Isolation of pure primary term human cytotrophoblasts and their differentiation into syncytiotrophoblast-like cells as an ex vivo model of the human placenta.

The placenta plays a fundamental role in fetal growth and maintenance of pregnancy. Its cellular components include a large multinucleated syncytiotrophoblast (STB) and its progenitor, cytotrophoblasts (CTBs), both of which perform vital functions in the human placenta. Primary cytotrophoblasts isolated from term human placentas that spontaneously fuse and differentiate into syncytiotrophoblast-like cells in vitro have been utilized to investigate the functions of the syncytiotrophoblast and placenta with multiple modifications. Although recent advances have enabled the use of trophoblast stem cell-derived organoids as a model for villous trophoblasts, primary CTBs offer several advantages, including spontaneous differentiation, easy access to materials (from term-delivered human placentas), and simple methodology. Here, we present a precise step-by-step process for isolating pure CTBs from term human placenta based on previously reported placenta digestion, density centrifugation, and CTB purification using anti-HLA-A, B, C antibody. Subsequently, we provide a method to improve CTB viability and differentiation into STB-like cells using epidermal growth factor (EGF) and a ROCK inhibitor (Y-27632) that ensures long-term and stable cultures without altering their proliferation. Because these cells can grow on standard tissue culture plates, this model can be easily utilized for various placental investigations, including innate immune responses, drug resistance, and STB metabolism. Employing this approach considerably enhances our understanding of placental functions, which are key to maternal and offspring health.

Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5 nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.

Generation of Bidimensional and Three-Dimensional Muscle Culture Systems.

Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.

Comprehensive identification, isolation, and culture of human breast cell types.

Tissues are formed and shaped by cells of many different types and are orchestrated through countless interactions. Deciphering a tissue's biological complexity thus requires studying it at cell-level resolution, where molecular and biochemical features of different cell types can be explored and thoroughly dissected. Unfortunately, the lack of comprehensive methods to identify, isolate, and culture each cell type from many tissues has impeded progress. Here, we present a method for the breadth of cell types composing the human breast. Our goal has long been to understand the essence of each of these different breast cell types, to reveal the underlying biology explaining their intrinsic features, the consequences of interactions, and their contributions to the tissue. This biological exploration has required cell purification, deep-RNA sequencing-and a thorough dissection of the genes and pathways defining each cell type. Whereas the molecular analysis is presented in an adjoining article, we present here an exhaustive cellular dissection of the human breast and explore its cellular composition and histological organization. Moreover, we introduce a novel FACS antibody panel and rigorous gating strategy capable of isolating each of the twelve major breast cell types to purity. Finally, we describe the creation of primary cell models from nearly every breast cell type-some the first of their kind- and submit these as critical tools for studying the dynamic cellular interactions within breast tissues and tumors. Together, this body of work delivers a unique perspective of the breast, revealing insights into its cellular, molecular, and biochemical composition.

MC4R Localizes at Excitatory Postsynaptic and Peri-Postsynaptic Sites of Hypothalamic Neurons in Primary Culture.

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites.

In vitro impact of Bisphenol A on the immune functions of primary cultured hemocytes of Pacific abalone (Haliotis discus hannai).

This study investigated the toxic effects of Bisphenol A (BPA) on the Pacific abalone (Haliotis discus hannai) using in vitro assays with primary cultured hemocytes. The abalone hemocytes were exposed to BPA concentrations up to 100 µM to assess cytotoxicity. Subsequently, hemocytes were exposed to sublethal BPA concentrations (LC20 = 2.3 µM and LC50 = 5.8 µM) for 48 h, and we evaluated the cellular immune responses of hemocytes via flow cytometry. Results showed no significant differences between LC20 and control groups, but LC50 exposure significantly reduced phagocytosis and oxidative capacities while increasing nitric oxide production. These findings suggest that BPA exposure negatively affects the immune system of the Pacific abalone, which makes them more susceptible to infections and other stressors in their natural environment. The study also implies that in vitro assays utilizing primary cultured abalone hemocytes may serve as effective proxies for quantifying the cytotoxic effects of chemical pollutants.

Pre-clinical phaeochromocytoma and paraganglioma models: Cell lines, animal models, and a human primary culture model.

