Long-term Culture Of Primary Cells Research Articles

Next-Generation Modeling of Cancer Using Organoids.

In the last decade, organoid technology has become a cornerstone in cancer research. Organoids are long-term primary cell cultures, usually of epithelial origin, grown in a three-dimensional (3D) protein matrix and a fully defined medium. Organoids can be derived from many organs and cancer types and sites, encompassing both murine and human tissues. Importantly, they can be established from various stages during tumor evolution and recapitulate with high accuracy patient genomics and phenotypes in vitro, offering a platform for personalized medicine. Additionally, organoids are remarkably amendable for experimental manipulation. Taken together, these features make organoids a powerful tool with applications in basic cancer research and personalized medicine. Here, we will discuss the origins of organoid culture, applications in cancer research, and how cancer organoids can synergize with other models of cancer to drive basic discoveries as well as to translate these toward clinical solutions.

Effects of hepatocyte growth factor on porcine mammary cell growth and senescence.

The porcine mammary glands share morphological and physiological similarities with human ones, making primary porcine mammary cells (PMC) suitable for biomedical research and a potential cellular therapeutic for breast cancer xenogeneic cell immunotherapy. Primary cells isolated from tissues remain the physiological functions of origin tissues but their self-renewal ability is restricted and cells acquire senescence during in vitro expansion. To overcome these drawbacks, here we sought to establish an approach to efficiently increase PMC's in vitro growth. We studied the effects of the hepatocyte growth factor (HGF) to maintain the expansion capacity of porcine mammary cells and identify the possible mechanisms. HGF could allow for the increase in vitro proliferation capacity of primary epithelial cells isolated from tissue samples. To effectively produce cells for biomedical research and xenogeneic cell therapy, we planned to study the effects of HGF and its potential mechanisms of action to stimulate cell growth for PMC expansion. After HGF treatment, the growth, cell cycle, senescence and the cell marker gene expression of PMCs were analyzed in standard 10% FBS and low serum 1% FBS containing medium. HGF significantly enhanced the cell proliferation by shifting the cell cycle population from G1 phase into S phase to increase cell division, reduced the senescent cells and reprogrammed gene expression profiles. We demonstrated that HGF could maintain the expansion capacity of PMCs by increasing cell growth and anti-senescence capability, suggesting its potential application in optimizing the long-term culture of primary cells. Adding a specific growth factor such as HGF in culture allows enhanced expansion of heterogeneous cell populations from normal porcine mammary glandular tissues in vitro. We believe that this cell culture approach will efficiently provide cells for studying mammary cell function and supply cells for therapeutic uses.

Blockade of thymic stromal lymphopoietin (TSLP) receptor inhibits TSLP-driven proliferation and signalling in lymphoblasts from a subset of B-precursor ALL patients

PurposeThe cytokine thymic stromal lymphopoietin (TSLP) and its receptor TSLPR are involved in intercellular communication in the course of allergic inflammation and have recently been implicated in the development of various malignancies including B cell precursor acute lymphoblastic leukemia (BCP-ALL). We studied TSLPR expression, TSLP-induced signal transduction and its antibody-mediated inhibition in long-term cultures of primary cells derived from B-precursor ALL patients. MethodsTSLPR expression was determined by flow cytometry and Western blot analysis, cell proliferation, signal transduction via the JAK/STAT pathway was analysed by Western blot detection of STAT tyrosine phosphorylation and by measuring TSLP-dependent activation of a STAT-specific reporter gene construct. For inhibition studies a recently introduced antagonistic antibody to the TSLPRα-subunit was used. ResultsTSLPR surface expression was observed in leukemic lymphoblasts from two out of ten patients with BCP-ALL. Upon TSLP stimulation, the cells with the highest TSLPR expression level showed enhanced proliferation and JAK/STAT-mediated gene regulation in a dose-dependent manner. By employment of an inhibitory antibody to the TSLPR, both TSLP-triggered cell proliferation and STAT transcription factor activation were specifically inhibited. ConclusionsThese results suggest that blockade of the TSLPR might be a therapeutic option for a subset of BCP-ALL patients.

Noncoding RNA control of cellular senescence.

Senescent cells accumulate in normal tissues with advancing age and arise by long-term culture of primary cells. Senescence develops following exposure to a range of stress-causing agents and broadly influences the physiology and pathology of tissues, organs, and systems in the body. While many proteins are known to control senescence, numerous noncoding (nc)RNAs are also found to promote or repress the senescent phenotype. Here, we review the regulatory ncRNAs (primarily microRNAs and lncRNAs) identified to-date as key modulators of senescence. We highlight the major senescent pathways (p53/p21 and pRB/p16), as well as the senescence-associated secretory phenotype (SASP) and other senescence-associated events governed by ncRNAs, and discuss the importance of understanding comprehensively the ncRNAs implicated in cell senescence.

Primary cell cultures from sea urchin ovaries: a new experimental tool

In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

A Metabolic Screening Study of Trichostatin A (TSA) and TSA-Like Histone Deacetylase Inhibitors in Rat and Human Primary Hepatocyte Cultures

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, Calpha-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amide-linked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance.

Presumptive insect circadian pacemakers in vitro: immunocytochemical characterization of cultured pigment-dispersing hormone-immunoreactive neurons of Leucophaea maderae.

The accessory medulla with its associated pigment-dispersing hormone-immunoreactive neurons appears to be the pacemaker that controls the circadian locomotor activity rhythm of the cockroach Leucophaea maderae. To permit studies at the level of individual, identified, pacemaker neurons, we developed specific long-term primary cell cultures of fully differentiated adult neurons of the accessory medulla. As judged from soma diameter distribution, the cultures contain an unbiased representation of apparently all neuronal types of the accessory medulla. The cultured cells survive and grow processes for more than 2 months with or without additional hemocyte coculturing. However, a strong positive effect on initial outgrowth was observed with hemocyte coculturing. At least six different morphological cell types of the accessory medulla could be distinguished in vitro. Among these only one cell type, the monopolar type C cell, was recognized in vitro with an antiserum against the neuropeptide pigment-dispersing hormone. Thus, the identifiable monopolar type C cells are candidates for circadian pacemaker neurons and will be the focus of further physiological characterizations.

Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich

Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well the in vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells.