ABSTRACT
Per- and polyfluoroalkyl substances (PFAS), ubiquitous environmental contaminants, pose significant challenges to ecosystems and human health. While cell cultures have emerged as new approach methodologies (NAMs) in ecotoxicity research, metabolomics is an emerging technique used to characterize the small-molecule metabolites present in cells and to understand their role in various biological processes. Integration of metabolomics with cell cultures, known as cell culture metabolomics, provides a novel and robust tool to unravel the complex molecular responses induced by PFAS exposure. In vitro testing also reduces reliance on animal testing, aligning with ethical and regulatory imperatives. The current review summarizes key findings from recent studies utilizing cell culture metabolomics to investigate PFAS toxicity, highlighting alterations in metabolic pathways, biomarker identification, and the potential linkages between metabolic perturbations. Additionally, the paper discusses different types of cell cultures and metabolomics methods used for studies of environmental contaminants and particularly PFAS. Future perspectives on the combination of metabolomics with other advanced technologies, such as single-cell metabolomics (SCM), imaging mass spectrometry (IMS), extracellular flux analysis (EFA), and multi-omics are also explored, which offers a holistic understanding of environmental contaminants. The synthesis of current knowledge and identification of research gaps provide a foundation for future investigations that aim to elucidate the complexities of PFAS-induced cellular responses and contribute to the development of effective strategies for mitigating their adverse effects on human health.
Subject(s)
Environmental Pollutants , Fluorocarbons , Metabolomics , Humans , Fluorocarbons/toxicity , Fluorocarbons/metabolism , Environmental Pollutants/toxicity , Cell Culture Techniques/methods , AnimalsABSTRACT
Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.
Subject(s)
Oligochaeta , Serum Albumin, Bovine , Humans , Animals , Culture Media, Serum-Free , Culture Media/chemistry , Hot Temperature , Cell Culture Techniques/methods , HeLa Cells , Vitamins , Cells, CulturedABSTRACT
It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.
Subject(s)
Rhodobacter sphaeroides , Rhodobacter sphaeroides/metabolism , Serum Albumin, Bovine/metabolism , Cell Culture Techniques/methods , Dietary Supplements , Amino Acids/metabolism , Arginine/metabolism , Glycine/metabolismABSTRACT
Plant stem cell cultures have so far been established in only a few plant species using cambial meristematic cells. The presence of stem cells or stem cell-like cells in other organs and tissues of the plant body, as well as the possibility of de novo generation of meristematic cells from differentiated cells, allow to consider the establishment of stem cell cultures in a broader range of species. This study aimed to establish a stem cell culture of the medicinal plant Calendula officinalis L. Callus tissues were induced from leaf and root explants, and already at this stage, stem and dedifferentiated cells could be identified. The cell suspension cultures established both from the root- and leaf-derived calli contained a high proportion of stem cells (92-93% and 72-73%, respectively). The most effective combination of growth regulators for the development of stem cells in calli as well as cell cultures was 1.0 mg/L 2,4-D and 0.5 mg/L BAP. The highest amount of stem cells (5.60-5.72 × 105) was in cell suspension derived from the roots. An effective protocol for the establishment of marigold stem cell suspension culture was developed. The ratio of root-derived stem cells against dedifferentiated cells exceeded 90%.
Subject(s)
Calendula , Plants, Medicinal , Cell Culture Techniques/methods , Plant Leaves , Stem CellsABSTRACT
3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and downregulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions.
Subject(s)
Prostatic Neoplasms , Spheroids, Cellular , Humans , Male , Cell Culture Techniques/methods , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Drug Evaluation, PreclinicalABSTRACT
The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix-cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.