While the establishment of human phaeochromocytoma and paraganglioma (PPGL) cell lines has proven to be particularly difficult over several decades of research, there are other reliable pre-clinical PPGL models currently available. This review provides a summary of these models, together with our recently established personalised drug screening platform using patient-derived PPGL primary cultures. Such currently available PPGL models include murine and rat PPGL cell lines, of which only one cell line (PC12) is publicly accessible through a cell repository, and PPGL animal models, of which the patient-derived xenograft models are promising but complex to establish. We have developed next-generation implementation of human PPGL primary cultures, enabling reliable and personalised drug screening and an individualised analysis of tumour drug responsivity based on the tumour's unique genetic, biochemical, immunohistochemical and clinical profile. Overall, reliable PPGL models, including patient-derived primary culture models, are essential to advance pre-clinical research in the field of PPGLs.

Adenosine Stimulates Beating of Neonatal Brain-Derived Cilia through Adenosine A2B Receptor on the Cilia and Activation of Protein Kinase A Pathway.

Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the substances and pathways which regulate their beating have not been well studied. Here, we used primary cultured cells derived from neonatal mouse brain that possess motile cilia and found that adenosine (ADO) stimulates ciliary beating by increasing the ciliary beat frequency (CBF) in a concentration-dependent manner, with the ED50 value being 5 µM. Ciliary beating stimulated by ADO was inhibited by A2B receptor (A2BR) antagonist MRS1754 without any inhibition by antagonists of other ADO receptor subtypes. The expression of A2BR on the cilia was also confirmed by immunofluorescence. The values of CBF were also increased by forskolin, which is an activator of adenylate cyclase, whereas they were not further increased by the addition of ADO. Furthermore, ciliary beating was not stimulated by ADO in the presence of a protein kinase A (PKA) inhibitors. These results altogether suggest that ADO stimulates ciliary beating through A2BR on the cilia, and activation of PKA.

Potential Role of Pig UCP3 in Modulating Adipocyte Browning via the Beta-Adrenergic Receptor Signaling Pathway.

Adipose tissue plays an important role in regulating body temperature and metabolism, with white adipocytes serving as storage units for energy. Recent research focused on the browning of white adipocytes (beige adipocytes), causing thermogenesis and lipolysis. The process of browning is linked to the activation of uncoupling protein (UCP) expression, which can be mediated by the ß3 adrenergic receptor pathway. Transcriptional factors, such as peroxisome proliferator activated receptor γ (PPARγ) and PPARγ coactivator 1 alpha, play vital roles in cell fate determination for fat cells. Beige adipocytes have metabolic therapeutic potential to combat diseases such as obesity, diabetes mellitus, and dyslipidemia, owing to their significant impact on metabolic functions. However, the molecular mechanisms that cause the induction of browning are unclear. Therefore, research using animal models and primary culture is essential to provide an understanding of browning for further application in human metabolic studies. Pigs have physiological similarities to humans; hence, they are valuable models for research on adipose tissue. This study demonstrates the browning potential of pig white adipocytes through primary culture experiments. The results show that upregulation of UCP3 gene expression and fragmentation of lipid droplets into smaller particles occur due to isoproterenol stimulation, which activates beta-adrenergic receptor signaling. Furthermore, PPARγ and PGC-1α were found to activate the UCP3 promoter region, similar to that of UCP1. These findings suggest that pigs undergo metabolic changes that induce browning in white adipocytes, providing a promising approach for metabolic research with potential implications for human health. This study offers valuable insights into the mechanism of adipocyte browning using pig primary culture that can enhance our understanding of human metabolism, leading to cures for commonly occurring diseases.

Modulation of Apoptosis by Bovine Gammaherpesvirus 4 Infection in Bovine Endometrial Cells and the Possible Role of LPS in This Process.

The prevalent pathogens associated with bovine uterine infections are bacteria that appear to increase the host's susceptibility to secondary infections with other bacteria or viruses, among which BoGHV4 is the most frequently found. In this work, the study of the pathways of apoptosis induction was carried out on an experimental model of primary culture of endometrial cells, in order to know the implication of BoGHV4 and the presence of bacterial LPS in the pathogenesis of the bovine reproductive tract. For this, different staining techniques and molecular analysis by RT-PCR were used. The results obtained allowed us to conclude that the level of cell death observed in the proposed primary culture is directly related to the time of viral infection and the presence of LPS in BoGHV4 infection. The apoptosis indices in cells infected with BoGHV4 and BoGHV4 + LPS revealed a maximum that correlated with the appearance of cytopathic effects and the maximum viral titers in the model studied. However, morphological, biochemical, and molecular changes were evident during both early and late stages of apoptosis. These findings provide information on the factors that may influence the pathogenesis of BoGHV4 and help to better understand the mechanisms involved in virus infection.

