Human Cell Culture Model Research Articles

Cigarette Smoke and Cigarette Smoke Condensate Induce Inflammation and Cytotoxicity in Precision-Cut Lung Slices (PCLS)

COPD is characterised by emphysema, mucus hypersecretion and airway inflammation. COPD is the fourth leading cause of death worldwide. Major reason to develop COPD is cigarette smoke (Cs). Therefore, a human relevant Cs model is needed. Currently several human cell culture models and non-human animal models are well-established. Most are lacking the human microenvironment, which is necessary to induce inflammatory, destructive and cytotoxic effects of Cs. Thus, the project's aim is to establish features of COPD in the 3D model of Precision-Cut Lung Slices (PCLS) of different species by using Cs and cigarette smoke condensate (Csc).

Cell sources for invitro human liver cell culture models.

Invitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic invitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells invitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for invitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality invitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

Abstract 4261: Novel tumor microenvironment (TME) restored 3D cell culture model and humanized patient-derived xenograft model for cancer research and drug evaluation

Abstract New anticancer drug development is an expensive and time consuming process, which typically can take more than 10 years and cost over a billion dollars. Although a majority of late discovery drug candidates demonstrate potency in preclinical models, most will ultimately fail to achieve efficacy in clinical trials. While recent strides have been made to improve preclinical models, the continued widespread use of traditional models creates translational challenges during clinical development. An intrinsic challenge for drug developers is to close the gap between preclinical modeling and translational efficiency for patients. Accurately modeling core attributes of the human tumor microenvironment (TME) in preclinical models is key for identifying novel targets, lowering toxicity and improving translational success. Even for advanced patient-derived xenograft (PDX) in vivo models, the cytokines generated by murine stromal cells fail to replicate many important paracrine networks, which can mislead preclinical pharmacology outcome data. To address this gap, we have begun to establish and characterize nearly 1,000 novel patient tumor samples, spanning over a dozen cancer indications, into robust human TME-restored 3D cell culture models and humanized patient derived xenograft models. Our goal is to improve overall patient-derived tumor model initiation success rates and subsequent translational pharmacology. Citation Format: Jing Zhang, Dabing Yang, Huilin Wang, Qiong Song, Hui Li, JP Shaw, Tianjing Deng. Novel tumor microenvironment (TME) restored 3D cell culture model and humanized patient-derived xenograft model for cancer research and drug evaluation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4261.

RECONSTRUCTING ALZHEIMER AMYLOID AND TAU PATHOLOGY IN 3D CELL CULTURES FROM STEM CELLS

Alzheimer’s disease (AD) affects 5.4 million individuals in the US with no effective therapeutic option available except for symptomatic treatments. Transgenic mouse models with FAD mutations have been used to study AD and for drug discovery. However, in these AD mouse models, deposition of beta-amylod does not lead to neurofibrillary tangle formation. Thus, there are profund limitations for their use as disease model. We previoulsy developed a novel 3D human neural cell culture model that recapitulates amyloid-β (Aβ) plaques and tangles (from endogenous tau) by expressing FAD mutations that lead to the deposition of beta-amyloid in roughly six weeks (Choi et al., Nature, 2014; Kim et al. Nature protocols, 2015). More recently, we have made 3D cultures using single-clonal human neural stem cells in a 96-well FACS-assisted single cell cloning protocol. These are being used for drug discovery aimed at preventing plaque and tangle formation. The clonal homogenous FAD cells secrete pathogenic Aβ species leading to plaques and phospho-tau/tangle pathology and exhibit dystrophic neurites, LDH release, and caspase 3 activation. They are currently being used for drug screening. The new single-clonal FAD cell lines, together with 3D culture technology, provide a novel model for studying AD neuropathogenesis and drug discovery.

Cell Models and Their Application for Studying Adipogenic Differentiation in Relation to Obesity: A Review.

Over the last several years, the increasing prevalence of obesity has favored an intense study of adipose tissue biology and the precise mechanisms involved in adipocyte differentiation and adipogenesis. Adipocyte commitment and differentiation are complex processes, which can be investigated thanks to the development of diverse in vitro cell models and molecular biology techniques that allow for a better understanding of adipogenesis and adipocyte dysfunction associated with obesity. The aim of the present work was to update the different animal and human cell culture models available for studying the in vitro adipogenic differentiation process related to obesity and its co-morbidities. The main characteristics, new protocols, and applications of the cell models used to study the adipogenesis in the last five years have been extensively revised. Moreover, we depict co-cultures and three-dimensional cultures, given their utility to understand the connections between adipocytes and their surrounding cells in adipose tissue.

