Organotypic Tissue Culture Research Articles

Modeling Esophagitis Using Human Three-Dimensional Organotypic Culture System

Esophagitis, whether caused by acid reflux, allergic responses, graft-versus-host disease, drugs, or infections, is a common condition of the gastrointestinal tract affecting nearly 20% of the US population. The instigating agent typically triggers an inflammatory response. The resulting inflammation is a risk factor for the development of esophageal strictures, Barrett esophagus, and esophageal adenocarcinoma. Research into the pathophysiology of these conditions has been limited by the availability of animal and human model systems. Three-dimensional organotypic tissue culture (OTC) is an innovative three-dimensional multicellular invitro platform that recapitulates normal esophageal epithelial stratification and differentiation. We hypothesized that this platform can be used to model esophagitis to better understand the interactions between immune cells and the esophageal epithelium. We found that human immune cells remain viable and respond to cytokines when cultured under OTC conditions. The acute inflammatory environment induced in the OTC significantly affected the overlying epithelium, inducing a regenerative response marked by increased cell proliferation and epithelial hyperplasia. Moreover, oxidative stress from the acute inflammation induced DNA damage and strand breaks in epithelial cells, which could be reversed by antioxidant treatment. These findings support the importance of immune cell-mediated esophageal injury in esophagitis and confirms the utility of the OTC platform to characterize the underlying molecular events in esophagitis.

Mutations in SULT2B1 Cause Autosomal-Recessive Congenital Ichthyosis in Humans

Ichthyoses are a clinically and genetically heterogeneous group of genodermatoses associated with abnormal scaling of the skin over the whole body. Mutations in nine genes are known to cause non-syndromic forms of autosomal-recessive congenital ichthyosis (ARCI). However, not all genetic causes for ARCI have been discovered to date. Using whole-exome sequencing (WES) and multigene panel screening, we identified 6 ARCI-affected individuals from three unrelated families with mutations in Sulfotransferase family 2B member1 (SULT2B1), showing their causative association with ARCI. Cytosolic sulfotransferases form a large family of enzymes that are involved in the synthesis and metabolism of several steroids in humans. We identified four distinct mutations including missense, nonsense, and splice site mutations. We demonstrated the loss of SULT2B1 expression at RNA and protein levels in keratinocytes from individuals with ARCI by functional analyses. Furthermore, we succeeded in reconstructing the morphologic skin alterations in a 3D organotypic tissue culture model with SULT2B1-deficient keratinocytes and fibroblasts. By thin layer chromatography (TLC) of extracts from these organotypic cultures, we could show the absence of cholesterol sulfate, the metabolite of SULT2B1, and an increased level of cholesterol, indicating a disturbed cholesterol metabolism of the skin upon loss-of-function mutation in SULT2B1. In conclusion, our study reveals an essential role for SULT2B1 in the proper development of healthy human skin. Mutation in SULT2B1 leads to an ARCI phenotype via increased proliferation of human keratinocytes, thickening of epithelial layers, and altered epidermal cholesterol metabolism.

In Vitro Systems Toxicology assessment of acute exposure of an aerosol from a candidate modified‐risk tobacco product on organotypic human small airways tissue culture

