Optimal Culture Conditions Research Articles

Germination Performance of Physalis peruviana L. Seeds under Thermal and Water Stress Conditions

Physalis peruviana holds significant economic value, making it crucial to determine optimal cultivation conditions, particularly concerning seed germination under varying water and temperature conditions. Therefore, this study aimed to assess the impacts of heat and water stress on the germination and vigor of P. peruviana seeds. The study was divided into two trials: the first examined the effect of constant temperatures of 10, 15, 20, 25, 30, 35, and 40 °C and alternating temperatures of 20 °C during the dark period and 30 °C during the light period (control) on seed germination and vigor. The second trial investigated germination and vigor under different water and thermal conditions using various osmotic potentials (0; −0.3; −0.6; and −0.9 MPa) and two temperatures (constant 30 °C and alternating 20 °C during the dark period and 30 °C during the light period). Both trials used a 16 h photoperiod. The germination tests revealed optimal (30 °C), moderate (20/30 °C), minimal (20 °C), and inhibited (40 °C) temperatures for the species’ germination. It was found that the first germination count could be conducted on the seventh day after sowing. Low water availability had negative effects on seed germination and vigor, especially at osmotic potentials below −0.45 and −0.61 MPa, combined with temperatures of 30 and 20/30 °C, respectively. Severe water stress, with osmotic potentials equal to or below −0.9 MPa, completely inhibited seed germination and vigor. Reduction in water potential and increased temperature resulted in a significant decrease in the percentage, speed, and quality of P. peruviana germination. These findings indicate that the species does not tolerate extreme temperatures, whether low (less than 15 °C) or high (greater than 35 °C), nor water stress with osmotic potentials lower than −0.45 MPa, much less the combination of these factors.

Screening and Identification of Soil Selenium-Enriched Strains and Application in Auricularia auricula.

Selenium (Se) is an essential trace element for human physiological metabolism. The application of organic Se as a source to cultivate Se-rich plants for micronutrient supplementation has been receiving increasing attention. In our study, a bacterial strain named H1 was isolated from the soil in Heilongjiang Province, China, and under optimal culture conditions, the unit Se content could reach 3000 μg·g-1 and its 16S ribosomal DNA sequence seemed to be a new molecular record of an Enterobacter species. After the domestication of Se tolerance and Se-rich experiments, H1 can be used as a Se source for cultivation of Se-rich Auricularia auricula. The results showed that soluble protein, soluble sugar, free amino acid and vitamin C contents in Auricularia auricula were notably increased by 28.7%, 21.8%, 32.5% and 39.2% under the treatment of Se concentration of 0.24 mg·kg-1, respectively. These findings enhance our understanding that H1 is more conducive to Se uptake and nutrient accumulation.

Systematic optimization of culture media for maintenance of human induced pluripotent stem cells using the response surface methodology

The application of human induced pluripotent stem cells (hiPSCs) provides tremendous opportunities in cell therapy. However, culturing these cells faces many practical challenges, including costs associated with cell culture media and the optimization of cell culture conditions. Providing an optimized culture platform for hiPSCs to maintain pluripotency and self-renewal and generate cost-effective and robust therapeutics is an immediate requirement. This study used the design of experiments and the response surface methodology, a powerful statistical tool, to generate empirical models for predicting the optimal culture conditions of the hiPSCs. Pluripotency and cell proliferation were applied as read-outs to determine the optimal concentration of basic fibroblast growth factor (bFGF) and cell density. One model was defined to predict pluripotency and cell proliferation in terms of the predictor variables of the bFGF concentration and cell seeding density. Predicted culture conditions to maximize maintaining cell pluripotency were successfully validated. The present study's findings provide a novel approach that can potentially allow controllable hiPSC culture routine in translational research.

Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing.

The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct Escherichia coli (E. coli) strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using E. coli BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing E. coli strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.

In Vitro Propagation Technology for the Endangered Aquatic Species Nymphoides coronata.

