Optimal Medium Research Articles

Application of the Knife-Edge Technique on Transition Metal Dichalcogenide Monolayers for Resolution Assessment of Nonlinear Microscopy Modalities.

We report application of the knife-edge technique at the sharp edges of WS2 and MoS2 monolayer flakes for lateral and axial resolution assessment in all three modalities of nonlinear laser scanning microscopy: two-photon excited fluorescence (TPEF), second- and third-harmonic generation (SHG, THG) imaging. This technique provides a high signal-to-noise ratio, no photobleaching effect and shows good agreement with standard resolution measurement techniques. Furthermore, we assessed both the lateral resolution in TPEF imaging modality and the axial resolution in SHG and THG imaging modality directly via the full-width at half maximum parameter of the corresponding Gaussian distribution. We comprehensively analyzed the factors influencing the resolution, such as the numerical aperture, the excitation wavelength and the refractive index of the embedding medium for the different imaging modalities. Glycerin was identified as the optimal embedding medium for achieving resolutions closest to the theoretical limit. The proposed use of WS2 and MoS2 monolayer flakes emerged as promising tools for characterization of nonlinear imaging systems.

Comparative genomic analysis and optimization of astaxanthin production of Rhodotorula paludigena TL35-5 and Rhodotorula sampaioana PL61-2.

Astaxanthin is a powerful antioxidant known to enhance skin, cardiovascular, eye, and brain health. In this study, the genome insights and astaxanthin production of two newly isolated astaxanthin-producing yeasts (TL35-5 and PL61-2) were evaluated and compared. Based on their phenotypic and genotypic characteristics, TL35-5 and PL61-2 were identified as basidiomycetous yeasts belonging to Rhodotorula paludigena and Rhodotorula sampaioana, respectively. To optimize astaxanthin production, the effects of cultural medium composition and cultivation conditions were examined. The optimal conditions for astaxanthin production in R. paludigena TL35-5 involved cultivation in AP medium containing 10 g/L glucose as the sole carbon source, supplemented with 1.92 g/L potassium nitrate, pH 6.5, and incubation at 20°C for 3 days with shaking at 200 rpm. For R. sampaioana PL61-2, the optimal medium composition for astaxanthin production consisted of AP medium with 40 g/L glucose, supplemented with 0.67 g/L urea, pH 7.5, and the fermentation was carried out at 20°C for 3 days with agitating at 200 rpm. Under their optimal conditions, R. paludigena TL35-5 and R. sampaioana PL61-2 gave the highest astaxanthin yields of 3.689 ± 0.031 and 4.680 ± 0.019 mg/L, respectively. The genome of TL35-5 was 20,982,417 bp in length, with a GC content of 64.20%. A total of 6,789 protein-encoding genes were predicted. Similarly, the genome of PL61-2 was 21,374,169 bp long, with a GC content of 64.88%. It contained 6,802 predicted protein-encoding genes. Furthermore, all essential genes involved in astaxanthin biosynthesis, including CrtE, CrtYB, CrtI, CrtS, and CrtR, were identified in both R. paludigena TL35-5 and R. sampaioana PL61-2, providing evidence for their ability to produce astaxanthin.

Microalgae Isolated from Singapore Mangrove Habitat as Promising Microorganisms for the Sustainable Production of Omega-3 Docosahexaenoic Acid

Docosahexaenoic acid (DHA, C22:6n-3) is an omega-3 fatty acid with beneficial effects for human health. In view of its increasing demand, DHA traditionally produced by marine fisheries will be insufficient, and an alternative sustainable source is urgently required. Here, we report the isolation and characterization of four novel microalgae strains, PLU-A, B, C and D, with a high DHA content of up to 45% from decayed mangrove samples collected from a coastal area in Singapore. Phylogenetic analysis revealed that these isolates were clustered with Schizochytrium sp. TK6 (OK244290.1) and were identified as Schizochytrium sp. strains. A medium optimization with Schizochytrium sp. PLU-D found a glucose-to-yeast extract ratio of 4:1 to be optimal for high biomass and lipid accumulation of up to 70% in shake flasks. In fed-batch fermentation scale-up with the Schizochytrium sp. PLU-D strain, this translates to 175 g/L dry biomass, 94 g/L lipid and 36.2 g/L DHA. Accordingly, the DHA titer obtained is superior to most of the scale-up production reported thus far, while the DHA content is comparable to two other commercially available DHA algae oils. These results suggest that Schizochytrium sp. PLU-D has high potential to be applied for the sustainable production of DHA.

