7 research outputs found
Recommended from our members
A multi-well plate model of reactive gliosis for high throughput screening of potential CNS therapies
Reactive astrogliosis is an important feature of CNS damage and disease which involves changes in astrocyte phenotype and morphology. In particular, CNS trauma can lead to the formation of a glial scar, a three dimensional (3D) mesh of astrocyte processes that can form a physical and chemical barrier to neuronal regeneration, which is a potential target for CNS drug and cell therapy. However, reactive gliosis is difficult to isolate and monitor in typical animal models of CNS damage. In monolayer culture systems astrocytes adopt a highly reactive phenotype, limiting the range of available models suitable for research in this area.
Our previous 3D cell culture systems allow astrocytes to be maintained with a relatively unreactive phenotype until stimulated, whereupon a classical reactive astrocyte response can be monitored. The aim of the current work is to adapt this approach in order to develop a multi-well plate system to provide a reliable, consistent model of reactive gliosis for high throughput screening and research. Once baseline viability and phenotype of primary rat astrocytes were investigated in these models, reactivity was triggered using treatments such as TGFβ1, as seen in Figure 1, hypoxia and low glucose. Outputs included confocal microscopy and 3D image analysis, Western blotting and RT-PCR to quantify markers such as GFAP and CSPG in test and control gels. Using GFP-labelled astrocytes permitted monitoring of cytoplasmic volume and shape, giving an additional measure of astrocyte hypertrophic response within stimulated conditions. The robust protocol that we have developed can form a basis to investigate astrocyte biology in a highly controlled environment, and to model phenotypic features of astrocytes in both damaged and undamaged CNS. A reproducible multi-well plate system will provide an experimental platform which allows potential CNS therapies to be screened at high-throughput, and the effects of potential modulators of astrocyte reactivity to be investigated simply and systematically
Recommended from our members
Adapting aligned, stabilised 3D tissues for large-scale neurobiological research
Recreating the 3D environment of the CNS using hydrogel matrices allows neurons and glial cells in vitro to behave similarly to their counterparts in vivo, providing a relevant tool for neurobiological studies. The overall aim is to develop robust 3D CNS tissue models engineered by a process of glial cell self-alignment and subsequently stabilised. Furthermore, these models have been developed for multi-well plate format at a scale suitable for high throughput screening. CNS tissue equivalents can be used to assess numerous aspects of the CNS in a reproducible, controllable and consistent manner
Recommended from our members
Development of stabilised aligned CNS co-culture technology to model the behaviour of a range of neural cell types
The overall aim is to develop robust culture models that recreate the 3D environment of the CNS, thereby allowing neurons and glial cells in vitro to behave similarly to their counterparts in vivo. A simple, consistent and physiologically relevant model system, which uses a multiwell plate format and can potentially be used at a scale suitable for commercial R&D, has been developed. The model uses an engineered neural tissue which is prepared by a process of initial glial cell self-alignment within a tethered 3D collagen hydrogel and subsequent stabilisation of the gel. Stabilisation is acheived using RAFT technology (www.raft3dcellculture.com), which entails partial removal of interstitial fluid thereby increasing matrix and cell density. A CNS co-culture system suitable for widespread adoption will require various combinations of cells to suit specific neuroscience research requirements. Both primary neuronal and glial cell types and relevant cell lines were used to establish engineered neural tissues which were then assessed using a range of measures including neural cell survival, morphology, proliferation, differentiation and sensitivity to stabilisation. In a bid to determine the complexity of the CNS model, neuron-glial interactions, markers for myelination and reactive gliosis were also investigated. The highly organised nature of the cells and extracellular matrix in this anisotropic system facilitates quantitative analysis of cellular features including neurite length and myelination. Initial studies reveal the new model system can be assembled quickly and reliably using various primary neural cells or cell lines, the approach can be scaled down to facilitate increased throughput, and the co-cultures exhibit characteristic behaviours that mimic in vivo scenarios
Recommended from our members
Optimising glial cell alignment in 3D culture to facilitate the development of neural tissue models for CNS research
Aim: The overall aim is to develop robust models for neuroscience research, with neural cells arranged in a hydrogel matrix to resemble living nervous system tissue. We create engineered neural tissue by a process of initial glial cell self-alignment within tethered 3D collagen gels and the aligned glia then support and direct neuronal growth to recreate the anisotropy of an organised CNS tract.
Method: A two-stage approach was developed to determine the glial cell seeding density to achieve consistent predictable alignment regardless of the cell source. (1) Contraction profiles were established using C6 glioma cells at densities from 0.1 to 6 million cells/ml in freefloating round collagen gels, in simple 24-well and 96-well plate assays. (2) Chosen seeding densities were then used to assess the degree of cellular alignment using tethered rectangular collagen gels.
Results: By combining data from the contraction profiles and alignment assays, the relationship between the % contraction of the free-floating round gels and the extent of cellular alignment in the tethered rectangular gels was established.
Conclusion: We have shown that contraction profiles in simple multiwell plate assays, using a small number of cells, can efficiently assess glial cells from different sources and determine the optimal seeding density for generating robust anisotropic engineered neural tissue
Recommended from our members
Adapting aligned stabilised hydrogels to facilitate large scale drug screening and neurobiological research.
Aim: The overall aim is to develop robust culture models that recreate the 3D environment of the CNS, allowing neurons and glial cells in vitro to behave similarly to their counterparts in vivo. This approach is being used to develop a simple, consistent model system which can be used at a scale suitable for drug screening, providing an experimental platform for neurobiological research.
Methods: Engineered neural tissue is created by a process of initial glial cell self-alignment within a tethered 3D collagen hydrogel. The aligned glia can then support and direct neuronal growth to recreate the anisotropic architecture of an organised CNS tract. RAFT technology (www.raft3dcellculture.com) is employed to stabilise the cellular hydrogels, through partial removal of interstitial fluid thereby increasing matrix and cell density. Initial experiments characterised the cell and matrix parameters required for reliable, consistent cellular alignment in large (1ml) moulds using C6 glioma cells, then scaled down moulds were assessed in terms of cellular alignment and support of neurite growth.
Results: Miniaturised moulds can be used to generate anisotropic cellular collagen gels, which can be stabilised using RAFT. Glial cell alignment in our scaled down system displayed similar patterns as in the larger standard system, with over 50% angles of deviation in both middle and side regions being less than 30°.
Discussion: The results indicate that engineered neural tissue models can be scaled down without compromising glial cell alignment and stabilisation. This approach may be beneficial for the development of an experimental platform for widespread adoption as it minimises the cell numbers required for generating robust anisotropic engineered neural tissue and could also lead to an increase in throughput to match the requirements for drug discovery screening
Optimising contraction and alignment of cellular collagen hydrogels to achieve reliable and consistent engineered anisotropic tissue
Engineered anisotropic tissue constructs containing aligned cell and extracellular matrix structures are useful as in vitro models and for regenerative medicine. They are of particular interest for nervous system modelling and regeneration, where tracts of aligned neurons and glia are required. The self-alignment of cells and matrix due to tension within tethered collagen gels is a useful tool for generating anisotropic tissues, but requires an optimal balance between cell density, matrix concentration and time to be achieved for each specific cell type. The aim of this study was to develop an assay system based on contraction of free-floating cellular gels in 96-well plates that could be used to investigate cell-matrix interactions and to establish optimal parameters for subsequent self-alignment of cells in tethered gels. Using C6 glioma cells, the relationship between contraction and alignment was established, with 60-80% contraction in the 96-well plate assay corresponding to alignment throughout tethered gels made using the same parameters. The assay system was used to investigate the effect of C6 cell density, collagen concentration and time. It was also used to show that blocking α1 integrin reduced the contraction and self-alignment of these cells, whereas blocking α2 integrin had little effect. The approach was validated by using primary astrocytes in the assay system under culture conditions that modified their ability to contract collagen gels. This detailed investigation describes a robust assay for optimising cellular self-alignment and provides a useful reference framework for future development of self-aligned artificial tissue
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