Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

Summary

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

Abstract

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (>90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

Introduction

The hippocampus was first described by a Bolognese anatomist, Giulio Cesare Aranzio, in the 1500's¹. In naming this newfound structure, Aranzio was likely inspired by its uncanny resemblance to the seahorse of the genus Hippocampus¹. The hippocampus is involved in stress responses but is widely known for its role in learning and memory. More specifically, the hippocampus is responsible for the encoding and retrieval of declarative and spatial memory¹.

The hippocampus, or hippocampus proper, is divided into the CA1 (cornu ammonis), CA2, and CA3 subfields¹. Compared to the rest of the nervous system, the hippocampus has several unique defining characteristics, including its plasticity and potential for ongoing neurogenesis². Neurogenesis is the process of the proliferation and differentiation of neural stem cells, followed by their integration into the pre-existing neuronal network. Neurogenesis is restricted to the subgranular zone of the dentate gyrus and subventricular zone of the lateral ventricles (and the olfactory bulbs)³. While neurogenesis is abundant in embryogenesis, it is a lifelong process^3,4. As such, this discussion will focus on adult neurogenesis in the hippocampus.

The subventricular and subgranular zones are neurogenic niches containing ependymal and vascular cells, as well as immature and mature lineages of neural stem cells⁵. Microglia contribute to these niches as immune cells to regulate neurogenesis⁶. Neural progenitor cells are nonstem cell progenies of neural stem cells⁷. Three types of neural progenitors are present in the subventricular zone: radial glia-like type B cells, type C transit-amplifying progenitors, and type A neuroblasts^3,8. The slowly dividing type B neural progenitor cells in the subventricular zone can differentiate into rapidly dividing type C cells⁸. Subsequently, type C cells differentiate into type A cells⁸. These neuroblasts migrate through the rostral migratory stream to the olfactory bulb before differentiating into interneurons or oligodendrocytes⁹. These olfactory bulb interneurons are key to olfactory short-term memory, and associative learning, whereas the oligodendrocytes myelinate axons of the corpus callosum⁹. The majority of adult neurogenesis occurs in the subgranular zone of the dentate gyrus, where radial type 1 and nonradial type 2 neural progenitors are found³. Most neural progenitor cells are destined to become dentate granule neurons and astrocytes¹⁰. Connected by gap junctions, astrocytes form networks to modulate plasticity, synaptic activity, and neuronal excitability⁵. As the primary excitatory neuron of the dentate gyrus, granule cells provide input from the entorhinal cortex to the CA3 region¹¹.

Neural stem cell populations can be isolated using immunomagnetic or immunofluorescent isolation strategies^12,13. Neural tissue is particularly difficult to dissociate; efforts to do so often result in samples with poor cell viability and/ or fail to produce the necessary single-cell suspension for downstream analysis. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination of techniques^14,15. In a study evaluating neural dissociation methods, the viability and quality of manual mechanical dissociation by pipette trituration versus combinations of pipette trituration and digestion with various enzymes were compared¹⁵. Quality was graded based on the amount of cell clumps and DNA or subcellular debris in the prepared suspension¹⁵. Suspensions of glial tumors subjected to manual mechanical dissociation alone had significantly lower cell viability than treatments with dispase or a combination of DNase, collagenase, and hyaluronidase¹⁵. Volovitz et al. acknowledged the variation in viability and quality between the different methods and emphasized that inadequate dissociation may reduce the accuracy of downstream analysis¹⁵.

In a separate study, the authors compared over 60 different methods and combinations of dissociation of cultured neuronal cells¹⁴. These methods included eight different variations of manual mechanical dissociation by pipette trituration, a comparison of incubation with five individual enzymes at three different intervals, and various combinations of mechanical dissociation with enzymatic digestion or the combination of two enzymes¹⁴. None of the mechanical methods yielded a single-cell suspension¹⁴. Four of the single enzyme treatments, ten of the combination enzymatic treatments, and four of the combinations of mechanical dissociation with enzymatic digestion yielded a single-cell suspension¹⁴. Enzymatic digestion with TrypLE followed by Trypsin-EDTA most effectively dissociated samples¹⁴. Incidentally, samples treated with TrypLE and/or Trypsin-EDTA tended to form gelatinous clumps¹⁴. While this study was performed on cultured cells, it speaks to the shortcomings of pipette trituration or enzymatic digestion alone.

Side-by-side comparisons of manual versus automated mechanical dissociation are lacking. However, one group ran flow cytometry to compare manual and semi-automated mechanical dissociation of whole mouse brains in conjunction with commercial papain or trypsin enzymatic dissociation kits¹⁶. Processing with the dissociator more consistently yielded viable cells¹⁶. Following dissociation, the authors also isolated Prominin-1 cells, neuronal precursor cells, and microglia¹⁶. For two of the three isolated cell populations, the purity of the isolated cells was slightly higher when samples were processed with the dissociator, as compared to manually¹⁶. Reiß et al. noted that person-to-person variability in pipetting technique hinders reproducibility of viable cell population yield in tissue dissociation¹⁶. The authors concluded that automated mechanical dissociation standardizes sample processing¹⁶.

The method of dissociation outlined in this manuscript is a combination of fully automated mechanical dissociation and enzymatic digestion, using solutions accompanying a commercial adult brain dissociation kit¹⁷. Unlike standard protocols, this optimized protocol reduces sample manipulation, yields a highly viable single-cell suspension, and is intended for processing minimal amounts of starting tissue.

Protocol

Experiments were conducted in accordance with the ethical standards approved by the Institutional Animal Care and Use Committee at UAMS. 6-month-old female C57Bl6/J wild-type mice were purchased and group-housed (4 mice per cage) under a constant 12 h light/dark cycle.

1. Preparation of reagents

1. Prepare fixable live/dead stain stock solution. Reconstitute the fluorescent stain with 20 µL of dimethyl sulfoxide (DMSO).
2. Wrap the vial in foil, label it as "Reconstituted", and store it at -20 °C for up to six months.
3. Prepare a 0.9% saline solution with heparin. Dilute the contents of one vial of heparin sodium (10,000 USP units per 10 mL) in 1 L of double-distilled water (ddH₂O).
4. Prepare enough for approximately 45 mL per animal and store at 4 °C for up to one week.
5. Make 1% paraformaldehyde (PFA).
   1. In a fume hood, heat a hot plate to 50 °C. In a Microwave, heat 100 mL of ddH₂O in a glass beaker to approximately 60 °C. Add a magnetic stir bar and transfer to the hot plate.
   2. In the fume hood, weigh out 1 g of PFA and add to the beaker of ddH₂O. Add 0.1125 g of NaOH crystals and mix until dissolved (5-10 min).
   3. Add 0.4 g of NaPO_4- monobasic and mix until dissolved (2-5 min). Vacuum filter the solution and adjust pH to 7.4 with HCl and NaOH.
   4. Cool on ice or at 4°C for 30 min before storing.
      NOTE: Aliquots of 1.5 mL can be stored at -20 °C for one year. Avoid freeze-thaw cycles. If, after thawing, the solution becomes cloudy or a precipitate has formed, the solution should not be used.
      CAUTION: Toxic, flammable. Always work with PFA under a ventilated hood wearing proper personal protective equipment.
6. Resuspend lyophilized Enzyme A with 1 mL of Buffer A. Do not vortex the solution.
   NOTE: Enzyme A and Buffer A as well as Buffers A, Y, and Z are reagents in the commercial Adult Brain Dissociation Kit¹⁷.
7. Divide Enzyme P into aliquots of 50 µL and resuspend Enzyme A into 10 µL aliquots. Per kit instructions, store at -20 °C for up to six months. Avoid freeze-thaw cycles.

2. Day of experiment

1. Cool the tabletop centrifuge to 4 °C.
2. Place aliquot(s) of PFA in the fridge for gradual thawing.
3. Place the reconstituted live/dead stain in the dark (e.g., a drawer) to thaw at room temperature.
4. Prepare the bovine serum albumin (BSA) Buffer. Add 0.5 g of BSA to 100 mL of 1x Dulbecco's phosphate-buffered solution without calcium and magnesium (D-PBS), pH 7.2.
5. Add a stir bar and mix on a stir plate for 30 min. Transfer to 50 mL conical tubes and store at 4 °C.
   NOTE: Always use freshly prepared BSA buffer.
6. Prepare live/dead stain working dilution. Add 1 µL of the reconstituted live/dead stain stock solution to 360 µL D-PBS and store it in the dark (e.g., a drawer or box) at room temperature. Prepare 50 µL of the working dilution per sample.

3. Perfusion

1. Place the saline solution with heparin on ice.
2. Turn on oxygen, set the flowmeter indicator ball on the small animal anesthesia vaporizer system to 1 L/min. Ensure there is adequate oxygen pressure and isoflurane.
3. Adjust the vaporizer dial to 3.5% (for induction and maintenance).
4. Prime the perfusion pump lines with the saline/heparin solution. Set the speed to 6 mL/min.
5. Place the mouse in the induction chamber, turn on the breather, and wait several minutes until the mouse is unresponsive. Confirm sufficient depth of anesthesia through the absence of pedal withdrawal to noxious pinch.
6. Place the mouse on its back on the dissection tray with its nose in the nose cone. Perform a secondary confirmation of full anesthetization through the absence of pedal withdrawal to noxious pinch. Pin all four paws to the tray.
7. Spray the animal's abdomen with 20% ethanol.
8. Using forceps, pinch the lower abdomen and lift the skin. Use scissors to cut through fur and skin to the bottom of the ribcage.
9. Make two diagonal incisions from below the ribcage toward each shoulder.
10. Carefully resect the diaphragm (avoiding the lungs and heart). Resect the ribcage to expose the heart.
11. Carefully sever any connective tissue around the heart.
    NOTE: Steps 3.10-3.11 are critical; perform with proficiency and dexterity.
12. Use the scissors to clip the right atrium (dark lobe on the upper left of the heart). Turn off the flow of isoflurane to the breather.
13. Hold the heart steady with forceps. With the bevel of the butterfly needle facing up, pierce the left ventricle while keeping the needle level and parallel to the animal.
14. Hold the needle in place, turn on the pump, and perfuse at least 30 mL of the saline/heparin solution until the fluid leaving the heart is opaque and the liver and lungs pale in color.
    NOTE: Steps 3.13-3.14 are critical; perform with proficiency and dexterity.
15. Turn off the pump, remove the needle, and transfer the mouse to the dissection area.

4. Dissection

1. Using large surgical scissors, decapitate the head.
2. Cut the fur from the back of the head up to the eyes. Peel the skin back to expose the skull.
3. Clip the skull between the eyes. Make two cuts at the back of the skull, at the 10 and 2 o'clock positions, then make one long cut (keep tips up to avoid damaging the brain) along the midsagittal line of the skull to the original cut between the eyes.
4. Use forceps to peel the two halves of the skull away to the sides. Use a spatula to remove the brain and place it into a 60 mm glass Petri dish on ice filled with cold D-PBS (Figure 1).
5. Use a scalpel or razor to separate each hemisphere. Then remove the olfactory bulbs and cerebellum.
6. Use forceps to remove the midbrain until the hippocampus is exposed.
7. Secure the brain with forceps. Using a second set of forceps, gently tease the hippocampus out of each hemisphere, and transfer both hippocampi to a labeled 1.5 mL tube containing cold D-PBS.
8. Place the sample tube containing the two hippocampi from the mouse on ice.

5. Prepare Enzyme Mix 1 and 2 for each sample

NOTE: For volumes greater than 2 mL, use a 10 mL serologic pipette; for volumes, 200 µL-2 mL, use a 1000 µL pipette; for volumes, 21-199 µL, use a 200 µL pipette; for volumes, 2-20 µL, use a 20 µL pipette; for volumes under 2 µL, use a 0-2 µL pipette.

1. For each sample, thaw one aliquot each of Enzyme P and Enzyme A at room temperature.
2. For Enzyme mix 1, combine 50 µL of Enzyme P and 1900 µL of Buffer Z in a labeled C Tube (Table of Materials).
3. For Enzyme mix 2, add 20 µL of Buffer Y to the thawed 10 µL aliquot of Enzyme A.

6. Adult brain dissociation protocol¹⁷

NOTE: When working with samples, tubes should be placed in a tube rack at room temperature while BSA and D-PBS remain on ice unless otherwise noted.

1. Switch on the dissociator.
2. Use forceps to transfer the hippocampi tissue pieces to the C Tube.
3. Transfer 30 µL of Enzyme mix 2 into the C Tube. Twist the cap until tension is felt, then tighten until it clicks.
4. Place the C Tube upside down into a position of the dissociator; the sample will be assigned the Selected status (Figure 2). Secure the heater over the C Tube.
5. Press the folder icon, select Favorites folder, scroll to and select the 37C_ABDK_02 program. Click on OK to apply the program to all selected C tubes, then tap on Start (Figure 2).
6. Label one 50 mL conical tube per sample.
7. Place a 70 µm cell strainer on each 50 mL conical tube and wet with 2 mL of BSA buffer.
8. Upon completion of the program, remove the heater and the C tube from the dissociator.
9. Add 4 mL of BSA buffer to the sample and apply the mixture to the cell strainer on the 50 mL conical tube.
10. Add 10 mL of D-PBS to the C Tube, close it, and swirl the solution gently. Apply it to the cell strainer on the 50 mL conical tube.
11. Discard the cell strainer and the C Tube. Centrifuge the suspension at 300 x g for 10 min at 4 °C. Then, aspirate and discard the supernatant.