Efficient In Vitro Regeneration System and Comparative Transcriptome Analysis Offer Insight into the Early Development Characteristics of Explants from Cotyledon with Partial Petiole in Small-Fruited Pepper (Capsicum annuum).

In our research, we utilized six small-fruited pepper germplasms as materials, selected cotyledons with the petiole and hypocotyls as explants, and conducted in vitro regeneration studies. Our outcomes specify that the most suitable explant is cotyledon with the petiole, and the suitable genotype is HNUCA341. The optimal medium for inducing and elongating adventitious buds for this genotype is Murashige and Skoog medium (MS) + 9.12 μM Zeatin (ZT) + 0.57 μM 3-Indoleacetic acid (IAA), with a bud induction rate of 44.4%. The best rooting induction medium is MS + 1.14 μM IAA, with a rooting rate of 86.7%. Research on the addition of exogenous hormones has revealed that the induction speed of buds in small-fruited pepper (HNUCA341) in the combination of ZT and IAA hormones (abbreviated as ZI) is quicker, and the induction effect is better. The histological observations indicate that ZI treatment accelerates the initiation of explant division and differentiation, causing a shorter duration of vascular-bundle tissue production. The plant hormone signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including ARR9 (LOC107843874, LOC107843885), ARR4 (LOC107848380, LOC107862455), AHK4 (LOC107870540), AHP1 (LOC107839518), LAX2 (LOC107846008), SAUR36 (LOC107852624), IAA8 (LOC107841020), IAA16 (LOC107839415), PYL4 (LOC107843441), and PYL6 (LOC107871127); these significantly enriched genes may be associated with in vitro regeneration. In addition, the carbon metabolism pathway and plant mitogen-activated protein kinase (MAPK) signaling pathway are also significantly enriched in KEGG. The results of the Gene Ontology (GO) analysis revealed that differentially expressed genes related to carbon metabolism and fixation, photosynthesis and MAPK signaling pathways were upregulated under ZI treatment. It was found that they might be associated with enhanced regeneration in vitro. Furthermore, we also screened out differentially expressed transcription factors, primarily from the MYB, bHLH, AP2/ERF, and NAC families. Overall, our work accumulated important data for the in-depth analysis of the molecular mechanism of in vitro regeneration of pepper, and provides valuable germplasm for establishing an efficient stable pepper genetic-transformation system based on tissue culture.

Production optimization and antioxidant potential of exopolysaccharide produced by a moderately halophilic bacterium Virgibacillus dokdonensis VITP14

This study aimed to enhance the extracellular polymeric substances (EPS) production of Virgibacillus dokdonensis VITP14 and explore its antioxidant potential. EPS and biomass production by VITP14 strain were studied under different culture parameters and media compositions using one factor at a time method. Among different nutrient sources, glucose and peptone were identified as suitable carbon and nitrogen sources. Furthermore, the maximum EPS production was observed at 5% of inoculum size, 5 g/L of NaCl, and 96 h of fermentation. Response surface methodology was employed to augment EPS production and investigate the optimal levels of nutrient sources with their interaction. The strain was observed to produce actual maximum EPS of about 26.4 g/L for finalized optimum medium containing glucose 20 g/L, peptone 10 g/L, and NaCl 50 g/L while the predicted maximum EPS was 26.5 g/L. There was a nine fold increase in EPS production after optimization study. Additionally, EPS has exhibited significant scavenging, reducing, and chelating potential (>85%) at their higher concentration. This study imparts valuable insights into optimizing moderately halophilic bacterial EPS production and evaluating its natural antioxidant properties. According to findings, V. dokdonensis VITP14 was a promising isolate that will provide significant benefits to biopolymer producing industries.