Subject(s)
Cell Culture Techniques , Ovarian Neoplasms , Female , Humans , Cell Culture Techniques/methods , Reproducibility of Results , Drug Evaluation, Preclinical/methods , Drug Discovery , Organoids , Ovarian Neoplasms/pathology , High-Throughput Screening Assays/methodsABSTRACT
Drug development and testing are a tedious and expensive process with a high degree of uncertainty in the clinical success and preclinical validation of manufactured therapeutic agents. Currently, to understand the drug action, disease mechanism, and drug testing, most therapeutic drug manufacturers use 2D cell culture models to validate the drug action. However, there are many uncertainties and limitations with the conventional use of 2D (monolayer) cell culture models for drug testing that are primarily attributed due to poor mimicking of cellular mechanisms, disturbance in environmental interaction, and changes in structural morphology. To overcome such odds and difficulties in the preclinical validation of therapeutic medications, newer in vivo drug testing cell culture models with higher screening efficiencies are required. One such promising and advanced cell culture model reported recently is the "three-dimensional cell culture model." The 3D cell culture models are reported to show evident benefits over conventional 2D cell models. This review article outlines and describes the current advancement in cell culture models, their types, significance in high-throughput screening, limitations, applications in drug toxicity screening, and preclinical testing methodologies to predict in vivo efficacy.
Subject(s)
Cell Culture Techniques , High-Throughput Screening Assays , Drug Evaluation, Preclinical/methods , Cell Culture Techniques/methods , High-Throughput Screening Assays/methods , Cell Culture Techniques, Three Dimensional , Drug DevelopmentABSTRACT
Obesity, and its consequences for human health, is a huge and complicated problem that has no simple solution. The constant search for natural and safe compounds with systemic action that can be used for obesity prophylactics and treatment is hampered by the limited availability and variable quality of biomass of wild medicinal plants. Plant cell biotechnology is an alternative approach for the sustainable production of vegetative biomass or individual phytochemicals with high therapeutic potential. In this study, the suspension cell biomass of the medicinal plants, Dioscorea deltoidea Wall., Tribulus terrestris L., and Panax japonicus (T. Nees) C.A. Mey, produced in 20 L and 630 L bioreactors, were tested for therapeutic effects in rat models with alimentary-induced obesity. Three-month intake of water infusions of dry cell biomass (100 mg/g body weight) against the background of a hypercaloric diet reduced weight gain and the proportion of fat mass in the obese animals. In addition, cell biomass preparation reduced the intracellular dehydration and balanced the amounts of intra- and extracellular fluids in the body as determined by bioimpedance spectroscopy. A significant decrease in the glucose and cholesterol levels in the blood was also observed as a result of cell biomass administration for all species. Hypocholesterolemic activity reduced in the line P. japonicus > D. deltoidea > T. terrestris/liraglutide > intact group > control group. By the sum of parameters tested, the cell culture of D. deltoidea was considered the most effective in mitigating diet-induced obesity, with positive effects sometimes exceeding those of the reference drug liraglutide. A safety assessment of D. deltoidea cell phytopreparation showed no toxic effect on the reproductive function of the animals and their offspring. These results support the potential application of the biotechnologically produced cell biomass of medicinal plant species as safe and effective natural remedies for the treatment of obesity and related complications, particularly for the long-term treatment and during pregnancy and lactation periods when conventional treatment is often contraindicated.
