Culture Substrate Research Articles

Upcycling Fishing Net Waste and Metal Oxide from Electroplating Waste into Alga Cultivation Structures with Antibacterial Properties

Plastic waste, especially discarded fishing nets, and electroplating sludges pose significant environmental challenges, impacting marine ecosystems and contributing to pollution. In alga cultivation, invasive microorganisms often hinder growth, necessitating strategies to combat these issues. This study aimed to develop recycled substrates for alga cultivation by repurposing fishing nets and enhancing their surfaces with antibacterial properties using copper oxide (CuO). Additionally, it explores the reuse of CuO from electroplating sludge, providing a sustainable solution that addresses both marine and industrial waste while supporting healthy alga development. Recycled substrates were produced, with different proportions of pure CuO and sludge (1 and 2 wt%) incorporated on the surface. These compositions were processed by hot compression molding and then the antibacterial activity was characterized using a qualitative and quantitative method. The results indicate the possibility of recycling fishing net into new substrates to alga cultivation and the functionalization of their surface using CuO as an antibacterial agent. The antibacterial tests showed a better activity for pure CuO compared to the residual sludge, and better for the higher surface concentration of 2 wt%. Despite the limited bacterial inhibition observed, there is an opportunity for reusing these sludges, typically disposed of in landfills, to obtain specific antibacterial agents that can be applied to the surface of substrates for algal growth.

Cell confinement by micropatterning induces phenotypic changes in cancer-associated fibroblasts

Recent advances in single-cell studies have revealed the vast transcriptomic heterogeneity of cancer-associated fibroblasts (CAFs), with each subset likely having unique roles in the tumor microenvironment. However, it is still unclear how different CAF subsets should be cultured in vitro to recapitulate their in vivo phenotype. The inherent plasticity of CAFs, or their ability to dynamically change their phenotype in response to different environmental stimuli, makes it highly challenging to induce and maintain a specific CAF state in vitro. In this study, we investigated how cell shape and confinement on two-dimensional culture substrates with different stiffnesses influence CAF transcriptomic profile and phenotype. Using micropatterning of polyacrylamide hydrogels to induce shape- and confinement-dependent changes in cell morphology, we observed that micropatterned CAFs exhibited phenotypic shifts towards more desmoplastic and inflammatory CAF subsets. Additionally, micropatterning enabled control over a range of CAF-specific markers and pathways. Lastly, we report how micropatterned and non-micropatterned CAFs respond differently to anti-cancer drugs, highlighting the importance of phenotype-oriented therapy that considers for CAF plasticity and regulatory networks. Control over CAF morphology offers a unique opportunity to establish highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity, heterogeneity, and development of novel therapeutic targets. Statement of SignificanceCancer-associated fibroblasts (CAFs) are the dominant stromal cell type in many cancers, and recent studies have revealed that they are highly heterogeneous and comprise several subpopulations. It is still unclear how different subsets of CAFs should be cultured in vitro to recapitulate their in vivo phenotype. In this study, we investigated how cell shape and confinement affect CAF transcriptomic profile and phenotype. We report that micropatterned CAFs resemble desmoplastic and inflammatory CAF subsets observed in vivo and respond differently to anti-cancer drugs as compared to non-patterned CAFs. Control over CAF morphology enables the generation of highly robust CAF phenotypes in vitro, facilitating deeper understanding of CAF plasticity and heterogeneity.

