developed for 500 L or 2000 L single-use bioreactors using engineering approaches. A main scaling criterion, vvm (volume of gas per volume of liquid per minute), was studied for scale-down model development after analyzing bioreactor hydrodynamic environment and gas transfer characteristics. Two different processes with distinguished peak cell densities (12–14 vs. 20–25 × 106 cells/mL) for a monoclonal antibody (mAb) were evaluated. We demonstrated that scaling-down using similar vvm as the criterion is feasible in reproducing large-scale gas transfer characteristics for both cell densities, though more challenging in high-density conditions. Furthermore, a range of pCO2 levels could be generated through vvm modulations. The same vvm approach was successfully applied to two other mAbs. Practical aspects of ambr®250 operations, especially the impact of background air sparge rate and antifoam addition on dissolved oxygen control and gas transfer, were also discussed. Understanding and managing gas transfer gaps between small- and large-scale bioreactors is expected to streamline scale-up effort, and equally importantly scale-down activities for process characterization.

Development of a scalable agitation suspension culture differentiation platform for generating erythroid cells from O-negative human induced pluripotent stem cells

Background & Aim Universal red blood cells (RBCs) derived from differentiation of O-negative (neg) human induced pluripotent stem cells (hiPSCs) can potentially supplement the emergency transfusion needs of the healthcare industry. Given that each unit of blood requires 1 trillion RBCs, there is a need to develop efficient bioprocesses that could allow for large-scale differentiation and generation of very high numbers of RBCs. While many groups have described means to differentiate hiPSCs into erythroid cells, most of these have not yet been demonstrated to be amenable for scale-up. Methods, Results & Conclusion Using multifactorial process optimization, we have developed a continuous suspension agitation culture platform for differentiating hiPSCs-microcarrier aggregates towards erythroid cells. We have efficiently scaled-up the process starting from 5 ml ultra-low attachment 6 well plates to 125 ml spinner culture flasks. The best performing hiPSC line was efficiently differentiated, generating close to 1.5 billion erythroblasts in 100 ml culture volumes with peak cell densities of erythroblasts approaching 1.5 × 107 cells/ml. By flow cytometry, as much as 60% of hiPSC derived erythroblasts were shown to be enucleated following an optimized scalable co-culture system that we developed. Functional (oxygen binding, hemoglobin characterization and membrane deformability) and transcriptomics evaluation show minimal differences between hiPSC derived RBCs as compared to cord-blood and adult derived RBCs, making them potentially useful for future transfusion applications. The agitation suspension culture differentiation bioprocess developed by our group could serve as a key platform for future scale-up of hiPSCs differentiation towards RBCs and other hematopoietic lineages in controlled bioreactors.

Escape band in Escherichia coli chemotaxis in opposing attractant and nutrient gradients

It is commonly believed that bacterial chemotaxis helps cells find food. However, not all attractants are nutrients, and not all nutrients are strong attractants. Here, by using microfluidic experiments, we studied Escherichia coli chemotaxis behavior in the presence of a strong chemoattractant (e.g., aspartate or methylaspartate) gradient and an opposing gradient of diluted tryptone broth (TB) growth medium. Our experiments showed that cells initially accumulate near the strong attractant source. However, after the peak cell density (h) reaches a critical value [Formula: see text], the cells form a "escape band" (EB) that moves toward the chemotactically weaker but metabolically richer nutrient source. By using various mutant strains and varying experimental conditions, we showed that the competition between Tap and Tar receptors is the key molecular mechanism underlying the formation of the escape band. A mathematical model combining chemotaxis signaling and cell growth was developed to explain the experiments quantitatively. The model also predicted that the width w and the peak position [Formula: see text] of EB satisfy two scaling relations: [Formula: see text] and [Formula: see text], where l is the channel length. Both scaling relations were verified by experiments. Our study shows that the combination of nutrient consumption, population growth, and chemotaxis with multiple receptors allows cells to search for optimal growth condition in complex environments with conflicting sources.

Effects of Formic and Levulinic Acids on Butyric Acid Synthesis by Clostridium tyrobutyricum in Xylose Media

Abstract. Weak acids released during hydrolysis of lignocellulosic biomass are potential inhibitors of microorganism fermentation. In this study, the effects of formic and levulinic acids on butyric acid synthesis by were investigated. With the addition of 1.2 to 4.8 g L-1 of formic acid, increased lag time, decreased cell density, and lower butyric acid productivity were observed. Up to 15% and 56% reduction in peak cell density and butyric acid productivity, respectively, were caused by formic acid addition, whereas there was no significant difference in butyric acid yield between the control and formic acid treated groups (except for the 2.4 g formic acid L-1 treatment). Levulinic acid did not show any notable effects on within the investigated concentration range (0 to 4.8 g L-1). Overall, showed strong tolerance of both formic and levulinic acids, but neither of these acids could be metabolized by the microbe.HighlightsFormic acid had dosage-dependent inhibitory effects on C. tyrobutyricum.Levulinic acid had no effects on cell growth or butyrate synthesis.Neither formic acid nor levulinic acid was metabolized by C. tyrobutyricum.C. tyrobutyricum showed strong tolerance to formic acid and levulinic acid. Keywords: Butyric acid, Clostridium tyrobutyricum, Formic acid, Levulinic acid, Lignocellulosic hydrolysate, Xylose.