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, stroke⁷, multiple sclerosis⁸, traumatic brain injury⁹, neuropathic pain¹⁰, spinal cord injury¹¹ and brain tumors such as gliomas¹².

Studies related to CNS homeostasis and diseases utilize rodent microglia due to a dearth of cost efficient and time efficient human primary microglia isolation protocols¹³. Rodent microglia resemble primary human microglia in expression of genes such as Iba-1, PU.1, DAP12 and M-CSF receptor and have been effective in understanding normal as well as diseased brain¹³. Interestingly, the expression of several immune related genes such as TLR4, MHC II, Siglec-11 and Siglec-3 varies between human and rodent microglia¹³. The expression of several genes also varies in temporal expression and in neurodegenerative diseases in both species^14,15. These significant differences make human microglia an essential model to study microglia function in homeostasis and disease. Primary human microglia can also be an effective tool for preclinical screening of potential drug candidates¹⁶. The above mentioned reasons underline the growing need for cost effective protocols for isolation of primary human microglia.

We have developed a protocol for isolation of primary human microglia from adult human brain tissue collected as a result of surgical window created for tumor resections or other surgical resections. The method here is considerably different from existing methods. We were able to isolate and culture microglia after a transit time of about 75 minutes from the tissue collection site to starting the isolation protocol in the laboratory. We have used the supernatant of L929 fibroblast cells to promote the growth of isolated microglia. This method specifically focuses on the culture and development of only primary microglia. The resulting culture prepared is about 80% microglia. While other protocols provide a enriched culture of microglia by density gradient centrifugation, flow cytometry and magnetic beads, the protocol is a rapid, simple, robust and cost effective way to culture primary human microglia^17,18,19,20. The ability to utilize surgically removed live adult brain tissue instead of fixed brain tissues from cadavers proves an added advantage of this method in contrast to existing procedures^18,21.

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 different location.
   NOTE: Prepare aCSF in autoclaved distilled water. Filter it with 0.22 µm syringe filter in the laminar hood. This can be stored for 1 month at 4 °C.
2. Wipe the collection tube carefully with 70% alcohol and transfer to an aseptic laminar air flow chamber.
3. Discard aCSF carefully and weigh tissue in an aseptic condition. Tissue weight is essential to calculate the volume of trypsin-EDTA needed for subsequent steps.
4. Keep the tissue in fresh warm aCSF at 37 °C for 5 minutes. This step is critical to avoid cell death.
5. Discard aCSF and wash tissue once with 1x PBS (phosphate buffered saline) at 37 °C. Ensure all blood is washed away with repeated PBS washes (as needed).
6. Incubate the tissue in warm PBS, at 37 °C, for 5 min.
7. Discard PBS carefully and transfer tissue to a sterilized Petri dish. PBS may be removed with a pipette. This will prevent any loss of tissue.
8. Dice the tissue into small (at least 1 mm³) pieces using a sterile scalpel. Finely diced tissue provides higher tissue surface area for tissue dissociation by trypsin-EDTA ensuring higher yield.
9. Transfer the diced tissue to a 50 mL tube containing 10 mL/g tissue of 0.25% trypsin-EDTA and mix by pipetting through a 10 mL serological pipette. Add 2 mL of trypsin/EDTA to a Petri dish and wash the plate thoroughly with the help of pipette. Add this trypsin back to the falcon tube. This minimizes loss of tissue and cells while dicing.
10. Incubate the tube on a shaker for 30 minutes at 37 °C at 250 rpm. This step increases the dissociation of cells from tissue.
11. At the end of the incubation, add 10 mL of neutralizing medium (50% DMEM/50% F12 with glutamine, 1% penicillin-streptomycin, 10% FBS) to neutralize trypsin. Mix with a 10 mL serological pipette. The amount of neutralizing media added should be equal to the amount of trypsin used.
12. Centrifuge the tube at 2,000 x g at 4 °C for 10 minutes.
13. Discard the supernatant and re-suspend the pellet in 1 mL of culture medium (50% DMEM/50% F12 with glutamine, 1% penicillin-streptomycin, 20% L929 supernatant, 10% FBS).
    NOTE: L929 cells are culture in DMEM (DMEM with glutamine, 1% penicillin-streptomycin, 10% FBS). ATCC recommended culture method should be followed for cell culture. Supernatant must be collected from the culture flask which is at least 75% confluent. It can be collected in bulk and stored at -80 °C to prevent degradation of growth factors. It is recommended to add L929 supernatant separately in flasks instead of adding to the stock culture medium.
14. Plate the cells in a T-25 flask, suited for adherent cells, and add 4 mL of additional culture medium. Incubate the flask at 37 °C with 5% CO₂. Carefully shake the flask to homogenously disperse the tissue. Avoid bringing the media to the neck of the flask, while shaking, as this may increase the chances of contamination.

2.Cell culture (Day 2)

1. Collect the media from the T-25 culture flask prepared on day 0 in three 1.5 mL centrifuge tubes by collecting equal volume of media in each tube. Wash the flask once with 1 x PBS. Shake the flask gently to remove any remnant tissue fragments left. Avoid harsh shaking of the flask as any remnant fragments will not adversely affect the culture. Add 5 mL fresh culture media to the flask.
2. Centrifuge the collected media at 1,466 x g (4000 rpm) at 4 °C for 4 minutes.
3. Discard the supernatant from each tube and add 1 mL of culture medium to one of the tubes. Mix thoroughly with pipette. Serially add the mixed media with cells to other tubes. Mix thoroughly with pipette and pool the cells in one tube.
4. Plate the cells in a separate T-25 flask, suited for adherent cells. Add 4 mL of culture medium and incubate the flask at 37 °C with 5% CO₂.

3.Cell culture (Day 4)

1. Discard the media from both flasks and add fresh 5 mL of culture media to the flask.
2. Incubate the flask at 37 °C with 5% CO₂ for 2 days.

4. Cell Culture (Day 6)

1. Cells will be ready for further experiments.

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