Subject(s)
Dioscorea , Lipid Metabolism Disorders , Panax , Plants, Medicinal , Tribulus , Humans , Female , Rats , Animals , Diet, High-Fat/adverse effects , Dioscorea/chemistry , Hypoglycemic Agents/pharmacology , Tribulus/chemistry , Biomass , Liraglutide , Plant Extracts/pharmacology , Plant Extracts/chemistry , Cell Culture Techniques/methods , Plants, Medicinal/chemistry , Obesity/drug therapyABSTRACT
Three-dimensional (3D) culture platforms have been adopted in a high-throughput screening (HTS) system to mimic in vivo physiological microenvironments. The automated dispenser has been established commercially to enable spotting or distributing non-viscous or viscous biomaterials onto microplates. However, there are still challenges to the precise and accurate dispensation of cells embedded in hydrogels such as Alginate- and Matrigel-extracellular matrices. We developed and improved an automated contact-free dispensing machine, the ASFA SPOTTER (V5 and V6), which is compatible with 96- and 384-pillar/well plates and 330- and 532-micropillar/well chips for the support of 3D spheroid/organoid models using bioprinting techniques. This enables the distribution of non-viscous and viscous biosamples, including chemical drugs and cancer cells, for large-scale drug screening at high speed and small volumes (20 to 4000 nanoliters) with no damage to cells. The ASFA SPOTTER (V5 and V6) utilizes a contact-free method that minimizes cross-contamination for the dispensation of encapsulated tissue cells with highly viscous scaffolds (over 70%). In particular, the SPOTTER V6 does not require a washing process and offers the advantage of almost no dead volume (defined as additional required sample volume, including a pre-shot and flushing shot for dispensing). It can be successfully applied for the achievement of an organoid culture in automation, with rapid and easy operation, as well as miniaturization for high-throughput screening. In this study, we report the advantages of the ASFA SPOTTER, which distributes standard-sized cell spots with hydrogels onto a 384-pillar/well plate with a fast dispensing speed, small-scale volume, accuracy, and precision.
Subject(s)
High-Throughput Screening Assays , Neoplasms , Humans , High-Throughput Screening Assays/methods , Cell Culture Techniques/methods , Drug Evaluation, Preclinical/methods , Hydrogels , Spheroids, Cellular , Tumor MicroenvironmentABSTRACT
The discrepancies between the findings in preclinical studies, and in vivo testing and clinical trials have resulted in the gradual decline in drug approval rates over the past decades. Conventional in vitro drug screening platforms employ two-dimensional (2D) cell culture models, which demonstrate inaccurate drug responses by failing to capture the three-dimensional (3D) tissue microenvironment in vivo. Recent advancements in the field of tissue engineering have made possible the creation of 3D cell culture systems that can accurately recapitulate the cell-cell and cell-extracellular matrix interactions, as well as replicate the intricate microarchitectures observed in native tissues. However, the lack of a perfusion system in 3D cell cultures hinders the establishment of the models as potential drug screening platforms. Over the years, multiple techniques have successfully demonstrated vascularization in 3D cell cultures, simulating in vivo-like drug interactions, proposing the use of 3D systems as drug screening platforms to eliminate the deviations between preclinical and in vivo testing. In this review, the basic principles of 3D cell culture systems are briefly introduced, and current research demonstrating the development of vascularization in 3D cell cultures is discussed, with a particular focus on the potential of these models as the future of drug screening platforms.
Subject(s)
Bioprinting , Bioprinting/methods , Cell Culture Techniques/methods , Tissue Engineering , Drug Evaluation, Preclinical/methods , Cell Culture Techniques, Three DimensionalABSTRACT
The neuroglial extracellular matrix (ECM) provides critical support and physiological cues for the proper growth, differentiation, and function of neuronal cells in the brain. However, in most in vitro settings that study neural physiology, cells are grown as monolayers on stiff surfaces that maximize adhesion and proliferation, and, therefore, they lack the physiological cues that ECM in native neuronal tissues provides. Macromolecular crowding (MMC) is a biophysical phenomenon based on the principle of excluded volume that can be harnessed to induce native ECM deposition by cells in culture. Here, we show that MMC using two species of Ficoll with vitamin C supplementation significantly boosts deposition of relevant brain ECM by cultured human astrocytes. Dopaminergic neurons cocultured on this astrocyte-ECM bed prepared under MMC treatment showed longer and denser neuronal extensions, a higher number of pre ad post synaptic contacts, and increased physiological activity, as evidenced by higher frequency calcium oscillation, compared to standard coculture conditions. When the pharmacological activity of various compounds was tested on MMC-treated cocultures, their responses were enhanced, and for apomorphine, a D2-receptor agonist, it was inverted in comparison to control cell culture conditions, thus emulating responses observed in in vivo settings. These results indicate that macromolecular crowding can harness the ECM-building potential of human astrocytes in vitro forming an ultra-flat 3D microenvironment that makes neural cultures more physiological and pharmacological relevant.
