Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Summary

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Abstract

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 10⁶ neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

Introduction

Understanding spinal cord pathology requires the use of various models, both on the macroscopic and microscopic levels. Large and small animal models^1,2,3 are used for in vivo investigations of spinal cord disease and injury. While studying these issues in vivo has its merits, analysis of the spinal cord is limited to whole spinal cord homogenate or to tissue sections⁴. This creates some ambiguity when trying to isolate specific responses and targets in the spinal cord among its resident neurons and surrounding glia. The increasing availability of genetically manipulated mice allows for more detailed investigations of the biology at cellular and molecular levels. Thus, a neonatal mouse model is used here, allowing for the study of the unique properties and biology of spinal cord neurons in vitro.

The isolation and maintenance of neurons in vitro is not particularly straightforward. There is a relative abundance of techniques for neuron isolation from the cortical tissue of adult rodents that seem to result in a substantial number of isolated neurons (i.e. millions)^5,6,7. In contrast, the yield of neurons from spinal cord tissue is lower^8,9,10, in part due to the smaller mass of tissue. Furthermore, in mice, there is a relative paucity of techniques for the isolation of neonatal spinal cord neurons, and existing methods are limited by lower neuron yields (i.e. hundreds)⁹ or laborious and resource-heavy techniques requiring the isolation of embryonic mice¹⁰.

In this protocol, we use a technique that allows for the cost- and resource-effective isolation of a substantial number of neurons from the spinal cords of neonatal mice. As is common in previously published techniques, we use papain as an enzymatic protease, allowing for the release of neurons from the spinal cord tissue^5,6 . In addition, we use a density gradient for refined cell separation, which has previously been shown to be effective^6,10. While the medium in which the cells are incubated can vary, in our experience and as previously published¹¹, supplementation with fresh B27 culture medium supplement has proven to be critical for neuron longevity. The neurons are typically viable for up to 10 days, allowing for treatment to be carried out.

Protocol

The care and treatment of animals in this procedure were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Colorado.

1. Preparing Solutions

1. Prepare and store all solutions at appropriate temperatures, as shown in Table 1.

2. Coating Wells and Slides

NOTE: Neurons do not adhere well to plastic or glass surfaces.

1. One day prior to the isolation of the neurons, coat the wells of a sterile 24-well culture plate with 0.5 mL of Poly-D-Lysine (PDL; Table 2) and leave it in a laminar flow hood O/N.
   NOTE: The preferred method is to use 24-well plates and to coat only the center wells, as evaporation from the peripheral wells tends to be more accelerated and to lead to inconsistent results. Additionally, glass slides can be placed inside the wells prior to coating. The neurons will attach to the coated glass slides and can be removed later for further microscopic imaging.
2. On the day of isolation, remove the PDL from the wells. Wash with sterile water for a few minutes before removing any remaining water and allowing it to dry for 1 h (see below).
3. After drying, coat the slides with laminin (Table 2).
   NOTE: Approximately 10 μL of laminin (10 µg/µL) is enough to coat each well of a sterile 24-well plate. This is mixed with 340 μL of medium (total volume: 350 μL) in order to cover the entire well. Let this sit for 2 h at RT in a culture hood and then aspirate prior to coating the cells.

3. Harvesting Spinal Cords

NOTE: All instruments should be autoclaved (135 °C and 30 psi for 4 x 7 min cycles) for sterility.

1. Euthanize 1-3 day-old C57BL/6 mouse pups in a chamber with isoflurane. Wait 30 s after the cessation of movement and pinch the leg to confirm a lack of response.
2. Separate the head from the body using scissors, with pup in the prone position.
3. Stabilize the hind legs or tail and arms on the procedure table, with dorsal side facing the user.
4. Cut the skin off using curved iris scissors.
5. Cut the spinal cord from the lumbar region just above the hips and proceed to cut both sides of the thorax to separate it from the body.
   NOTE: This requires the careful dissection of the spinal cord from visceral organs to avoid inadvertent damage to other organs (Figure 1a).
6. Wash sequentially for 10 s in 3 x 10 cm Petri dishes containing 5 mL of 0.2 μm filter-sterilized Phosphate-Buffered Saline (PBS) to remove excess tissue.
7. Insert a 22 G needle and syringe filled with 5 mL of filter-sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth Petri dish (Figure 1b).
8. Collect the spinal cord in a 15 mL tube with 5 mL of HABG (Table 1) on ice. Use care to avoid crushing the spinal cord.
9. Repeat steps 3.1-3.8 for each pup in the litter.
   NOTE: Ideally, this process should take less than 30 min per spinal cord to ensure healthy neuron isolation.

