Culture Methodology Research Articles

P0961 Establishing a new cellular therapy for patients with Ulcerative Colitis

Abstract Background Inflammatory Bowel Diseases (IBD), which include Crohn’s Disease and Ulcerative Colitis (UC), show an accelerated incidence rate, affecting up to 1% in Western countries, and an increased burden in healthcare costs, reaching up to 5.6 billion euros per year. Although there are several treatment options, the remission rates are around 30-60%, with primary non-response and loss of response to be the main reasons for changing treatment plans. Thus, there is a need for new therapeutic strategies targeting these groups of non-responding patients. Towards this, we are developing a novel cellular therapy for UC, using autologous transplantation of intestinal epithelial organoids. Methods Here, colonic epithelial cells will be isolated, expanded under GMP conditions, and transplanted into ulcerated colonic regions of patients with UC, to promote and accelerate mucosal healing. This initial strategy targets patients with localized ulceration unresponsive to first- and second-lines of treatments. Results We have established a robust protocol for expanding intestinal epithelial organoids. These organoids contain all major cell type populations that orchestrate the intestinal epithelium. We have successfully transplanted both murine and human colonic organoids into NOG mice. After successful engraftment onto the damaged intestinal mucosa, they promote and accelerate mucosal healing. Currently, we are establishing scale-up culturing methodologies, in order to expand our cell cultures to a number of cells that would be enough to promote mucosal healing during transplantation in humans. Finally, we are refining the cell culture conditions to be GMP-approved, and thus, registering our final product as acceptable to be used during human clinical trials. Conclusion Our approach could offer a new therapeutic option for patients with UC that do not respond to current drug regimens and are in need for an effective treatment.

Knowledge hiding and social exchange theory: a systematic review and meta-analysis.

The literature on the antecedents and consequences of knowledge hiding remains fragmented, limiting its practical applications. Social exchange theory (SET), one of the most widely adopted sociological frameworks, offers unique insights into the dynamics of knowledge hiding. This study synthesizes the application of SET in analyzing the nomological framework of knowledge hiding through a systematic literature review and meta-analysis. A meta-analysis was conducted based on the random-effects model and the meta-analytic structural equation modeling method, incorporating 66 primary studies with a total of 20,603 participants. Additionally, we examined the mediating role of knowledge hiding by linking key antecedents and consequences. Moreover, an exploratory analysis was conducted to investigate the moderating effects of national culture and research methodology, providing evidence to justify the true heterogeneity in the pairwise relationships between knowledge hiding and its antecedents. The research results generally support most pairwise relationships between knowledge hiding and its correlates, which were theoretically developed based on SET. This study is the first attempt to explore the explanatory power of SET in analyzing the knowledge-hiding phenomenon, and whether the establishment of a knowledge exchange loop contributes to a deeper understanding of this dyadic construct.

Navigating stem cell culture: insights, techniques, challenges, and prospects.

Stem cell research holds huge promise for regenerative medicine and disease modeling, making the understanding and optimization of stem cell culture a critical aspect of advancing these therapeutic applications. This comprehensive review provides an in-depth overview of stem cell culture, including general information, contemporary techniques, encountered problems, and future perspectives. The article begins by explaining the fundamental characteristics of various stem cell types, elucidating the importance of proper culture conditions in maintaining pluripotency or lineage commitment. A detailed exploration of established culture techniques sheds light on the evolving landscape of stem cell culture methodologies. Common challenges such as genetic stability, heterogeneity, and differentiation efficiency are thoroughly discussed, with insights into cutting-edge strategies and technologies aimed at addressing these hurdles. Moreover, the article delves into the impact of substrate materials, culture media components, and biophysical cues on stem cell behavior, emphasizing the intricate interplay between the microenvironment and cell fate decisions. As stem cell research advances, ethical considerations and regulatory frameworks become increasingly important, prompting a critical examination of these aspects in the context of culture practices. Lastly, the article explores emerging perspectives, including the integration of artificial intelligence and machine learning in optimizing culture conditions, and the potential applications of stem cell-derived products in personalized medicine. This comprehensive overview aims to serve as a valuable resource for researchers and clinicians, fostering a deeper understanding of stem cell culture and its key role in advancing regenerative medicine and biomedical research.