Growth and Yield Performance of Pleurotus ostreatus Cultivated on Agricultural Residues

Food insecurity and malnutrition are among the major problems in most developing nations recently. Mushroom cultivation is one of the promising strategies to overcome these challenges. The growth and productivity of mushrooms differ because of their wide range of cultivation substrates. Cultivating Pleurotus ostreatus on suitable substrates is one of the key factors affecting its growth and productivity. This study was, therefore, conducted to investigate the effect of cultivation substrates, namely straws of tef (Trt1), barley (Trt2), and wheat (Trt3), husks of faba bean (Trt4) and field pea (Trt5), and sawdust (Trt6) alone, and their mixture (1:1, w/w) (Trt7) on the growth and yield of P. ostreatus. Mycelial colonization, primordial formation, and days to first harvest were faster (13.00, 19.67, and 22.67 days) for the P. ostreatus cultivated on Trt7 whereas those grown on Trt6 were delayed (18.00, 27.00, and 29.67 days), respectively. Trt7 gave a higher (67.33) fruiting body/bunch and total yield (2001.70 g/bag). Biological efficiency was also significantly (p < 0.05) higher for Trt7 (238.64%). Strong relationships between cap diameter and mushroom yield (r = 0.84***), number of bunches (r = 0.76***), number of fruiting bodies (r = 0.80***), stipe length (r = 0.83***), and total yield (r = 0.84***) were among significant positive correlations observed. In conclusion, cultivating P. ostreatus on the Trt7 (mixed substrate) is recommended rather than using either of the residues alone.

Direct activation of Toll-like receptor 2 signaling stimulated by contact with the interfacial structures of chitin nanofibers

The innate immune system, which eliminates pathogens and abnormal cells, is involved in the pathogenesis of various diseases and infections, where Toll-like receptors (TLRs) play a critical regulatory role. In this study, we investigated the potential of chitin nanofiber (CtNF) to induce an immune response, which is expected to act as an agonist of TLR2. Crab-derived CtNF, surface-deacetylated CtNF, and surface-carboxylated cellulose NF were employed as TLR2-mediated immune stimulator, signal regulator, and cell adhesion promoter, respectively, to fabricate cell culture scaffolds for HEK293 cells with TLR2 and human monocyte THP-1 cells with or without TLR2. Surface deacetylation of CtNF drastically diminished the immunological response of HEK293 cells, suggesting that the N-acetyl groups on the solid CtNF surface were pivotal for TLR2-mediated stimulation. A comparison of wild-type and TLR2-KO THP-1 cells on cell culture substrates with N-acetyl groups ranging from 0 to 1.39 mmol g−1 revealed that immune signaling for nuclear factor-κB and interferon regulatory factor pathways was strongly dependent on the surface N-acetyl group content. The immunostimulatory level at the interface of solid CtNF and immune cells could be regulated by simply mixing CtNF and surface-deacetylated CtNF, which is a significant advantage for its potential use as a novel immunostimulant.

Graphene SU-8 Platform for Enhanced Cardiomyocyte Maturation and Intercellular Communication in Cardiac Drug Screening.

Cell culture substrates designed for myocardial applications are pivotal in promoting the maturation and functional integration of cardiomyocytes. However, traditional in vitro models often inadequately mimic the diverse biochemical signals and electrophysiological properties of mature cardiomyocytes. Herein, we propose the application of monolayer graphene, transferred onto SU-8 cantilevers integrated with a microelectrode array, to evaluate its influence on the structural, functional, and electro-mechano-physiological properties of cardiomyocytes. The monolayer graphene, prepared using chemical vapor deposition, is adeptly transferred to the target substrates via thermal release tape. The electrical conductivity of these graphene-enhanced SU-8 substrates is about 1600 S/cm, markedly surpassing that of previously reported cell culture substrates. Immunofluorescence staining and Western blot analyses reveal that the electrically conductive graphene significantly enhances cardiomyocyte maturation and cardiac marker expression compared to bare SU-8 substrates. Cardiomyocytes cultured on graphene-transferred substrates exhibit conduction velocity approximately 3.4 times greater than that of the control group. Such improvements in cardiac marker expression, mechano-electrophysiological performance lead to better responsiveness to cardiovascular drugs, such as Verapamil and Isoproterenol. While the graphene monolayer does not fully replicate the complex environment found in native cardiac tissue, its use on SU-8 substrates offers a feasible approach for accelerating cardiomyocyte maturation and facilitating drug screening applications.