4. Isolating Neurons

NOTE: The following step should be performed in a laminar flow hood. Familiarity with basic sterile technique is expected.

1. Tissue Mincing
   1. Take the tube containing the spinal cords and shake lightly to suspend the tissue.
   2. Pour tissue from the tube into a 60 mm glass Petri dish and dice with a razor blade to create fine pieces ~0.5 mm in size.
   3. Transfer the tissue with a wide-bore pipette into a 15 mL tube containing 5 mL of HABG.
   4. Place it in a 30 °C water bath for 30 min to allow the cells to equilibrate at this temperature. Keep the cells on a shaker just enough to allow them to be suspended in the fluid.
      NOTE: This step is done to avoid shocking the cells upon transfer from ice to the digestion medium. Keeping the cells at 30 °C helps to decrease cell death associated with an otherwise increased metabolism at 37 °C.
2. Prepare the digestion medium (Table 1).
3. Prepare the density gradient (Table 1).
   1. Prepare each of the 4 layers in 4 separate 15 mL tubes, as outlined in Table 1.
   2. Add 1 mL from each layer into a new 15 mL tube. Start with layer 1 at the bottom and sequentially add until reaching layer 4 at the top. Avoid disturbing the layers while adding.
4. Wash the PDL-coated plates from step 2.2. Wash with sterile water for a few minutes before removing any remaining water and allowing them to dry for 1 h.
5. Transfer the tissue to digestion medium.
   1. Remove the tissue-containing tube from the shaker water bath at 30 °C and allow it to settle for a few minutes.
   2. Remove the digestion medium tube from the 37 °C water bath and aspirate it into a leur-lock syringe.
   3. Aspirate off the excess HABG from the tissue-containing tube.
   4. Use a leur-lock 0.2 μm filter on the syringe to add digestion medium to the tissue-containing tube.
   5. Place the tube in a 30 °C water bath for 30 min. Keep the cells shaking just enough to allow them to be suspended in the fluid.
      NOTE: It is important not to keep the cells in the digestion medium for too long or to let the temperature get too high, which could lead to excessive digestion and result in the tissue becoming suspended in a gelatinous mixture.
6. During this period, coat the laminin as in step 2.3.
7. Perform trituration (i.e. separating the cells from the tissue).
   1. Remove the tube from the shaking 30 °C water bath and allow it to settle for a few min.
   2. Aspirate excess digestion medium.
   3. Suspend the tissue in 2 mL of HABG.
   4. Using a narrow-bore pipette, triturate 10x for 45 s.
      NOTE: This is probably the single most crucial step and can significantly decrease the yield if not done properly.
      1. Aspirate the tissue into the pipette and immediately empty the contents back.
      2. Avoid introducing air, as it will significantly decrease the viable yield.
         NOTE: The ideal pipette is a 9" glass pipette. The tip of the pipette should be fire polished to smooth out rough surfaces. It should then be siliconized by placement in a 1:20 solution of dichlorodimethylsilane (DMDCS) in chloroform and left O/N. The pipette should then be removed and allowed to air dry. Subsequently, it should be autoclaved for sterilization.
         Caution: DMDCS and chloroform are highly flammable, and siliconizing should be carried out in a fume hood.
   5. Aspirate the top 2 mL of supernatant and place it into a new 15 mL tube labeled "collection."
   6. Repeat steps 4.7.3-4.7.5 two additional times (the cell collection tube should have 6 mL by the end).
   7. Slowly transfer the collection tube contents into the gradient tube prepared in step 4.2, avoiding the disruption of the gradient.
8. At this point, remove the previously prepared neurobasal medium (Table 1) from the refrigerator and allow it to warm in a 37 °C bath.
9. Purify the neurons.
   1. Centrifuge the gradient tube for 15 min at 800 x g and 22 °C.
   2. Collect the desired layer(s) with a pipette (Figure 2) and place in a new 15 mL tube. For the highest-purity neuron isolation (i.e. >90%), collect layer 3. For more yield with less purity (i.e. >70-80%), collect layers 2 & 3.
   3. Dilute out the density gradient by adding 5 mL of HABG to the newly collected layers.
   4. Centrifuge at 200 x g for 2 min at 22 °C.
   5. Discard the supernatant, re-suspend in 5 mL of HABG, and flick the pellet to suspend the cells.
   6. Centrifuge at for 2 min 200 x g at 22 °C.
   7. Discard the supernatant, resuspend in 3 mL of neurobasal medium, and flick the pellet to resuspend the cells.
10. Count the cells.
    1. Take 10 μL of the solution, now with cells in neurobasal medium, and mix with 10 μL of Trypan blue.
    2. Place 10 μL of this mixture in a glass counting chamber.
    3. Using a standard glass counting chamber, count the number of cells in each of the four 4 x 4 quadrants. Add all of the cells counted (n), multiply by 2 (dilution factor), divide by 4 (number of quadrants counted), multiply by 3 (volume of neurobasal medium), and multiply by 10⁴ to obtain the concentration of cells in cells/mL.
       