The primary site of morphological changes in the airways associated with chronic cigarette smoking is the small airway epithelium. The small airway refers to bronchi of less than 2 mm in diameter. It is the initial site of Chronic Obstructive Pulmonary Disease (COPD). Mechanistic investigations of the effect of inhaled substances on the small airway tissue are difficult to conduct because biopsy samples are not readily obtainable. Recently, a human small airway epithelium culture model grown at the air liquid interface, SmallAir™ (Epithelix, Switzerland) was established. The model is reconstituted from primary small airway epithelial cells isolated from the distal lungs. The morphology of the model mimics the characteristics of in vivo human small airway epithelial tissue. It offers a relevant alternative for an in‐vitro toxicity assessment of inhalation exposure. Using the newly developed human organotypic small airway model (reconstituted from a healthy, non‐smoker donor), this study assessed the biological impact of an aerosol generated from a candidate modified risk tobacco product, the tobacco heating system 2.2 (THS2.2). The biological impact of the THS2.2 aerosol was compared with that of the smoke generated from the reference cigarette 3R4F at comparable nicotine concentrations. A comprehensive set of biological endpoints were measured at various time points after exposure including cytotoxicity, culture histology, cytochrome P450 (CYP) 1A1/1B1 activity, secreted pro‐inflammatory mediators concentrations, and global mRNA/miRNA alterations. Following a 28‐min exposure to 3R4F smoke, various inflammatory markers (MMP‐9, IL‐6 and IL‐8) and genes (MGST2, GPX2, AKR1B1) known to be involved in the pathogenesis of COPD were upregulated. A significantly lower impact of THS2.2 aerosol exposure was observed not only on these COPD‐related molecular entities, but also on cytotoxicity, secreted pro‐inflammatory mediators, and global alterations of mRNA and miRNA as compared with those of 3R4F smoke at comparable nicotine concentrations. Using a network‐based Systems Toxicology approach, the gene expression profile 4 h after 3R4F smoke exposure at 0.15 mg nicotine/L elicited the highest biological impact (taken as 100% impact). By comparison, the impact of THS2.2 aerosol exposure at comparable nicotine concentration was only 14.6%. Collectively, this study shows that the biological impact of THS2.2 aerosol was significantly lower than that of 3R4F smoke at similar nicotine concentrations on the in vitro small airway cultures.Support or Funding InformationPMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH‐2000 Neuchâtel, Switzerland (Part of Philip Morris International group of companies).

The role of polypeptide compounds in mechanism of CNS plasticity in patients with hereditary pathology of peripheral motor neuron

Synaptic pruning is a physiological mechanism of neuroplasticity, which is regulated through synthesis of growth polypeptides, neurotrophins. The role of neurotrophins in the mechanism of synaptic pruning in patients with hereditary pathology of peripheral motor neuron was studied in a clinical experimental trial. It was found that patients had elevated levels of regulatory growth polypeptides, which led to the axon growth inhibition effect in organotypic tissue cultures. Thus, neurotrophin overexpression can be considered as a factor preventing synaptic pruning and contributing to further process of neurological degeneration in nerve tissue in patients with hereditary pathology of peripheral motor neuron.

Organotypic Models of Lung Cancer.

Human lung cancer is one of the leading causes of death worldwide, with nearly 2 million of new cases diagnosed each year, often too late for a successful therapeutic intervention. In this chapter, organotypic models of lung cancer will be reviewed. Ex vivo tissue explants, spheroids, organoids, and novel bioengineering approaches are currently being used to study human lung cancer. Although there is no ideal method that will fully recapitulate the complex human lung architecture, the three-dimensional (3D) organotypic models described here represent a major advance from classical two-dimensional (2D) tissue culture models. Organotypic tissue cultures are better at predicting in vivo tissue responses to anticancer drugs or carcinogenic toxins. In addition, there is also a possibility to use these systems as part of personalized medicine (e.g., to assist clinicians in selecting appropriate therapeutic choices based on individual patient-derived tissue responses to drugs in vitro). There is still a need for improvement in the 3D culture systems, such as automation for high-throughput testing and reduction of costs. However, given the large number of failed clinical trials due to safety or efficacy reasons, more attention should be given to these more physiologically relevant 3D organotypic tissue culture models.

Method for organotypic tissue culture in the aged animal

Organotypic slicing of brain tissue from young rodents has been used as a powerful model system for biomedical research [1–3]. Organotypic slicing complements cell culture and in vivo studies in multiple facets. This system can be useful for investigating manipulation of cellular signaling pathways without the hindrance of the blood-brain barrier while sacrificing fewer animals in the process. It also allows for preserved cellular connectivity and local intact circuitry which is a drawback of isolated cell cultures. Studies on age-related diseases have mainly used embryonic or early postnatal organotypic slice tissue. Excluding synaptic plasticity studies that are usually carried-out over a few hours and use adult mice or rats, a handful of studies performed on adult animals have had success for survival of slices [4,5]. Here we describe a method for culturing organotypic slices with high viability from hippocampus of aged mice and rabbits.•Our method permits slices from mice as old as 16 months and rabbits as old as years of age to survive ex vivo up to 8 weeks [6–9]. Such a slice system may be relevant to investigating age-related brain diseases.