Design, characterization, and modeling of microcirculation systems with integrated oxygenators

Abstract. Here, we describe a microfluidic system for hypoxia assays on human cell culture models. These systems are developed to replace or reduce animal testing in biomedical basic research. The presented system uses a gas-permeable membrane as a gas–liquid interface and a micropump for media actuation to influence the oxygen content in two cell culture chambers. To apply well-defined hypoxic conditions to the cells, a good understanding of the mass transport phenomena is necessary. Therefore, a complete network model of the microfluidic system is presented. This model is validated by means of micro-particle image velocimetry (µPIV) and optical oxygen measurement with fluorescence lifetime detection. Finally, the impact of several process parameters, e.g., the gas permeability of the pump, is discussed using the developed model.

Hypoxia and tissue destruction in pulmonary TB

BackgroundIt is unknown whether lesions in human TB are hypoxic or whether this influences disease pathology. Human TB is characterised by extensive lung destruction driven by host matrix metalloproteinases (MMPs),...

Effect Of 30 Or 11.5 Volts Of Pulse Stimulation On Mitochondrial Density In Vitro

We have previously shown an increase in mitochondrial density using an exercise mimetic electrical pulse stimulation (EPS) (30V; single bipolar pulses of 2ms and 1Hz continuously) applied to an in vitro human primary cell culture model. PURPOSE: To determine if continuous EPS of 11.5V, 2ms single bipolar pulses at 1Hz is a valid in vitro exercise mimetic to effectively increase mitochondrial density and insulin action of human primary myotubes. METHODS: Primary cultures were established from myoblast extracted from 5 healthy lean Caucasian male donors (23 ± 1.9 y; BMI 24.2 ± 0.6 kg/m2). Myoblasts were pooled, grown and differentiated into myotubes. EPS was applied to fully differentiated myotubes at either 11.5V or 30V, using single bipolar pulses of 2ms and 1Hz continuously for 24 hours. Control groups were maintained without stimulation. Protein was extracted from EPS stimulated and unstimulated (control) myotubes for western immunobloting of mitochondrial density measured by total OXPHOS, and insulin action measured by Akt phosphorylation. RESULTS: 24 hours of EPS at 30V resulted in ~1.7 fold greater OXPHOS content compared to the unstimulated control, while 11.5V resulted in 0.82 fold lesser OXPHOS content compared to control. After 24 hours of EPS, there was a 0.9 fold and 0.5 fold difference in the Akt phosphorylation using 30V and 11.5V respectively, compared to corresponding unstimulated control cells. CONCLUSION: 30V of EPS resulted in a greater mitochondrial density, an evident adaptation to regular physical activity, and greater Akt phosphorylation compared to 11.5V stimulation, suggesting electrical pulse stimulation of continuous single bipolar pulses of 2ms at 30V and 1Hz, is a more effective exercise mimetic model for in vitro human myotubes than 11.5V.

Comparative genome-wide screens reveal novel regulators of Cytotoxic Lymphocyte-mediated cell death.

Abstract Cytotoxic Lymphocytes (CLs) are key players in the effector branch of the immune response, detecting and eliminating transformed and pathogen-infected cells. While the physiological roles of CL-mediated apoptosis are well established, many regulators of these apoptotic pathways in target cells remain poorly understood. We performed comparative genome-wide haploid genetic screens in a human cell culture model for CL killing and resistance in order to identify positive and negative regulators of target cell survival during CL-mediated killing. These regulators include genes encoding glycosylphosphatidylinositol (GPI) anchor synthesis enzymes and immune surveillance receptors. In order to identify further potential components of these pathways, we performed an additional resistance screen in a GPI-anchored protein deficient haploid cell line. We identified both common and disparate regulators between unaltered target cells and GPI-anchored protein deficient target cells. These disparate regulators underline the importance of GPI-anchored proteins in CL target recognition and killing. Some gene knockouts enriched by CL-mediated killing in both screens are also mutated in cancer. Further study of the regulators identified by these screens will advance our understanding of how the many killing pathways used by CLs shape the interaction between the immune system and cancer.

075 Ephrin-A loss in cutaneous squamous cell carcinoma progression and metastasis

075 Ephrin-A loss in cutaneous squamous cell carcinoma progression and metastasis

078 Dsg2 enhances spontaneous BCC formation in Ptc1+/−mice

078 Dsg2 enhances spontaneous BCC formation in Ptc1+/−mice

Growth factor restriction impedes progression of wound healing following cataract surgery: identification of VEGF as a putative therapeutic target.

