A mesh microelectrode array for non-invasive electrophysiology within neural organoids.

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

Spatiotemporal Changes of Cerebral Monocarboxylate Transporter 8 Expression.

Background: Mutations of monocarboxylate transporter 8 (MCT8), a thyroid hormone (TH)-specific transmembrane transporter, cause a severe neurodevelopmental disorder, the Allan-Herndon-Dudley syndrome. In MCT8 deficiency, TH is not able to reach those areas of the brain where TH uptake depends on MCT8. Currently, therapeutic options for MCT8-deficient patients are missing, as TH treatment is not successful in improving neurological deficits. Available data on MCT8 protein and transcript levels indicate complex expression patterns in neural tissue depending on species, brain region, sex, and age. However, information on human MCT8 expression is still scattered and additional efforts are needed to map sites of MCT8 expression in neurovascular units and neural tissue. This is of importance because new therapeutic strategies for this disease are urgently needed. Methods: To identify regions and time windows of MCT8 expression, we used highly specific antibodies against MCT8 to perform immunofluorescence labeling of postnatal murine brains, adult human brain tissue, and human cerebral organoids. Results: Qualitative and quantitative analyses of murine brain samples revealed stable levels of MCT8 protein expression in endothelial cells of the blood-brain barrier (BBB), choroid plexus epithelial cells, and tanycytes during postnatal development. Conversely, the neuronal MCT8 protein expression that was robustly detectable in specific brain regions of young mice strongly declined with age. Similarly, MCT8 immunoreactivity in adult human brain tissue was largely confined to endothelial cells of the BBB. Recently, cerebral organoids emerged as promising models of human neural development and our first analyses of forebrain-like organoids revealed MCT8 expression in early neuronal progenitor cell populations. Conclusions: With respect to MCT8-deficient conditions, our analyses not only strongly support the contention that the BBB presents a lifelong barrier to TH uptake but also highlight the need to decipher the TH transport role of MCT8 in early neuronal cell populations in more detail. Improving the understanding of the spatiotemporal expression in latter barriers will be critical for therapeutic strategies addressing MCT8 deficiency in the future.

Dewaxed ECM: A simple method for analyzing cell behaviour on decellularized extracellular matrices.

Decellularization techniques have been used on a wide variety of tissues to create cell-seedable scaffolds for tissue engineering. Finding a suitable decellularization protocol for a certain type of tissue can be laborious, especially when organ perfusion devices are needed. In this study, we report a quick and simple method for comparing decellularization protocols combining the use of paraffin slices and two-dimensional cell cultures. We developed three decellularization protocols for adult murine kidney that yielded decellularized extracellular matrices (ECMs) with varying histological properties. The resulting paraffin-embedded ECM slices were deparaffinized and reseeded with murine embryonic stem cells (mESCs). We analyzed cell attachment four days post seeding via determination of cell numbers, and used quantitative Real-Time PCR 13 days post seeding to measure gene expression levels of two genes associated with renal development, Pax2 and Pou3f3. The three decellularization protocols produced kidney-matrices that showed clearly distinguishable results. We demonstrated that formerly paraffin-embedded decellularized ECMs can effectively influence differentiation of stem cells. This method can be used to identify optimal decellularization protocols for recellularization of three-dimensional tissue-scaffolds with embryonic stem cells and other tissue-specific cell types.

ECM modulated early kidney development in embryonic organ culture.

The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases.

A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization.

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle.

FAS (CD95) mutations are rare in gastric MALT lymphoma but occur more frequently in primary gastric diffuse large B-cell lymphoma.

A loss of FAS (CD95) function has been proposed to constitute an important step in early mucosa-associated lymphoid tissue (MALT) lymphoma development and FAS mutations have been recognized in malignant lymphomas, in particular at extranodal sites. Since primary gastric lymphomas frequently exhibit resistance to FAS-mediated apoptosis, we investigated whether FAS is mutated in 18 gastric MALT lymphomas and 28 diffuse large B-cell lymphomas (DLBCL). We detected seven mutations in five lymphomas, one MALT lymphoma and four DLBCL; two DLBCL had two mutations. The MALT lymphoma exhibited a point mutation in the splice donor region of intron 3. Three DLBCL had missense mutations in exon 2, which encodes a signal peptide and a portion of the extracellular FAS ligand-binding domain. One DLBCL carried a point mutation in the splice donor region of intron 8, which would result in exon skipping. Two DLBCL harbored a missense mutation in exon 9, which encodes the intracellular death domain. The two death domain mutations inhibited FAS ligand-induced apoptosis in a dominant-negative mode, when transiently expressed in human T47D breast carcinoma and Jurkat T cells. A signal peptide and an extracellular domain mutation, however, failed to inhibit apoptosis in these transfection assays. They are likely to reduce apoptosis in lymphoma cells solely by a loss of function. In summary, our data show that FAS mutations are rare in primary gastric MALT lymphomas (5.6%) but occur in a subset of primary gastric DLBCL (14.3%) and suggest that these mutations contribute to the pathogenesis of gastric lymphomas by rendering lymphocytes resistant to apoptosis.