High Cell Density Research Articles

Pilot-scale process development for recombinant adeno-associated virus (rAAV) production based on high-density Sf9 cell culture

BackgroundIn recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production.MethodsIn this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established.ResultsHere, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 106 cells/mL and the rAAV titre increasing to 20 × 1011 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level.ConclusionsThis successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production.

Multivariate Environmental Factors and Seasonal Spatial Dynamics Affecting the Phytoplankton Community in Yazhou Bay, South China Sea

This study investigated phytoplankton and water environmental factors in Yazhou Bay, South China Sea, during the winter, spring, and summer of 2023. It examined phytoplankton community structure, subgroup heterogeneity, and key environmental drivers. Phytoplankton abundance ranged from 0.08 to 14.30 × 10⁴ cells·L−1, with high concentrations in estuary and nearshore zones. In summer, currents carry phytoplankton offshore, with stratification leading to high sedimentation in southern offshore waters. RDA results indicated that in winter and spring, inorganic nitrogen mainly influences phytoplankton distribution, while silicate is the primary factor in summer. Although seasonal differences in total phytoplankton abundance are minimal, significant horizontal and vertical distribution variations exist. Diverse preferences of different phytoplankton species for temperature, salinity, nitrogen, and phosphorus result in high species diversity. The Shannon–Wiener diversity index (H′) averages 3.96 ± 0.09, and the Pielou evenness index (J) averages 0.82 ± 0.01. Dominant species include Pseudo-nitzschia pungens, Skeletonema costatum, and Rhizosolenia sinica. Influenced by external oceanic water masses, estuary input, and islands, phytoplankton subgroups show regional and seasonal variations. Despite recorded harmful algal blooms (HABs) in adjacent waters, Yazhou Bay’s high biodiversity and low cell density suggest a low HAB risk, though future risks due to climate change and human activities remain.

Experimental determination of maximum shear stress in Mobius® Breez perfusion microbioreactors and comparative analysis with stirred tank bioreactors

Perfusion processes have experienced increased popularity in recent years due to their ability to sustain high cell densities and productivities in biopharmaceutical production, offering advantages over traditional batch and fed-batch cultivation methods. The Mobius® Breez microbioreactor significantly reduces experimental effort by downsizing the classical volume of perfusion bioreactors to the mL range and thus represents a valuable tool for process development. However, miniaturization has raised questions regarding comparability with traditional bioreactors in terms of the physical environment, such as hydrodynamic shear stress. Therefore, the maximum hydrodynamic shear stress, cultivation performance, and membrane-wall contact were evaluated to elucidate the system's behavior. Findings reveal two distinct operational conditions, distinguished by the presence or absence of membrane-wall contact, resulting in varying levels of hydrodynamic stress. Conditions lacking membrane contact demonstrate stress levels within safe operating thresholds for CHO cells, while those involving membrane contact exceed these thresholds, potentially leading to cell damage. Through the identification of critical frequencies of membrane motion, this study offers insights for optimizing microbioreactor operation and enhancing overall bioprocess efficiency.

Optimizing Fermentation Strategies for Enhanced Tryptophan Production in Escherichia coli: Integrating Genetic and Environmental Controls for Industrial Applications

Tryptophan is an essential aromatic amino acid widely used in the pharmaceutical, agricultural, and feed industries. Microbial fermentation, mainly using Escherichia coli, has become the preferred method for its production due to sustainability and lower costs. Optimizing tryptophan production requires careful control of various fermentation parameters, including nutrients, pH, temperature, and dissolved oxygen (DO) levels. Glucose, as the primary carbon source, must be fed at controlled rates to avoid metabolic overflow, which leads to by-product accumulation and reduced production efficiency. Nitrogen sources, both organic (such as yeast extract) and inorganic (like ammonium), influence biomass growth and tryptophan yield, with ammonium levels requiring careful regulation to avoid toxic accumulation. Phosphate enhances growth but can lead to by-product formation if used excessively. pH is another critical factor, with an optimal range between 6.5 and 7.2, where enzyme activity is maximized. Temperature control promotes growth and production, particularly between 30 °C and 37 °C. High DO levels increase tryptophan titers by boosting the pentose phosphate pathway and reducing by-products like acetate. Furthermore, surfactants and supplements such as betaine monohydrate and citrate help alleviate osmotic stress and enhance precursor availability, improving production efficiency. Careful manipulation of these parameters allows for high-density cell cultures and significant tryptophan accumulation, making microbial fermentation competitive for large-scale production.