Subject(s)
Cell Culture Techniques , Extracellular Matrix , Cell Culture Techniques/methods , Cell Differentiation , Coculture Techniques , Humans , Macromolecular SubstancesABSTRACT
Classic preclinical investigations on the mechanisms and effects of photodynamic therapy (PDT) are typically performed in two-dimensional cell cultures that have some, albeit limited, relevance to cancer biology. Bioengineered three-dimensional (3D) culture models of cancer are gaining traction in translational oncology as microtumors recapitulate the tumor architectures and cellular heterogeneity more faithfully than conventional 2D cultures. These 3D models bridge a gap between highly relevant but low-throughput in vivo animal models and high-throughput two-dimensional cultures with low clinical relevance, and thus hold promise as preclinical testing platforms in PDT research. Here, we discuss the potential applications of organotypic cancer models for PDT research and provide two well-established methodologies for generating 3D cultures of cancer: a liquid-suspended spheroid model and an adherent microtumor culture model grown on extracellular matrix scaffolds. Particular emphasis is given to harvesting the cultures for the purpose of immunoblotting and flow cytometry.
Subject(s)
Neoplasms , Photochemotherapy , Animals , Cell Culture Techniques/methods , Extracellular Matrix , Neoplasms/drug therapyABSTRACT
INTRODUCTION: Colorectal cancer (CRC) is one of the most common and deadly tumors worldwide. CRC in vitro and in vivo models that recapitulate key features of human disease are essential to the development of novel and effective therapeutics. However, two-dimensional (2D) in vitro culture systems are considered too simple and do not represent the complex nature of the human tumor. However, three-dimensional (3D) models have emerged in recent years as more advanced and complex cell culture systems, able to closely resemble key features of human cancer tissues. AREAS COVERED: The authors' review the currently established in vitro cell culture models and describe the advances in the development of 3D scaffold-free models to study CRC. The authors also discuss intestinal spheroids and organoids. As well as in vitro models for drug screening and metastatic CRC (mCRC). EXPERT OPINION: The ideal CRC in vitro model is not yet established. Spheroid-based 3D models represent one of the most used approaches to recapitulate the tumor environment, overcoming some limitations of 2D models. Mouse and patient-derived organoids are more advanced models that can mimic more closely the characteristics and properties of CRC, with the possibility of including cells derived from patients with metastatic CRC.
Subject(s)
Colorectal Neoplasms , Organoids , Animals , Cell Culture Techniques/methods , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Drug Discovery/methods , Drug Evaluation, Preclinical , Humans , Mice , Spheroids, CellularABSTRACT
Intact (whole) cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) is an established method for biotyping in clinical microbiology as well as for revealing phenotypic shifts in cultured eukaryotic cells. Intact cell MALDI-TOF MS has recently been introduced as a quality control tool for long-term cultures of pluripotent stem cells. Despite the potential this method holds for revealing minute changes in cells, there is still a need for improving the ionization efficiency or peak reproducibility. Here we report for the first time that supplementation by fine particles of black phosphorus to the standard MALDI matrices, such as sinapinic and α-cyano-4-hydroxycinnamic acids enhance intensities of mass spectra of particular amino acids and peptides, presumably by interactions with aromatic groups within the molecules. In addition, the particles of black phosphorus induce the formation of small and regularly dispersed crystals of sinapinic acid and α-cyano-4-hydroxycinnamic acid with the analyte on a steel MALDI target plate. Patterns of mass spectra recorded from intact cells using black phosphorus-enriched matrix were more reproducible and contained peaks of higher intensities when compared to matrix without black phosphorus supplementation. In summary, enrichment of common organic matrices by black phosphorus can improve discrimination data analysis by enhancing peak intensity and reproducibility of mass spectra acquired from intact cells.
