Transcriptome Profiling of Mouse Embryonic Fibroblast Spontaneous Immortalization: A Comparative Analysis.

Cell immortalization, a hallmark of cancer development, is a process that cells can undergo on their path to carcinogenesis. Spontaneously immortalized mouse embryonic fibroblasts (MEFs) have been used for decades; however, changes in the global transcriptome during this process have been poorly described. In our research, we characterized the poly-A RNA transcriptome changes after spontaneous immortalization. To this end, differentially expressed genes (DEGs) were screened using DESeq2 and characterized by gene ontology enrichment analysis and protein-protein interaction (PPI) network analysis to identify the potential hub genes. In our study, we identified changes in the expression of genes involved in proliferation regulation, cell adhesion, immune response and transcriptional regulation in immortalized MEFs. In addition, we performed a comparative analysis with previously reported MEF immortalization data, where we propose a predicted gene regulatory network model in immortalized MEFs based on the altered expression of Mapk11, Cdh1, Chl1, Zic1, Hoxd10 and the novel hub genes Il6 and Itgb2.

Development and characterization of an immortalized nasopharyngeal epithelial cell line to explore airway physiology and pathology in yak (Bos grunniens).

Airway epithelial cells play a crucial role in investigating the physiological and pathological mechanisms of the respiratory tract in yaks, a species whose unique respiratory system has garnered extensive interest. Despite this growing interest, there currently are no available airway epithelial cell lines from yaks, underscoring the crucial need to establish a yak respiratory epithelial cell line. Therefore, our objective was to isolate a population of primary yak nasopharyngeal epithelial cells (pYNE) and transform them into immortalized yak nasopharyngeal epithelial cells (iYNE), assessing their suitability as an in vitro model. Employing a combined method of physical elimination and differential adhesion, we successfully isolated a population of high-purity pYNE, and developed an iYNE line through pCI-neo-hTERT plasmid transfection. Karyotype and transmission electron microscopy analyses confirmed that pYNE and iYNE share identical morphologies and structures. Gel electrophoresis and real-time PCR analyses demonstrated that pYNE and iYNE expressed similar levels of KRT18 and CDH1 genes (p ≥ 0.541). Notably, iYNE expressed a significantly high level of TERT gene expression (p < 0.001). Immunofluorescence analysis demonstrated that both cell types expressed Pan-Cytokeratin, ZO-1, and E-cadherin proteins. Furthermore, immunoblotting analysis indicated significantly higher levels of hTERT and Ki67 proteins in iYNE (p < 0.001), and similar levels of Cluadin-3 and Occludin proteins (p ≥ 0.103). Proliferation curve analysis highlighted iYNE's serum-dependency and significantly enhanced proliferation capacities (p < 0.001). Additionally, pYNE and iYNE cells demonstrated comparable susceptibilities to infectious bovine rhinotracheitis virus (IBRV). These findings collectively suggest that the developed iYNE retains the evaluated physiological characteristics of pYNE, making it an appropriate in vitro model. This advancement will facilitate further investigation into the respiratory physiological and pathological mechanisms in yaks.

Development of a Bmi1+ Cardiac Mouse Progenitor Immortalized Model to Unravel the Relationship with Its Protective Vascular Endothelial Niche.

The adult mammalian heart has been demonstrated to be endowed with low but real turnover capacity, especially for cardiomyocytes, the key functional cell type. The source, however, of that turnover capacity remains controversial. In this regard, we have defined and characterized a resident multipotent cardiac mouse progenitor population, Bmi1+DR (for Bmi1+ Damage-Responsive cells). Bmi1+DR is one of the cell types with the lowest ROS (Reactive Oxygen Species) levels in the adult heart, being particularly characterized by their close relationship with cardiac vessels, most probably involved in the regulation of proliferation/maintenance of Bmi1+DR. This was proposed to work as their endothelial niche. Due to the scarcity of Bmi1+DR cells in the adult mouse heart, we have generated an immortalization/dis-immortalization model using Simian Vacuolating Virus 40-Large Antigen T (SV40-T) to facilitate their in vitro characterization. We have obtained a heterogeneous population of immortalized Bmi1+DR cells (Bmi1+DRIMM) that was validated attending to different criteria, also showing a comparable sensitivity to strong oxidative damage. Then, we concluded that the Bmi1-DRIMM population is an appropriate model for primary Bmi1+DR in vitro studies. The co-culture of Bmi1+DRIMM cells with endothelial cells protects them against oxidative damage, showing a moderate depletion in non-canonical autophagy and also contributing with a modest metabolic regulation.