Installment of Automation and Modernized Culture Methods Updates a Drug-Sensitivity Screening Platform and Paves the Way to Next-Generation of Precision Medicine

Since the field of drug discovery has seen a rise in the use of primary tumor specimens (PTS) derived from patients with hematological malignancies as live materials in ex vivo drug treatments in late 2010s, they have discovered many intriguing phenotypes of AML cells that can lead to therapeutic insights of this disease category. However, when we consider the fact that current methods in ex vivo drug screenings are not using the most updated culture and cell processing methodology, it is highly expected that modernization of cell processing and analyzing procedures will enable us to detect phenotypes of PTS at a higher resolution. Herein, we have installed our own updated methods in multimodal categories. Even though the number of primary tumor specimens in the screening cohort is not comparable to the ones in other studies using AML PTS, (24 cases of AML, 3 cases of CML, and 1 case of MPN, while we ongoingly recruit more cells), our approach is characterized by its full-automation both in cell processing and data acquisitions using multicolor flow cytometry, as well as updated ex vivo culture methods after optimization using different culture media. We harvested cells for flow cytometry as of day 0, 3, and 6. In a monotherapy section, 24 compounds (6 conventional chemotherapy drugs, 10 epigenetic inhibitors, 4 kinase inhibitors including a FLT3 inhibitor, 3 signal transduction inhibitors, and Venetoclax) were included, while 3 different combination therapies (IDR+ AraC, Ven + 5-Aza, and Ven + AraC) were performed using 6x6 matrix layouts. Drugs were prepared in 384-well plates using a computationally controlled drug dispenser. Using those updated systems, we could discover; 1) Those fully-automated high-throughput platform needs their own optimizations regarding seeding numbers, liquid handling methods, and optimal plastic vessels with low inherent variations of results. 2) PTS are well tolerable to up to 6 days of ex vivo culture 3) Choosing optimal time points for each of the drugs ensures more accurate representation of their drug activities. For example, epigenetic inhibitors have tendency to show better results on day 6 rather than day 3. 4) Multicolor flow cytometry provides previously unseen differences in drug activity profiles upon different clusters, which had not been visible from bulk-grade sensitivity profiling, such as colorimetric viability assay. Those specimens were genetically profiled by whole genome/ exome sequencing, as well as other multi-omics analyses (Bulk RNA seq, methylome analysis, and scRNA seq). 5) Optimizations of ex vivo culture methods not only ensure our better quality of these 6-day drug effects, but also extend the possibility of PTS for further applications by allowing leukemia stem cell fractions to be maintained in vitro for longer periods of time. They include long-term culture and ex vivo expansion of leukemic stem cells using the most updated serum-free liquid culture, and further gene modifications using various vectors. Collectively, our platform for DSS confers next-generation use of primary patient-derived biomaterials so that even a small- to middle-sized group can conduct these complexed assays to test their hypotheses based on primary specimens, which had been possible only in high-volume multi-institutional alliances.

The AIDS Memorial Quilt as Radical Public History

Radical public history relies on reflective practices that include formal and experientially acquired knowledge within a broad interdisciplinary framework. Using material culture methodologies from history and anthropology, we present the AIDS Memorial Quilt as an example of radical public history in action. We recount the history and meaning of the Quilt and share quantitative survey information based on college students’ responses to six Quilt blocks displayed with supplemental informational banners. Embracing a core pillar of radical public history—shared authority—our authors include interdisciplinary academic and non-academic community partners whose combined skills, lived experiences, and expertise are critical to this movement and this paper. We finish with insights for those wishing to host Quilt blocks in their community.

Predominance of Trichophyton soudanense as Agent of Dermatophytoses in Cape Verdean School-Age Children.