11. Seed the cells on culture plates
    1. Dilute the cell suspension to 300,000 cells in 1 mL of neurobasal medium.
       NOTE: Based on the concentration of cells obtained in the above steps, additional neurobasal medium is added to obtain a final concentration of 3*10⁵ cells/mL. The equation is C₁V₁=C₂V₂, where C₁ is the initial concentration of cells obtained from the harvest; V₁ is 3 mL; and C₂ is 3*10⁵ cells/mL, as discussed above. The equation is solved for V₂. Add the volume of neurobasal medium necessary to make the total volume of cells in solution equal to V₂.
    2. Shake gently to distribute the cells in solution and add 1 mL to each well in the coated 24-well plates.

Results

Using this technique, a single litter (4-10 pups) allows for the isolation of 1-2.5 10⁶ neurons suitable for seeding onto culture plates. Typically, 4-8 wells are seeded at the concentration mentioned above (i.e. 300,000 cells/mL). Figure 3 demonstrates the appearance of neurons at this concentration after a week in culture at low- (a) and high- (b) magnification light microscopy. However, we have also bee...

Discussion

This technique allows for the reliable culture of spinal cord neurons. Once proficiency in the technique is achieved, it takes approximately 3.5 h to complete. We have been able to carry out the isolation of neurons from 2 separate litters (16 mice total) in approximately 4 h. The key step in feasibility is being able to proficiently extract the spinal cords from the mice. The yield allows for plating several wells and for the ability to test the neurons under various conditions. We have been able to treat the neurons af...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hibernate A Medium - 500 mL	Thermo-Fisher	A1247501	https://www.thermofisher.com/order/catalog/product/A1247501
Hibernate A Minus Calcium - 500 mL	Brainbits	HA-Ca	http://www.brainbitsllc.com/hibernate-a-minus-calcium/
Glutamax 100X - 100 mL	Thermo-Fisher	35050061	https://www.thermofisher.com/order/catalog/product/35050079
B27 Supplement 50X - 10 mL	Thermo-Fisher	17504044	https://www.thermofisher.com/order/catalog/product/17504044
Papain, Lyophilized - 100 mg	Worthington	LS003119	http://www.worthington-biochem.com/pap/cat.html
Neurobasal A Medium - 500 mL	Thermo-Fisher	10888022	https://www.thermofisher.com/order/catalog/product/10888022
Penicillin-Streptomycin (10,000 U/mL)	Thermo-Fisher	15140122	https://www.thermofisher.com/order/catalog/product/15140122
Poly-D-Lysine (PDL) hydrobromide - 5 mg	Sigma-Aldrich	P6407-5MG	http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en®ion=US
Mouse Laminin - 1 mg	Thermo-Fisher	23017015	https://www.thermofisher.com/order/catalog/product/23017015
Trypan Blue - 20 mL	Sigma-Aldrich	T8154-20ML	http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en®ion=US
OptiPrep Density Gradient Medium - 250 mL	Sigma-Aldrich	D1556-250ML	http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en®ion=US
Dichlorodimethylsilane (DMDCS, Sigma Silicoat)	Sigma-Aldrich	440272-100ML	http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en®ion=US
Chloroform	Sigma-Aldrich	288306-1L	http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en®ion=US
Glass Pippette - 9"	Sigma-Aldrich	13-678-20C	http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en®ion=US
Pipette bulb - 5 mL	Sigma-Aldrich	Z186678-3EA	http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en®ion=US&cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1
BRAND® Petri dish, glass - 60 x 15 mm	Sigma-Aldrich	BR455717-10EA	http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en®ion=US
Sterile 24-Well Cell Culture Plate	Sigma-Aldrich	M8812-100EA	http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en®ion=US
Hausser Hemacytometer (glass counting chamber)	Fischer Scientific	02-671-6	https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716
Glass Slides - 12 mm sterile cover glass - uncoated	Neuvitro	GG-12-1.5-Pre	http://www.neuvitro.com/german-coverslip-12mm-diameter.htm
NeuN Rabbit Monoclonal Antibody - 100 µL	Abcam	ab177487	After fixing in paraformaldehyde (PFA) and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluted 1:200 for 18 h in 4 °C
MAP-2 Mouse Monoclonal Antibody - 50 µL	Abcam	ab11267	After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluted 1:500 for 18 h in 4 °C