Subject(s)
Phosphorus/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Amino Acids/analysis , Amino Acids/chemistry , Cell Culture Techniques/methods , Cell Line , Human Embryonic Stem Cells , Humans , Peptides/analysis , Peptides/chemistry , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standardsABSTRACT
In order to overcome the weakness of conventional approaches for cell culture, and provide cells with more in vivo-like microenvironment for studying hepatotoxicity of drugs, "multiple-in-one" strategy was adopted to fabricate a 3D scaffold of silk fibroin/hydroxyapatite/poly lacticco-glycolic acid (SF/HA/PLGA), where HepG2 cells were cultivated and the toxicity of drugs to the cells was investigated. The prepared 3D scaffold proves to bear proper porosity, excellent mechanical property, steady pH environment and good biocompatibility for cell culture. Furthermore, the validity of the developed 3D-SF/HA/PLGA-scaffold based platform was verified by probing the toxicity of a known drug-induced liver injury (DILI) concern acetaminophen (APAP) to HepG2 cells. Eventually, an application of the platform to dioscin (a medicinal plant extract) reveals the hepatotoxicity of dioscin, which involves the inhibition of the expression of CYP3A4 mRNA in the cells. The developed 3D-SF/HA/PLGA-scaffold platform may become a universal avenue for safety evaluation of drugs.
Subject(s)
Acetaminophen/toxicity , Cell Culture Techniques/methods , Composite Resins/chemistry , Drug Evaluation, Preclinical/methods , Tissue Scaffolds/chemistry , Cell Differentiation , Cell Proliferation , Chemical and Drug Induced Liver Injury , Cytochrome P-450 Enzyme System/metabolism , Diosgenin/analogs & derivatives , Diosgenin/toxicity , Glucose/metabolism , Hep G2 Cells , Humans , Pharmaceutical PreparationsABSTRACT
Human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs), hold great promise for drug discovery, toxicology studies, and regenerative medicine. Here, we describe standardized protocols and experimental procedures that combine automated cell culture for scalable production of hPSCs with quantitative high-throughput screening (qHTS) in miniaturized 384-well plates. As a proof of principle, we established dose-response assessments and determined optimal concentrations of 12 small molecule compounds that are commonly used in the stem cell field. Multi-parametric analysis of readouts from diverse assays including cell viability, mitochondrial membrane potential, plasma membrane integrity, and ATP production was used to distinguish normal biological responses from cellular stress induced by small molecule treatment. Collectively, the establishment of integrated workflows for cell manufacturing, qHTS, high-content imaging, and data analysis provides an end-to-end platform for industrial-scale projects and should leverage the drug discovery process using hPSC-derived cell types.
Subject(s)
Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Cell Culture Techniques/methods , Cell Differentiation/physiology , Drug Evaluation, Preclinical , High-Throughput Screening Assays/methods , HumansABSTRACT
Three-dimensional (3D) culture systems opened up new horizons in studying the biology of tissues and organs, modelling various diseases, and screening drugs. Producing accurate in vitro models increases the possibilities for studying molecular control of cell-cell and cell-microenvironment interactions in detail. The Notch signalling is linked to cell fate determination, tissue definition, and maintenance in both physiological and pathological conditions. Hence, 3D cultures provide new accessible platforms for studying activation and modulation of the Notch pathway. In this review, we provide an overview of the recent advances in different 3D culture systems, including spheroids, organoids, and "organ-on-a-chip" models, and their use in analysing the crucial role of Notch signalling in the maintenance of tissue homeostasis, pathology, and regeneration.