Dermatophytoses are infectious skin diseases of public health importance because of their transmissibility and high prevalence, especially among children. This is the first study aiming to estimate and report the burden of dermatophytoses on school-age children on the island of Santiago in Cape Verde, an African country that is an archipelago. A total of 249 students attending the afternoon shift of three elementary schools in the city of Achada Igreja were examined. Of these, 60 had suspected lesions of dermatophyte infection. However, from the samples collected from these 60 students, including hair, nails, and skin scrapings, only 18 dermatophyte isolates were obtained, corresponding to a point prevalence of 7.2%. Morphological species identification demonstrated three different species: Trichophyton soudanense, Trichophyton rubrum, and Trichophyton violaceum; re-identification by sequencing the internal transcribed spacer (ITS) 1 and 2 regions of ribosomal DNA, and the 5.8S rDNA encoding gene (ITS-5.8S region), revealed T. soudanense as the most prevalent species, with only one case of T. rubrum. This is the first epidemiological data describing dermatophytoses and dermatophytes in Cape Verde among school-age children on one of the archipelago islands. It reinforces the need of using culture and accurate identification methodologies when gathering epidemiological data on dermatophytoses.

Thin microporous polydimethylsiloxane membrane prepared by phase separation and its applications for cell culture

Animal experiments are often required for biological studies. However, in vitro cell culture models, such as cell-culture inserts and microphysiological systems, can provide a suitable alternative, making them essential tools in cell biology research, including the simulation of an organ environments closely related to the human body. Cell-culture inserts with porous membranes assist in recreating in vivo cell culture environments to study and process cell-culture assays. However, conventional cell culture membranes typically made of polyethylene terephthalate or polycarbonate cannot accommodate cell types that require deformable substrates. As such, this paper introduced a novel approach using spin-casting-assisted polymer-blend phase separation to create thin, flexible, and highly porous membranes for cell culture applications. Polydimethylsiloxane (PDMS) was selected as the material for the porous membrane, and polystyrene (PS) was used as a counter pair to induce phase separation with PDMS. PDMS facilitated the necessary reversible deformations during cell culture owing to its low elastic modulus. The thickness of the membrane and connectivity of the phase-separated PS domains can be adjusted, facilitating the fine-tuning of the pore size and density to improve the membrane performance. Therefore, this study successfully fabricated thin microporous PDMS membranes with improved performance over standard membranes for cell-culture inserts, namely a higher porosity, flexibility, and softness. The results of this study can enhance cell culture methodologies and contribute to a deeper understanding of cellular processes.

Comparison of targeted next-generation sequencing and traditional microbial culture in the diagnosis of pulmonary infections

This study investigated the diagnostic potential of targeted next-generation sequencing (tNGS) for pulmonary infections. The positivity rate of tNGS was significantly higher than that of traditional microbial culture (92.6 % vs 25.2 %, χ2 = 378.272, P < 0.001). The proportion of two or more species of pathogens detected using tNGS exceeded that detected using microbial culture (χ2 = 337.283, P < 0.001). There were inconsistencies between the results of the tNGS antibiotic resistance gene and the drug susceptibility test resistance phenotype. The tNGS technique demonstrates rapid and effective capabilities in identifying bacteria, fungi, viruses, and specific pathogens, with a detection sensitivity that surpasses that of conventional culture methodologies. Microbial drug resistance genotypes detected by tNGS cannot accurately predict drug resistance phenotypes and require further improvement or integration with traditional microbial culture to establish a foundation for effective clinical treatment.

Challenges in Adopting Agile Methodologies in the Software Engineering Processes Management: a Literature Review