Secondary visual loss occurs in millions of patients due to a wound-healing response, known as posterior capsule opacification (PCO), following cataract surgery. An intraocular lens (IOL) is implanted into residual lens tissue, known as the capsular bag, following cataract removal. Standard IOLs allow the anterior and posterior capsules to become physically connected. This places pressure on the IOL and improves contact with the underlying posterior capsule. New open bag IOL designs separate the anterior capsule and posterior capsules and further reduce PCO incidence. It is hypothesised that this results from reduced cytokine availability due to greater irrigation of the bag. We therefore explored the role of growth factor restriction on PCO using human lens cell and tissue culture models. We demonstrate that cytokine dilution, by increasing medium volume, significantly reduced cell coverage in both closed and open capsular bag models. This coincided with reduced cell density and myofibroblast formation. A screen of 27 cytokines identified nine candidates whose expression profile correlated with growth. In particular, VEGF was found to regulate cell survival, growth and myofibroblast formation. VEGF provides a therapeutic target to further manage PCO development and will yield best results when used in conjunction with open bag IOL designs.

Inhibition of HSV-1 Replication by Gene Editing Strategy.

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)

PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors and in vitro differentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCs in vitro. Knock-down of PMM2 by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.

Output-driven feedback system control platform optimizes combinatorial therapy of tuberculosis using a macrophage cell culture model

Tuberculosis (TB) remains a major global public health problem, and improved treatments are needed to shorten duration of therapy, decrease disease burden, improve compliance, and combat emergence of drug resistance. Ideally, the most effective regimen would be identified by a systematic and comprehensive combinatorial search of large numbers of TB drugs. However, optimization of regimens by standard methods is challenging, especially as the number of drugs increases, because of the extremely large number of drug-dose combinations requiring testing. Herein, we used an optimization platform, feedback system control (FSC) methodology, to identify improved drug-dose combinations for TB treatment using a fluorescence-based human macrophage cell culture model of TB, in which macrophages are infected with isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible green fluorescent protein (GFP)-expressing Mycobacterium tuberculosis (Mtb). On the basis of only a single screening test and three iterations, we identified highly efficacious three- and four-drug combinations. To verify the efficacy of these combinations, we further evaluated them using a methodologically independent assay for intramacrophage killing of Mtb; the optimized combinations showed greater efficacy than the current standard TB drug regimen. Surprisingly, all top three- and four-drug optimized regimens included the third-line drug clofazimine, and none included the first-line drugs isoniazid and rifampin, which had insignificant or antagonistic impacts on efficacy. Because top regimens also did not include a fluoroquinolone or aminoglycoside, they are potentially of use for treating many cases of multidrug- and extensively drug-resistant TB. Our study shows the power of an FSC platform to identify promising previously unidentified drug-dose combinations for treatment of TB.

Early Detection of NephrotoxicityIn VitroUsing a Transparent Conducting Polymer Device

Electrical methods for monitoring cell toxicity are becoming increasingly popular because of their amenability for longer-term, continuous, and label-free monitoring. Microfabrication techniques are also opening the way for miniaturization of devices and integration with microphysiometer or lab-on-chip–type devices. In the present work, we demonstrate the use of a transparent conducting polymer device, termed the “organic electrochemical transistor” (OECT), which we have used to monitor the effects of a subset of known nephrotoxicants (cisplatin, tobramycin, and gentamycin) on cell integrity in vitro. Based on a similar principle to traditional electronic impedance spectroscopy, the OECT provides an even more sensitive way to detect small changes in ionic currents in an electrolyte, as a result of inherent amplification of signal provided thanks to the transistor format. The device monitored the ability of the human renal proximal tubular epithelial cells, a human primary cell culture model of the kidney proximal tubule, to act as a barrier to ion flow (analogous to transepithelial resistance), even though this was not possible with a commercially available instrument (cellZscope). Further, the device demonstrated extremely rapid nephrotoxicity even at low concentrations, the transparency of the device allowed in situ monitoring of cells on the device, as well as immunofluorescence staining of key biomarkers of kidney tubule function: KIM-1 and ZO-1. Correlation of electrical and optical data was demonstrated as a powerful tool for validation of the method. In summary, the OECT is presented as an extremely versatile tool for highly sensitive nephrotoxicity monitoring, amenable for future integration into microfabricated lab-on-chip platforms.

Visualizing tropoelastin in a long-term human elastic fibre cell culture model.

Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network. Future investigations will allow the tracking of TE produced under various conditions. In tissue engineering applications the fluorescent fibre network can be visualized under various conditions or it serves as a tool for investigating fibre degradation processes in disease-in-a-dish-models.

Abstract A51: Investigation of the DEK oncogene as a blood biomarker for breast cancer