Systematic insights into cell density-dependent transcriptional responses upon medium replacements

Understanding the molecular mechanisms governing transfection efficiency and particle secretion in high cell density cultures is critical to overcome the cell density effect upon transient gene expression. The effect of different conditioned media in HEK293 transcriptome at low and high cell density is explored. A systematic pair-wise comparative study was performed to shed light on the effect on previous phenotypical characteristics of different media conditions: fresh, exhausted and media depleted from extracellular vesicles (EVs) as well as associated proteins and RNAs. The obtained results suggest that restorative effects observed in transfection efficiency when employing EV-depleted media may arise predominantly from physicochemical alterations rather than cellular processes. A significant downregulation of genes associated with nucleocytoplasmic transport for the conditions involving the use of exhausted or EV-depleted media was observed. Moreover, upregulation of histone-related genes in EV-depleted media suggest a role for histone signaling in response to cellular stress or growth limitations, thereby highlighting the potential of manipulating histone levels as a promising strategy to enhance transient transfection. It was also corroborated that the accumulation of extracellular matrix proteins upon cell growth may inhibit transfection by an already-known competitive effect between cell membrane-bound and free proteoglycans. Proteomic characterization of EV-depleted media further unveiled enrichment of pathways associated with infection response and double-strand DNA breaks, suggesting that HEK293 cells undergo an induced infection-like state that disrupts cellular processes. Importantly, the study reveals that EV-depleted media stimulates virion release pathways underscoring the complex interplay between extracellular vesicles and viral budding.

Dual-directional regulation of extracellular respiration in Shewanella oneidensis for intelligently treating multi-nuclide contamination

Radionuclide contamination has become a global environmental concern due to the high mobility and toxicity of certain isotopes. Bioreduction mediated by electrochemically active bacteria (EAB) with unique extracellular electron transfer (EET) capability is recognized as a promising approach for nuclear waste treatment. However, it is difficult to achieve bidirectional regulation of EET pathway through traditional genetic manipulation, limiting the bioremediation application of EAB. Here, we designed and optimized a novel Esa quorum sensing (EQS) system for highly efficient gene expression and interleaved cellular functional output. By promoting dimethyl sulfoxide reductase at low cell density and increasing the synthesis of electron conductive complex and flavins at high cell density, the EQS system dynamically switched the extracellular respiratory pathway of Shewanella oneidensis MR-1 according to cell density. The engineered strain exhibited precise switching and substantial improvement in the extracellular remediation of multiple nuclides, sequentially increasing the reduction of iodine IO3- and uranium U(VI) by 2.51- and 2.05-fold compared with the control, respectively. Furthermore, a mobile bacterial biofilm material was fabricated for collecting uranium precipitates coupled with U(VI) reduction. This work clearly demonstrates that EQS system contributes to the bidirectional regulation of EET pathway in EAB, providing an effective and refined strategy for bioremediation of multi-nuclide contamination.

Enzymatic modification of wheat starch by a novel maltotetraose-forming amylase from Atopomonas hussainii to retard retrogradation and improve bread quality

To retard starch retrogradation and improve bread quality, a novel maltotetraose-forming amylase (AhMFA) from Atopomonas hussainii was expressed in Komagataella phaffii. After high cell density fermentation, the enzyme activity reached a maximum level of 3032 U mL−1. AhMFA showed optimal activity at pH 6.0 and 55 °C, respectively. After raw wheat starch was treated with AhMFA at 55 °C for 1 h, the relative crystallinity decreased from 24.5 % to 20.8 % without changing the A-type crystalline pattern. The side chain components with A, B1 and B2 chains were reduced to 27.5 %, 44.9 %, and 13.8 %, respectively. The retrogradation enthalpy of wheat starch decreased significantly by 67.8 %. Moreover, the decreased Mixolab parameters (C5 and C5 − C4) indicated that AhMFA reduced starch retrogradation of wheat dough. After addition of AhMFA (3 ppm), the specific volume of bread increased by 29.5 % and its hardness decreased by 46.1 % compared to the control. The AhMFA-added bread exhibited good anti-staling properties with 43.7 % less hardness than the control after storage at 4 °C for 4 days. This study provided a novel maltotetraose-forming amylase for starch modification to retard retrogradation and improve bread quality.