Objective: To identify and select the main challenges in adopting agile methodologies in the software engineering processes management, that is, to analyze the most recurring challenges involving the application of agile methodologies in organizational environments. Theoretical Framework: Software Development is marked by a dynamic environment in constant change, where organizations must experiment with new values, manifested in flexibility, adaptation, intelligence, innovation and proactivity, using innovative tools, as conditions to become efficient and competitive. Method: The research was conducted based on bibliographical review, whose strategy was the analysis of a group of authors who constituted the theoretical framework to substantiate and explain the categories of this study, centered on agile methodologies. Results and Discussion: The results revealed that organizations encounter obstacles of different natures to implement agile methodologies in their environment, making project development difficult, thus not having the changes considered with good receptivity among employees, given the ingrained values and culture, becoming challenges for the organization. Implications of the Research: Given this evidence, it is concluded that organizational culture has as its primary factor resistance to change, as being the organization's main challenge for the acceptance and implementation of agile methodologies, indicating that there is no compatibility between organizational culture and agile methodologies. Originality/Value: This study contributes to identifying factors that become challenges to the successful implementation of agile methodologies in organizations, such as changes in organizational culture.

Prevalence and Antibiotic Resistance of Ureaplasma urealyticum in Sperm Cultures: A Retrospective Analysis", Abidjan, Côte d’Ivoire

Aims: Mycoplasmas are bacteria of the urogenital tract often associated with various infections and infertility. In terms of treatment, acquired resistance to antibiotics has been reported. The aim of this study was to investigate changes in the prevalence and antibiotic resistance of Ureaplasma urealyticum strains isolated during sperm culture, in order to help update therapeutic protocols for the treatment of U. urealyticum-associated infections Place and Duration of Study: These were men received at the INHP from January 2018 to December 2021 for sperm culture Methodology: This is a retrospective study in which the culture results of 907 semen samples received in the laboratory between 2018 and 2021 were analysed. Identification, indicative counts and antibiotic susceptibility testing of U. urealyticum were performed using the Biosynex Mycoplasma kit. Results: 283 strains of U. urealyticum were isolated between 2018 and 2021, with an average prevalence of 31.2%. In 2018, only minocycline, josamycin and roxithromycin were active on all the strains tested, while fluoroquinolones and clindamycin showed high levels of resistance, with rates of 75.9% and 86.1% respectively. From 2019 onwards, resistance spread to all antibiotics, with increasing levels of resistance, with the exception of minocycline, which remained active on all strains. The highest resistance rates were observed in 2021 and were 74.6% and 96.6% for pristinamycin and clindamycin respectively. Conclusion: Despite the high level of resistance, minocycline and josamycin have shown relatively stable antibacterial activity and could be proposed as the molecules of choice for the treatment of U. urealyticum infections. Closer monitoring of the development of antibiotic resistance in these bacteria is therefore necessary.

Adipogenic differentiation of hematopoietic lineage cells isolated from adipose tissue of humans.

We previously discovered some adipocytes in the major white fat depots of mice and humans arise from bone marrow-derived cells of hematopoietic lineage rather than conventional mesenchymal precursors, termed bone marrow-derived adipocytes (BMDA). Here we aimed to determine if hematopoietic lineage cells isolated from adipose tissue and circulation of humans could undergo adipogenic differentiation in vitro, thereby establishing an in vitro model for studies of BMDA. We hypothesized that hematopoietic lineage cells isolated from adipose tissue, but not circulation, of humans would demonstrate adipogenic potential. Participants included younger (20-50 years) and older (>50-75 years) men and women, BMI 20-37 kg/m2. Subcutaneous abdominal adipose tissue biopsies were obtained and stromal cell populations identified by flow cytometry. Sorted cells underwent in vitro cultivation via traditional mesenchymal culture methodology (mesenchymal lineage) or a novel 3D-fibrin clot followed by traditional adherent culture (hematopoietic lineage) for assessment of proliferation and differentiation capacity. We found hematopoietic lineage cells isolated from the adipose tissue stroma, but not the circulation, were capable of proliferation and multilineage (adipogenic and osteogenic) differentiation in vitro. We provide a new investigative tool that can be used to perform translational studies of BMDAs and provide initial evidence that hematopoietic lineage cells isolated from the adipose tissue of humans can undergo hematopoietic-to-mesenchymal transition with multilineage differentiation potential in an in vitro environment.

