Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation
December 8th, 2017
Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.
The overall goal of this 3D cell culture protocol is to model the glial scar event of the central nervous system using key indicator cells:microglia, astrocytes and oligodendrocytes.This method can help answer key questions in the neuro-inflammation field, such as the potential for glial cells to enhance regenerative strategies and unravel the glial scar.The main advantage of this technique is that glial biology can be rapidly characterized in a 3D micro-environment with systematic and high-through put manipulations.To prepare cover slips for seeding, begin by adding one milliliter of 3-propyl methacrylate to 49 milliliters of distilled water to make a 2%solution.Then place 18 millimeter glass cover slips into the solution and rock for one hour at room temperature.After one hour, rinse the cover slips by serially dipping each cover slip in three beakers of 100 milliliters of deionized water.Then dry the cover slips in a vacuum with a desiccator and oven at 40 degrees Celsius overnight.The next day, use forceps to quickly immerse individual cover slips in 70%ethanol.Then, without drying, drop each cover slip into a well of a 12-well plate.Next, wash each well with one milliliter of sterile deionized water.Then add one milliliter of sterile poly
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Overview
This article presents a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. It details the processes of primary cell culture, hydrogel synthesis, and imaging techniques.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Tissue Engineering
Background
- 3D cell culture systems are essential for studying glial cells.
- Rat brain-derived glia cells play a crucial role in neurobiology.
- Hydrogels provide a supportive environment for cell growth.
- Imaging techniques are vital for analyzing cell morphology.
Purpose of Study
- To develop a reliable protocol for culturing glial cells in 3D.
- To enhance understanding of glial cell interactions.
- To facilitate advanced imaging of cultured cells.
Methods Used
- Primary cell culture of rat brain-derived glia cells.
- Synthesis of methacrylated hyaluronic acid (HAMA) hydrogel.
- Photo-polymerization of HAMA for cell encapsulation.
- Sample processing for confocal and scanning electron microscopy.
Main Results
- Successful encapsulation of glial cells in HAMA hydrogel.
- Demonstrated viability and functionality of cultured cells.
- High-quality imaging of cell morphology achieved.
- Protocol provides a foundation for future glial cell studies.
Conclusions
- The developed protocol is effective for 3D culture of glial cells.
- This method can be applied to various neuroscience research areas.
- Future studies can build on this framework for advanced investigations.