Loss of keratin 14 expression from immortalized keratinocytes by promoter methylation.

Immortalized keratinocytes can offer a low-cost experimental platform for human skin research, with increased cell yield compared to primary cultures. However, the usefulness of these surrogate cell models is highly dependent on their ability to retain the phenotypic attributes of the parent cells. Keratins K14 and K5 are the hallmarks of undifferentiated, mitotically active basal keratinocytes. We observed occasional progressive loss of K14 expression in growing keratinocyte cell lines, with persistent retention of K5 and an epithelial phenotype, and investigated possible reasons for this. We show that K14 repression occurs by DNA promoter methylation of KRT14 gene and is compounded by histone deacetylation and by the presence of EGF. In vivo, keratinocytes shut down K14 synthesis as they commit to terminal differentiation and move from the basal to spinous layer, but by laser-capture microdissection of human epidermis we could detect no evidence of increased selective KRT14 methylation in this normal process. Loss of K14 expression suggests that epidermal identity of cultured keratinocytes can be compromised in certain tissue culture situations, possibly due to the immortalization method and persistent EGF supplementation.

Immortalization of Mesenchymal Stem Cell Lines from Sheep Umbilical Cord Tissue.

Mesenchymal stem cells (MSCs) possess significant differentiation potential, making them highly promising in medicine and immunotherapy due to their regenerative capabilities and exosome secretion. However, challenges such as limited cell divisions and complex testing hinder large-scale MSC production. In this study, we successfully established an immortalized MSC line by transfecting the human telomerase reverse transcriptase (TERT) gene into MSCs isolated from pregnant sheep umbilical cords. This approach effectively inhibits cell senescence and promotes cell proliferation, enabling the generation of umbilical cord mesenchymal stem cells (UCMSCs) on a larger scale. Our findings demonstrate that these transfected TERT-UCMSCs exhibit enhanced proliferative capacity and a reduced aging rate compared to regular UCMSCs while maintaining their stemness without tumorigenicity concerns. Consequently, they hold great potential for medical applications requiring large quantities of functional MSCs.

An efficient method for immortalizing mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inefficient or alter the physiological properties of the cells. Here, we describe an optimized protocol for immortalizing MEFs via CRISPR-mediated deletion of the Tp53 gene. This method is highly efficient and consistently generates immortalized MEFs, or iMEFs, within 14 days. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted to immortalize other mouse primary cell types.

Activation of cellular antioxidative stress and migration activities by purified components from immortalized stem cells from human exfoliated deciduous teeth.

Although stem cell-based regenerative medicine has been extensively studied, it remains difficult to reconstruct three dimensional tissues and organs in combination with vascular systems in vitro. One clinically successful therapy is transplantation of mesenchymal stem cells (MSC) into patients with graft versus host disease. However, transplanted cells are immediately damaged and destroyed because of innate immune reactions provoked by thrombogenic inflammation, and patients need to take immunosuppressive drugs for the immunological regulation of allogeneic cells. This reduces the benefits of stem cell transplantation. Therefore, alternative therapies are more realistic options for clinical use. In this study, we aimed to take advantage of the therapeutic efficacy of MSC and use multiple cytokines released from MSC, that is, stem cells from human exfoliated deciduous teeth (SHEDs). Here, we purified components from conditioned media of immortalized SHED (IM-SHED-CM) and evaluated the activities of intracellular dehydrogenase, cell migration, and antioxidative stress by studying the cells. The immortalization of SHED could make the stable supply of CM possible. We found that the fractionated component of 50-100 kD from IM-SHED-CM had higher efficacy than the original IM-SHED-CM in terms of intracellular dehydrogenase and cell migration in which intracellular signal transduction was activated via receptor tyrosine kinases, and the glutathione peroxidase and reductase system was highly active. Although antioxidative stress activities in the fractionated component of 50-100 kD had slightly lower than that of original IM-SHE-CM, the fraction still had the activity. Thus, the use of fractionated components of 50-100 kD from IM-SHED-CM could be an alternative choice for MSC transplantation because the purified components from CM could maintain the effect of cytokines from SHED.

Comparison of primary and passaged tumor cell cultures and their application in personalized medicine.