Nymphoides coronata is an endangered aquatic plant species with significant medicinal and ecological importance. To preserve N. coronata from going extinct, we need to provide seedlings and efficient multiplication techniques so that it can be extensively studied. This study aimed to identify the most suitable sterilization treatment, growth medium, and substrate for the cultivation and propagation of N. coronata. Ethanol sterilization, fungicide treatment, and sterile water washing were the most important sterilization steps. A combination of 6-benzylaminopurine (6-BA) and indoleacetic acid (IAA) was the most suitable medium for bud induction and shoot proliferation. The use of α-naphthaleneacetic acid (NAA) increased the rooting rate and rooting time compared to indole-3-butyric acid (IBA). Increasing the concentration of NAA from 0.5 to 1.0 mg/L increased the rooting rate from 78 to 100% and reduced the rooting time from 7 to 5 days. The survival rate of N. coronata seedlings was 100% in a mixture of red soil and sand (1:1, w/w). As a result, the procedure mentioned above could potentially be used to safely propagate this rare species on a large scale. These findings provide valuable insights into the optimal conditions for the successful cultivation and propagation of N. coronata, which can contribute to the conservation and sustainable use of this important rare plant species.

Tenebrio molitor Larvae Feeding Strategy to Degrade Polypropylene Components from Disposable Masks

<p><strong>Abstract.</strong>The surge in mask consumption during the COVID-19 pandemic led to the widespread use of disposable polypropylene masks for protection. However, the accumulation of used masks poses environmental challenges due to the slow degradation of polypropylene. Here, we investigate the potential of <em>Tenebrio molitor</em> larvae in degrading polypropylene masks, exploiting their synthetic polymer-breaking enzymes and associated microflora. In this study, we implemented various feeding strategies and established specific feed compositions to assess <em>T. molitor</em> larvae's consumption behavior towards polypropylene masks. Over a 21-day observation period, we noted a consumption rate of 0.4% of masks, with a 7.5% mortality rate among the larvae. The average daily consumption rate was 0.201 grams, resulting in a 2.3% increase in larval weight and the production of 0.4635 grams of feces. Our findings highlight the potential of <em>T. molitor</em> larvae as effective mask degraders. Optimizing cultivation conditions and feeding strategies may further enhance microbial diversity, potentially introducing more polypropylene degraders within <em>T. molitor</em>, thereby expediting mask degradation. This study emphasizes the promising role of <em>T. molitor</em> larvae in addressing the environmental challenges posed by the accumulation of polypropylene masks.</p><p><strong>Keywords: </strong></p><p>Mask, Degrade, <em>Tenebrio molitor</em>, Microflora, Polypropylene</p>

Murine bone-derived mesenchymal stem cells undergo molecular changes after a single passage in culture

The rarity of the mesenchymal stem cell (MSC) population poses a significant challenge for MSC research. Therefore, these cells are often expanded in vitro, prior to use. However, long-term culture has been shown to alter primary MSC properties. Additionally, early passage primary MSCs in culture are often assumed to represent the primary MSC population in situ, however, little research has been done to support this. Here, we compared the transcriptomic profiles of murine MSCs freshly isolated from the bone marrow to those that had been expanded in culture for 10 days. We identified that a single passage in culture extensively altered MSC molecular signatures associated with cell cycling, differentiation and immune response. These findings indicate the critical importance of the MSC source, highlighting the need for optimization of culture conditions to minimize the impact on MSC biology and a transition towards in vivo methodologies for the study of MSC function.

Особенности высокорослого среднеспелого сорта сои Ягуар с признаком ускоренного прироста надземной биомассы

The tall, middle maturing soybean cultivar Jaguar with a trait of accelerated formation of aboveground biomass was obtained as a result of individual selection in F4 from the hybrid combination Vilana × D-7/10 by tall growth (> 150 cm) of the stem, large seeds, middle maturity and lodging resistance. Based on the results of variety testing in 2020–2023, the soybean cultivar Jaguar exceeded the standard cultivar Vilana by 0.26 t/ha of yield. With an average vegetative period of 123 days at the latitude of Krasnodar (45°), the height of plants of this cultivar is 138 cm, varying over the years from 120 to 154 cm. In optimal moisture conditions, the weight of 1000 seeds is 160–190 g. The phenomenon of accelerated formation of aboveground biomass of plants of the cultivar Jaguar y in optimal cultivation conditions is confirmed by a 44% more intense daily increase in plant height and a 55% more intense daily increase in aboveground biomass compared to these indicators for the standard cultivar Vilana. The new tall, middle maturing soybean variety Jaguar, with a trait of accelerated formation of aboveground biomass, is characterized by increased responsiveness to additional moisture by increasing the plant height to ultra-high, increasing the number of nodes, beans and seeds, seed size and weight, and yield. It has increased resistance of tall plants to tilting and lodging due to excessive moisture due to the increased mechanical strength of the stem. Due to its tall growth and high positive response to additional moisture supply, it is suitable for cultivation in the southern foothill and central zones of the Krasnodar region, with irrigation throughout the North Caucasus and Lower Volga regions of the Russian Federation, as well as in the republics of Central Asia and Transcaucasia.

