A live imaging‐friendly slice culture method using collagen membranes

Abstract

Aim Organotypic brain slice culture preserves the geographical position of neurons and neuronal circuits. The slice cultures also maintain both non‐neuronal cell types and the surrounding extracellular matrix. The interface method has been widely used for slice cultures, in which brain slices are placed on semiporous polytetrafluoroethylene (PTFE) membranes. However, a low optical transparency of PTFE membrane makes it difficult to perform live imaging of deep regions of slice cultures using an inverted microscope. To overcome the issue, we evaluated the suitability of using collagen membranes for slice cultures, especially focusing on live imaging of the cellular dynamics of green fluorescent protein (GFP)‐expressing microglia. Methods Entorhinohippocampal slices were cultured on either collagen or PTFE membranes. The influence of membrane type on the ability to observe deep regions of slice cultures was examined by live imaging using an inverted microscope. Results Collagen membranes were thinner and had better optical transparency compared with PTFE membranes. There were no differences in cell viability, density of neurons or microglia. The densify of visible short branches of microglia in live imaging was higher in collagen membranes than PTFE membranes. Conclusion Collagen membranes are suitable for live imaging of cellular dynamics in slice cultures using an inverted microscope.

Replacement of Mouse Microglia With Human Induced Pluripotent Stem Cell (hiPSC)-Derived Microglia in Mouse Organotypic Slice Cultures

“…The preparation and maintenance of slice cultures, including culture media, were performed as previously described (Kasahara et al, 2016 ; Ogaki et al, 2020 ). To prepare slice cultures, P6 mouse brains were sectioned into 400-μm-thick horizontal slices using a DTK-1500 vibratome (Dosaka, Kyoto, Japan) in aerated, ice-cold Gey’s balanced salt solution (GBSS) containing 36 mM glucose.…”

Assessing Microglial Dynamics by Live Imaging

“…Recently, Ogaki et al focused on the fact that PTEE membranes, which are frequently used to place slices in culture, make live imaging using inverted microscopy difficult due to the low light transmission of PTFE membranes ( 10 ) ( Table 3 ). The authors evaluated the performance of the collagen membrane by placing sections on the light-permeable collagen membrane instead of the PTFE membrane and culturing the slices.…”

“…In Bernier et al ( 9 ), the authors defined tiny protrusions near the tip of large process as filopodia, and provided the quantitative data of filopodia and large process. In Nimmerjahn et al ( 1 ) and Ogaki et al ( 10 ), short branches that emanated from the primary process of microglia were regarded as filopodia.…”

Ultra-thin and ultra-porous nanofiber networks as a basement-membrane mimic

“…[54][55][56] These membranes have disadvantages such as large thicknesses (>10 μm) and randomly distributed fibers and pores. Native extracellular matrix (ECM) membranes have also been developed, 43,48,59 such as vitrified collagen membranes, [44][45][46][47] but these membranes have low porosity due to the vitrification process and are rather thick (≥10 μm).…”