Cancer Research-Why 3-D Cultures Work

The concept of bringing your cancer cell assay to life with 3-D nanofibers is new. New approaches beg the question, "why change?". Traditional 2-D cultures are the standard and work well enough for many assays.

Change is driven by proof that the new solutions yield better results. I would like to share data showing the potential capabilities and benefits of switching to random and aligned nanofibers.

Increased drug sensitivity

Increased drug sensitivity of human A549 lung cancer cells when grown on random nanofibers or aligned nanofibers when compared to flat tissue culture plastic (TCP). This shows the significant problem of developing drugs using 2-D surfaces and explains why such a large amount of animal testing is required for pre-clinical drug development. Proliferation on 2-D TCPS is artificially high when compared to 3-D culture on nanofibers. Using a high-throughput 3-D nanofiber-based scaffold for in vitro drug screening can more accurately predict the in vivo response of drugs.

The ability to do high-resolution imaging through the nanofiber scaffold (through the bottom of the culture plate) is critical to validate cell/phenotype markers especially in high throughput screening and high content analysis.  Standard microscopes and automated plate readers using light, fluorescence, absorbance, or luminescence are compatible with the nanofiber plates.  Comparison of A549 cells on flat tissue culture polystyrene (A), randomly oriented nanofibers (B), and aligned nanofibers (C).

Cancer Cell Migration-More Like in vivo

Scanning electron microscope images of an ex-vivo human glioblastoma tumor sample cultured on aligned nanofibers showing the tumor dispersion along the nanofibers exactly how they would migrate in vivo along the white matter within the brain and central nervous system.

I will continue to post new developments with our 3-D culturing products. It is our goal to bring your cell based assays to life!

Neuron-specific class III beta-tubulin (TuJ1) Staining in hNT2.19 Neurons

Our Tuj-1 antibody is widely used and frequently published. It is proving a potent marker for confirming the differentiation of human neural progenitors to neurons.

In this study the authors use the marker for staining hNT2.19 Neurons: Mary J. Eaton, Yerko Berrocal, and Stacey Q. Wolfe. Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines. Pain Research and Treatment. Volume 2012 (2012), Article ID 356412, 31 pages. doi:10.1155/2012/356412. 
The results show great promise. They show hNT2 or hNT2-derived cell lines, such as hNT2.17 and hNT2.19, have great potential to permanently reverse symptoms of neuropathic pain following PNS and CNS injuries and can offer new hope to treat these intractable conditions to significantly improve human health. This includes neuropathic pain resulting from diabetic neuropathy and Spinal Cord Injury.

Images: Comparison of graft sites of hNT2.17 and hNT2.19 in the QUIS and severe contusive-SCI models, respectively, at 6 weeks after cell transplant. (a) Sagittal section of anti-GABA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for GABA (arrows) on the pial membranes. (b) Sagittal section of anti-NuMA-immunostained QUIS + hNT2.17 transplant lumbar spinal cord 6 weeks after grafting. Easily detectible hNT2.17 cells stain for NuMA (arrows) on the pial membranes in adjacent sections. The hNT2.19 were alternately injected into the subarachnoid space two weeks after severe contusive SCI. Cell graft sites were co-localized with 5HT (c) and the human-specific marker TuJ1(d) (neuron-specific class III β-tubulin). There are many surviving hNT2.19 (d) grafted cells visible on the pial surface, which stain for TuJ1 (arrows) at the end of the experiment, 56 days after SCI and about 6 weeks after cell transplant. Adjacent sections with the same grafted hNT2.19 (c) are labeled for 5HT (arrows). Magnification bar = 20 μm.

Protocol: Modified methods for staining spinal cord sections for the human neuron-specific class III beta-tubulin (TuJ1) to identify grafted hNT2.19 neurons after grafting have previously been described [51]. The sections were washed with 0.1 M PBS pH 7.4 and permeabilized with 0.4% Triton-X-100 in 0.1 M PBS, 10% normal goat serum (NGS) for one hour. The sections were then incubated overnight at 4°C in the primary anti-TuJ1 antibody (1 : 100 DPBS), and the permeabilizing solution, followed by a one-hour incubation at room temperature with the secondary antibody solution, biotinylated mouse IgG raised in goat (Vector; 1 : 200), a Peroxidase ABC reporter in 0.1 M PBS (Vector) and “VIP” substrate (Vector). Some sections were stained in the absence of primary antibody and served as the negative controls.

I will continue to post new applications for our stem cell research reagents.

Neuromics' Cortical Neurons & Kinetic NeuroTrack Assays

Providing tools that insure excellent Cell Based Assays is a cornerstone of our business strategy. Lauren McGillicuddy and her team at Essen Bioscience have been using our E18 Primary Rat Cortical Neurons to develop NeuroTrak^TM assays enabling kinetic quantification of neurite dynamics (initiation, branching, extension, retraction). NeuroTrack is one of several CellPlayer^TM assays that can be run in IncuCyte Zoom^TM.

The proof is in the results and these show the both the potency of the cells and the powerful capablities of the IncuCyte ZOOM hardware and software:

Images:  Neurite outgrowth of rat E18 cortical neurons in a 96-well microplate at 24, 48 and 120 hours. top: 20x HD phase image of primary neurons  bottom: Image segmentation of neurites (light blue) and cell body cluster (raspberry).

Images: Neurite outgrowth of rat E18 cortical neurons in a 96-well microplate. Left: 20x HD phase image of primary neurons, 96 hours post plating ; Middle: Image segmentation of neurites (yellow) and cell body cluster (raspberry); Right: Concentration and time dependent inhibition of neurite outgrowth with the protein kinase C inhibitor, Ro-31-8220 (mean ± SD; n=6 per condition).

Check out this cool  assay animation!

I will continue to keep you posted on new assays and related methods using our primary neuron and astroglial cells.

Focus on Vision Systems Research

New Reagents, Publications and Data

We are intensifying our focus on Vision System Research with the addition of new antibodies. We are offering 50 USD off. They include: PSD-95/SAP90, Rhodopsin (A531), Rhodopsin (B630) and select Calcium (Ca2+) Signaling-Binding antibodies.

Image: Rhodopsin staining of pig retinal sections (green) and counter-stained with NF-M (red) and DNA (blue). Rhodopsin is most abundant in the outer segments of retina (OS), NF-M is abundant in the optic nerve fiber layer (ONFL), but seen in processes and neurons in other regions also. Other layers are pigmented epithelium (PE), outer and inner nuclear layers (ONL, INL), outer and inner plexiform layers (OPL, IPL) and ganglion cell layer (GCL). Inset: Bovine retinal extracts blotted with rhodopsin. Protocols on data-sheet.
Our ability to effectively serve researchers is confirmed by the growing parade of publications. I would like to highlight a recent publication by Dr. Sal Salvatore and his colleagues. It features use of our Shank 1a antibody. They are the first to show Shank 1 expression in the mammalian retina revealing Shank 1 immunoreactivity within both synaptic layers of the retina: Salvatore L. Stella Jr, Alejandro Vila, Albert Y. Hung, Michael E. Rome, Uyenchi Huynh, Morgan Sheng, Hans-Juergen Kreienkamp, Nicholas C. Brecha. Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina. PLoS ONE 7(9): e43463. doi:10.1371/journal.pone.0043463.

Images: Shank 1A immunoreactivity is in both the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the mouse YFP-16 line retina. A–C: A. Image of a retinal section immunostained for Shank 1A. B. Mouse YFP-16 line vertical retinal section. C. Shank 1A (red) immunolabeling and YFP (yellow). Shank1A expression is restricted to the OPL and IPL. A regular pattern of Shank 1A immunolabeling appears in the OPL, which is indicative of cone photoreceptor terminals. D–E: High magnification zoom of the OPL demonstrates that Shank 1A puncta (red) are distal to the dendrite tips (yellow) of YFP labeled cone bipolar cells, suggesting that Shank 1A is expressed presynaptic to the YFP cone bipolar cell dendrite. G–L: High magnification zoom of the IPL demonstrates that Shank 1A puncta are likely expressed postsynaptically to bipolar cell terminals. G. Shank 1A immunoreactive puncta. H. YFP labeled neurons and processes within the IPL region. I. PKCα labeled rod bipolar cell axons and terminals. J. Combined Shank 1A (red) and PKCα (blue) immunolabeling illustrate that shank 1A puncta are postsynaptic to rod bipolar cell terminals in the IPL. K. Combined Shank 1A (red) immunolabeling and YFP (yellow) in the IPL demonstrate that Shank 1A puncta are postsynaptic to cone bipolar cell terminals in the IPL. L. Combined triple fluorescent image of Shank 1A (red), PKCα (blue), and YFP (yellow) in the IPL. OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer. Scale bars = 10 µm. doi:10.1371/journal.pone.0043463.g001

More Pubs
Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018

...a goat polyclonal anti–platelet-derived growth factor receptor-α (PDGFR-α) antibody (1:100; Neuromics, Edina, MN)
Raoul Torero Ibad, Jinguen Rheey, Sarah Mrejen, Valérie Forster, Serge Picaud, Alain Prochiantz, and Kenneth L. Moya. Otx2 Promotes the Survival of Damaged Adult Retinal Ganglion Cells and Protects against Excitotoxic Loss of Visual Acuity In Vivo. The Journal of Neuroscience, 6 April 2011, 31(14): 5495-5503; doi: 10.1523/​JNEUROSCI.0187-11.2011...For antibody neutralization experiments, anti-Otx2 antibody (Neuromics) was dialyzed against PBS, and then Otx2 (25 ng in 500 mul) was incubated with anti-Otx2 (0.5 mug) in culture medium at 37C...

Hoon Shim, Chih-Ting Wang, Yen-Lin Chen, Viet Q. Chau, Kevin G. Fu, Jianqi Yang, A. Rory McQuiston, Rory A. Fisher, and Ching-Kang Chen. Defective Retinal Depolarizing Bipolar Cells (DBCs) in Regulators of G-protein Signaling (RGS) 7 and 11 Double Null Mice. JBC Papers in Press. Published on February 27, 2012 as Manuscript M112.345751. The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.345751...Animals were sacrificed by CO2 inhalation and the eyeballs were immediately enucleated. After removal of cornea and lens the resulting eyecups were immersionfixed in 4% paraformaldehyde in 1X PBS at room temperature for 15 minutes. This short fixation time ensured good mGluR6 and Gb5 signals at the OPL. After cryoprotection in 30% sucrose in 1X PBS, the eyecups were embedded in TBS (Richard Allan Scientific, Kalamazoo, MI), sectioned at 20μm thickness, and stained...

I will keep you posted on progress.

ASIC3 and Osteoarthritis

ASIC3 modulates pain and disease progression

Neuromics' foundation is built on providing reagents for pain researchers. I have posted the twists and turns via key publications and related data. Here's yet another success story with one of our Pain and Inflammation Research Antibodies.

Acid sensing ion channels (ASICs) are sodium-selective ion channels activated by low extracellular pH, and belong to the degenerin/epithelial Na+ channel superfamily. ASIC3  is the most sensitive to such a pH change [2,3], abundantly expressed in dorsal root ganglia (DRG) [4], and strongly correlated with pain. Here researchers show the role of ASIC3 in osteoarthritis: Masashi Izumi, Masahiko Ikeuchi, Qinghui Ji, Toshikazu Tani. Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis. Journal of Biomedical Science 2012, 19:77 doi:10.1186/1423-0127-19-77.
Highlights: OA was induced via intra-articular mono-iodoacetate (MIA) injection, and pain related behaviors were evaluated including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test. OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Images: Fast Blue labeling and immunohistochemistry staining for ASIC3 : (a-b) Naïve- model, (c-d) OA-model, (e-f) APETx2 administration to OA-model in early phase. Photos in each row are the same DRG. In (b),(d),(f), large arrows indicate Fast Blue labeled, ASIC3 immunoreactive (ASIC3-ir) DRG cells, while ASIC3-ir cells that were not labeled by Fast Blue are indicated by small arrowheads. More than 100 FB-labeled neurons were analyzed from 4 rats in each group. The percentage of ASIC3-ir knee joint afferents was 18 ± 3% (mean ± SD) in naïve models, 46 ± 4% in OA-models (p = 0.003), and 20 ± 5% in the early-phase APETx2 group (p = 0.006), respectively. Scale bar: 50 μm

Protocol: The [DRG] sections were blocked in 3% normal goat serum for 1 h, then incubated in primary antibody of ASIC3 (Neuromics; Edina, MN, GP 14015, 1:500) overnight in a humid chamber. The next day, the sections were incubated in the secondary antibody (Vector; Burlingame, CA, FI-7000, 1:500, FITC tagged) for 2 h. All antisera used were diluted in PBS containing 1% normal goat serum and 0.05% Triton X-100. Before, between, and after each incubation step, the sections were washed 3 times for 5 min in PBS. Finally, all sections were mounted with Vectashield (Vector, Burlingame, CA).
1. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 1997, 386:173–177. 2. Lingueglia E: Acid-sensing ion channels in sensory perception. J Biol Chem 2007, 282:17325–17329. note: see http://neuromics.net/weblog/post/tag/dr-eric-lingueglia/ for research using our siRNA transfectio reagent for ASIC3 gene expression analysis. 
3. Wemmie JA, Price MP, Welsh MJ: Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci 2006, 29:578–586.
4. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 2001, 21:8026–8033.

hMSCs+Petaka=Excellent SC Cultures

I have been promoting the capabilities of our new Petaka^TM Cell Culturing Systems. My confidence that you will be delighted with these capabilities is based, in part, on the success Dr. Jim Musick, CEO, Vitro Biopharma had culturing our UCB derived Human Mesenchymal Stem Cells.

These cells work "hand in glove" with Petaka. The system enabled Jim to:

• Culture the cells with out the addition of CO₂ or humidity (no environmental dehydration)-no incubator required.
• Use less growth media
• Drive the cells into dormancy enabling them to be maintaned, shipped and stored at Room Temperature.