The Photocleavable Protein PhoCl-Based Dynamic Hydrogels.

Dynamic protein hydrogels have attracted increasing attention owing to their tunable physiochemical and mechanical properties, customized functionality, and biocompatibility. Among the different types of dynamic hydrogels, photoresponsive hydrogels are of particular interest. Here, we report the engineering of a photoresponsive protein hydrogel by using the photocleavable protein PhoCl. We employed the well-developed SpyTag and SpyCatcher chemistry to engineer PhoCl-containing covalently cross-linked hydrogels. In the hydrogel network, PhoCl, which can be cleaved into two fragments upon violet irradiation, is employed as a dynamic structural motif to regulate the cross-linking density of the hydrogel network. The resultant PhoCl-containing hydrogels showed photoresponsive viscoelastic properties. Upon violet irradiation, the PhoCl hydrogels soften, leading to an irreversible reduction in the storage moduli. However, no gel-sol transition was observed. Leveraging this light-induced stiffness change, we employed this hydrogel as a cell culture substrate to investigate the mechanobiological response of NIH-3T3 fibroblast cells. Our results showed that 3T3 cells can change their morphologies in response to the stiffness change of the PhoCl hydrogel substrate dynamically, rendering PhoCl-based hydrogels a useful substrate for other mechanobiological studies.

Effects of biochar, N-enriched biochar and urea on tomato seed germination, vegetative growth, and fruit traits.

Agronomic uses of biochar have been intensely explored in the last 15 years. Recently, a new generation of biochar-based fertilizers has been developed. Raw biochar (BCH), nitrogen-enriched biochar (N + BCH) or urea were added to a coir fiber-based substrate for tomato cultivation, to assess seed germination, growth and fruiting of two cultivars (Cuarenteno and Moneymaker). BCH stimulated seed germination and early radicle growth, possibly because of the presence of karrikins detected in both BCH and N-BCH (0.039 and 0.044 mg kg-1, respectively). However, BCH reduced growth in adult plants in both cultivars, probably because of ammonium retention, causing low-N-stress-related symptoms such as accumulation of flavonoids in the leaf. Urea was toxic for seed germination because of the fast release of ammonium, but caused a positive effect on adult plant growth and yield, increasing chlorophyll in both cultivars, quantum yield and ascorbic acid in cv. Cuarenteno, and decreasing flavonoids and peroxide in leaves of both cultivars. Unlike urea, N + BCH showed a positive impact on plant growth and yield, but without releasing high amounts of ammonium or negatively affecting seed germination. Nitrogen-rich amendments reduced phosphorus and increased iron leaf content in both cultivars. BCH can be effectively used as a growth medium constituent in nurseries for seedling production, whereas N + BCH offers a promising alternative to urea or other nitrogen mineral fertilizers for crop cultivation. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pleurotus spp.-an effective way in degradation mycotoxins? A comprehensive review.

Mycotoxins-secondary metabolites produced by filamentous fungal species-occur as a global problem in agriculture due to the reduction in crop quality and the negative effects on human and animal health. There is a need to develop environment-friendly methods of detoxification. In recent years, a number of biological methods for the removal/degradation of mycotoxins have been described. One of them-particularly interesting due to its high effectiveness-is mycoremediation, which involves the ability of Pleurotus spp. mushrooms to remove toxic contaminants from the environment and food. Pleurotus spp. biosynthesizes ligninolytic enzymes, such as laccase and manganese peroxidase that are the main factors of enzymatic degradation of various pollutants, including mycotoxins. The degradation process of mycotoxins (especially aflatoxins) with the participation of isolated enzymes reaches approximately 30-100%, depending on the culture conditions, substrate, and mediators used. In the food industry, their application may include, among others, the detoxification of animal feed from mycotoxins or fermentation products (e.g., juices and wines). While these applications are promising, they require further research to expand toxicological knowledge and optimize their use. This review presents current research on this new and very promising topic related to the use of edible Pleurotus spp. mushrooms in the process of biological degradation of toxic fungal metabolites.