7. Debris removal

1. Resuspend the pellet with 1550 µL of cold D-PBS and transfer the suspension to a labeled 15 mL conical tube.
2. Add 450 µL of cold Debris Removal Solution and pipette up and down (do not vortex).
   NOTE: Debris Removal Solution is a reagent in the commercial Adult Brian Dissociation Kit¹⁷.
3. Gently overlay 1 mL of cold D-PBS on top of the cell suspension, keeping the tip against the wall of the conical tube. Repeat until the total overlay is 2 mL.
   NOTE: This step is critical; perform with proficiency and dexterity.
4. Centrifuge at 3000 x g for 10 min at 4 °C with full acceleration and full brake.
   NOTE: If the phases are not clearly separated, repeat steps 7.2-7.3. Centrifuge a final time at 1000 x g for 10 min at 4 °C.
5. The suspension should now consist of three distinct layers (Figure 3). Aspirate the topmost layer. Sweep the pipette tip back and forth to aspirate the white middle layer. Remove as much of the middle layer as possible without disturbing the bottommost layer.
   NOTE: This step is critical; perform with proficiency and dexterity.
6. Add 2 mL of cold D-PBS and pipette up and down to mix.
7. Centrifuge at 1000 x g for 10 min at 4 °C with full acceleration and full brake. Aspirate and discard the supernatant. Resuspend the pellet in 1 mL of BSA buffer.
   ​NOTE: Cells can be resuspended in the appropriate buffer then magnetically labeled and isolated in preparation for single-cell sequencing at this point.

8. Cell count

1. Perform cell counting as per the manufacturer's protocol of available cell counter (one option is noted in the Table of Materials)

9. Live/dead stain

1. Centrifuge the remaining 900 µL (from 7.7) at 1000 x g for 10 min at 4 °C with full acceleration and full brake.
2. While the sample is spinning, label one flow tube per sample and wrap it in foil to limit light exposure.
3. Aspirate and discard the supernatant.
4. Resuspend the pellet in 50 µL of diluted live/dead stain (previously prepared).
   NOTE: This step should be performed in a low-light setting. Turn off overhead room lights to achieve this.
5. Transfer each sample to the corresponding labeled flow tube and incubate at room temperature for 8-10 min in the dark (e.g., a drawer or box).
6. Add 500 µL of BSA buffer and centrifuge at 1000 x g for 10 min at 4 °C with full acceleration and full brake.
7. Aspirate and discard the supernatant.
   ​NOTE: The pellet may not be visible; leave a small amount of buffer behind so as not to unintentionally aspirate the pellet. Cells can be resuspended in the appropriate buffer, blocked, and stained with cell-specific antibodies at this point. See Supplemental File 1 for sample protocol¹⁸.

10. Fixation (optional)

1. Resuspend the pellet in 200 µL of 1% PFA (previously prepared). Incubate for 15 min at 4 °C.
2. Wash by adding 500 µL of D-PBS and centrifuge at 300 x g for 10 min at 4 °C.
3. Aspirate the supernatant.
   NOTE: The pellet may not be visible; leave a small amount of buffer behind so as not to unintentionally aspirate the pellet.
4. Resuspend the pellet in 200 µL of D-PBS and store at 4 °C for up to 3 days.

11. Flow cytometry

1. Label the filter caps on the new tubes.
2. Using a 1 mL pipette, pipette each sample onto the filter cap.
3. Centrifuge briefly at 200 x g at 4 °C, only allowing the centrifuge to reach 200 x g before stopping the run.
4. Proceed to flow cytometry core for downstream analysis.

Results

Samples were processed with a flow cytometer at a core facility, and the resulting data were evaluated with a software package for flow analysis. Previously, compensation controls were analyzed-the live/dead stain and negative control. If multiple fluorochromes are used, fluorescence minus one (FMO) controls and single-stain controls should be prepared for each antibody. Compensation for spectral overlap for the experimental samples was calculated based on the analyzed controls. For cell population identification, a hier...