Helicobacter pylori positive oral squamous cell carcinoma demonstrate higher pathological tumor staging and poorer overall survival

BackgroundHelicobacter pylori (H pylori), a bacterium characterized by its spiral shape and gram-negative nature, impacts approximately half of the global population, showing a greater prevalence in developing nations. There are various factors that contribute to the pathogenicity of H pylori in the gastric mucosa, leading to gastric ulcer, gastritis and gastric cancers. The relationship between H pylori and gastric cancers has been well documented. The association between Oral Squamous Cell Carcinoma (OSCC) and H pylori still remains a grey field. The study aimed to evaluate the presence of H pylori in OSCC. Materials and MethodsThe study consisted of 46 case samples and 21 controls. The case samples comprised of histopathologically confirmed cases of OSCC obtained from patients undergoing wide local excision. Fresh tissue samples were collected during cryosection and stored in eppendorf tubes. The control samples were collected from the gingiva and buccal mucosa of apparently healthy patients with no history of habits, undergoing procedures such as gingivectomy and impaction. All the cases and controls were subjected to immunohistochemistry for Helicobacter pylori antibody. The cases demonstrating Helicobacter pylori in immunohistochemistry further underwent additional Real-Time- Polymerase Chain Reaction (RT-PCR) and culture methodology for subsequent confirmation. Results15/46 cases (32.6 %) showed positive immunohistochemical expression of H pylori in OSCC, while all the twenty-one controls were negative (p value 0.001). Out of the 15 cases tested using culture methodology, a total of 7 cases, representing 46.7 % of the sample, were positive for the presence of H pylori (p- value 0.003). Similar statistically significant results were also obtained for 16S rRNA gene with RT- PCR. Furthermore, H pylori positive cases were frequently found in higher pathological tumor staging. A significant increase in overall survival rate was evident among the H pylori negative cases. ConclusionHelicobacter pylori was significantly expressed in OSCC tissues when compared to healthy tissues. Immunohistochemical analysis of the presence of H pylori in FFPE OSCC samples yielded more positive results when compared to culture and PCR methodology. We opine that in OSCC, H pylori may have a role in the faster progression of the disease, rather than merely a ‘chance spectator’.

The Transformative Role of 3D Culture Models in Triple-Negative Breast Cancer Research.

Advancements in cell culturing techniques have allowed the development of three-dimensional (3D) cell culture models sourced directly from patients' tissues and tumors, faithfully replicating the native tissue environment. These models provide a more clinically relevant platform for studying disease progression and treatment responses compared to traditional two-dimensional (2D) models. Patient-derived organoids (PDOs) and patient-derived xenograft organoids (PDXOs) emerge as innovative 3D cancer models capable of accurately mimicking the tumor's unique features, enhancing our understanding of tumor complexities, and predicting clinical outcomes. Triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive nature, propensity for early metastasis, and limited treatment options. TNBC PDOs and PDXOs have significantly contributed to the comprehension of TNBC, providing novel insights into its underlying mechanism and identifying potential therapeutic targets. This review explores the transformative role of various 3D cancer models in elucidating TNBC pathogenesis and guiding novel therapeutic strategies. It also provides an overview of diverse 3D cell culture models, derived from cell lines and tumors, highlighting their advantages and culturing challenges. Finally, it delves into live-cell imaging techniques, endpoint assays, and alternative cell culture media and methodologies, such as scaffold-free and scaffold-based systems, essential for advancing 3D cancer model research and development.

An advanced culture methodology suitable for the self-assemble and tissue-fragment derived intrahepatic cholangiocarcinoma organoids

An advanced culture methodology suitable for the self-assemble and tissue-fragment derived intrahepatic cholangiocarcinoma organoids

Phytosynthesis of silver nanoparticles using aqueous sandalwood (Santalum album L.) leaf extract: Divergent effects of SW-AgNPs on proliferating plant and cancer cells.

