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An efficient method for the isolation of highly functional primary neurons
Easily and reproducibly isolate and culture primary neurons.
Hai-Yan Wu, Ph.D.;
Joanna Geddes, B.S.;
Kay Opperman, Ph.D.;
Barbara Kaboord, Ph.D.;
Primary neuron cultures are powerful model systems used to study neuron morphology and differentiation, synaptic function and neurotransmitter release, neurotoxicity during pre-clinical drug development, and disease modeling. Because primary neuron cultures eliminate many of the complications present in whole animal models, there are advantages to using them in electrophysiological recording and stimulation experiments, pharmacological manipulations, and high-resolution microscopic analyses [1,2,3]. However, primary neurons are highly sensitive to isolation conditions and their culture or growth environment. Because methods differ from lab to lab, cell yields, viability, and maturation rates are highly variable and make it difficult to compare results and repeat experiments between laboratories.
In this article, we report a method for isolating neuronal cells using a gentle tissue digestion enzyme formulation. We compared the efficacy of our new method to existing protocols for the digestion of embryonic-Day-17 to 19 (E17-19) whole mouse cortexes and the subsequent cultivation of the isolated neurons. Our new method has been commercialized as the Thermo Scientific Pierce Primary Neuron Isolation Kit (Part No. 88280). The kit is a significant improvement over existing trypsin protocols to generate high-yield, high-viability, high-quality neuronal cultures as manifested by their morphological characteristics, cell-specific protein expression and the extent of synaptic scaling.
RESULTS and DISCUSSION:
Isolating primary neurons from brain tissue requires the controlled use of proteases to digest intercellular protein junctions followed by gentle mechanical disruption to liberate individual cells. This enzymatic digestion process has significant impact on the overall isolation efficiency and viability of the isolated neurons. To determine the best digestion enzyme for brain tissue, a panel of proteases at various concentrations was screened to determine which enzyme or enzyme combination was most efficient at liberating healthy neuronal cells (data not shown). The best candidate was selected and compared to the standard literature procedure. Primary neurons were isolated from mouse embryonic cortical tissue at E17-19 using an optimized gentle enzymatic digestion method described in the Pierce Primary Neuron Isolation Kit (Figure 1) or a traditional, do-it-yourself (DIY) trypsin-based method . Cell yield and cell viability were determined from cell suspensions prepared from one pair of mouse cortices (cortexes). We obtained approximately a 2-fold increase in cell yield compared to the trypsin method (Figure 2). Cell viability from our method (94-96%) is consistently higher than that from trypsin protocols (83-92%). The procedure works well with both mouse and rat tissue from various sections of the brain including cortex and hippocampus (Table 1).
Figure 1. Schematic diagram of the primary neuron isolation procedure. Hereafter in this article, this procedure for the Thermo Scientific Pierce Primary Neuron Isolation Kit (Part No. 88280) will be called the “Pierce Neuron Kit”.
Figure 2. Primary neuronal cell yield and viability immediately after isolation. Primary neurons were isolated from mouse embryonic cortical tissue at E17-19 using either the Pierce Neuron Kit (see Methods) or a trypsin-based DIY method. Cell yield and cell viability were determined from cell suspensions prepared from one pair of mouse cortexes in a total volume of 1.5mL. n=10 mouse cortical pairs from five independent experiments. Data represent the mean ± SD.
Table 1. Cell yield and viability from a typical isolation with the Pierce Neuron Kit. Results are for one pair of cortices or three pairs of hippocampi in 1.5mL cell suspension. Viability was determined by trypan blue exclusion.
|Mouse cortical neuron
||4.5 x 106
|Mouse hippocampal neuron
||3.6 x 106
|Rat cortical neuron
||4.0 x 106
|Rat hippocampal neuron
||4.0 x 106
Since most primary neurons are not utilized immediately but are cultured for 1-3 weeks to re-establish dendritic processes and active synapses, it is important to assess the health and composition of the cultures over time. Cell morphology was examined by phase contrast microscopy at Day 1, Day 14, and Day 28. A few rounded cell bodies with short projections were observed in the culture at Day 1 (Figure 3, top). By Day 14 and 28, an extensive, intertwined network of dendrites had developed which was further confirmed by expression of the pre-synaptic marker protein synaptophysin and post-synaptic protein PSD95 at synaptic terminals (Figure 3, bottom right). As a further indication of good health, axon morphology was visualized in Day 7 cultured neurons transfected with GFP. Neurons growing in our optimized support media with neuronal growth supplements exhibited well differentiated axons bearing extensive axon arbors (Figure 3, bottom left).
Figure 3. Phase contrast images of primary neuron cultures at different developmental stages in culture after isolation with the Pierce Neuron Kit. Top: Phase-contrast images of mouse cortical cultures after 1, 14 and 28 days. Bottom: GFP transfected cultured neurons at Day 7 (left) and Immunostained culture neurons at Day 28 (right) with postsynaptic density protein 95 (PSD95, green), and synaptophysin (red). The inset image indicates positive staining of synaptophysin and PSD95 at synaptic terminals, indicative of differentiated and functional neurons.
Cell viability and purity were also evaluated after one and seven days in culture. To determine cell viability in cultured neurons, propidium iodide (PI, red), a nuclear fluorescent dye that can be excluded from viable cells, was used to reveal dead cells in cultures (Figure 4A). Despite the presence of some PI-labeled cells at Day 1 in cultures prepared with both our method and a trypsin-based method, the ratio of PI-labeled cells to total cells was 25% in neuron cultures prepared by trypsin-based DIY method, much higher than the ratio obtained in cultures prepared by our method (Figure 4B). To study the purity of the neuron cultures, cells were immunostained with an antibody specific to neuronal marker protein microtubule-associated protein 2 (MAP2, green) and glial cell marker glial fibrillary acidic protein (GFAP, red) (Figure 4D). Neuron purity was calculated as the ratio of total GFAP negative cells to total cells indicated by nuclear staining. At Day 1, about 90% purity was observed in cultures prepared by our method, while cultures prepared by the trypsin-based method were only 80% pure (Figure 4E). Since isolated neurons from both methods are grown in the same neuronal culture media and supplements, at Day 7 we show that the cell viability and cell purity in both cultures were comparable (Figure 4C and 4F).
A. Day 1 Images
B,C. Cell Viability
D. Day 7 Images
E,F. Cell Purity
Figure 4. Neuron viability and purity over time after isolation with the Pierce Neuron Kit. A. Representative images of cortical neurons at Day 1 in culture. Neurons were immunostained with an antibody specific to MAP2; dead cells were visualized using propidium iodide (PI; red). Nuclei were visualized using Hoechst 33342 (Blue). Images were taken at 20X magnification using a Carl Zeiss™ microscope (AxioVision™ Rel. 4.7). B and C. Cell viability at Day 1 and Day 7 was calculated as the ratio of total PI negative cells to total cells indicated by nuclear staining. A total of 200 cells were analyzed from two independent experiments. Data represent the mean ± SD. D. Representative images of cortical neurons at Day 7 in a medium density culture. Neurons were immunostained using antibodies specific to MAP2 (green) and GFAP (red); Nuclei were visualized using Hoechst 33342 (Blue). Images were taken at 20X magnification using a Carl Zeiss microscope (AxioVision Rel. 4.7). E and F. Cell purity at Day 1 and Day 7 was calculated as the ratio of total GFAP negative cells to total cells indicated by nuclear staining. A total of 200 cells were analyzed from two independent experiments. Data represent the mean ± SD.
Neuronal morphology is an important indicator of neuronal function. It is thought that neurons with more complex and enriched dendrite branches better integrate synaptic inputs and communicate as networks [5,6,7]. To characterize neuronal morphology in culture, Sholl analysis was applied to assess neuronal dendrite complexity. Neurons at Day 7 were transfected with GFP to label individual neurons. During the course of 21 days in culture, neurons from both preparation methods exhibited intricately branched dendritic arbors studded with protrusions that included mature spines and few filopodia (Figure 5A). However, a Sholl analysis showed the presence of more intricately branched dendritic arbors in neuronal cultures prepared by our method than those neurons isolated by the trypsin DIY method.
A. Phalloidin Labeling, Day 21
B,C. Sholl Analysis
Figure 5. Dendritic complexity measurements of primary neuron cultures. A. Representative images of dendritic segments labeled with Phalloidin at Day 21 in culture. B. Representative illustration of an application of Sholl analysis to a GFP labeled neuron. C. Sholl analysis of total number of dendritic intersection crossing 70-80µm radius ring from cell body (n=20 neurons).
Another indication of neuronal function is the degree of observed synaptic scaling, an indication of neuronal homeostatic synaptic plasticity. The degree of synaptic scaling was visualized by immunofluorescent staining with antibodies against synaptic marker proteins. Bright and densely-spaced immunoreactive puncta corresponding to the excitatory N-Methyl-D-aspartic acid receptor I (NR1), pre-synaptic vesicle protein synaptophysin, and post-synaptic density protein 95 (PSD95) were greater in neuronal cultures prepared with our new method compared to those cultures prepared using a trypsin method (Figure 6A). The stronger immunoreactivity of NR1, synaptophysin, and PSD95 observed in neurons prepared by our method implies a higher protein expression level in those neuronal cultures. To quantitatively measure synaptic protein expression, synaptosomes were isolated from Day 15 cultured neurons using Syn-PER Synaptic Protein Extraction Reagent (Part No. 87793). The total protein yield in synaptosome suspensions prepared from our neuronal cultures is about 33% higher compared to samples from neuronal cultures prepared by literature methods (Figure 6B), confirming a greater extent of synapse formation and synaptic protein expression in our culture.
A. Cortical Neurons, Day 22
B. Synaptic Protein Yield, Day 15
Figure 6. Synaptic scaling in cultured neurons. A. Representative images of cultured neurons immunostained for the synaptic proteins NR1and synpatophysin (top), as well as PSD95 (bottom) in Day 22 primary cultured cortical neurons. More intricately branched dendritic arbors and higher intensity immunoreactive puncta for synaptic proteins, NR1, synaptophysin, and PSD95 are present in neurons isolated and cultured using the Pierce Primary Neuron Isolation Kit compared to those isolated using a do-it-yourself (DIY) trypsin method. Images were taken at 20X magnification using a Carl Zeiss microscope (AxioVision Rel. 4.7). B. A higher synaptic protein yield is obtained from cultures prepared using our method. Synaptic protein extracts were prepared from Day 15 cortical neurons using Syn-PER Reagent (Part No. 87793). The protein yield in synaptosome suspensions was quantified using the Pierce BCA Protein Assay (Part No. 23225).
To show the functional utility of our cultured primary neurons, we investigated an excitotoxic response at the molecular level using the NMDA receptor agonist N-Methyl-D-aspartic acid (NMDA). NMDA-induced over-activation of the NMDA receptor causes the cysteine protease calpain to cleave calcineurin A, a Ca2+/Calmodulin (CaM)-dependent phosphatase, into a truncated but constitutively active form which plays an important role in excitotoxic neurodegeneration . Consistent with the literature reports, the calpain-mediated truncated CaNA clearly appeared in both cultured neurons prepared by our method and the trypsin-based method following stimulation by NMDA. These truncated CaNA products are mediated by calpain activation as ALLM, a calpain inhibitor, significantly reduced the truncated Western blot signals. These results indicate that the neuron cultures isolated and cultured using our method can be used as a powerful model system to study cellular mechanisms in neurobiology and neuropathology.
Figure 7. Synaptic protein changes in response to an NMDA-induced neuroexcitotoxic model. Cultured neurons at Day 15 were incubated with 500μM NMDA for 2 hours. Where indicated, the calpain inhibitor II (ALLM, 25μM) was added 30 minutes before NMDA stimulation. Neurons were collected 2 hours after NMDA stimulation, and samples were subjected to Western blot analysis to detect changes in CaNA. β-Actin was used as a loading control.
We have provided a standardized, reproducible, and easily adaptable method for the isolation and culture of primary neurons from embryonic mouse/rat cortex and hippocampus. Neurons isolated and cultured using our method (i.e., the Pierce Primary Neuron Isolation Kit) are appropriately polarized, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form numerous, functional synaptic connections. They can be used as a model system for molecular and cellular biology studies of neuronal development and function, especially for visualizing the subcellular localization of endogenous or expressed proteins and protein trafficking.
Primary cortical neuron isolation:
Freshly micro-dissected whole mouse cortexes at embryonic-day 17-19 (E17-19) were incubated with Pierce Neuronal Isolation Enzyme (with papain)(Part No. 88285) for 30 minutes and washed twice with Hanks Balanced Salt Solution (HBSS). The tissue was disrupted in serum-supplemented neuronal culture medium by pipetting up and down 20 times with a pipette fitted with a 1000µL tip to generate a single cell suspension. The trypsin-based procedure was identical, except that trypsin was used instead of papain . Total cell yield was determined using an Invitrogen™ Countess™ Automated Cell Counter (Life Technologies, Inc.) and cell viability was determined by trypan blue exclusion assay.
Immunostaining of primary cortical neurons:
Neurons at the indicated days in culture were fixed with 4% paraformaldehyde, permeablilized with 0.1% Triton™ X-100 in HBSS for 10 minutes at room temperature, and blocked with 3% BSA in HBSS for 30 minutes at room temperature. Cells were then labeled with primary antibodies overnight at 4°C and subsequently with the corresponding secondary antibodies at room temperature for 1 hour, then washed twice with HBSS.
Synpatic protein extraction from primary neurons:
Synaptic protein lysates were prepared from Day 15 cultured neurons using Syn-Per Synaptic Protein Extraction Reagent (Part No. 87793) per the instructions. Protein concentrations were determined using Pierce BCA Protein Assay Kit (Part No. 23225).
Equal quantities of total protein (10-20μg/lane) were resolved on denaturing 2-10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 3% bovine serum albumin and incubated with primary antibody overnight at 4°C. Blots were incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature and then washed. Bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Part No. 34080) and exposed to film.
Time-pregnant CD-1 mice were obtained from Charles River Laboratories and housed in the University of Illinois College of Medicine at Rockford animal facility. Experiments were performed exactly as approved by the Animal Care and Use Committee at the University of Illinois College Of Medicine in Rockford, IL, and conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
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