Microbial allies: exploring fungal endophytes for biosynthesis of terpenoid indole alkaloids.

Terpenoid indole alkaloids (TIAs) are natural compounds found in medicinal plants that exhibit various therapeutic activities, such as antimicrobial, anti-inflammatory, antioxidant, anti-diabetic, anti-helminthic, and anti-tumor properties. However, the production of these alkaloids in plants is limited, and there is a high demand for them due to the increasing incidence of cancer cases. To address this research gap, researchers have focused on optimizing culture media, eliciting metabolic pathways, overexpressing genes, and searching for potential sources of TIAs in organisms other than plants. The insufficient number of essential genes and enzymes in the biosynthesis pathway is the reason behind the limited production of TIAs. As the field of natural product discovery from biological species continues to grow, endophytes are being investigated more and more as potential sources of bioactive metabolites with a variety of chemical structures. Endophytes are microorganisms (fungi, bacteria, archaea, and actinomycetes), that exert a significant influence on the metabolic pathways of both the host plants and the endophytic cells. Bio-prospection of fungal endophytes has shown the discovery of novel, high-value bioactive compounds of commercial significance. The discovery of therapeutically significant secondary metabolites has been made easier by endophytic entities' abundant but understudied diversity. It has been observed that fungal endophytes have better intermediate processing ability due to cellular compartmentation. This paper focuses on fungal endophytes and their metabolic ability to produce complex TIAs, recent advancements in this area, and addressing the limitations and future perspectives related to TIA production.

Optimizing surface finish in FDM-printed polycarbonate spur gears through abrasive flow finishing: insights from physics and material science perspectives

In recent times, the usage of polymers has experienced notable growth across diverse manufacturing sectors. Polymeric gears, integral to automation, material handling systems, toys, and household appliances, have become ubiquitous. Although additive manufacturing techniques, especially Three-Dimensional (3D) printing, offer versatile applications, they grapple with challenges, notably poor surface finishing attributed to layer accumulation. This work explores the field of abrasive flow machining (AFM) in experimental settings using FDM-printed polymeric gears. The AFM medium concoction involves coal ash powder as the foundational material, EDM oil as the carrier fluid, and the infusion of glycerin as additives. Rigorous investigations were undertaken to pinpoint the optimal viscosity of the AFM medium and refine process parameters with a central focus on enhancing surface quality. A Taguchi L9 Design of Experiment (DOE) was meticulously crafted for parameter optimization using the Minitab statistical software. The investigation established a functional relationship between the output parameter (surface roughness) and key input variables (layer thickness, abrasive percentage, abrasive mesh size, and finishing time). The maximum level of AFM media optimization was attained at 33% abrasive concentration, 220 abrasive mesh size, and 60% liquid synthesizer. Additionally, the results of the investigation showed that a media viscosity of 0.50 Pa-sec, layer thickness of 0.1, and culminating time of 45 min were the optimal values for the most % improvement in surface roughness. The initial surface roughness underwent a profound reduction from 12.30 μm to 0.30 μm, marking an exceptional improvement of 97.56%. This inquiry contributes significant insights into the refinement of AFM parameters for elevating the surface finish of FDM-printed polymeric gears, promising enhanced performance across diverse applications.

Analytically monitored cultivation of Arthrospira platensis on food waste extracts to compose a sustainable, low-prized, Zarrouk-based medium

ABSTRACT A sustainable, low-cost medium based on food waste for cultivating the cyanobacterium Arthrospira platensis (A. platensis) was investigated. Zarrouk medium components, especially phosphate, were substituted with extracts from beetroot peel, brewer’s grains, and walnut press cake. Ion concentrations of magnesium, calcium, iron, potassium, sodium, phos-phate, nitrate, sulfate, and chloride were analyzed to determine the optimal medium composition. Walnut press cake and brewer’s grains extracts showed high potential due to their magnesium, calcium, and phosphate content. Brewer’s grains extracts also provided glucose as an additional carbon source. Cultivations in shaking flasks and a photobioreactor (PBR) monitored cell growth through optical density and biomass concentration. The excess of magnesium and reduced phos-phate concentrations positively affected biomass production. A. platensis’s ability to metabolize glucose resulted in 68% higher biomass production. Growth in brewer’s grains extract required no additional phosphate. PBR cultivation showed A. platensis consumed significant magnesium and nitrate, achieving higher biomass concentrations with optimized gas exchange and light reflection systems.