Физиологические свойства перспективного грибного штамма антагониста возбудителя сухой гнили подсолнечника Т-5 Trichoderma sp.

Dry rot is one of the most dangerous and wide spreading disease on sunflower in last years. Due to the growing demand for environmentally friendly production of agricultural products, the demand for competitive microbiological products is increasing. Since 2020, there are studied and developed environmentally friendly microbiological preparations of a multifunctional type of action against the dry rot pathogen on sunflower Rhizopus oryzae Went & Prins. Geerl. at the V.S. Pustovoit All-Russian Research Institute of Oil Crops. The physiological qualities of a promising antagonist strain of the dry rot pathogen T-5 Trichoderma sp. isolated as a result of stepwise screening were studied during stationary cultivation on liquid nutrient media in order to develop elements of a technological regimen for the production of a microbiological preparation in a preparative form wettable powder. Optimal conditions for surface cultivation of the fungus T-5 Trichoderma sp. on a liquid nutrient medium were determined as following: temperature +25 °C; acidic reaction of the medium (pH 3); glucose and starch as carbon source; source of nitrogen nutrition is corn extract. The media by Rudakova and No. 1 were identified as the best complicated liquid nutrient media.

Biological Characteristics, Domesticated Cultivation Protocol, Antioxidant Activity, and Protective Effects against Cellular Oxidative Stress of an Underutilized Medicinal Mushroom: Fomitopsis palustris.

Brown-rot fungus is one of the important medicinal mushrooms, which include some species within the genus Fomitopsis. This study identified wild macrofungi collected from a broad-leaved tree in Liaoning Province as Fomitopsis palustris using both morphological and molecular methods. To elucidate the potential medicinal and economic value of F. palustris, we conducted single-factor and orthogonal tests to optimize its mycelium culture conditions. Subsequently, we completed liquid culture and domestic cultivation based on these findings. Furthermore, crude polysaccharides were extracted from the cultivated fruiting bodies of F. palustris and their antioxidant activity was evaluated using chemical methods and cell-based models. The results showed that the optimal culture conditions for F. palustris mycelium were glucose as the carbon source, yeast extract powder as the nitrogen source, pH 6.0, and a temperature of 35 °C. Moreover, temperature was found to have the most significant impact on mycelial growth. The liquid strains were fermented for 6 days and then inoculated into a cultivation substrate composed of broadleaf sawdust, resulting in mature fruiting bodies in approximately 60 days. The crude polysaccharides extracted from the cultivated fruiting bodies of F. palustris (FPPs) possess in vitro scavenging abilities against DPPH radicals and OH radicals, as well as a certain ferric-reducing antioxidant power. Additionally, FPPs effectively mitigated H2O2-induced oxidative stress in RAW264.7cells by enhancing the intracellular activity of antioxidant enzymes such as SOD and CAT, scavenging excess ROS, and reducing MDA levels. This study provides preliminarily evidence of the potential medicinal and economic value of F. palustris and offers initial data for the future development and utilization of this species.

Optimization of culture conditions for keratinase production of Bacillus tropicus KRKJVR 5 by using RSM (Response Surface Methodology)

This study aimed to optimize the production of keratinase by Bacillus tropicus KRKJVR 5 using response surface methodology (RSM). The factors considered were temperature (20 to 40°C), pH (5 to 9), inoculum size (0.5 to 2.5%), incubation period (24 to 120 hours) and agitation speed (RPM) (50 to 350). A total of 32 experimental runs were conducted based on the central composite design (CCD) model, with observed keratinase activity ranging from 2.00 to 13.33 U/mL. The highest keratinase activity (13.33 U/mL) was observed under the conditions of 30°C temperature, pH 7.0, 1.5% inoculum size, 72 hours incubation period and 150 RPM. The lowest activity (2.00 U/mL) was recorded at extreme conditions of low pH (3.0) and high temperature (50°C), highlighting the sensitivity of keratinase production to these factors. The regression analysis revealed a significant model with an R-squared value of 93.78%, indicating a strong fit to the data. The ANOVA results further supported the significance of these factors, with the overall model being highly significant (F-Value = 8.30, P-Value = 0.000). The contour plots demonstrated the optimal conditions for maximizing keratinase activity. The study effectively identified the optimal conditions for keratinase production by Bacillus tropicus KRKJVR 5 using response surface methodology. The results indicate that a temperature of 30°C, pH of 7.0, inoculum size of 1.5%, incubation period of 72 hours, and agitation speed of 150 RPM are ideal for producing high levels of keratinase. These findings provide valuable insights for scaling up the process for industrial applications, emphasizing the importance of precise control over culture conditions to achieve high enzyme yields.

Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02h before methanol feeding, 1.16% methanol concentration and 40.36μL glucose feeding amount(for 20mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

Pathogen-epithelium interactions and inflammatory responses in Salmonella Dublin infections using ileal monolayer models derived from adult bovine organoids

Salmonella enterica serovar Dublin (S. Dublin) is an important enteric pathogen affecting cattle and poses increasing public health risks. Understanding the pathophysiology and host–pathogen interactions of S. Dublin infection are critical for developing effective control strategies, yet studies are hindered by the lack of physiologically relevant in vitro models. This study aimed to generate a robust ileal monolayer derived from adult bovine organoids, validate its feasibility as an in vitro infection model with S. Dublin, and evaluate the epithelial response to infection. A stable, confluent monolayer with a functional epithelial barrier was established under optimized culture conditions. The model’s applicability for studying S. Dublin infection was confirmed by documenting intracellular bacterial invasion and replication, impacts on epithelial integrity, and a specific inflammatory response, providing insights into the pathogen-epithelium interactions. The study underscores the utility of organoid-derived monolayers in advancing our understanding of enteric infections in livestock and highlights implications for therapeutic strategy development and preventive measures, with potential applications extending to both veterinary and human medicine. The established bovine ileal monolayer offers a novel and physiologically relevant in vitro platform for investigating enteric pathogen-host interactions, particularly for pathogens like S. Dublin.

Biodegradation of Polyurethane by Fungi Isolated from Industrial Wastewater-A Sustainable Approach to Plastic Waste Management.

Polyurethane (PU) is a type of polymer, which exists in various forms in the environment. Very few studies are available concerning the structure or enzymatic mechanism of the microbial community, which can degrade PU. Degradation of PU remains a difficult problem with respect to the environmental and biological disciplines. This study mainly focused on identifying the micro-organisms able to degrade polyurethane and confirming the degradation by performing a plate assay, Sturm test and scanning electron microscopy. Optimal culture conditions for maximum PU degradation were also analyzed through classical methods. A soil burial test was conducted by placing polyurethane films in the soil for one month, and the microbe growing on the surface of polyurethane films-with a maximum degradation of 55%-was isolated and identified as Aspergillus versicolor (ARF5). The culture medium was also optimized with different physical and chemical parameters for maximum PU degradation. The presence of CO2 as a by-product of PU biodegradation was confirmed through the Sturm test.

A Comprehensive Study on the Micropropagation of Costus igneus: Media Composition, Growth, and Development

Costus igneus, commonly known as insulin plant, is an ornamental plant valued for its spiral ginger- like foliage and medicinal properties. Micropropagation is an efficient method for rapid multiplication of Costus igneus. This study aims to determine the optimal media composition and culture conditions for in vitro propagation of C. igneus. Axillary bud explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators including auxins (indole-3-acetic acid, indole-3-butyric acid and naphthaleneacetic acid) and cytokinins (6- benzylaminopurine and kinetin). Microshoots cultured on MS + 1.5 mg/L BAP showed the highest shoot proliferation rate (98%) and maximum number of shoots per explant (12.6). Elongated microshoots were rooted on half-strength MS medium supplemented with different auxins. The highest rooting percentage (95%) and maximum number of roots per shoot (9.8) were observed on medium containing 2.5 mg/L IBA. The regenerated plantlets were acclimatized and successfully transferred to pots with 80% survival rate. Morphological and phytochemical analysis showed no significant differences between the in vitro propagated and mother plants. This study demonstrates the potential of micropropagation for large scale production of quality planting material of C. igneus. Further studies on genetic and epigenetic stability are recommended to validate this protocol for commercial applications.