The biogenic approach for the synthesis of metal nanoparticles provides an efficient eco-friendly alternative to chemical synthesis. This study presents a novel route for the biosynthesis of silver nanoparticles using aqueous sandalwood (SW) leaf extract as a source of reducing and capping agents under mild, room temperature synthesis conditions. The bioreduction of Ag+ to Ago nanoparticles (SW-AgNPs) was accompanied by the appearance of brown color, with surface plasmon resonance peak at 340-360 nm. SEM, TEM and AFM imaging confirm SW-AgNP's spherical shape with size range of 10-32 nm. DLS indicates a hydrodynamic size of 49.53 nm with predominant negative Zeta potential, which can contribute to the stability of the nanoparticles. FTIR analysis indicates involvement of sandalwood leaf derived polyphenols, proteins and lipids in the reduction and capping of SW-AgNPs. XRD determines the face-centered-cubic crystalline structure of SW-AgNPs, which is a key factor affecting biological functions of nanoparticles. This study is novel in using cell culture methodologies to evaluate effects of SW-AgNPs on proliferating cells originating from plants and human cancer. Exposure of groundnut calli cells to SW-AgNPs, resulted in enhanced proliferation leading to over 70% higher calli biomass over control, enhanced defense enzyme activities, and secretion of metabolites implicated in biotic stress resistance (Crotonyl isothiocyanate, Butyrolactone, 2-Hydroxy-gamma-butyrolactone, Maltol) and plant cell proliferation (dl-Threitol). MTT and NRU were performed to determine the cytotoxicity of nanoparticles on human cervical cancer cells. SW-AgNPs specifically inhibited cervical cell lines SiHa (IC50-2.65 ppm) and CaSki (IC50-9.49 ppm), indicating potential use in cancer treatment. The opposing effect of SW-AgNPs on cell proliferation of plant calli (enhanced cell proliferation) and human cancer cell lines (inhibition) are both beneficial and point to potential safe application of SW-AgNPs in plant cell culture, agriculture and in cancer treatment.

Impact of microglia isolation and culture methodology on transcriptional profile and function.

Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate five different microglia isolation methods-three variants of microglia isolation from neonatal mice and two variants of microglia isolation from adult mice-on transcriptional profile and response to HMGB1. Microglia from neonatal mice, age 0-3days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8weeks included isolation by adherence properties followed by 7days of culture with 100ng/mL GM-CSF and 100ng/mL M-CSF (Vijaya et al. in Front Cell Neurosci 17:1082180, 2023), or acute isolation using CD11b beads (Bordt et al. in STAR Protoc 1:100035, 2020. https://doi.org/10.1016/j.xpro.2020.100035 ). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1β, and IFNβ concentration in supernatant by ELISA and assessing gene expression patterns using bulk mRNA sequencing. All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription and secretion of TNFα, IL1β, and IFNβ in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from neonates than from adult mice, with sparse changes among the three MGC culturing conditions. Additionally, cultured microglia derived from adult and microglia derived from MGCs from neonates display transcriptional signatures corresponding to an earlier developmental stage. These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

Deep Learning Powered Identification of Differentiated Early Mesoderm Cells from Pluripotent Stem Cells.

Pluripotent stem cells can be differentiated into all three germ-layers including ecto-, endo-, and mesoderm in vitro. However, the early identification and rapid characterization of each germ-layer in response to chemical and physical induction of differentiation is limited. This is a long-standing issue for rapid and high-throughput screening to determine lineage specification efficiency. Here, we present deep learning (DL) methodologies for predicting and classifying early mesoderm cells differentiated from embryoid bodies (EBs) based on cellular and nuclear morphologies. Using a transgenic murine embryonic stem cell (mESC) line, namely OGTR1, we validated the upregulation of mesodermal genes (Brachyury (T): DsRed) in cells derived from EBs for the deep learning model training. Cells were classified into mesodermal and non-mesodermal (representing endo- and ectoderm) classes using a convolutional neural network (CNN) model called InceptionV3 which achieved a very high classification accuracy of 97% for phase images and 90% for nuclei images. In addition, we also performed image segmentation using an Attention U-Net CNN and obtained a mean intersection over union of 61% and 69% for phase-contrast and nuclear images, respectively. This work highlights the potential of integrating cell culture, imaging technologies, and deep learning methodologies in identifying lineage specification, thus contributing to the advancements in regenerative medicine. Collectively, our trained deep learning models can predict the mesoderm cells with high accuracy based on cellular and nuclear morphologies.