Discussion

Several steps in this neural dissociation protocol require proficient technique and dexterity–perfusion, supernatant aspiration, and myelin removal. Throughout the perfusion process, the internal organs must remain intact (aside from removing the diaphragm and clipping the heart); this includes avoiding the upper chambers of the heart with the butterfly needle. While the amount of saline with heparin needed varies, transparent fluid flowing from the heart indicates the process is complete. The brain must be complet...

Materials

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tubes	Fisher Scientific	02-682-003	Basix, assorted color
15 mL Falcon Tubes	Becton Dickinson Labware Europe	352009	Polystyrene
25 mL Serological Pipets	Fisher Scientific	14-955-235
5 mL Round Bottom Polystyrene Test Tube	Falcon	352052
500 mL Vacuum Filter/ Storage Bottle System	Corning	431097
70 μm cell strainer	Fisher Scientific	08-771-2
Adult Brain Dissociation Kit	Miltenyi Biotec	130-107-677	Contains Enzyme P, Buffer Z, Buffer Y, Enzyme A, Buffer A, Debris Removal Solution
Aluminum Foil	Fisher Scientific	01-213-105
Anti-ACSA-2-PE-Vio770, mouse, clone REA969	Miltenyi Biotec	130-116-246
Anti-Myelin Basic Protein	Sigma-Aldrich	M3821-100UG
Anti-PSA-NCAM-PE, human, mouse and rat, Clone 2-2B	Miltenyi Biotec	130-117-394
BD LSRFortessa	BD
BSA	Sigma-Aldrich	A7906-50G
CD11b-VioBlue, mouse, Clone REA592	Miltenyi Biotec	130-113-810
CD31 Antibody	Miltenyi Biotec	130-111-541
Ceramic Hot Plate Stirrer	Fisher Scientific	11-100-100SH
Dimethyl Sulfoxide	Fisher Scientific	BP231-100
Ethanol	Pharmco by Greenfield Global	111000200
Falcon 50 mL Conical Centrifuge Tubes	Fisher Scientific	14-432-22
Fine Scissors - Sharp	Fine Science Tools	14060-09	Perfusion
FlowJo	BD		(v10.7.0)
gentleMACS C Tubes	Miltenyi Biotec	130-093-237
gentleMACS Octo Dissociator with Heaters	Miltenyi Biotec	130-096-427
Gibco DPBS (1X)	ThermoFisher Scientific	14190144
Glass Beaker	Fisher Scientific	02-555-25A
Heparin sodium	Fresenius Kabi	504011
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit	ThermoFisher	L34965
Magnetic Stir Bar	Fisher Scientific	14-513-51
Noyes Spring Scissors	Fine Science Tools	15012-12	Dissection
Paraformaldehyde	Sigma-Aldrich	441244-3KG	Prilled, 95%
Pipette tips GP LTS 20 µL 960A/10	Rainin	30389270
Pipette Tips GP LTS 250 µL 960A/10	Rainin	30389277
Pipette tips RT LTS 1000 µL FL 768A/8	Rainin	30389213
Rainin Pipet-Lite XLS (2, 20, 200, 1000 μL)	Rainin	30386597
RBXMO FITC XADS	Fisher Scientific	A16167
Round Ice Bucket with Lid	Fisher Scientific	07-210-129
Round-Bottom Tubes with Cell Strainer Cap	Falcon	100-0087
S1 Pipet Fillers	ThermoFisher Scientific	9541
Spatula & Probe	Fine Science Tools	10090-13	Dissection & Perfusion
Surflo Winged Infusion Set 23 G x 3/4"	Termuno	SV-23BLK	Butterfly needle
Test Tube Rack	Fisher Scientific	14-809-37
Thermo Scientific Legend XTR Centrifuge	ThermoFisher	discontinued	Or other standard table top centrifuge
Variable-Flow Peristaltic Pump	Fisher Scientific	13-876-2	Low-flow model
VetFlo Starter Kit for Mice	Kent Scientific	VetFlo-MSEKIT	Anesthesia mask, tubing, induction chamber, charcoal canisters
VetFlo Vaporizer Single Channel Anesthesia System	Kent Scientific	VetFlo-1210S	0.2–4 LPM
Vi-CELL XR Cell Viability Analyzer	Beckman Coulter Life Sciences	731196	Cell Counting
Vi-CELL XR 4 Bags of Sample Vials	Beckman Coulter Life Sciences	383721	Cell Counting