Study on the main physicochemical characteristics of different plant cultivation substrates and their effects on standard roses

Study on the main physicochemical characteristics of different plant cultivation substrates and their effects on standard roses

Study of the Degradation and Utilization of Cellulose from Auricularia heimuer and the Gene Expression Level of Its Decomposition Enzyme

Auricularia heimuer is a wood-rotting edible mushroom, and with the continuous development of the industry, the research on its grass-rotting cultivation is becoming more and more important. In this study, A. heimuer was cultivated using herbaceous substrate (reed) completely replacing the traditional woody substrate (oak), and the correlation between the relative expression of cellulase gene, cellulase activity, cellulose degradation and yield of different strains of A. heimuer were studied by combining qRT-PCR technology at different growth stages. The results showed that the cellulose degradation were positively correlated with the yield of reed and sawdust substrate at two growth stages, and were positively correlated with three cellulase activities. The relative expression of four cellulase genes were positively correlated with enzyme activity. There were inter-strain differences in the expression of the enzyme genes, which were basically consistent with the trend of the enzyme activity of the strains; g5372 and g7270 were more actively expressed in the mycelium period, while g9664 and g10234 were more actively expressed in the fruiting period. The results of SEM showed that the mycelium of A15 and A125 were different in their ability to degrade and utilize lignocellulose in reed substrate. The parental hybridization test further verified that qRT-PCR could be used as a rapid method to evaluate the cellulose degradation ability of A. heimuer strains. Seven strains (A12, A15, A184, A224, Z6, Z12, and Z18) with high cellulose degradation ability were screened. This study provides a reference for further understanding the role of A. heimuer cellulase genes in the degradation and metabolism of cellulose and for breeding new varieties more suitable for herbaceous substrate cultivation.

Optimizing food waste bioconversion with sodium selenite-enhanced Lucilia sericata maggots: a sustainable approach for chicken feed production and heavy metal mitigation.

Recycling food waste by feeding it to insects can result in the continuous production of high-quality animal feed protein and organic fertilizer. However, the bioconversion efficiency and safety of using insects as feed protein for animal breeding are important factors limiting the development of this technology. Therefore, we aimed to optimize the efficiency of bioconversion of food waste using Lucilia sericata maggot (LSM). Sodium selenite (SS) was used to improve the quality and safety of each trophic-level organism. The results showed that an SS concentration of 15mgkg-1 w.w. in the food waste culture substrate (SS15), the yield and quality of the obtained LSMs were optimal. The total selenium (Se) content of LSMs was 82.4 ± 1.16mgkg-1 d.w., and non-inorganic Se accounted for 96.4% ± 2.01% of the total Se content. Additionally, the conversion efficiency of food waste was 18.7% higher than that in the control group (p < 0.05). When SS15 was used to raise maggots as a protein substitute for fish meal (commercial feed), the weight of the chickens and the crude protein content were 1.09-1.26times and 1.09-1.13times, respectively (p < 0.05), in comparison with the corresponding findings obtained with the use of ordinary maggots and commercial feed. In this group, glutathione peroxidase, superoxide dismutase, catalase, and immunoglobulin A and G activities were significantly higher than those obtained with the other feeds (p < 0.05). During this cyclic utilization process, the total Se content in chickens (0.31 ± 0.05mgkg-1 w.w. in the breast, 0.19 ± 0.01mgkg-1 w.w. in the leg, and 0.57 ± 0.01mgkg-1 w.w. in the liver) significantly increased (p < 0.05). Meanwhile, the Cu and Zn contents in the LSMs and chickens increased, whereas cadmium, lead, chromium, and nickel absorption was inhibited (p < 0.05). Health risk assessment based on the levels of Se and heavy metals showed that Se-enriched chickens produced using this method can be safely consumed.