IMPLEMENTATION OF IN VITRO ASSAY FOR EVALUATION OF BIOCORRECTIVE ACTION OF BIOACTIVE SUBSTANCES AN ANIMAL ORIGIN

Slaughter products are good source of bioactive substances with different biological effects. Nowadays, approximately 220 functional peptides consisting of 2–10 amino acid residues have been identified. In this study, the biological activity of medium-molecular-weight substances (5–30 kDa, Mw) and low-molecular-weight substances (less than 5 kDa, Mw) extracted from aorta of Sus scrofa were investigated in vitro. Medium-molecular-weight and low-molecular-weight fractions possessed a tissue specific effect on organotypic tissue cultures of chicken embryos and laboratory rats. Biological activity depended on molecular weight. Medium-molecular-weight fractions possessed higher effect on aortas explants of chicken embryo: area index of cellular monolayer was 56.6±5.2 % (area index of the monolayer was 37.47±3.27 % for low-molecularweight fraction) in comparison with control explants. The low-molecular-weight fractions stimulated aortic tissues explants of aging rats — area index exceeded area index formedium-molecularweight fractions by more than 30%.

Role of Thyroxine in the Development of Keratoconus.

Keratoconus (KC) is a corneal ectasia whose pathophysiology is still mostly unknown. We investigated whether thyroid gland dysfunction (TGD) is associated with the development of KC. We first conducted an epidemiological study, examining the prevalence of TGD among patients with KC. Then, we compared tear thyroxine (T4) in TGD and immunohistochemical staining of its receptors (T4Rs) between patients with KC and controls. The significance of T4 for corneal metabolism was studied in organotypic tissue cultures from monkey corneas. We found that TGD prevalence among patients with KC is 13.6%, which is higher than its prevalence in the general population (about 2%). Tear T4 was higher in KC, and keratocyte T4Rs were elevated in KC compared with controls. Furthermore, core proteins such as collagen and cytokeratins were equally altered both in KC and in the cultured corneas substituted with T4. Our data implicate a crucial role of T4 in KC pathophysiology, which is most likely mediated by T4Rs.

Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice Cultures

Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity, and metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral (AAV) vectors as a versatile tool to transduce human mature adipocytes in organotypic three-dimensional tissue cultures.

Comparative systems toxicology assessment of the Tobacco Heating System 2.2 and reference cigarettes (3R4F), on human organotypic respiratory tissue cultures

Comparative systems toxicology assessment of the Tobacco Heating System 2.2 and reference cigarettes (3R4F), on human organotypic respiratory tissue cultures

Modulation of the transducer function of Na+,K+-ATPase: new mechanism of heart remodeling.

Endogenous digitalis-like factors were found in the mammalian and human blood. It was the starting point for the elucidation of the new non-pumping function of the Na+,K+-ATPase. It was previously well known that Na+,K+-ATPase is a pharmacological target receptor for cardiac glycosides (J.C. Skou. 1957. Biochim. Biophys. Acta, 23: 394-401). We have investigated the trophotropic effects of such agents as ouabain, epinephrine, norepinephrine, atenolol, and comenic acid using the organotypic tissue culture combined with the reconstruction of optical cross sections and confocal microscopy. It was shown that the growth zone of organotypic culture forms a multidimensional structure. Our results indicate that the cardiac glycoside ouabain applied in endogenous concentrations (10-8, 10-10 mol/L) can modulate transducer function of Na+,K+-ATPase and control the cell growth and proliferation. It was also shown that Src-kinase is involved in "endogenous" ouabain activated intracellular pathways as a series unit. Epinephrine (10-9-10-14 mol/L) and comenic acid (10-6-10-10 mol/L) were demonstrated to modulate the growth of 10- to 12-day-old chicken embryo cardiac tissue explants in a dose-dependent manner. Epinephrine and comenic acid regulate growth and proliferation of the cardiac tissue via receptor-mediated modulation Na+,K+-ATPase as a signal transducer. The trophotropic effects of the investigated agents specifically control the heart remodeling phenomenon.

Systems Toxicology Assessment of the Biological Impact of a Candidate Modified Risk Tobacco Product on Human Organotypic Oral Epithelial Cultures.