Addressing bioreactor hiPSC aggregate stability, maintenance and scaleup challenges using a design of experiment approach

BackgroundStem cell-derived therapies hold the potential for treatment of regenerative clinical indications. Static culture has a limited ability to scale up thus restricting its use. Suspension culturing can be used to produce target cells in large quantities, but also presents challenges related to stress and aggregation stability.MethodsUtilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power.ResultsExpansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1–1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion.ConclusionThis study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.

Optimization of growth media and light requirement for obtaining maximum biomass and yield of mushroom (Cordyceps militaris)

Optimization of growth media and light requirement for obtaining maximum biomass and yield of mushroom (Cordyceps militaris)

Design and optimization of gradient fibrous media using the method of moments

The goal of this work is to optimize the deposition mass distribution of polydisperse particles inside the fibrous media by modifying the distribution of the fiber packing density across filter depth. Two types of gradient fibrous media with linear and exponential fiber packing density distribution along the flow direction were derived. The method of moments was employed to study the transport and deposition characteristics of polydisperse aerosol particles inside the fibrous media. The reliability of the calculated results by the method of moments was confirmed by the numerical simulations based on the discrete phase model (DPM). Results indicate that the gradient fibrous media allows more particles to transport and deposit into the interior of the fibrous media, which improves the uniformity of particle deposition distribution across filter depth. There exists an optimal fiber packing density distribution ratio for the gradient fibrous media where the non-uniformity of deposition mass distribution of polydisperse particles across filter depth is minimized. In this way, uniform deposition distribution along the flow direction can be generally achieved by optimizing the fiber packing density distribution, which will contribute to the optimal design of microstructures for the fibrous filter.

Multiobjective optimization of porous medium for efficient transpiration cooling of hypersonic vehicles using genetic algorithm

Transpiration cooling has been proven an effective method for reducing heat flux on the surfaces of high-speed vehicles. This study investigates the effects of porous medium properties, employing a black-box optimization method to determine the optimal length, thickness, and porosity for a porous medium in a transpiration cooling system on a flat plate under hypersonic laminar flow. The objectives include optimizing thermal effectiveness, coolant consumption, and the weight and cost of the porous material. A multiobjective genetic optimization algorithm is directly integrated with a Computational Fluid Dynamics (CFD) solver, and 1562 CFD simulations were conducted to identify the optimal configuration. The results demonstrate that the length and porosity of the porous medium more significantly impact thermal effectiveness than the thickness. Furthermore, the optimization identified a configuration for the porous medium that, when compared to the original case, shows reductions in length, thickness, and porosity of 3.5%, 11%, and 29%, respectively. Additionally, there were average improvements in thermal effectiveness and coolant consumption of 4.56% and 3.9%, respectively, while the weight and cost of the porous material increased by 3.73% and 3.65%, respectively.

Bunian People: Myths, Perceptions and Impressions of Behavior and Culture in Minangkabau

Minangkabau has a unique and diverse culture, starting from art, culture, songs, fairy tales, poetry, beliefs, characterizations and visual characters. One of the unique things about Minangkabau is that it is a belief that is widely known by the public and has been discussed from time to time until now. One of the interesting beliefs in Minangkabau is about the Urang Bunian mythology, however, the lack of understanding of the target audience and the lack of optimal media in conveying information about Urang Bunian visualizations are the main factors for the author to design the Urang Bunian character design in Minangkabau culture through the Art Book media. Urang Bunian was made in the form of an Art Book media which contains about creating character designs from the information obtained. Apart from the Art Book media, the delivery of this information is also supported by several other media which help the main media to introduce the subject to the target audience, namely posters, stickers, t-shirts, tote bags, holograms, x-banners, mini x-banners. The design method used is the 5w+1h approach, namely what, who, when, where, why and how. By using the 5w+1h theory, all aspects summarized in the character design design process can be created through Art Book media