Corn cobs as growing media for production of cucumber plants in agriculture sector: a new trend

Peat and perlite are considered traditional growing substrates in soilless culture. The high cost of both and the urgent need for agricultural waste recycling prompted scientists and soilless culture growers to find alternative growing substrates provide optimal growth conditions and help dispose waste safely. In this study, corn cobs (CB) were tested as growing media and compared with peat moss (PM) and perlite (P). The organic wastes tested: CB, PM, and P were examined with washed sand (WS) at five treatments (WS, CB, CB + peat, CB + P and CB + sand). Cucumber (Cucumis sativus L.) plants were cultivated using 5 kg pots that contained the investigated substrates. Vegetative growth parameters, early fruiting stage, photosynthetic pigments, and cucumber yield had the highest values with CB and CB + P substrates, while the lowest ones were found in WS and CB + WS. CB resulted in greater flowering with the number of fruits per plant and yield being 39.70%, 37.33%, and 33.67%, 29.88 in both seasons, higher than the lowest substrate (WS). Although adding P to CB showed the highest values of measured growth characteristics, its high cost decreased net profit. Furthermore, CB characteristics as a growing media qualify it for use in cucumber soilless culture production. The use of this replacement media, which contains crushed CB, could reduce (but not eliminate) the need for PM and P in soilless culture. Also, using pyrolyzed biomass in growing media provides another way to convert biomass residues into valuable products.

Transformative Biomaterials in Burn Care: Utilizing Scaffolds, Hydrogels, and Chitosan for Optimized Healing and Scar Reduction

With over a million patients per year in the USA alone, burns are among the most common injuries in the world. In addition to preventing difficulties during burn injuries, the growing impact of scaffolds, hydrogels, cell scaffolds, and cell sheets with healing boosting elements expedite triggers and strengthens re-epithelialization and wound healing, which limits the creation of scars. While superficial partial-thickness and superficial burns typically heal without surgery, severe burns require special care, including topical antimicrobial surgery or bandages. Usually composed of polymeric biomaterials, scaffolds offer the structural support needed for cell adhesion and the subsequent formation of tissue. The term "tissue engineering triad" often refers to cells, scaffolds, and growth-stimulating signals. This may be accomplished in several ways by combining different polymers. Simple (planar) 3D structures may be produced using conventional and rapid prototyping techniques, but complicated structures have been successfully produced by carefully controlling the Molds and processing parameters. For biomedical and tissue engineering applications, chitosan has been widely employed (skin, bone, cartilage, and vascular grafts to substrates for mammalian cell culture). Additionally, it is renewable, biocompatible, biodegradable, nonantigenic, bioactive, and nontoxic.

Biosensor for integrin inhibition of mammalian cell adhesion and migration using micropatterned cell culture substrate and retroreflective optical signaling

Integrins are a family of transmembrane receptors that play a crucial role in cell adhesion and migration. Integrins can uniquely transduce biochemical signals bidirectionally across the membrane and physically link the cell-cell and cell-extracellular matrix (ECM) with ligand bonds. The arginyl-glycyl-aspartic acid (RGD) peptide motif is present in the ECM as a minimal recognition sequence for integrins. To leverage this property in cell-based therapy, RGD variants, such as cyclic-type RGDfK (c(RGDfK)), which share a similar structure with RGD but exhibit a higher affinity for integrins, have been developed. However, because most evaluation methods for newly developed RGD variants focus on affinity strength, tools for cellular effects are required. In this study, we developed a new platform that integrates micropatterned three-dimensional cell culture substrates with a non-spectroscopic optical analysis system to quantitatively analyze the effects of RGD variants on cell adhesion and migration. The specially micropatterned substrate provides a cell adhesive and migration area to provide a restricted analysis area. Owing to the characteristics of retroreflective Janus particles (RJPs), a non-spectroscopic optical analysis system provides long-term stable optical verification properties and a simple optical setup. These techniques were integrated to quantitatively determine the integrin inhibitory effect of various concentrations of RGD variant. To demonstrate the efficacy of the developed cellular level RGD variant testing platform, the model cell line L929 fibroblast and model RGD variant c(RGDfK) were analyzed ranging from 0 to 10 μM. The results showed that the developed system could effectively and quantitatively analyze the effects of RGD variants on cells across various concentrations.