Cigarette smoke (CS) has been reported to increase predisposition to oral cancer and is also recognized as a risk factor for many conditions including periodontal diseases, gingivitis, and other benign mucosal disorders. Smoking cessation remains the most effective approach for minimizing the risk of smoking-related diseases. However, reduction of harmful constituents by heating rather than combusting tobacco, without modifying the amount of nicotine, is a promising new paradigm in harm reduction. In this study, we compared effects of exposure to aerosol derived from a candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, with those of CS generated from the 3R4F reference cigarette. Human organotypic oral epithelial tissue cultures (EpiOral, MatTek Corporation) were exposed for 28 min to 3R4F CS or THS2.2 aerosol, both diluted with air to comparable nicotine concentrations (0.32 or 0.51 mg nicotine/L aerosol/CS for 3R4F and 0.31 or 0.46 mg/L for THS2.2). We also tested one higher concentration (1.09 mg/L) of THS2.2. A systems toxicology approach was employed combining cellular assays (i.e., cytotoxicity and cytochrome P450 activity assays), comprehensive molecular investigations of the buccal epithelial transcriptome (mRNA and miRNA) by means of computational network biology, measurements of secreted proinflammatory markers, and histopathological analysis. We observed that the impact of 3R4F CS was greater than THS2.2 aerosol in terms of cytotoxicity, morphological tissue alterations, and secretion of inflammatory mediators. Analysis of the transcriptomic changes in the exposed oral cultures revealed significant perturbations in various network models such as apoptosis, necroptosis, senescence, xenobiotic metabolism, oxidative stress, and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) signaling. The stress responses following THS2.2 aerosol exposure were markedly decreased, and the exposed cultures recovered more completely compared with those exposed to 3R4F CS.

Using Organotypic Epithelial Tissue Culture to Study the Human Papillomavirus Life Cycle.

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.

Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells

Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation.

P755. Effect of carboxylic acids of gut microbial origin on host cell proliferation in organotypic tissue cultures

P755. Effect of carboxylic acids of gut microbial origin on host cell proliferation in organotypic tissue cultures

Assessment of the Tobacco Heating System 2.2, a candidate Modified Risk Tobacco Product, on human organotypic nasal and bronchial epithelial tissue culture using systems toxicology approach

Assessment of the Tobacco Heating System 2.2, a candidate Modified Risk Tobacco Product, on human organotypic nasal and bronchial epithelial tissue culture using systems toxicology approach

Embryonic and mature astrocytes exert different effects on neuronal growth in rat ventral mesencephalic slice cultures.

One obstacle with grafting of dopamine neurons in Parkinson’s disease is the insufficient ability of the transplant to reinnervate the host striatum. Another issue is the prospective interaction between the donor fetal tissue and the adult astrocytes of the host. To study nerve fiber growth and its interaction with immature/mature astrocytes, ventral mesencephalic (VM) organotypic rat tissue cultures from embryonic days (E) 12, E14, and E18 were studied up to 35 days in vitro (DIV), and co-cultures of E14 VM tissue and mature green fluorescent protein (GFP)-positive astrocytes were performed. Generally, nerve fibers grew from the tissue slice either in association with a monolayer of migrated astroglia surrounding the tissue (glial-associated), or distal to the astroglia as non-glial-associated outgrowth. The tyrosine hydroxylase (TH)-positive glial-associated nerve fiber outgrowth reached a plateau at 21 DIV in E12 and E14 cultures. In E18 cultures, TH-positive neurons displayed short processes and migrated onto the astrocytes. While the non-glial-associated nerve fiber outgrowth dominated the E14 cultures, it was found absent in E18 cultures. The GFP-positive cells in the VM and GFP-positive astrocyte co-cultures were generally located distal to the monolayer of migrated fetal astrocytes, a few GFP-positive cells were however observed within the astrocytic monolayer. In those cases TH-positive neurons migrated towards the GFP-positive cells. Both the non-glial- and glial-associated nerve fibers grew onto the GFP-positive cells. Taken together, the glial-associated growth has limited outgrowth compared to the non-glial-associated nerve fibers, while none of the outgrowth types were hampered by the mature astrocytes.

A critical assessment of the scientific basis, and implementation, of regulations for the safety assessment and marketing of innovative tobacco-related products.