A Novel Timing Equation for Predicting Optimal Contrast Medium Enhancement in Abdomen CT Scan Procedure

Abdominal CT (Computed Tomography) scans are crucial for diagnosing a wide range of abdominal conditions by providing detailed images of the abdominal organs. The aim of this study is to develop and validate a novel timing equation for predicting optimal contrast medium enhancement in abdominal CT procedures. Utilizing a quantitative research design, data was retrospectively collected from 155 patients who underwent CT scans with contrast media, focusing on variables such as age, gender, weight, creatinine levels, and injection parameters. Statistical analysis, including multi-linear regression and ANOVA, was conducted to derive predictive equations for arterial, venous, and delayed times. The results indicated that Location of the cannula significantly influence arterial time, age is significantly influence venous enhancement time, while weight was the only significant predictor of delayed time, demonstrating the need for patient-specific timing in CT scans. In conclusion, the proposed timing equation could enhance diagnostic accuracy and patient care by optimizing contrast enhancement in abdominal CT procedures.

Development of Mycological Medium using Tomato Juice Extract as Principal Base

Study’s Novelty/Excerpt This study introduces an approach to mycological media development by utilizing Tomato Juice Extract (TJA) as a primary base, highlighting its rich nutrient profile and potential for sustainable, cost-effective fungal cultivation. The research uniquely demonstrates the influence of various TJA concentrations on fungal growth, revealing selective preferences for certain fungi and emphasizing the importance of medium optimization. These findings provide significant insights into the potential of natural sources in microbiological practices, laying the groundwork for future studies to further optimize TJA and explore its practical applications in mycology. Full Abstract This project focused on developing mycological media using the primary base of Tomato Juice Extract (TJA). Tomatoes contain a rich mix of vitamins, minerals, and organic acids, creating an ideal environment for cultivating various fungi. Various concentrations of Tomato Juice Agar (TJA) and Potato Dextrose Agar (PDA) were prepared (200 mL, 300 mL, and 400 mL). TJA was created by combining tomato filtrate with different sugar concentrations (10g, 15g, 20g), keeping agar agar powder constant at 20 g. Samples of soil from refuse dumpsite and spoiled bread were inoculated onto the prepared TJA and PDA (control). The total fungal counts, isolation, and identification were determined using PDA and TJA. Microscopic analyses, serial dilutions, and colony characterization provided a detailed assessment. TJA exhibited total fungal counts of 2-3 propagules/g for refuse dumpsite soil and 3-4 propagules/ml for spoiled bread. PDA yielded counts of 3-6 propagules/g for refuse dumpsite soil and 3-5 propagules/ml for spoiled bread. Various fungi, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Rhizopus stolonifer, Penicillium. digitanum, and Fusarium oxyporum were identified. TJA showed selective preferences for Aspergillus niger and Rhizopus stolonifer. This study successfully developed mycological media with tomato juice extract, yielding promising fungal cultivation results. Different TJA concentrations influenced fungal growth, thus emphasizing the importance of selecting the appropriate medium. The absence of growth of some of the test fungal isolates on TJA and exclusive isolation of Fusarium oxysporum on PDA shows medium-specific preferences. Tomato juice extract aligns with sustainability and cost-effectiveness, offering an alternative to traditional components. Valuable insights into concentration-dependent fungal growth guide future experimentation and optimization. Tomato juice extract as a mycological medium base introduces a sustainable alternative with unique nutritional profiles. Concentration-dependent fungal preferences revealed the significance of medium optimization. These findings encourage the use of natural sources for more sustainable microbiological practices. While laying innovative mycological media groundwork, further research is needed to optimize tomato juice agar fully. Mycological media development with tomato juice extract offers practical benefits in cost-effectiveness and sustainability.