Inhibition of PIM kinase in tumor associated macrophages suppresses inflammasome activation and sensitizes prostate cancer to immunotherapy.

Immunotherapy has changed the treatment paradigm for many types of cancer, but immune checkpoint inhibitors (ICIs) have not shown benefit in prostate cancer (PCa). Chronic inflammation contributes to the immunosuppressive prostate tumor microenvironment (TME) and is associated with poor response to ICIs. The primary source of inflammatory cytokine production is the inflammasome. Here, we identify PIM kinases as important regulators of inflammasome activation in tumor associated macrophages (TAMs). Analysis of clinical data from a cohort of treatment naïve, hormone responsive PCa patients revealed that tumors from patients with high PIM1/2/3 display an immunosuppressive TME characterized by high inflammation (IL-1β and TNFα) and a high density of repressive immune cells, most notably TAMs. Strikingly, macrophage-specific knockout of PIM reduced tumor growth in syngeneic models of prostate cancer. Transcriptional analyses indicate that eliminating PIM from macrophages enhanced the adaptive immune response and increased cytotoxic immune cells. Combined treatment with PIM inhibitors and ICIs synergistically reduced tumor growth. Immune profiling revealed that PIM inhibitors sensitized PCa tumors to ICIs by increasing tumor suppressive TAMs and increasing the activation of cytotoxic T cells. Collectively, our data implicate macrophage PIM as a driver of inflammation that limits the potency of ICIs and provides preclinical evidence that PIM inhibitors are an effective strategy to improve the efficacy of immunotherapy in prostate cancer.

Dynamically cultured, differentiated bovine adipose-derived stem cell spheroids as building blocks for biofabricating cultured fat

Cultured or cultivated meat, animal muscle, and fat tissue grown in vitro, could transform the global meat market, reducing animal suffering while using fewer resources than traditional meat production and no antimicrobials at all. To ensure the appeal of cultured meat to future customers, cultured fat is essential for achieving desired mouthfeel, taste, and texture, especially in beef. In this work we show the establishment of primary bovine adipose-derived stem cell spheroids in static and dynamic suspension culture. Spheroids are successfully differentiated using a single-step protocol. Differentiated spheroids from dynamic cultures maintain stability and viability during 3D bioprinting in edible gellan gum. Also, the fatty acid composition of differentiated spheroids is significantly different from control spheroids. The cells are cultured antibiotic-free to minimize the use of harmful substances. This work presents a stable and bioprintable building block for cultured fat with a high cell density in a 3D dynamic cell culture system.

Bioprinted High-Cell-Density Laminar Scaffolds Stimulate Extracellular Matrix Production in Osteochondral Co-Cultures.

Many tissues have a laminar structure, but there are limited technologies for establishing laminar co-cultures for in vitro testing. Here, we demonstrate that collagen-alginate-fibrin (CAF) hydrogel scaffolds produced using the reactive jet impingement bioprinting technique can produce osteochondral laminar co-cultures with well-defined interfaces between cell types and high cell densities to support cell-cell interaction across the interfaces. The influence of cell density and the presence of the two cell types on the production of extracellular matrix (ECM) and the emergent mechanical properties of gels is investigated using IHC, ELISA, gel mass, and the compression modulus. The results indicate that high-cell-density cultures and co-cultures with these specific cell types produce greater levels of ECM and a more biomimetic in vitro culture than low-cell-density cultures. In laminar scaffolds produced using TC28a2 chondrocytes and SaoS-2 osteoblasts, both cell density and the presence of the two cell types enhance ECM production and the mechanical properties of the cultures, presenting a promising approach for the production of more biomimetic in vitro models.