Thermoresponsive Brush Coatings for Cell Sheet Engineering with Low Protein Adsorption above the Polymers' Phase Transition Temperature.

Thermoresponsive polymer coatings on cell culture substrates enable noninvasive cell detachment and cell sheet fabrication for biomedical applications. Optimized coatings should support controlled culture and detachment of various cell types and allow chemical modifications, e.g., to introduce specific growth factors for enhanced gene expression. Furthermore, the sterilization and storage stability of the coatings must be assessed for translational attempts. Poly(glycidyl ether) (PGE) brush coatings with short alkoxy side chains provide a versatile platform for cell culture and detachment, but their polyether backbones are susceptible to oxidation and degradation. Thus, we rationally designed potential alternatives with thermoresponsive glycerol-based block copolymers comprising a stable polyacrylate or polymethacrylate backbone and an oligomeric benzophenone (BP)-based anchor. The resulting poly(ethoxy hydroxypropyl acrylate-b-benzophenone acrylate) (pEHPA-b-BP) and poly(ethoxy hydroxypropyl methacrylate-b-benzophenone methacrylate) (pEHPMA-b-BP) block copolymers preserve the short alkoxy-terminated side chains of the PGE derived structure on a stable, but hydrophobic, aliphatic backbone. The amphiphilicity balance is maintained through incorporated hydroxyl groups, which simultaneously can be used for chemical modification. The polymers were tailored into brush coatings on polystyrene surfaces via directed adsorption using the BP oligomer anchor. The resulting coatings with thickness values up to ∼3 nm supported efficient adhesion and proliferation of human fibroblasts despite minimal protein adsorption. The conditions for cell sheet fabrication on pEHPA-b-BP were gentler and more reliable than on pEHPMA-b-BP, which required additional cooling. Hence, the stability of pEHPA-b-BP and PGE coatings was evaluated post gamma and formaldehyde (FO) gas sterilization. Gamma sterilization partially degraded PGE coatings and hindered cell detachment on pEHPA-b-BP. In contrast, FO sterilization only slowed detachment on PGE coatings and had no adverse effects on pEHPA-b-BP, maintaining their efficient performance in cell sheet fabrication.

Optimization of Magnetic Biochar Preparation Process, Based on Methylene Blue Adsorption.

The search for low-cost and effective adsorbents for the removal of organic dyes from contaminated water is urgently needed. The substantial amount of waste mushroom cultivation substrates generated in practical production can serve as an ideal material for the preparation of adsorbents. In this study, we investigated the main control parameters affecting the performance of magnetic mushroom substrate biochar and optimized the process of preparing biochar by using the Plackett-Burman and central composite design methods. Various analytical techniques including SEM, EDX, BET, and VSM were used to characterize the biochar. The results indicate that the carbonization temperature had the most significant impact on the yield and adsorption performance of biochar. Under the conditions of a carbonization temperature of 600 °C, a carbonization retention time of 1 h, and an impregnation ratio of 0.1, the yield and methylene blue adsorption value of magnetic biochar were 42.54% and 2297.04 μg/g, respectively, with a specific surface area of 37.17 m2/g. This biochar effectively removed methylene blue from the solution, demonstrating a high economic efficiency for wastewater treatment and pollution control. Furthermore, the adsorption-desorption cycle studies revealed its excellent stability and reusability. Additionally, based on the response surface methodology, a three-dimensional surface model of the adsorption performance of magnetic biochar under different carbonization conditions was established, providing a theoretical basis for the preparation of magnetic biochar from agricultural wastes.

Using Polycaprolactone Nanofibers for the Proof-of-Concept Construction of the Alveolar-Capillary Interface.