The association of TNF-alpha secretion and mtDNA copy number in CD14+ monocytes of patients with obesity and CHD.

Introduction: Mitochondrial dysfunction may be one of the causes of inflammatory activation of monocytes and macrophages, which leads to excessive secretion of inflammatory mediators and the development of chronic inflammation. Aims: The study was aimed to evaluate the secretion of inflammatory cytokine tumor necrosis factor-α (TNF-α) in the primary culture of monocytes, and to analyze its relationship with the number of mitochondrial DNA (mtDNA) copies in the blood of patients with coronary heart disease (CHD) and obesity. Materials and methods: 108 patients with obesity and concomitant CHD and a control group of 25 participants were included in the study. CD14+ monocytes were isolated by a standard method in a ficoll-urographin gradient, followed by separation using magnetic particles. The number of mtDNA copies was estimated using qPCR. Results: It was demonstrated that the number of mtDNA copies was significantly increased in groups of patients with CHD and obesity + CHD in comparison with control group. mtDNA copy number positively correlated with basal and LPS-stimulated TNF-α secretion, the most significant correlation was found in the group of patients with CHD and obesity. Conclusion: Thus, the change in mtDNA copy number in CD14+ monocytes which indicates the presence of mitochondrial dysfunction, confirm the direct involvement of mitochondria in the violation of the inflammatory response of monocytes revealed in this study as an increased secretion of inflammatory cytokine TNF-α.

Exploring the impact of culture techniques and patient demographics on the success rate of primary culture of human periodontal ligament stem cells.

Background/purpose: Periodontal ligament stem cells (PDLSCs) have the potential for regenerating periodontal tissue. The study aims to investigate the impact of demographics (ages, gender, disease) and culture techniques (shipping storage time and culture method) on the success of primary culture. Materials and methods: PDLSCs were collected from 51 teeth of 26 patients and cultured via outgrowth (OG) and enzymatic digestion (ED) methods. Cells characteristics were confirmed by flow cytometry, MTT, and ARS. The primary culture success rate was evaluated with a serial chi-square test to determine the relationship with culture technique (ED/OG and ≤4 h/prolonged culture) and patient demographics (Young/Old, Female/Male, and Health/Periodontitis). Results: The overall success rate of Health group (69.7%) was higher than Periodontitis (38.9%). Culturing within 4 h possessed a higher success rate (71.8%) than prolonged group (16.7%) regardless of patient demographics, and using OG method (81.5%) revealed more promising. Subgroup analysis of 39 cases (culture within 4 h) found that the success rate of OG was higher than ED in the Old group (87.5%-25.0%) and in the Periodontitis group (83.3%-25.0%). Conclusion: Primary culturing of PDLSCs within 4 h and using the outgrowth method led to higher success rates regardless of patient demographics. It can achieve successful PDLSCs culture of older patients or patients with periodontal disease by appropriate culture technique.

Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex.

Primary neuronal culture is a valuable in vitro model for analyzing the molecular mechanisms underlying the development and function of neural circuits. In contrast to neurons in vivo, primary cultured neurons can easily be transfected with genes of interest or treated with chemicals such as agonists and inhibitors of a specific target molecule. Furthermore, time-dependent morphological changes, such as the acquisition of neuronal polarity, axon elongation, and dendrite branch formation, can be analyzed by using primary neuronal cultures. Here, we describe a method for preparing a primary culture of neurons from the developing cerebral cortex, together with a method for gene transfer to primary cultured cortical neurons.

Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons.

Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.

In Vitro Culture of White Adipose Tissue.