Effect of Hydroxyapatite Nanowires on Formation and Bioactivity of Osteoblastic Cell Spheroid.

Compared with traditional high-density cell spheroids, which are more prone to core necrosis, nanowires effectively improve the biological activity of core cells in spheroids, emanating more innovations for optimizing the internal cell survival environment and providing differentiation signals. In this study, hydroxyapatite nanowires (HAW), which provide numerous material exchange channels for internal cells by interpenetrating into cell spheroids, were added to osteoblast precursor (MC3T3-E1) cell spheroids. HAW, synthesized using the hydrothermal method, was used as a regulatory material to prepare uniformly sized 3D composite spheroids with good biological activity. Subsequently, material characterization and biocompatibility tests were performed on HAW, and the biological activity and osteogenic differentiation ability of the cell spheroids were tested. Notably, in 2D coculture, HAW displayed a certain attraction to MC3T3-E1 cells and promoted cell aggregation toward it. The content of HAW determined whether composite cell spheroids can form aggregated spherical structures, and incorporation of HAW alleviated core necrosis and enhanced the osteogenic phenotype. In summary, these findings indicate that the prepared HAW-bone cell composite spheroids can potentially be used as building blocks for the construction of large high-density biomimetic tissues and organoids using 3D bioprinting technology.

Immunomodulatory role of tumor microenvironment on oncological outcomes in advanced laryngeal cancer

BackgroundThe study evaluated the prognostic impact of the immune microenvironment in LSCC with markers of major immune cells to identify the key determinants of short-term disease-free survival (ST DFS) and reveal factors related to disease progression.MethodsThe study cohort included 61 patients who underwent total laryngectomy, 83.6% of whom were male with a mean age of 64.3 years at the time of surgery. Twenty-five patients had long term DFS (over 5 years), 8 – had moderate DFS (between 2 and 5 years), and 28 had short-term DFS (less than 2 years). Immunohistochemical staining and evaluation were performed on samples collected after the laryngectomy.ResultsThe samples’ assessment revealed that the mean expression of all analysed markers was the highest both in stroma and the tumor compartment for short term DFS (ST DFS) patients. Analysis confirmed that a high stromal density of CD8 cells (p = 0.038) significantly correlated with DFS, and that the increased presence of CD57 cells (p = 0.021) was significantly associated with ST DFS. Moreover, the high density of CD68 cells in the tumor epithelial compartment had a negative prognostic impact on DFS (p = 0.032). Analysis of overall survival in the studied cohort with Kaplan-Meyer curves revealed that a high stromal density of CD68 cells was a significant negative predictor of OS (p = 0.008).ConclusionsThe observed associations of CD68 cells infiltration with progression and prognosis in patients with LSCC provide potential screening and therapeutic opportunities for patients with unfavourable outcomes.

Preparation of Thermosensitive Lignocellulose Hollow Fiber Membrane Grafted With PNIPAAm and Its Application as a Cell Culture Carrier in a RSOC Dynamic Culture.

Currently, the cells, which are urgently required for large-scale application in biomedical-related fields, harvested by traditional trypsin digestion are usually subject to repeated digestion, leading to a reduction of cell activity. In this study, poly (N-isopropylacrylamide) (PNIPAAm) was grafted onto the lignocellulose hollow fiber membranes (HFMs) with cerium ammonium nitrate (CAN) as the initiator to prepare thermosensitive HFMs, which was combined with a rotation system of culture (RSOC) to achieve dynamic culture and non-destructive harvesting of cells from the HFMs. The results of ATR-FTIR, elemental analysis, and SEM confirmed the successful preparation of PNIPAAm-grafted-HFMs, which also showed good biocompatibility to apply for cell culture carriers. In cooling detachment, the HFMs-0.01 group could completely detach the cells within 1h with a cell separation efficiency of more than 90%. The laminin (LN) and fibronectin (FN) harvested by cooling detachment of P8 generation PC12 cells reached 0.0531±0.0032 and 2.5045±0.0001pg/cell, respectively, which were significantly higher than that by trypsin digestion. In addition, the cells on the thermosensitive HFMs proliferated fastest in RSOC at 30rpm with higher glucose consumption and lactate metabolism than in static conditions. Moreover, the cells that had dynamic detachment at 20rpm had the highest cell density and activity. Therefore, the thermosensitive HFMs could be applied as cell culture carriers in RSOC for cell culturing at 30rpm and harvesting at 20rpm, which would provide considerable potential for large-scale cell culture in vitro.