Passaged cell lines represent currently an integral component in various studies of malignant neoplasms. These cell lines are utilized for drug screening both in monolayer cultures or as part of three-dimensional (3D) tumor models. They can also be used to model the tumor microenvironment in vitro and in vivo through xenotransplantation into immunocompromised animals. However, immortalized cell lines have some limitations of their own. The homogeneity of cell line populations and the extensive passaging in monolayer systems make these models distant from the original disease. Recently, there has been a growing interest among scientists in the use of primary cell lines, as these are passaged directly from human tumor tissues. In this case, cells retain the morphological and functional characteristics of the tissue from which they were derived, an advantage often not observed in passaged cultures. This review highlights the advantages and limitations of passaged and primary cell cultures, their similarities and differences, as well as existing test systems that are based on primary and passaged cell cultures for drug screening purposes.

Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) hold promise in regenerative medicine owing to their multipotent capabilities resembling mesenchymal stem cells (MSCs). Despite their potential, SHED have not been extensively investigated because their limited lifespan and unavailability of cell-lines pose challenges for therapeutic applications. This study investigated the effect of ectopic human telomerase reverse transcriptase (hTERT) expression on SHEDs' proliferation while preserving stemness and genomic integrity. METHODS: Deciduous teeth were collected from children aged 6-10 years. After isolation and characterization, the SHED were transduced with pBabe-puro-hTERT retrovirus to establish SHED cell-line, which was evaluated and compared with pBabe-puro (mock control) for stemness, multipotency and growth attributes through flow cytometry, trilineage differentiation, and growth kinetics. We also estimated hTERT gene expression, genomic integrity, and validated cell-line through STR analysis. RESULTS: Following hTERT transduction, SHED displayed elevated hTERT gene expression while retaining fibroblast-like morphology and mesenchymal stem cell markers. Moreover, after hTERT transduction cellular shape remained same along with increased replicative lifespan and proliferation potential. SHED-hTERT cells exhibited multi-potency and maintained stemness, as evidenced by surface marker expression and multilineage differentiation. Furthermore, genomic integrity was not affected by hTERT integration, as confirmed by STR analysis and CDKN2A gene assessment. CONCLUSION: Ectopic hTERT expression in SHED successfully prolonged their replicative lifespan and improved their ability to proliferate and migrate, while preserving their stemness, multipotency and genomic integrity, suggesting minimal carcinogenic risk. Establishment of SHED cell-line holds potential in regenerative medicine applications, especially in cell-based drugs and tissue engineering experiments.

Comparative Analysis of Primary Ovarian Cancer Cells and Established Cell Lines as a New Tool for Studies on Ovarian Cancer Cell Complexity.

Primary cancer cells reflect the genetic background and phenotype of a tumor. Immortalized cells with higher proliferation activity have an advantage over primary cells. The aim of the study was to immortalize the primary ovarian cancer (OvCa) cells using the plasmid-carrying human telomerase reverse transcriptase (hTERT) gene and compare their phenotype and biological activity with the primary cells. The primary OvCa3 A and OvCa7 A cells were isolated from the ascitic fluid of two high-grade serous ovarian cancer patients and were characterized using immunocytochemical methods, flow cytometry, real-time RT-PCR, Western blot, metabolic activity, and migratory potential. Both immortalized ovarian cancer cell lines mirrored the phenotype of primary cancer cells, albeit with modifications. The OvCa3 A hTERT cells kept the mesenchymal stem cell phenotype of CD73/CD90/CD105-positivity and were CD133-negative, whereas the cell population of OvCa7 A hTERT lost CD73 expression, but almost 90% of cells expressed the CD133 characteristic for the CSCs phenotype. Immortalized OvCa cells differed in gene expression level with respect to Sox2 and Oct4, which was associated with stemness properties. The OvCa7 A hTERT cells showed higher metabolic and migratory activity and ALDH1 expression than the corresponding primary OvCa cells. Both primary and immortalized cell lines were able to form spheroids. The newly established unique immortalized cell line OvCa7 A hTERT, with the characteristic of a serous ovarian cancer malignancy feature, and with the accumulation of the p53, Pax8, and overexpression of the CD133 and CD44 molecules, may be a useful tool for research on therapeutic approaches, especially those targeting CSCs in ovarian cancer and in preclinical 2D and 3D models.

Establishment of an immortalized yak granulosa cell line: in vitro tool for understanding the molecular processes of ovarian follicle development.

