Immortalized Cell Lines Research Articles

Immunofluorescent Staining of Human Hepatic Multicellular Spheroids: A Model for Studying Liver Diseases.

In vitro studies on liver diseases, such as non-alcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma, are traditionally performed in two-dimensional (2D) cultures of isolated primary cells or immortalized cell lines. However, this approach has limitations, as 2D cultures inadequately replicate the cell-cell and cell-extracellular matrix interactions found in three-dimensional (3D) environments. To overcome this limitation, various 3D models, such as spheroids, have been developed. These spheroids serve as simplified biomimetic in vitro models for studying liver diseases. They can be generated using a variety of cells from healthy and pathological tissues, including liver cancer. Here, we present a comprehensive protocol for performing immunofluorescent staining and confocal imaging on whole human hepatic multicellular spheroids, utilizing primary cells or cell lines. The immunofluorescence technique is a potent tool to understand the spatial distribution of different cell types within the spheroids and define the interactions that occur among these cells.

Production and biological properties of nano porous glass microparticles for anticancer drug carrier

Nanoporous glass (NPG) microparticles were produced by conventional melt-quenching followed by acid-alkali leaching to get material for anticancer drug carriers. NPG exhibited a positive zeta potential of 34 mV after [3-(2-aminoethylamino) propyl] trimethoxysilane treatment. The specific surface area and the total pore volume of NPG were 47.3 m2/g and 0.692 cm3/g, respectively. The 5-Fluorouracil (5FU) loading capacity of NPG was measured as 18.2 ± 0.2 mg5FU/gNPG. The drug release rate was monitored for 120 hours. To evaluate the cytotoxic effects of NPG on both MCF-7 breast cancer cells and MCF-12A, an immortalized cell line, the study employed the 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium5-carboxanilide inner salt (XTT) assay. The XTT results revealed that NPG showed a time and concentration-dependent cytotoxic effect. It is anticipated that NPG is a safe and effective material for drug delivery systems for in vitro and a promising alternative material for in vivo applications.

Monitoring Mitophagy Dynamics in Live Cells.

The purpose of this protocol is to provide a comprehensive, stepwise guide for assessing mitophagy flux utilizing a live-cell mt-KEIMA approach. The proposed protocol is sensitive, reproducible, quantitative, and easy to perform. While mitophagy has been extensively studied, current methodologies primarily focus on terminal measurements, neglecting the dynamic aspect of this process. Hence, the introduction of this straightforward live-cell mitophagy tracing protocol enables real-time monitoring of the dynamics of mitochondrial selective autophagy, thereby enhancing the ability to draw conclusions regarding key regulators and the reversibility of the process. The assay employs a lentiviral approach to induce mt-KEIMA expression in primary or immortalized cell lines. Subsequently, the respective mitophagy reporter cells are observed using a live-cell imaging system at specific time intervals, and further quantification allows the detection of mitophagy flux. This protocol has proven efficacious in investigating mitophagy flux, including responses to chemical inducers or genetically modified cells over time. Notably, this approach is well-suited for large throughput screening of chemicals or appropriate gene-editing libraries that may influence mitophagy responses in cells.

CRISPR-Cas9-Induced Gene Editing in Primary Human Monocytes.

Monocytes play important and diverse roles in both homeostatic and inflammatory immune responses. The CRISPR-Cas9 system in lentiviral vectors has been widely used to manipulate specific genes of immortal monocyte cell lines to study monocyte functions. However, human primary monocytes are refractory to this method with low gene knockout (KO) efficiency. In this chapter, we developed an in vitro gene-editing procedure for primary human monocytes with a consistent and high-gene KO efficiency via a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single-guide RNA (sgRNA). This method can be adapted to study the functions of targeted signaling molecules involved in modulating monocyte polarization in primary human monocytes.

Adipose mesenchymal stem cell-derived exosomes promote skin wound healing in diabetic mice by regulating epidermal autophagy.