White adipose tissue (WAT) plays a crucial endocrine organ that regulates blood glucose and lipid levels, satiety, and inflammation. Before the described technique, primary white adipocytes could not be stably cultured in vitro. The lack of a reliable primary culture model impeded research in WAT metabolism and drug development. We have developed a novel technique for WAT primary culture called "sandwiched white adipose tissue" (SWAT). SWAT overcomes the natural buoyancy of adipocytes by sandwiching minced WAT between sheets of adipose-derived stromal cells. The resulting constructs are viable for at least 8 weeks in culture. SWAT maintains the intact extracellular matrix, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

A landscape of patient-derived cancer-associated fibroblast signals in endometrial cancers.

In conversation with endometrial tumor cells, the endometrial cancer-associated fibroblasts (CAFs) are the "partners in crime" of uterine neoplasm's highly heterogeneous tumor microenvironment (TME). We designed a laboratory-friendly method to culture endometrial CAFs on a patient-to-patient basis for studying the CAF-TME and CAF-tumor cell interaction(s). Here, we present a comprehensive characterization of endometrial CAFs derived from patients' tumor tissues (T) and tumor-adjacent normal tissues (N). We used more than 80 T and N from 53 consecutive consented patients with endometrial cancers at the Avera Cancer Institute. We derived TCAF and NCAF in a non-enzymatic feeder-layer culture and characterized their expression of markers by qRT-PCR, flow cytometry, immunocytochemistry, immunofluorescence, and Western blot. Although similar in the expression pattern of EpCAM-/CK18-/vimentin+ as in ovarian CAFs, endometrial NCAFs, and TCAFs characteristically presented dual morphology in culture. Endometrial CAFs were EpCAM-/CK18-/CD45-/CD31-/SMA+/TE-7+/PDGFRA+/CXCL12+/Meflin+/CD155+/CD90+ with patient-specific positivity for S100A4/FAP/PD-L1/CD44. Endometrial CAFs expressed mRNAs for signaling proteins of several pathways and receptor-ligands, including (1) cell cycle pathway, (2) TGF pathway, (3) FGF pathway, (4) Wnt-beta-catenin pathway, (5) HER pathway, (6) tyrosine kinase receptor ligands, and (7) steroid receptors. We tested the hypoxic response of CAFs to show that endometrial CAFs upregulate MMP1 in a HIF-1a-independent manner. In trying to delineate the relationship between expressions of CAF markers and T-cells in the tumor tissue, we observed that FAP-positive CAFs that are derived from CD4/CD8 positive tumor tissue expressed CXCL12 mRNA. The data indicate the role of the CXCL12-CXCR4 pathway of the CAF-rich stroma in the lymphocytic infiltration of the tumor. We demonstrate that endometrial CAFs can be cultured in an enzymatic-digestion-independent manner, and their signaling landscape can be mapped toward understanding CAF-TME dialogue. Our data will help unearth the functional relevance of endometrial CAFs in the context of clinical outcomes and designing CAF-inclusive therapy in the future.

In vitro culture and tissue-derived specific expression of melanocytes from ovary of adult Silky Fowl.

The presence of a significant number of melanocytes in the ovary and follicular membrane of Silky Fowl suggests their potential involvement in follicle development. Currently, there is a lack of available data regarding to the isolation of primary melanocytes from adult chickens. To date, primary melanocytes and their in vitro culture system have been successfully conducted in the peritoneum of chicken embryos. Herein, melanocytes from silky fowl ovaries were isolated and identified. Silky Fowl ovaries were obtained by mixed digestion of 0.1% collagenase II and 0.25% trypsin-EDTA. Melanocytes could be further purified and cultured up to 5 generations in vitro. RNA-seq analysis was used to investigate whether there were differences in the functional status of melanocytes in different tissues and developmental stages. Consequently, differential gene expressions between peritoneal and ovarian melanocytes were compared. These findings demonstrated that the Silky Fowl ovary had higher expression levels of genes involved in the production of sexual hormones and melanogenesis, while those of melanocytes derived from the peritoneum were involved in amino acid metabolism, lipid synthesis, and overall metabolic rates. This suggests that the role of melanocytes is dependent on the origin tissue and developmental stage, and is tightly connected to the function of the specific source tissue from which the cells were derived. This study provides a method for isolating adult melanocytes and serve as a basis for further investigate the effect of SFOM on germ cells.

FABP2 is Involved in Intestinal α-Synuclein Pathologies.

