Obtaining Human Microglia from Adult Human Brain Tissue

Summary

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Abstract

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

Introduction

The central nervous system (CNS) is constructed of a complex network of neurons and glial cells¹. Among the glial cells, microglia function as the innate immune cells of the CNS^2,3. Microglia are responsible for maintaining homeostasis in the healthy CNS⁴. Microglia also play an important role in neurodevelopment, by pruning synapses². Microglia are central to the pathophysiology of several neurological diseases including but not restricted to; Alzheimer's disease⁵, Parkinson's disease⁶

Protocol

All tissues were acquired after ethical clearance from the institute ethics committees of Indian Institute of Technology Jodhpur and All India Institute of Medical Sciences (AIIMS) Jodhpur.

1.Tissue acquisition and processing (Day 0)

1. Collect the tissue in a 50 mL tube containing 10 mL of ice cold artificial cerebrospinal fluid (aCSF) (2 mM CaCl₂∙2H₂O, 10 mM glucose, 3 mM KCl, 26 mM NaHCO₃, 2.5 mM NaH₂PO₄, 1 mM MgCl₂∙6H₂O, 202 mM sucrose)²⁰. Ensure that the tube is kept on ice if the tissue needs to be transferred to a....

Results

By using the above-mentioned protocol (Figure 1), we were able to isolate primary human microglia from live surgically resected brain tissues. Cultured cells were stained with Ricinus communis agglutinin-1 (RCA-1) lectin for microglia (green) and with Glial fibrillary acidic protein (GFAP) for astrocytes (red) (Figure 2) as previously described^22,23,

Discussion

Microglia ensure homeostasis in the normal brain and play central roles in the pathophysiology of various neurological diseases⁴. Microglia are central to neurodevelopment and formation of synapses². Microglial studies have proven pivotal in understanding the development and progression of diverse neurological diseases⁴. Rodent microglia are the prevalent model of choice for primary microglial studies, even though, rodent microglia are different from.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibiotic-Antimycotic solution	Himedia	A002
Calcium chloride	Sigma	223506
Centrifuge (4 °C)	Sigma	146532
Centrifuge tubes	Abdos	P10203
CO2 incubator	New Brunswik	Galaxy 170 S
D-Glucose	Himedia	GRM077
DMEM medium with glutamine	Himedia	AL007S
Fetal bovine serum	Himedia	RM9955
Flacon tube (50 ml)	Thermo Fsiher Scientific	50CD1058
Fluorescein Ricinus communis agglutinin-1	Vector	FL-1081
Fluorescent microscope	Leica	DM2000LED
Fluoroshield with DAPI	Sigma	F6057
GFAP antibody	GA5	3670S
Incubator shaker	New Brunswik Scientific	Innova 42
L929 cell line	ATCC	NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC CCL-1)
Laminar air flow	Thermo Fsiher Scientific	1386
Magnesium chloride	Himedia	MB040
Monosodium phosphate	Merck	567545
Nutrient Mixture F-12 Ham Medium	Himedia	Al106S
Petri dish	Duran Group	237554805
Phosphate buffered saline	Himedia	ML023
Potassium chloride	Himedia	MB043
Serological pipette	Labware	LW-SP1010
Sodium bicarbonate	Himedia	MB045
Sucrose	Himedia	MB025
Syringe filter (0.2μ, 25 mm diameter)	Axiva	SFPV25R
T-25 tissue culture flasks suitable for adherent cell culture.	Himedia	TCG4-20X10NO
Trypsin-EDTA (0.25%)	Gibco	25200-056