Adipose mesenchymal stem cell-derived exosomes (ADSC-Exos) have great potential in the field of tissue repair and regenerative medicine, particularly in cases of refractory diabetic wounds. Interestingly, autophagy plays a role in wound healing, and recent research has demonstrated that exosomes are closely associated with intracellular autophagy in biogenesis and molecular signaling mechanisms. Therefore, this study aimed to investigate whether ADSC-Exos promote the repair of diabetic wounds by regulating autophagy to provide a new method and theoretical basis for the treatment of diabetic wounds. Western blot analysis and autophagy double-labelled adenovirus were used to monitor changes in autophagy flow in human immortalized keratinocyte cell line (HaCaT) cells. ADSC-Exos were generated from ADSC supernatants via ultracentrifugation. The effectiveness of ADSC-Exos on HaCaT cells was assessed using a live-cell imaging system, cell counting kit-8 and cell scratch assays. The cells were treated with the autophagy inhibitor bafilomycin A1 to evaluate the effects of autophagy on cell function. The recovery of diabetic wounds after ADSC-Exo treatment was determined by calculating the healing rates and performing histological analysis. High-throughput transcriptome sequencing was used to analyze changes in mRNA expression after the treatment of HaCaT cells with ADSC-Exos. ADSC-Exos activated autophagy in HaCaT cells, which was inhibited by high glucose levels, and potentiated their cellular functions. Moreover, ADSC-Exos in combination with the autophagy inhibitor bafilomycin A1 showed that autophagy defects further impaired the biological function of epidermal cells under high-glucose conditions and partially weakened the healing effect of ADSC-Exos. Using a diabetes wound model, we found that ADSC-Exos promoted skin wound healing in diabetic mice, as evidenced by increased epidermal autophagy and rapid re-epithelialization. Finally, sequencing results showed that increased expression of autophagy-related genes nicotinamide phosphoribosyltransferase (NAMPT), CD46, vesicle-associated membrane protein 7 (VAMP7), VAMP3 and eukaryotic translation initiation factor 2 subunit alpha (EIF2S1) may contribute to the underlying mechanism of ADSC-Exo action. This study elucidated the molecular mechanism through which ADCS-Exos regulate autophagy in skin epithelial cells, thereby providing a new theoretical basis for the treatment and repair of skin epithelial damage by ADSC-Exos.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Deer antler stem cells immortalization by modulation of hTERT and the small extracellular vesicles characters.

Deer antler stem cells (AnSCs) exhibit properties of both embryonic and mesenchymal stem cells, with superior self-renewal and proliferation, which drive rapid antler growth and regeneration. AnSCs and their derived small extracellular vesicles (sEVs) hold promising potential for applications in regeneration medicine. Due to the restricted proliferative capacity inherent in primary cells, the production capacity of AnSCs and their sEVs are limited. Human telomerase reverse transcriptase (hTERT) is the most important telomerase subunit, hTERT gene insertion has been successfully employed in generating immortalized cell lines. In this study, we successfully established immortalized AnSCs by transducing the hTERT gene using lentivirus. Compared to primary AnSCs, hTERT-AnSCs demonstrated extended passage potential and accelerated proliferation rates while maintaining the mesenchymal stem cell surface markers CD44 and CD90. Additionally, hTERT-AnSCs retained the capacity for osteogenic, adipogenic, and chondrogenic differentiation. sEVs derived from hTERT-AnSCs exhibited a particle size distribution similar to that of AnSCs, both displaying a cup-shaped morphology and expressing CD81, ALIX, and TSG101, while notably lacking GM130 expression. We successfully isolated primary stem cells from deer antler and established the immortalized hTERT-AnSCs. Remarkably, this cell line maintains its stem cell characteristics even after 40 passages. The sEVs derived from these cells exhibit identical morphological and structural features to those of primary AnSCs. This research provides essential technical support for the application of AnSCs and their sEVs in regenerative medicine.

Enhancing pre-clinical research with simplified intestinal cell line models

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.

Forskolin induces FXR expression and enhances maturation of iPSC-derived hepatocyte-like cells.

The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases, their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients, enabling the study of disease-associated mutations and, when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately, the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study, we optimized our previously published protocol by fine-tuning the initial cell number, exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple, cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB, HNF4α, and CYP3A4) and protein (ALB) expression, as well as significantly elevated inducible CYP3A4 activity.

Generating Patient-Derived HCC Cell Lines Suitable for Predictive In Vitro and In Vivo Drug Screening by Orthotopic Transplantation.