A Review on Flower Bulb Micropropagation: Challenges and Opportunities

This comprehensive review scrutinizes tissue culture and micropropagation methodologies in geophytes, focusing on bulbous plants. The examination encompasses key stages, including somatic embryogenesis, bulb growth, dormancy breaking, and planting. Studies underscore the pivotal role of plant growth regulators (PGRs) in plant regeneration and bulb growth. Bioreactor systems for healthy plant regeneration, rooting methods, acclimatization strategies, and considerations for ex vitro survival are elucidated. The review also delves into somaclonal variation dynamics and acknowledges the burgeoning field of gene editing, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) studies, as a promising avenue for enhancing valuable compound content in geophytes. In addition to addressing challenges in flower bulb micropropagation, this review briefly highlights emerging opportunities, including the potential integration of artificial intelligence (AI) to optimize culture conditions, predict growth parameters, and enhance efficiency in bulb production. The conclusion emphasizes the necessity of a multifaceted approach integrating biochemistry, physiology, and molecular biology to address existing challenges and improve tissue culture protocols for diverse geophyte species. This review article also intends to highlight how tissue culture techniques could contribute to the development and valorization of flower bulbs in today’s scenario of the ornamental industry.

Lab-grown insect meat – Chemical and biological insights – A comprehensive review

Abstract Lab-grown insect meat is a promising alternative to traditional livestock for sustainable food production. This review paper aims to provide a comprehensive overview of the current state of knowledge regarding lab-grown insect meat, emphasizing key aspects such as life cycle assessment, insect cell culture history, technological advancements, and bio-robotics in insect cell culture. Comparisons and challenges between insect and mammalian/avian cell culture methodologies are presented. The nutritional content of edible insects (proximate, amino acid, mineral, and vitamin content) and the potential health benefits of consuming insect meat are discussed. The paper also explores embryonic and adult myogenesis processes in insect cells, as well as the significance of insect body fat and muscle cells in culture. Applications of insect cell culture in various fields, such as food production and pharmaceutical development, are presented. Moreover, the potential occurrence of mutations in lab-grown insect cells is examined. Lastly, the review addresses the drawbacks and limitations of insect labriculture, discussing factors such as scalability, cost-efficiency, and public acceptance. Overall, this comprehensive review provides essential insights into the chemical and biological aspects of lab-grown insect meat, paving the way for further research and development in this emerging field. This article is the first review article reporting the chemical and biological insights of lab-grown insect meat.

Yeast Biodiversity of Karst Waters: Interest of Four Culture Media and an Improved MALDI-TOF MS Database

Karst aquifers are a significant source of drinking water and highly vulnerable to pollution and microbial contamination. Microbiological regulations for the quality of drinking water mostly focus on bacterial levels and lack guidance concerning fungal contamination. Moreover, there is no standardised microbial analysis methodology for identifying fungi in water. Our main objective was to establish the most effective culture and identification methodology to examine yeast diversity in karst waters. We assessed the comparative efficacy of four culture media (CHROMagar Candida, dichloran glycerol 18% [DG18], dichloran rose Bengal chloramphenicol [DRBC], and SYMPHONY agar) for yeast isolation from karst water samples. Furthermore, we investigated the comprehensiveness of databases used in MALDI-TOF mass spectrometry (MALDI-TOF MS) for identifying environmental yeast species. In total, we analysed 162 water samples, allowing the identification of 2479 yeast isolates. We demonstrate that a combination of four culture media, each with distinct specifications, more efficiently covers a wide range of yeast species in karst water than a combination of only two or three. Supplementation of a MALDI-TOF MS database is also critical for analysing environmental microbial samples and improved the identification of yeast biodiversity. This study is an initial step towards standardising the analysis of fungal biodiversity in karst waters, enabling a better understanding of the significance of this environmental reservoir in relation to public health.