The Impact of Genotype and Controlled Environment Cultivation Parameters on Tomato-Leaf-Derived Exosome-like Nanoparticle Yield and Properties

Horticultural plant material offers several advantages for isolating exosomes and other natural plant-derived exosome-like nanoparticles (PDENs) due to the accessibility and affordability of plant material for widespread applications. This study aims to explore the impacts of the tomato genotype (‘Admiro’, ‘Roma’, ‘Brooklyn’, ‘Marmande’ and ‘Betalux’) and the main cultivation parameters in controlled environment agriculture on the yield and properties of their PDENs for pharmaceutical and cosmeceutical applications. The PDEN yield, size distribution, and antioxidative properties of young tomato seedlings were evaluated. The ‘Betalux’ tomato was distinguished by a remarkably higher nanoparticle concentration and a uniform size distribution and was selected for further experiments. The impact of cultivation temperature (18, 22, and 26 °C), nitrogen nutrition (0, 250, and 500 mg L−1), and the lighting photosynthetic photon flux density (PPFD; 150, 250, and 450 µmol m−2 s−1) on nanoparticle properties was investigated. Optimal conditions consisting of a temperature of 22 °C, 250 mg L−1 nitrogen nutrition, and 250 µmol m−2 s−1 lighting PPFD were used as a reference. Optimal temperature, nitrogen nutrition, and lighting intensity resulted in the highest nanoparticle yield, the most uniform particle distribution, and the highest impact of PDEN preparations on keratinocyte metabolic activity. Deviation from optimal cultivation conditions reduced the tomato biomass and the PDEN protein and yield.

Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C–H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs.Key points• The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase.• After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate.• The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

An in vitro 3-dimensional Collagen-based Corneal Construct with Innervation Using Human Corneal Cell Lines

To develop a 3-dimensional corneal construct suitable for invitro studies of disease conditions and therapies. Invitro human corneal constructs were created using chemically crosslinked collagen and chondroitin sulfate extracellular matrix and seeded with 3 human corneal cell types (epithelial, stromal, and endothelial) together with neural cells. The neural cells were derived from hybrid neuroblastoma cells and the other cells used from immortalized human corneal cell lines. To check the feasibility and characterize the constructs, cytotoxicity, cell proliferation, histology, and protein expression studies were performed. Optimized culture condition permitted synchronized viability across the cell types within the construct. The construct showed a typical appearance for different cellular layers, including healthy appearing, phenotypically differentiated neurons. The expected protein expression profiles for specific cell types within the construct were confirmed with western blotting. An invitro corneal construct was successfully developed with maintenance of individual cell phenotypes with anatomically correct cellular loci. The construct may be useful in evaluation of specific corneal disorders and in developing different corneal disease models. Additionally, the construct can be used in evaluating drug targeting and/or penetration to individual corneal layers, testing novel therapeutics for corneal diseases, and potentially reducing the necessity for animals in corneal research at the early stages of investigation. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

The Effect of Inhibiting the Wingless/Integrated (WNT) Signaling Pathway on the Early Embryonic Disc Cell Culture in Chickens.

The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal-Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.

Caki-1 Spheroids as a 3D Model for Studying Free Fatty Acid-Induced Proximal Tubule Lipotoxicity

Three-dimensional (3D) multicellular spheroid shows a biological complexity resembling native tissue in comparison to conventional monolayer cultures, making it a better model system to assess drug effcacy and cytotoxicity. The main aim of this study was to examine the usefulness of Caki-1, a human proximal tubule (PT) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent changes in morphology and functionality. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact 3D spheroids with uniform morphology, good circularity, and increased diameter from days 2-10. There was no change when the spheroids were incubated with lipid-free bovine serum albumin (BSA, 0.5%) from day 6 to 10. Chronic exposure to palmitate (300 μM) resulted in dispersed and flattened spheroid morphology, with a larger number of dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in caspase-3 activity at the external layers of the spheroids, suggesting that the detached cells underwent apoptosis. Simultaneous addition of oleate (200 μM) prevents palmitate-induced spheroid disintegration and cell death in Caki-1 3D culture. Mechanistically, palmitate triggered endoplasmic reticulum (ER) stress and autophagy dysfunction, as evidenced by significantly increased protein levels of ER stress markers AFT4 and CHOP, and autophagy substrate p62. Oleate upregulated adipose differentiation-related protein (ADRP, a lipid droplet-associated protein), and promoted lipid droplet formation in palmitate-treated Caki-1 cells. These results indicate that Caki-1 3D spheroids are a simple and reproducible in vitro system for lipotoxicity study. It can potentially be used for evaluating human renal proximal tubular toxicity and drug screening. NIH/NIGMS R16GM149499, NIH TL1DK136047. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.