Hepatocellular carcinoma (HCC) results in high mortality due to ineffective systemic therapy. Human immortalized cell lines are commonly used to study anti-tumor effects in the context of new anti-tumor therapies and tumor biology. As immortalized cell lines have limited biological relevance and heterogeneity compared to primary cells, patient-derived tumor tissues, and corresponding immune cells are the gold standards for studying the complexity of individual tumor entities. However, culturing primary HCC cells has a low success rate. Here, we aimed to establish a reproducible approach to preserve the patient-derived liver cancer cells for in vitro and in vivo studies. The underlying study aimed to establish an in vitro pre-screening platform to test treatment options' effectivity and dosage, e.g., for new substances, autologous modified immune cells, or combined therapies in HCC. We initially employed 15 surgical resection specimens from patients with different HCC entities for isolation and preservation. The isolated liver cancer cells from four HCC-diagnosed patients were used for orthotopic transplantation into the healthy liver of immunodeficient mice, allowing them to grow for six months before human liver cancer cells were isolated and cultured. As a result, we generated and characterized four new primary-like liver cancer cell lines. Compared to immortalized HCC cell lines, freshly generated liver cancer cells displayed individual morphologies and heterogeneous protein-level characteristics. We assessed their ability to proliferate, migrate, form spheroids, and react to common medications compared to immortalized HCC cell lines. All four liver cancer cell lines exhibit strong migration and colony-forming characteristics in vitro, comparable to extensively investigated immortalized HCC cell lines. Moreover, the four etiological different liver cancer cell lines displayed differences in the response to 5-FU, Sorafenib, Axitinib, and interferon-alpha treatment, ranking from non-responders to responders depending on the applicated medication. In sum, we generated individual patient-derived liver cancer cell lines suitable for predictive in vitro drug screenings and for xenograft transplantations to realize the in vivo investigation of drug candidates. We overcame the low cultivation success rate of liver cancer cells derived from patients and analyzed their potential to serve a pre-clinical model.

The Importance and Challenges of Primary Chicken Embryo Liver Cells in Studies of Poultry Viral Diseases: A Review

Primary chicken embryo liver (CEL) cells are derived from the liver tissue of chicken embryonated eggs (CEE) using an aseptic isolation technique and growth under a controlled atmosphere in an artificial environment for cell attachment and proliferation. Although this primary cultured cell has been established for more than six decades, utilization of primary cells is still the preferable medium nowadays as the “gold standard” due to several advantages over other diagnostic techniques. Cells provide better adaptability of the viruses and easily mimic the natural host environment with high virus titration. The volume of virus suspension could be increased by applying an immortal chicken embryo liver-derived cell line. The current review aimed to highlight the importance and challenges of using primary chicken embryo liver cells in poultry virus studies. Primary CEL cells are widely used as an alternative host for diagnosis of infectious poultry viruses, cultivation and passaging of virus isolates, and vaccine production. Yet, there are some challenges and limitations in handling this primary cell, which requires appropriate facilities and environment to sustain the rapid growth of confluent monolayer cells, as highlighted in this paper. The availability of specific pathogen-free CEE is a major concern due to limited resources globally, thus creating a challenge for vaccine manufacturers to upscale the cultured cells. Future improvement of primary cell culture preparation necessitates new technology by applying cellular microcarrier in the bioreactor machine for efficient cell growth and subsequent routine virus cultivation. This study can help the researchers understand the advantages of primary CEL cells and their applications due to their significant impact on poultry viruses.

Comparison of HepaRG and HepG2 cell lines to model mitochondrial respiratory adaptations in non‑alcoholic fatty liver disease.

Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non‑alcoholic steatohepatitis (NASH), there is a lack of invitro models of non‑alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for invitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA‑treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II‑driven respiration and β‑oxidation were linked to increased complex I and II activities in FFA‑treated HepaRG cells, but not in FFA‑treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA‑treated HepaRG cells than in FFA‑treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA‑treated HepaRG cells, but not in FFA‑treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed invitro model of early‑stage NASH.

Reversibly immortalization establishes a hair follicle stem cell line with hair follicle reconstruction ability.

