Recent advances in organotypic tissue slice cultures for anticancer drug development

Organotypic tissue slice culture is established from animal or patient tissues and cultivated in an in vitro ecosystem. This technique has made countless contributions to anticancer drug development due to the vast number of advantages, such as the preservation of the cell repertoire and immune components, identification of invasive ability of tumors, toxicity determination of compounds, quick assessment of therapeutic efficacy, and high predictive performance of drug responses. Importantly, it serves as a reliable tool to stratify therapeutic responders from nonresponders and select the optimal standard-of-care treatment regimens for personalized medicine, which is expected to become a potent platform and even the gold standard for anticancer drug screening of individualization in the near future.     

The C‐terminal domain of tetanus toxin protects motoneurons against acute excitotoxic damage on spinal cord organotypic cultures

The C‐terminal domain of tetanus toxin (Hc‐TeTx) has been suggested to act as a neuroprotective agent by activating signaling pathways related to neurotrophins and also to exert anti‐apoptotic effects. Here, we show the beneficial properties of the recombinant protein Hc‐TeTx to protect spinal motoneurons against excitotoxic damage. In vitro spinal cord organotypic cultures were used to assess acute glutamate excitotoxic damage. Our results indicate that Hc‐TeTx treatment improves motoneuron survival within a short therapeutical window (the first 2 h post‐injury). Within this interval, we found that p44/p42 MAP kinase (ERK1/2) and glycogen synthase kinase‐3 (GSK3β) signaling pathways play a crucial role in the neuroprotective effect. Moreover, we demonstrated that Hc–TeTx treatment initiate autophagy which is ERK1/2‐ and GSK3β‐dependent. These findings suggest a possible therapeutical tool to improve motoneuron survival immediately after excitotoxic insults or during the secondary injury phase that occurs after spinal cord trauma.     

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity ¹.Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.     

Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17–92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma.     

Development by three‐dimensional approaches and four‐dimensional imaging: To the knowledge frontier and beyond

Many advances have been taken on elucidating embryonic development and tissue homeostasis and repair by the use of experimental strategies that preserve the three‐dimensional (3D) organization and allow quantitative analysis of images over time (four‐dimensional). Ranging from the understanding about the relationship between blastomeres and the events that take place during gastrulation by the use of time‐lapse imaging through 3D cultures that mimic organogenesis, the advances in this area are of critical value. The studies on embryonic development without disrupting the original architecture and the development of 3D organoid cultures pave a new avenue for unprecedented experimental advances that will positively impact the emergence of new treatments applying regenerative principles for both tissue repair and organ transplant. Birth Defects Research (Part C) 105:1–8, 2015. © 2015 Wiley Periodicals, Inc.     

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2^+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2^+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2^+/+ and GluRδ2^+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2^+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2^+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2^+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2^+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

A comparison of primary oesophageal squamous epithelial cells with HET‐1A in organotypic culture

Background Information. Carcinoma of the oesophagus is the sixth leading cause of cancer death in the western world and is associated with a 5‐year survival of less than 15%. Recent evidence suggests that stromal—epithelial interactions are fundamental in carcinogenesis. The advent of co‐culture techniques permits the investigation of cross‐talk between the stroma and epithelium in a physiological setting. We have characterized a histologically representative oesophageal organotypic model and have used it to compare the most commonly used squamous oesophageal cell line, HET‐1A, with primary oesophageal squamous cells for use in studies of the oesophageal epithelium in vitro. Results. When grown in an organotypic culture with normal fibroblasts, the oesophageal carcinoma cell lines OE21 (squamous) and OE19 (adenocarcinoma) morphologically resembled the tumour of origin with evidence of stromal invasion and mucus production, respectively. However, HET‐1A cells, which were derived from normal squamous oesophageal cells, appeared dysplastic and failed to display evidence of squamous differentiation. By comparison with primary oesophageal epithelial cells, the HET‐1A cells were highly proliferative and did not express the epithelial markers E‐cadherin or CK5/6 (casein kinase 5/6), or the stratified epithelial marker ΔNp63, but did express the mesenchymal markers vimentin and N‐cadherin. Conclusion. Studies of epithelial carcinogenesis will benefit from culture systems which allow manipulation of the stromal and epithelial layers independently. We have developed an organotypic culture using primary oesophageal squamous cells and fibroblasts in which a stratified epithelium with a proliferative basal layer that stains strongly for ΔNp63 develops. This model will be suitable for the study of the molecular events in the development of Barrett's oesophagus. The most commonly used normal oesophageal squamous cell line, HET‐1A, does not have the characteristics of normal oesophageal squamous cells and should not be used in models of the normal oesophageal epithelium. Until more representative cell lines are available, future studies in oesophageal cancer will be reliant on the availability and manipulation of primary tissue.     

Clusterin increases post-ischemic damages in organotypic hippocampal slice cultures

Clusterin or apolipoprotein J is a heterodimeric glycoprotein which is known to be increased during tissue involution in response to hormonal changes or injury and under circumstances leading to apoptosis. Previous studies in wild-type (WT) and clusterin-null (Clu−/−) mice indicated a protective role of clusterin over-expression in astrocytes lasting up to 90 days post-ischemia. However, in in vitro and in vivo models of neonatal hypoxia-ischemia, clusterin exacerbates necrotic cell death. We developed recombinant forms of clusterin and examined their effect on propidium iodide uptake, neuronal and synaptic markers as well as electrophysiological recordings in hippocampal slice cultures from Clu−/− and WT mice subjected to oxygen-glucose deprivation (OGD). WT mice displayed a marked up-regulation of clusterin associated with electrophysiological deficits and dramatic increase of propidium iodide uptake 5 days post-OGD. Immunocytochemical and western blot analyses revealed a substantial decrease of neuronal nuclei and synaptophysin immunoreactivity that predominated in WT mice. These findings contrasted with the relative post-OGD resistance of Clu−/− mice. The addition of biologically active recombinant forms of human clusterin for 24 h post-OGD led to the abolishment of the ischemic tolerance in Clu−/− slices. This deleterious effect of clusterin was reverted by the concomitant administration of the NMDA receptor antagonist, d -2-amino-5-phosphonopentanoate. The present data indicate that in an in vitro model of ischemia characterized by the predominance of NMDA-mediated cell death, clusterin exerts a negative effect on the structural integrity and functionality of hippocampal neurons.