Our scientific, logistical, ethical and animal welfare-related concerns about the latest US Food and Drug Administration (FDA) regulations for existing and so-called 'new' tobacco products, aimed at reducing harmful exposures, are explained. Such claims for sales in the USA now have to be based on a wide range of information, a key part of which will increasingly be data on safety and risk. One of the pathways to achieve marketing authorisation is to demonstrate substantial equivalence (SE) with benchmark products, called predicates. However, the regulations are insufficiently transparent with regard to: a) a rationale for the cut-off date for 'old' and 'new' products, and for exempting the former from regulation; b) the scientific validity and operation of SE; c) options for product labelling to circumvent SE; d) the experimental data required to support, and criteria to judge, a claim; and e) a strategy for risk assessment/management. Scientific problems related to the traditional animal methods used in respiratory disease and inhalation toxicology, and the use of quantitative comparators of toxicity, such as the No Observed Adverse Effect Level, are discussed. We review the advantages of relevant in vitro, mechanism-based, target tissue-oriented technologies, which an advisory report of the Institute of Medicine of the US National Academy of Sciences largely overlooked. These benefits include: a) the availability, for every major site in the respiratory tract, of organotypic human cell-based tissue culture systems, many of which are already being used by the industry; b) the accurate determination of concentrations of test materials received by target cells; c) methods for exposure to particulate and vapour phases of smoke, separately or combined; d) the ability to study tissue-specific biotransformation; and e) the use of modern, human-focused methodologies, unaffected by species differences. How data extrapolation, for risk assessment, from tissue culture to the whole animal, could be addressed, is also discussed. A cost (to animal welfare)-benefit (to society, including industry and consumers) analysis was conducted, taking into account the above information; the potential for animal suffering; the extensive data already available; the existence of other, less hazardous forms of nicotine delivery; the fact that much data will be generated solely for benchmarking; and that many smokers (especially nicotine-dependents) ignore health warnings. It is concluded that, in common with policies of several tobacco companies and countries, the use of laboratory animals for tobacco testing is very difficult, if not impossible, to justify. Instead, we propose and argue for an integrated testing scheme, starting with extensive chemical analysis of the ingredients and by-products associated with the use of tobacco products and their toxicity, followed by use of in vitro systems and early clinical studies (involving specific biomarkers) with weight-of-evidence assessments at each stage. Appropriate adjustment factors could be developed to enable concentration-response data obtained in vitro, with the other information generated by the strategy, to enable the FDA to meet its objectives. It is hoped that our intentionally provocative ideas will stimulate further debate on this contentious area of regulatory testing and public safety.

Short peptides stimulate cell regeneration in skin during aging

The search for new safe and efficient low-molecular weight substances stimulating skin regeneration is an important objective of geriatric cosmetology. The present study addressed the effects of the peptides LK and AEDG at concentrations of 0.05–2.00 ng/mL on the proliferation of organotypic tissue cultures of skin from young and old animals. The application of peptides enhanced fibroblast proliferation in skin cell cultures from young and old animals by 29–45%. The effect on aging skin was observed in a narrower concentration range; in addition, it was less pronounced than the effect on skin cell cultures obtained from young animals. These data reveal the potential of LK and AEDG as components of cosmetic products that restore the structure of aging skin.

The effect of dipeptides consisting of leucine and lysine on cell proliferation in an organotypic culture of myocardium and spleen tissue from young and old rats

The effects of novel dipeptides consisting of leucine and lysine on organotypic tissue cultures of spleen and myocardium of young (3-month-old) and old (24-month-old) rats have been investigated. L-Leu-Lys dipeptide stimulated cell proliferation in spleen and myocardium explants from juvenile rats when applied at concentrations of 0.001–1 and 0.01–1 ng/mL, respectively. Stimulation of explants from old rats was observed at peptide concentrations ranging from 0.01 to 1 ng/mL. L-Lys-Leu dipeptide stimulated cell proliferation in all types of rat tissue explants, but the peptide concentrations required for stimulation were slightly higher in this case. The data on high biological activity of the novel dipeptides lay the foundation for further research on their effects on the whole-organism level and possible future application in geriatric practice to enhance lymphoid and myocardial tissue regeneration.