BACKGROUND: Recently, the hypothesis that pathological α-Synuclein propagates from the gut to the brain has gained attention. Although results from animal studies support this hypothesis, the specific mechanism remains unclear. This study focused on the intestinal fatty acid-binding protein (FABP2), which is one of the subtypes of fatty acid binding proteins localizing in the gut, with the hypothesis that FABP2 is involved in the gut-to-brain propagation of α-synuclein. The aim of this study was to clarify the pathological significance of FABP2 in the pathogenesis and progression of synucleinopathy. METHODS: We examined the relationship between FABP2 and α-Synuclein in the uptake of α-Synuclein into enteric neurons using primary cultured neurons derived from mouse small intestinal myenteric plexus. We also quantified disease-related protein concentrations in the plasma of patients with synucleinopathy and related diseases, and analyzed the relationship between plasma FABP2 level and progression of the disease. RESULTS: Experiments on α-Synuclein uptake in primary cultured enteric neurons showed that following uptake, α-Synuclein was concentrated in areas where FABP2 was localized. Moreover, analysis of the plasma protein levels of patients with Parkinson's disease revealed that the plasma FABP2 and α-Synuclein levels fluctuate with disease duration. The FABP2/α-Synuclein ratio fluctuated more markedly than either FABP2 or α-Synuclein alone, depending on the duration of disease, indicating a higher discriminant ability of early Parkinson's disease patients from healthy patients. CONCLUSIONS: These results suggest that FABP2 potentially contributes to the pathogenesis and progression of α-synucleinopathies. Thus, FABP2 is an important molecule that has the potential to elucidate the consistent mechanisms that lead from the prodromal phase to the onset and subsequent progression of synucleinopathies.

Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line.

The prevalence of alcohol-related hepatocellular carcinoma (HCC) has been increasing during the last decade. Cancer research requires cell lines suitable for both in vitro and in vivo assays. However, there is a lack of cell lines with a high in vivo metastatic capacity for this HCC subtype. Herein, a new HCC cell line was established, named HCC-ZJ, using cells from a patient diagnosed with alcohol-related HCC. The karyotype of HCC-ZJ was 46, XY, del (p11.2). Whole-exome sequencing identified several genetic variations in HCC-Z that occur frequently in alcohol-associated HCC, such as mutations in TERT, CTNNB1, ARID1A, CDKN2A, SMARCA2, and HGF. Cell counting kit-8 assays, colony formation assays, and Transwell assays were performed to evaluate the proliferation, migration, and sensitivity to sorafenib and lenvatinib of HCC-Z in vitro. HCC-ZJ showed a robust proliferation rate, a weak foci-forming ability, a strong migration capacity, and a moderate invasion tendency in vitro. Finally, the tumorigenicity and metastatic capacity of HCC-Z were evaluated using a subcutaneous xenograft model, an orthotopic xenograft model, and a tail-veil injection model. HCCZJ exhibited strong tumorigenicity in the subcutaneous xenograft and orthotopic tumor models. Moreover, HCC-ZJ spontaneously formed pulmonary metastases in the orthotopic tumor model. In summary, a new HCC cell line derived from a patient with alcohol-related HCC was established, which showed a high metastatic capacity and could be applied for in vitro and in vivo experiments during pre-clinical research.Highlights• An alcohol-related HCC cell line, HCC-ZJ, was established• HCC-ZJ was applicable for in vitro functional experiment and gene editing• HCC-ZJ was applicable for in vivo tumor growth and spontaneous metastasis models.

Navigating challenges: optimising methods for primary cell culture isolation.

Primary cell lines are invaluable for exploring cancer biology and investigating novel treatments. Despite their numerous advantages, primary cultures are laborious to obtain and maintain in culture. Hence, established cell lines are still more common. This study aimed to evaluate a range of techniques for isolating primary breast cancer cultures, employing distinct enzymatic compositions, incubation durations, and mechanical approaches, including filtration. Out of several protocols, we opted for a highly effective method (Method 5) that gave rise to a primary cell culture (BC160). This method combines mechanical disaggregation and enzymatic digestion with hyaluronidase and collagenase. Moreover, the paper addresses common issues in isolating primary cultures, shedding light on the struggle against fibroblasts overgrowing cancer cell populations. To make primary cell lines a preferred model, it is essential to elaborate and categorise isolation methods, develop approaches to separate heterogeneous cultures and investigate factors influencing the establishment of primary cell lines.