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma

ABSTRACT Background Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods Liver tissue samples were collected during 2008–2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells (“hot” cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with “hot” TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn “cold” into “hot” TIME in ICC.

Geometric constraint of mechanosensing by modification of hydrogel thickness prevents stiffness-induced differentiation in bone marrow stromal cells.

Extracellular matrix (ECM) stiffness is fundamental in cell division, movement and differentiation. The stiffness that cells sense is determined not only by the elastic modulus of the ECM material but also by ECM geometry and cell density. We hypothesized that these factors would influence cell traction-induced matrix deformations and cellular differentiation in bone marrow stromal cells (BMSCs). To achieve this, we cultivated BMSCs on polyacrylamide hydrogels that varied in elastic modulus and geometry and measured cell spreading, cell-imparted matrix deformations and differentiation. At low cell density BMSCs spread to a greater extent on stiff compared with soft hydrogels, or on thin compared with thick hydrogels. Cell-imparted matrix deformations were greater on soft compared with stiff hydrogels or thick compared with thin hydrogels. There were no significant differences in osteogenic differentiation relative to hydrogel elastic modulus and thickness. However, increased cell density and/or prolonged culture significantly reduced matrix deformations on soft hydrogels to levels similar to those on stiff substrates. This suggests that at high cell densities cell traction-induced matrix displacements are reduced by both neighbouring cells and the constraint imposed by an underlying stiff support. This may explain observations of the lack of difference in osteogenic differentiation as a function of stiffness.

The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes

BackgroundThe production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled.ResultsUsing different growth rates in the range of 0.075 and 0.4 h-1, highest surfactin titres of 36 g/L were reached at 0.25 h-1 with production yields YP/S of 0.21 g/g and YP/X of 0.7 g/g, while growth rates lower than 0.2 h-1 resulted in insufficient and slowed biomass formation as well as surfactin production (YP/S of 0.11 g/g and YP/X of 0.47 g/g for 0.075 h-1). In contrast, feeding rates higher than 0.25 h-1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1.ConclusionsOverall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h-1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates.

GefB, a GGDEF domain-containing protein, affects motility and biofilm formation of Vibrio parahaemolyticus and is regulated by quorum sensing regulators

Vibrio parahaemolyticus (V. parahaemolyticus) stands as the predominant etiological agent responsible for gastroenteritis associated with the consumption of seafood. Cyclic di-guanosine monophosphate (c-di-GMP), a secondary messenger in bacteria, controls multiple bacterial behaviors including pathogenesis, the development of biofilms, and motility. The protein GefB (VPA1478), characterized by the presence of a GGDEF domain, inhibits the swarming motility of V. parahaemolyticus. In this study, we showed that deletion of gefB remarkably reduced cellular c-di-GMP level and biofilm formation by V. parahaemolyticus, but significantly enhanced the swimming and swarming motility. In addition, GefB inhibited the polar and lateral flagellar genes but activated genes associated with exopolysaccharide production of V. parahaemolyticus. The data also demonstrated that vpa1477 and gefB are co-transcribed as a single transcriptional unit, designated as vpa1477-gefB. Transcription of vpa1477-gefB was under the collective regulation of the master quorum sensing (QS) regulators AphA and OpaR, which function at low (LCD) and high cell density (HCD), respectively. AphA positively regulated vpa1477-gefB transcription at LCD, whereas OpaR negatively regulated its transcription at HCD. The findings significantly enhance our comprehension of the metabolism and regulatory mechanisms of c-di-GMP in V. parahaemolyticus.