The alveolar-capillary interface is the key functional element of gas exchange in the human lung, and disruptions to this interface can lead to significant medical complications. However, it is currently challenging to adequately model this interface invitro, as it requires not only the co-culture of human alveolar epithelial and endothelial cells but mainly the preparation of a biocompatible scaffold that mimics the basement membrane. This scaffold should support cell seeding from both sides, and maintain optimal cell adhesion, growth, and differentiation conditions. Our study investigates the use of polycaprolactone (PCL) nanofibers as a versatile substrate for such cell cultures, aiming to model the alveolar-capillary interface more accurately. We optimized nanofiber production parameters, utilized polyamide mesh UHELON as a mechanical support for scaffold handling, and created 3D-printed inserts for specialized co-cultures. Our findings confirm that PCL nanofibrous scaffolds are manageable and support the co-culture of diverse cell types, effectively enabling cell attachment, proliferation, and differentiation. Our research establishes a proof-of-concept model for the alveolar-capillary interface, offering significant potential for enhancing cell-based testing and advancing tissue-engineering applications that require specific nanofibrous matrices.

Optimizing cultivation substrate moisture and calcium application for the comprehensive growth of cucumber under different air temperatures

Optimizing cultivation substrate moisture and calcium application for the comprehensive growth of cucumber under different air temperatures

Effects of Clump Size on the Pluripotency and Proliferation in the Passaging Process of Mouse Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are a promising cell source because of their pluripotency and self-renewal abilities. However, there is a risk of pluripotency loss during cell expansion. Particularly, cell passaging is associated with a higher risk of decreasing cell quality. There are two iPSC passaging methods: single-cell and clump passaging. Single-cell passaging is a rapid and simple method for cell manipulation, whereas clump passaging is superior for maintaining iPSC pluripotency. Therefore, clump passaging is a robust method for expanding iPSCs while maintaining their pluripotency. However, clump size control during clump passaging is difficult because colony fragmentation is performed manually by pipetting the colonies detached from the cell culture substrates. In this study, the effect of pipetting on iPSC colony fragmentation was evaluated and the relationship between iPSC clump size and pluripotency was clarified. An automated pipetting device was developed to standardize the clump passage process. The effect of clump size on the pluripotency and proliferative capacity of mouse iPSCs was investigated. Clump size was controlled by varying the number of pipetting cycles, and pluripotency and proliferation were assessed via alkaline phosphatase staining and flow cytometry. Our results revealed that a decrease in clump size corresponded to an increase in cell proliferation, while pluripotency maintenance was optimized under specific clump sizes. These results underscore the significance of clump size for stem cell quality, emphasizing the need for a balanced approach to maintain pluripotency while fostering proliferation in the cell expansion culture for iPSCs.

Bacterial Acyl Homoserine Lactones Triggered Non‐Native Substrate Hydroxylation Catalyzed by Directed‐Evolution‐Derived Cytochrome P450BM3 Mutants

AbstractWe report the directed evolution of cytochrome P450BM3 to efficiently utilize the bacterial quorum sensing signalling molecule N‐decanoyl homoserine lactone (C10‐HSL) as an effective decoy molecule. This represents the first important step in our endeavor to develop of a self‐sufficient decoy‐molecule system in whole‐cells that only necessitates the addition of culture medium and substrate to realize the hydroxylation of non‐native substrates. Following five rounds of directed evolution, mutant P450BM3, in the presence of C10‐HSL, catalyzed the hydroxylation of benzene at a rate of 475 min−1, the highest turnover rate recorded for any P450 enzyme, and achieving a 46% yield in a whole‐cell reaction system. High‐resolution X‐ray crystal structure analysis of a series of mutants narrates the directed evolution process, revealing how C10‐HSL is fixed in the binding pocket to permit binding of non‐native substrates. Finally, introduction of the C10‐HSL synthase gene ExpI into Escherichia coli, enabled the in situ production of C10‐HSL, realizing, for the first time, the hydroxylation of non‐native substrates without the need for the laborious synthesis and addition of decoy molecules.