Subject(s)
Cell Culture Techniques/methods , Drug Evaluation, Preclinical , Receptors, Notch/genetics , Humans , Microfluidics/methods , Organoids/cytology , Signal Transduction/genetics , Spheroids, Cellular/cytologyABSTRACT
Organoids have complex three-dimensional structures that exhibit functionalities and feature architectures similar to those of in vivo organs and are developed from adult stem cells, embryonic stem cells, and pluripotent stem cells through a self-organization process. Organoids derived from adult epithelial stem cells are the most mature and extensive. In recent years, using organoid culture techniques, researchers have established various adult human tissue-derived epithelial organoids, including intestinal, colon, lung, liver, stomach, breast, and oral mucosal organoids, all of which exhibit strong research and application prospects. Studies have shown that epithelial organoids are mainly applied in drug discovery, personalized drug response testing, disease mechanism research, and regenerative medicine. In this review, we mainly discuss current organoid culture systems and potential applications of this technique with human epithelial tissue.
Subject(s)
Adult Stem Cells/cytology , Cell Culture Techniques/methods , Drug Evaluation, Preclinical/methods , Epithelial Cells/cytology , Organoids , Cell Culture Techniques/trends , Drug Evaluation, Preclinical/trends , HumansABSTRACT
Transitional cell carcinoma (TCC) is the most common malignant tumor of the canine urinary tract and tends to have a poor prognosis due to its invasive potential. Recent studies have reported that up to 80% of canine urothelial carcinoma has the BRAF V595E mutation, which is homologous to the human V600E mutation. Activating the BRAF mutation is an actionable target for developing effective therapeutic agents inhibiting the BRAF/mitogen-activated protein kinase (MAPK) pathway in canine cancer as well as human cancer. We established novel canine TCC cell lines from two tumor tissues and one metastatic lymph node of canine TCC patients harboring the BRAF V595E mutation. Tumor tissues highly expressed the BRAF mutant and phosphorylated extracellular signal-related kinases (ERK)1/2 proteins. The derived cell lines demonstrated activated MAPK pathways. We also evaluated the cell lines for sensitivity to BRAF inhibitors. Sorafenib, a multiple kinase inhibitor targeting RAF/vascular endothelial growth factor receptor (VEGFR), successfully inhibited the BRAF/MAPK pathway and induced apoptosis. The established canine TCC cell lines responded with greater sensitivity to sorafenib than to vemurafenib, which is known as a specific BRAF inhibitor in human cancer. Our results demonstrated that canine TCC cells showed different responses compared to human cancer with the BRAF V600E mutation. These cell lines would be valuable research materials to develop therapeutic strategies for canine TCC patients.
Subject(s)
Carcinoma, Transitional Cell/veterinary , Cell Culture Techniques/veterinary , Proto-Oncogene Proteins B-raf/genetics , Urologic Neoplasms/veterinary , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Transitional Cell/drug therapy , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/pathology , Cell Culture Techniques/methods , Cells, Cultured , Dogs , Female , Mice , Mutation , Sorafenib/therapeutic use , Urologic Neoplasms/drug therapy , Urologic Neoplasms/genetics , Urologic Neoplasms/pathology , Xenograft Model Antitumor Assays/methodsABSTRACT
Plasmacytoid dendritic cells (pDCs) constitute a rare type of immune cell with multifaceted functions, but their potential use as a cell-based immunotherapy is challenged by the scarce cell numbers that can be extracted from blood. Here, we systematically investigate culture parameters for generating pDCs from hematopoietic stem and progenitor cells (HSPCs). Using optimized conditions combined with implementation of HSPC pre-expansion, we generate an average of 465 million HSPC-derived pDCs (HSPC-pDCs) starting from 100,000 cord blood-derived HSPCs. Furthermore, we demonstrate that such protocol allows HSPC-pDC generation from whole-blood HSPCs, and these cells display a pDC phenotype and function. Using GMP-compliant medium, we observe a remarkable loss of TLR7/9 responses, which is rescued by ascorbic acid supplementation. Ascorbic acid induces transcriptional signatures associated with pDC-specific innate immune pathways, suggesting an undescribed role of ascorbic acid for pDC functionality. This constitutes the first protocol for generating pDCs from whole blood and lays the foundation for investigating HSPC-pDCs for cell-based immunotherapy.