Small molecular weight alginate gel porogen for the 3D bioprinting of microvasculature.

In order to recreate the complexity of human organs, the field of tissue engineering and regenerative medicine has been focusing on methods to build organs from the bottom up by assembling distinct small functional units consisting of a biomaterial and cells. This bottom-up engineering requires bioinks that can be assembled by 3D bioprinting and that permit fast vascularization of the construct to ensure survival of embedded cells. To this end, a small molecular weight alginate (SMWA) gel porogen is presented herein. Alginate is a biocompatible biomaterial, which can be easily converted into small porogen gels with the procedure reported in this article. The SMWA porogen is mixed with photo-crosslinkable hydrogels and leached from the hydrogel post-crosslinking to increase porosity and facilitate vascularization. As a proof of concept, this system is tested with the commonly used biomaterial Gelatin Methacryloyl (GelMA). The SMWA porogen-GelMA blend is proven to be bioprintable. Incubating the blend for 20min in a low concentration phosphate buffered saline and sodium citrate solution significantly reduces the remaining porogen in the hydrogel . The intent to completely leach the porogen from the hydrogel was abandoned, as longer incubation times and higher concentrations of phosphate and citrate were detrimental to endothelial proliferation. Nonetheless, even with remnants of the porogen left in the hydrogel, the created porosity significantly improves viability, growth factor signaling, vasculogenesis, and angiogenesis in 3D bioprinted structures. This article concludes that the usage of the SMWA porogen can improve the assembly of microvasculature in 3D bioprinted structures. This technology can benefit the bottom-up assembly of large scaffolds with high cell density through 3D bioprinting by improving cell viability and allowing faster vascularization.

Epidermal growth factor induces the initiation of epithelial mesenchymal transition in high-density colorectal cancer cell culture.

Epithelial-mesenchymal transition (EMT) is a step in the process through which colorectal cancer cells metastasize by gaining the cellular mobility associated with mesenchymal cells. However, whether the EMT occurs in cells tightly bound to each other remains largely unknown. In this study, we examined the dual influence of intercellular contact and epidermal growth factor (EGF) signaling on the induction of EMT in SW480 human colon carcinoma cells. Stimulation of densely cultured SW480 cells with EGF initiated partial EMT, following which E-cadherin levels were reduced. In these cells, the transcriptional repression of E-cadherin was caused by ZEB1 binding to its promoter region. EGF signaling did not directly induce ZEB1 mRNA upregulation but contributed to ZEB1 protein stability by regulating proteasomal degradation. Our findings indicate that EGF can induce EMT in colorectal cancer cells in the presence of cell-cell contact and may be a potential therapeutic target for metastasis.

High photosynthesis rates in Brassiceae species are mediated by leaf anatomy enabling high biochemical capacity, rapid CO2 diffusion and efficient light use.

Certain species in the Brassicaceae family exhibit high photosynthesis rates, potentially providing a valuable route toward improving agricultural productivity. However, factors contributing to their high photosynthesis rates are still unknown. We compared Hirschfeldia incana, Brassica nigra, Brassica rapa and Arabidopsis thaliana, grown under two contrasting light intensities. Hirschfeldia incana matched B. nigra and B. rapa in achieving very high photosynthesis rates under high growth-light condition, outperforming A. thaliana. Photosynthesis was relatively more limited by maximum photosynthesis capacity in H. incana and B. rapa and by mesophyll conductance in A. thaliana and B. nigra. Leaf traits such as greater exposed mesophyll specific surface enabled by thicker leaf or high-density small palisade cells contributed to the variation in mesophyll conductance among the species. The species exhibited contrasting leaf construction strategies and acclimation responses to low light intensity. High-light plants distributed Chl deeper in leaf tissue, ensuring even distribution of photosynthesis capacity, unlike low-light plants. Leaf anatomy of H. incana, B. nigra and B. rapa facilitated effective CO2 diffusion, efficient light use and provided ample volume for their high maximum photosynthetic capacity, indicating that a combination of adaptations is required to increase CO2-assimilation rates in plants.