The yak, a unique species of cattle found exclusively on the western plateau of China, is a valuable source of livelihood for local residents. However, their low fecundity restricts the expansion of yak farming, whereas regional factors limit studies on yak breeding. Granulosa cells (GCs), which provide essential steroid hormones and growth factors for oocytes, have been the focus of many studies on the mechanisms of follicular growth and atresia. This study aimed to establish an immortalized cell line model that could serve as a tool for future studies on the mechanisms of ovarian follicle development in yaks. First, we isolated primary yak granulosa cells (yGCs) and evaluated their replicative senescence after continuous in vitro subculturing. Subsequently, an immortalized culture method for primary yGC was explored, and a new cell line model was established to study the mechanism of follicular development in vitro. We used a mammalian gene expression lentivirus vector to transfer the simian virus 40 large T antigen (SV40T) into primary yGC to obtain an immortalized cell line. The immortalized yGCs were morphologically identical to the primary yGCs, and cell proliferation and growth were normal within a limited number of generations. Follicle-stimulating hormone receptor (FSHR), a specific marker for GCs, was positively expressed in immortalized yGCs. Furthermore, the immortalized yGCs retained the ability of GCs to synthesize estradiol and progesterone and expressed genes related to steroid synthesis. The establishment of immortalized yGC opens up a myriad of possibilities for advancing our understanding of yak reproductive biology and improving yak breeding strategies.

Considerations for cultivated crustacean meat: potential cell sources, potential differentiation and immortalization strategies, and lessons from crustacean and other animal models.

Cultivated crustacean meat (CCM) is a means to create highly valued shrimp, lobster, and crab products directly from stem cells, thus removing the need to farm or fish live animals. Conventional crustacean enterprises face increasing pressures in managing overfishing, pollution, and the warming climate, so CCM may provide a way to ensure sufficient supply as global demand for these products grows. To support the development of CCM, this review briefly details crustacean cell culture work to date, before addressing what is presently known about crustacean muscle development, particularly the molecular mechanisms involved, and how this might relate to recent work on cultivated meat production in vertebrate species. Recognizing the current lack of cell lines available to establish CCM cultures, we also consider primary stem cell sources that can be obtained non-lethally including tissues from limbs which are readily released and regrown, and putative stem cells in circulating hemolymph. Molecular approaches to inducing myogenic differentiation and immortalization of putative stem cells are also reviewed. Finally, we assess the current status of tools available to CCM researchers, particularly antibodies, and propose avenues to address existing shortfalls in order to see the field progress.

Establishment of primary prostate epithelial and tumorigenic cell lines using a non-viral immortalization approach.

BACKGROUND: Research on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells. METHODS: Radical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells. RESULTS: Initial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent. CONCLUSION: Whereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.

Living Beyond Restriction: LBR promotes cellular immortalization by suppressing genomic instability and senescence.

Cellular immortalization is a complex process that requires multiple genetic alterations to overcome restricting barriers, including senescence. Not surprisingly, many of these alterations are associated with cancer; two tumor suppressor pathways, the cellular tumor antigen p53 and p16-Retinoblastoma (RB) pathways, are the best-characterized examples, but their mutations alone are known to be insufficient to drive full immortalization. En et al. identified a role for the lamin B receptor (LBR) in promoting cellular proliferation and immortalization in p53- and RB-deficient cells by maintaining their genome integrity and suppressing senescence. Thus, modulation of LBR could be exploited to treat cancer and potentially also to promote cell rejuvenation.

An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells.

The utilization of chicken oviductal epithelial cells (OECs) as a bioreactor to produce therapeutic proteins has shown promise, but the time taken to obtain transgenic offspring impedes efficient validation of protein production. To overcome this barrier, we focused on the immortalization of chicken OECs (cOECs) using retroviral vector-mediated c-MYC oncogene expression to establish an in vitro pre-validation system for chicken bioreactors. The resulting immortalized cOECs exhibited sustained proliferation, maintained a normal diploid chicken karyotype, and expressed key oviduct-specific genes (OVA, OVM, LYZ, AVD, and ESR1). Notably, hormonal administration of diethylstilbestrol (DES) or progesterone (P4) upregulated oviduct-specific genes in these cells. To enhance the utility of these immortalized cOECs as an in vitro validation system for chicken bioreactors, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was employed to knock-in (KI) an enhanced green fluorescence protein (EGFP) gene at the ovalbumin (OVA) locus. The resulting OVA EGFP KI immortalized cOECs secreted both EGFP and OVA proteins into the culture medium, with secretion enhanced under DES treatment. This successful integration of an exogenous gene into cOECs enhances their potential as a versatile in vitro validation system for chicken bioreactors. The established immortalized cOECs overcome previous challenges associated with long-term culture and maintenance, providing a reliable platform for efficient protein production validation. This study presents a comprehensive characterization of the immortalized cOECs, addressing critical limitations associated with in vivo systems and laying a foundation for the development of a streamlined and effective chicken bioreactor model.

Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-ß-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.

Development of a Mouse Experimental System for the In Vivo Characterization of Bioengineered Adipose-Derived Stromal Cells.

Human adipose-derived stromal cells (ADSCs) are an important resource for cell-based therapies. However, the dynamics of ADSCs after transplantation and their mechanisms of action in recipients remain unclear. Herein, we generated genetically engineered mouse ADSCs to clarify their biodistribution and post-transplantation status and to analyze their role in recipient mesenchymal tissue modeling. Immortalized ADSCs (iADSCs) retained ADSC characteristics such as stromal marker gene expression and differentiation potential. iADSCs expressing a fluorescent reporter gene were seeded into biocompatible nonwoven fabric sheets and transplanted into the dorsal subcutaneous region of neonatal mice. Transplanted donor ADSCs were distributed as CD90-positive stromal cells on the sheets and survived 1 month after transplantation. Although accumulation of T lymphocytes or macrophages inside the sheet was not observed with or without donor cells, earlier migration and accumulation of recipient blood vascular endothelial cells (ECs) inside the sheet was observed in the presence of donor cells. Thus, our mouse model can help in studying the interplay between donor ADSCs and recipient cells over a 1-month period. This system may be of value for assessing and screening bioengineered ADSCs in vivo for optimal cell-based therapies.

Upregulated expression of lamin B receptor increases cell proliferation and suppresses genomic instability: implications for cellular immortalization.

Mammalian somatic cells undergo terminal proliferation arrest after a limited number of cell divisions, a phenomenon termed cellular senescence. However, cells acquire the ability to proliferate infinitely (cellular immortalization) through multiple genetic alterations. Inactivation of tumor suppressor genes such as p53, RB and p16 is important for cellular immortalization, although additional molecular alterations are required for cellular immortalization to occur. Here, we aimed to gain insights into these molecular alterations. Given that cellular immortalization is the escape of cells from cellular senescence, genes that regulate cellular senescence are likely to be involved in cellular immortalization. Because senescent cells show altered heterochromatin organization, we investigated the implications of lamin A/C, lamin B1 and lamin B receptor (LBR), which regulate heterochromatin organization, in cellular immortalization. We employed human immortalized cell lines, KMST-6 and SUSM-1, and found that expression of LBR was upregulated upon cellular immortalization and downregulated upon cellular senescence. In addition, knockdown of LBR induced cellular senescence with altered chromatin configuration. Additionally, enforced expression of LBR increased cell proliferation likely through suppression of genome instability in human primary fibroblasts that expressed the simian virus 40 large T antigen (TAg), which inactivates p53 and RB. Furthermore, expression of TAg or knockdown of p53 led to upregulated LBR expression. These observations suggested that expression of LBR might be upregulated to suppress genome instability in TAg-expressing cells, and, consequently, its upregulated expression assisted the proliferation of TAg-expressing cells (i.e. p53/RB-defective cells). Our findings suggest a crucial role for LBR in the process of cellular immortalization.

Cell Immortality: In Vitro Effective Techniques to Achieve and Investigate Its Applications and Challenges.

Cells are very important to researchers due to their use in various biological studies in in vitro and in vivo settings. This importance stems from the short lifespan of most cells under laboratory conditions, which can pose significant challenges, such as the difficulties associated with extraction from the source tissue, ethical concerns about separating cells from human or animal models, limited cell passage ability, and variation in results due to differences in the source of the obtained cells, among other issues. In general, cells in laboratory conditions can divide into a limited number, known as the Hayflick limit, due to telomere erosion at the end of each cellular cycle. Given this problem, researchers require cell lines that do not enter the senescence phase after a limited number of divisions. This can allow for more stable studies over time, prevent the laborious work associated with cell separation and repeated cultivation, and save time and money in research projects. The aim of this review is to summarize the function and effect of immortalization techniques, various methods, their advantages and disadvantages, and ultimately the application of immortalization and cell line production in various research fields.

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.