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Isolation, Culture and Functional Characterization of Glia and Endothelial…
Isolation, Culture and Functional Characterization of Glia and Endothelial Cells From Adult Pig Brain
DISCUSSION
Primary glial and endothelial cell cultures have been beneficial
to study the function and dysfunction of these cells in vitro.
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Compare to other studies in protocols for purification and
culture of mature oligodendrocytes from adult pig
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Similar methodologies were
used to isolate and culture pig neural progenitor cells, astrocytes, microglia
none of the reported methods allows to purify all different glia and endothelial cells from an adult pig in parallel and gives access to freshly isolated as well as cultured cells
Summary: Provided inexpensive
and comprehensive method for primary glia and endothelial cell
culture,
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able to isolate and culture different types of glial cells and endothelial cells from a single pig brain in parallel.
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INTRODUCTION
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- Primary cultures of glial & endothelial cells crucial for basic & translational neuroscience research
- Better mimicks human central nervous system (CNS)
- Dysfunction of glia @ endothelial cells cause many human CNS diseases
- Adult pig brain tissue easily accessible (no need to kill the animal) & it is the closest to the human brain
- Pig is the closest to human as it shares similarity of organ functioning, anatomical localization & gene pool sharing
- However, few substantial differences occur between human and rodent glia in terms of its morphological & functional
- In this research article (Tanti et al., 2019), primary cultures of oligodendrocyte, microglia, astrocyte & endothelial were generated from same pig brain tissue & grown for 8 weeks.
MATERIAL AND METHODS
MATERIALS AND METHODS
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Cell Culture
- Preparation of the Plates/Flasks
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TNF-AlphaELISA
- In vitro Myelination Assay
- In vitro Tube Formation Assay
- Cytotoxicity Assay With AQP4 Positive NMO Serum
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RESULTS
ISOLATION OF DIFFERENT CELL FRACTION
- The flow cytometric analyses demonstrated the purity of the isolated cell fractions
- The CD11b expressed CD11b but MOG, CD31 or GFAP and the O4 sorted fraction are expressed
- CD31 which are endothelial cells expressed CD31 but not CD11b, MOG or GFAP
- The triple negative fraction expressed GFAP
- The results are confirmed by Reversed transcription PCR (rtPCR) with lineage specific probes
- After 2 weeks of culturing in a specific cell culture media, 95% of the cells from CD11b expressed CD11b and Iba1 but give negative result for other cells
- 95% of O4 sorted cell expressed the oligodendrocyte markers
80%of CD31 expressed CD31
- 60% from negatively selected fraction expressed astrocyre markers
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