Management Strategy for the Development of Pesantren's Hidden Curriculum in Schools

The curriculum at SMPIQu Al-Bahjah Cirebon faces a challenge in integrating the formal, official curriculum with the non-formal pesantren (Islamic boarding school) curriculum. This disconnect has resulted in the pesantren curriculum becoming somewhat "hidden" or underrepresented. The core issue addressed in this research is how to design, implement, and evaluate the development of this hidden pesantren curriculum. Using a qualitative research method, the study draws from both primary and secondary data, collected through observation and interviews. The findings reveal a five-step approach to developing the hidden curriculum. Implementation is achieved through structured activities such as work meetings, scheduling academic activities, enhancing the management information system (SIM), improving teaching staff and student resources, strengthening students' morals, and increasing proficiency in foreign languages. The evaluation process includes supervision, regular meetings, audits, and reporting. The study concludes that effective curriculum development hinges on a morals and creed-based education, improved administrative and management systems, optimization of media as a development tool, and active participation in competitions and events. This research contributes to the formulation of strategies for developing the hidden pesantren curriculum, offering practical benefits for all stakeholders involved.

Evaluation of 21 different media on shoot regeneration in 11 cultivars of citrus using juvenile tissue

Citrus is one of the most important global fruit crops, with oranges accounting for more than half the total production. Citrus can be genetically engineered for improved traits, but the process is severely limited by shoot regeneration rates, especially in commercial varieties. Although standardizing a transformation protocol is difficult due to genotype and season dependencies, use of an optimal mineral nutrient basal culture medium increases the success of recovering transgenic Citrus cells. The purpose of this study is to improve shoot regeneration from juvenile tissue in multiple Citrus genotypes. Explants of 11 Citrus cultivars were regenerated in 21 published media to determine the optimal media for each genotype. The number of shoots from juvenile tissue were counted 90 days after the explants were first cultured. The type of basal medium strongly affected the rates of shoot regeneration from Citrus juvenile tissue, and the effectiveness was negatively correlated with the level of ammonium. For each Citrus grouping, optimized media formulations were determined, and potential improvements were predicted. In general, medium R7100 appeared to be the most effective for regeneration of the Citrus genotypes tested.

An overview of light ray deflection calculation by magnetars in nonlinear electrodynamics

Due to the lack of experimental confirmation of the effects of nonlinear electrodynamics (NED) of vacuum on the electromagnetic wave, it remains the most pressing issue. One of the most optimal media for studying these effects, occurring in astrophysics, are magnetars. Various methods of studying the bending of a wave passing through the magnetospheres of magnetars play a key role in a deeper understanding of vacuum NED. This article reviews modern methods for determining the angle of bending of the electromagnetic wave under the effect of NED of vacuum and gravity, with special attention paid to NED of vacuum in intense electromagnetic fields, which is a characteristic feature of magnetars. The article discusses various methods and techniques used to measure these angles, as well as their potential astrophysical applications.

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization.

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely αA- and γ-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.

Construction of oral squamous cell carcinoma organoids invitro 3D-culture for drug screening.

Patient-derived organoids are potent pre-chemotherapy models. Due to limited research on diverse types of oral squamous cell carcinoma (OSCC) and construction efficiency, our goal was to optimize OSCC organoid models from various sites and assess drug responsiveness. We screened and optimized culture media, employing three-dimensional techniques to construct human-derived oral squamous cell carcinoma (OSCC) organoid models invitro. Morphological validation, immunofluorescence analysis, tissue origin verification, and Short Tandem Repeat (STR) sequencing confirmed the consistency between organoids and source tissues. These organoid models were then subjected to varying concentrations of anticancer drugs, with subsequent assessment of cell viability to calculate IC50 values. Twenty-nine surgical specimens yielded an 86.2% success rate in culturing 25 organoids invitro. Morphological consistency confirmed nuclear atypia and positive expression of K5, P40, and E-cadherin, indicating squamous epithelial origin. Cultured complex organoids included α-SMA+ tumour-associated fibroblasts and tumour stem cells expressing CD44 and Ki67. STR sequencing affirmed genomic homogeneity between cultured organoids and source tissues. Drug sensitivity testing revealed diverse responses among organoids, highlighting their value for assessing drug sensitivity. An efficient OSCC organoid culture system for personalized invitro drug sensitivity screening was established, laying the foundation for precise treatment development.