Abstract Breast cancer (BC) is one of the leading causes of death for women in the United States. This is due, in part, because women wait too long to get a diagnosis or the disease returns, sometimes years later, due to ineffective treatment. Therefore, it is critically important to identify biomarkers that can better inform clinicians while providing a screening test more amenable to patients than mammograms. We are investigating the feasibility of using DEK protein levels in patient plasma as a biomarker for disease stage and prognosis. DEK is a chromatin-organizing protein that functions in DNA replication and repair, transcription, and mRNA splicing. The DEK oncoprotein is highly upregulated in many types of cancer including breast, head and neck, and melanoma. Using human cell culture and murine models, our previous studies have shown that DEK is functionally important for the promotion of cellular growth, invasion/metastasis, drug resistance, and breast cancer stem cell maintenance. Work by the Markovitz laboratory indicated that DEK is secreted by macrophages as a pro-inflammatory signaling molecule and can be internalized by neighboring cancer cells to promote typical DEK functions. This led to our hypothesis that DEK may be present in the plasma of cancer patients. In fact, the detection of DEK protein in urine is currently being explored as a diagnostic biomarker for bladder cancer. To test this hypothesis, we collected peripheral blood from patients with newly diagnosed, untreated BC (irrespective of the clinical stage) and age- and ethnicity-matched normal healthy controls. Plasma was separated from the samples and subjected to DEK specific ELISA (Cusabio, Wuhan, China). Preliminary results demonstrate that DEK is indeed present in the plasma of BC patients and we are currently comparing these levels to normal healthy controls. Further analyses are ongoing to determine whether DEK levels correlate with pertinent clinical and pathological variables including age, tumor stage, and ER/PR/HER-2 status. In addition, we are examining if DEK levels can predict response to various treatment modalities and risk of relapse. These data will be important to verify DEK plasma measurements as a clinically useful test and may give insight to future personalized and targeted treatment strategies for BC. Citation Format: Arun Sendilnathan, Mahmoud Charif, Elizabeth Shaughnessy, Jaime D. Lewis, Neetu Radhakrishnan, Elyse Lower, Catherine Lanigan, Harriet Kumar, Trisha M. Wise-Draper, Lisa Privette Vinnedge. Investigation of the DEK oncogene as a blood biomarker for breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A51.

The Effect of Silver Carp Skin-Derived Peptides on the Activities of VEGF and Hsp70.1 Gene Transcription Promoters.

Studies conducted on human cell culture models have demonstrated that collagen-derived peptides can exert a beneficial effect in medicine. However, all these studies were conducted using animal collagen samples, most often originating from bovine or porcine skin. Currently attempts are being made to replace animal collagen with fish collagen. The aim of the study was to compare the effect of silver carp skin-derived peptide extract on the transcriptional activities of human VEGF and hsp70.1 gene promoters inserted into the plasmids with secreted alkaline phosphatase as a reporter gene. Changes in the activity of the promoters were investigated using a HEK293FT cell line transfected with pVEGF-SEAP or pHsp70-SEAP. The cells were cultured in dishes containing peptides separated using reverse-phase high performance liquid chromatography. The study demonstrated that the silver carp skin-derived peptide extract exerts both an inhibitory effect on the VEGF gene promoter and activating effect on the hsp70.1 gene promoter. Higher biological activity was recorded in the case of a freshly prepared peptide extract compared to one stored at 4°C for three months. The silver carp skin-derived collagen peptides influence VEGF and hsp70.1 gene promoters' transcriptional activity.

Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care Products on HaCaT Human Skin Cells

Background: Human skin is normally exposed to ionizing and UV radiations and on occasions it may also be subjected to beauty products or drugs that the host uses; all of which can generate reactive oxygen species (ROS). Objective: In this study, Calendula officinalis extract, which contains antioxidant compounds, was examined for its protective effects against the products that generate ROS- induced cytotoxic activity towards human skin cells. Methodology: The protection against cytotoxicity by Calendula officinalis extract was investigated in vitro using a dose-response on HaCaT human skin cells. The proprietary aqueous calendula extract C from biodynamically grown plant was examined. Protection against cytotoxicity was measured via the methyl tetrazolium cytotoxicity (MTT) assay. Cells were exposed to the calendula extracts for 48h before being exposed to personal care products consisting of two head lice treatments -Lice Breaker (Permethrin 1% w/w) and Organix Pyrethrum treatment (4 g/L Pyrethrins, 16 g/LPiperonyl Butoxide) and two beauty products -Nivea Visage Q10 plus Anti-Wrinkle face moisturizer + TiO2; and Nivea Visage Q10 Plus Anti-Wrinkle face moisturizer (without TiO2) for 1h. Results: The effect of different concentrations of calendula extract on HaCaT human skin cells in vitro was explored. Doses of 1% (v/v) [1.4 mg dry weight equiv/ml] or less showed no toxicity. Cells were also exposed to the calendula extract for 48 hours before being exposed to the four personal care products for 1h. using the MTT cytotoxicity assay; it was observed that extract of Calendula officinalis gave time-dependent and concentrationdependent protection against harmful products that induce cell killing. Pre-incubation with the calendula extract for 48 hours significantly increased survival relative to the population without the extract by 30% and 47.4%, respectively, following treatment with personal care products. Conclusion: This study demonstrates that calendula extract contains bioactive and free radical scavenging compounds that significantly protect against personal care products that generate oxidative stress cytotoxicity in a human skin cell culture model.