Hair follicle stem cells (HFSCs) play critical roles in the periodic regeneration of hair follicles. HFSCs are also a good model for stem cell biology research. However, no stable mouse HFSC cell line has been reported, which restricts the research and application of HFSCs. We isolated HFSCs from mouse hair follicles and immortalized them by inducing a reversible SV40 large T antigen. Through monoclonal screening, we identified a reversibly immortalized cell line, immortalized HFSC (iHFSC2). RNA sequencing, fluorescence-activated cell sorting, western blotting and immunofluorescence experiments revealed that the expression patterns of iHFSC2 and HFSC were similar at the protein and mRNA levels. After that, iHFSC2s were passaged and morphologically monitored for up to 40 times to detect their long-term culture potential. The long-term cultured iHFSC2 could regenerate hair follicles with complete hair follicle structure and HFSCs in the bulge area. This work successfully established an HFSC cell line with the ability of hair follicle reconstruction.

The dual role of the DREAM/G2M pathway in non-tumorigenic immortalization of senescent cells.

Anti-aging and tumorigenesis share common genes and pathways, and thus targeting these genes as part of anti-aging interventions carries the risk of tumorigenesis. It is essential to understand the gene signatures that balance tumorigenesis and aging. To achieve this goal, we analyzed RNA-sequencing data from three non-tumorigenic immortalized cell lines that spontaneously escaped from senescence. By single sample gene set enrichment assay (ssGSEA) and GSEA analysis, we found that both cell growth signaling (E2F targets, MYC targets) and tumor surveillance mechanisms (DNA repair, G2M checkpoint, mitotic spindle) were up-regulated in all three cell lines, suggesting that these genes are potential signatures for non-tumorigenic immortalization. Further analysis revealed that the 182 commonly up-regulated genes in these three cell lines overlapped with the DREAM/G2M pathway, which is known to be the upstream regulator of E2F, Myc targets, DNA repair, G2M checkpoint and mitotic spindle pathways in its cell cycle activation or inhibitory form. By western blotting, quantitative PCR and co-immunoprecipitation, we verified that both forms of the DREAM pathway are up-regulated in all three cell lines; this pathway facilitates control of cell cycle progression, supporting a new mechanism for non-tumorigenic immortalization. Thus, we propose that the DREAM/G2M pathway plays important dual roles with respect to preventing tumorigenesis in the process of immortalization. Our data might serve as the basis for the identification of new signature pathways or gene biomarkers for non-tumorigenic immortalization, and may aid in the discovery of new targets for tumor-free anti-aging drug screening.

Infection of PRRSV inhibits CSFV C-strain replication by inducing macrophages polarization to M1

It is a common sense that porcine reproductive and respiratory syndrome virus (PRRSV) infection could cause immune failure of classical swine fever (CSF) vaccine, and porcine alveolar macrophages (PAMs) are the target cells of both. To elucidate the role of macrophage polarization in PRRSV infection induced CSF vaccine failure, an immortal porcine alveolar macrophage line PAM39 cell line was used to investigate the effect of PRRSV or/and CSFV C-strain (CSFV-C) infection on macrophage polarization in vitro. Interestingly, PRRSV single infection or PRRSV co-infection with CSFV-C promoted PAM39 cells to M1, while CSFV-C single infection induced PAM39 cells to M2. After the construction of M1 and M2 PAM39 cells polarization models, M1 polarized PAM39 cells were found to inhibit the replication of CSFV-C, and Chinese medicine such as matrine, ginsenosides and astragalus polysaccharides could alleviate the polarization of PAM39 cells and the replication of CSFV-C. Furthermore, interferon (IFN)-γ and lipopolysaccharide (LPS) co-stimulation induced NF-κB activation while matrine treatment blocked M1 polarization-induced NF-κB pathway activation. These findings provided a theoretical basis for designing a new strategy to improve the immune effect of CSFV-C based on porcine alveolar macrophage polarization subtypes.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26nM), AUC 7.30 ± 0.32 (IC50 50.43nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Ex vivo discovery of synergistic drug combinations for hematologic malignancies

Combination therapies have improved outcomes for patients with acute myeloid leukemia (AML). However, these patients still have poor overall survival. Although many combination therapies are identified with high-throughput screening (HTS), these approaches are constrained to disease models that can be grown in large volumes (e.g., immortalized cell lines), which have limited translational utility. To identify more effective and personalized treatments, we need better strategies for screening and exploring potential combination therapies. Our objective was to develop an HTS platform for identifying effective combination therapies with highly translatable ex vivo disease models that use size-limited, primary samples from patients with leukemia (AML and myelodysplastic syndrome). We developed a system, ComboFlow, that comprises three main components: MiniFlow, ComboPooler, and AutoGater. MiniFlow conducts ex vivo drug screening with a miniaturized flow-cytometry assay that uses minimal amounts of patient sample to maximize throughput. ComboPooler incorporates computational methods to design efficient screens of pooled drug combinations. AutoGater is an automated gating classifier for flow cytometry that uses machine learning to rapidly analyze the large datasets generated by the assay. We used ComboFlow to efficiently screen more than 3000 drug combinations across 20 patient samples using only 6 million cells per patient sample. In this screen, ComboFlow identified the known synergistic combination of bortezomib and panobinostat. ComboFlow also identified a novel drug combination, dactinomycin and fludarabine, that synergistically killed leukemic cells in 35 % of AML samples. This combination also had limited effects in normal, hematopoietic progenitors. In conclusion, ComboFlow enables exploration of massive landscapes of drug combinations that were previously inaccessible in ex vivo models. We envision that ComboFlow can be used to discover more effective and personalized combination therapies for cancers amenable to ex vivo models.

Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators.

Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale-derived cells can be immortalized with a combination of human genes, mutant cyclin-dependent kinase 4 (CDK4R24C ), cyclin D1, and Telomerase Reverse Transcriptase (TERT)is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA-seq based on next-generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke-whale-derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.

A novel patient-derived immortalised cell line of myxofibrosarcoma: a tool for preclinical drugs testing and the generation of near-patient models

BackgroundMyxofibrosarcoma is a rare malignant soft tissue sarcoma characterised by multiple local recurrence and can become of higher grade with each recurrence. Consequently, myxofibrosarcoma represents a burden for patients, a challenge for clinicians, and an interesting disease to study tumour progression. Currently, few myxofibrosarcoma preclinical models are available.MethodsIn this paper, we present a spontaneously immortalised myxofibrosarcoma patient-derived cell line (MF-R 3). We performed phenotypic characterization through multiple biological assays and analyses: proliferation, clonogenic potential, anchorage-independent growth and colony formation, migration, invasion, AgNOR staining, and ultrastructural evaluation.ResultsMF-R 3 cells match morphologic and phenotypic characteristics of the original tumour as 2D cultures, 3D aggregates, and on the chorioallantoic membrane of chick embryos. Overall results show a clear neoplastic potential of this cell line. Finally, we tested MF-R 3 sensitivity to anthracyclines in 2D and 3D conditions finding a good response to these drugs.ConclusionsIn conclusion, we established a novel patient-derived myxofibrosarcoma cell line that, together with the few others available, could serve as an important model for studying the molecular pathogenesis of myxofibrosarcoma and for testing new drugs and therapeutic strategies in diverse experimental settings.

Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis invitro.

Dermatological research relies on the availability of suitable models that most accurately reflect the invivo situation. Primary keratinocytes obtained from skin reduction surgeries are not only limited by availability but have a short lifespan and show donor-specific variations, which hamper the understanding of general mechanisms. The spontaneously immortalized keratinocyte cell line HaCaT displays chromosomal aberrations and is known to differentiate in an abnormal manner. To overcome these issues, we validated different engineered immortalized cell lines created from primary human keratinocytes (NHK) as model systems to study epidermal function. Cell lines either immortalized by the expression of SV40 large T antigen and hTERT (NHK-SV/TERT) or by transduction with HPV E6/E7 (NHK-E6/E7) were analysed for their growth and differentiation behaviour using 2D and 3D culture systems and compared to primary keratinocytes. Both cell lines displayed a robust proliferative behaviour but were still sensitive to contact inhibition. NHK-E6/E7 could be driven into differentiation by Ca2+ switch, while NHK-SV/TERT needed withdrawal from any proliferative signal to initiate a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and functional epidermal barrier. However, only NHK-E6/E7 showed a degree of epidermal maturation and stratification that was comparable to primary keratinocytes.