Images: hMSCs in Petaka and cell dormancy and recovery.

Additional Resources:

Petaka Presentations-Petaka features and capabilities. Includes: culturing stem cells, other primary cells and cell lines; customizing phsiological conditions to match cells' in vivo environment and cell culture pausing for room temperature handling and shipping.

Petaka Cell Culturing Video Protocols
Petaka Cell Harvesting Video Protocols
Petaka Cell Maintenance, Shipping and Recovery Video Protocols

Petaka means better cultures at a lower cost. I will continue to post new data confirming this here.

TRPV1, SCI and Autonomic Dysreflexia

Acute autonomic dysreflexia (AD) is a reaction of the autonomic (involuntary) nervous system to overstimulation. It is characterised by severe paroxysmal hypertension (episodic high blood pressure) associated with throbbing headaches, profuse sweating, nasal stuffiness, flushing of the skin above the level of the lesion, bradycardia, apprehension and anxiety, which is sometimes accompanied by cognitive impairment.  

My friend Matt Ramer and his team at University of British Columbia use our antibodies as markers for sensory neurons. In this important study they use our TRPV1 (Neuromics, Edina, MN, USA; 1:2,000)...Substance P (Neuromics, 1:1,000) and   β-III-tubulin (Neuromics; 1:500) to label neuronal profiles in the DRG: Leanne M. Ramer, A. Peter van Stolk, Jessica A. Inskip, Matt S. Ramer and Andrei V. Krassioukov. Plasticity of TRPV1-expressing sensory neurons mediating autonomic dysreflexia following spinal cord injury. DOI=10.3389/fphys.2012.00257.
Highlights: Spinal cord injury (SCI) triggers profound changes in visceral and somatic targets of sensory neurons below the level of injury. Despite this, little is known about the influence of injury to the spinal cord on sensory ganglia. One of the defining characteristics of sensory neurons is the size of their cell body: for example, nociceptors are smaller in size than mechanoreceptors or proprioceptors. In these experiments, we first used a comprehensive immunohistochemical approach to characterize the size distribution of sensory neurons after high- and low-thoracic SCI. Male Wistar rats (300 g) received a spinal cord transection (T3 or T10) or sham-injury. At 30 days post-injury, dorsal root ganglia (DRGs) and spinal cords were harvested and analyzed immunohistochemically. In a wide survey of primary afferents, only those expressing the capsaicin receptor (TRPV1) exhibited somal hypertrophy after T3 SCI. Hypertrophy only occurred caudal to SCI and was pronounced in ganglia far distal to SCI (i.e., in L4-S1 DRGs). Injury-induced hypertrophy was accompanied by a small expansion of central territory in the lumbar spinal dorsal horn and by evidence of TRPV1 upregulation. Importantly, hypertrophy of TRPV1-positive neurons was modest after T10 SCI. Given the specific effects of T3 SCI on TRPV1-positive afferents, we hypothesized that these afferents contribute to autonomic dysreflexia (AD). Rats with T3 SCI received vehicle or capsaicin via intrathecal injection at 2 or 28 days post-SCI; at 30 days, AD was assessed by recording intra-arterial blood pressure during colo-rectal distension (CRD). In both groups of capsaicin-treated animals, the severity of AD was dramatically reduced.

Images: High-thoracic (T3) spinal cord injury had no effect on medium-to-large sized neurons in the L4/L5 DRG expressing heavy neurofilament (NF200). (A) NF200-positive neurons did not undergo SCI-induced hypertrophy, nor did the proportion of neurons expressing NF200 change. (B) Hypertrophy of TRPV1-expressing DRG neurons was not accompanied by increased co-localization of TRPV1 and NF200. Ganglia were harvested 3 months after sham-injury (gray) or complete T3 SCI (black). Arrow: DRG neuron immunopositive for both TRPV1 and NF200. Scale bar = 70 μm.

Conclusion: Previous work has identified numerous mechanisms that might contribute to induction and progression of AD, and the list of putative mechanisms includes injury-induced changes in the vasculature and multiple components of the spinal sensory-sympathetic circuitry caudal to SCI (Krenz and Weaver, 1998; Krassioukov et al., 1999; Krenz et al., 1999; Brock et al., 2006; McLachlan and Brock, 2006). In terms of sensory plasticity, prior findings demonstrate that severity of AD is closely correlated to the extent of intraspinal nociceptor sprouting (Cameron et al., 2006). However, this is the first study to demonstrate AD mediated by a specific subset of afferents that exhibit pronounced somatic, but only slight central, injury-induced plasticity. Given the array of pronounced changes in peripheral targets of sensory neurons after SCI, it is not surprising that they respond to injury. Plasticity occurring outside the CNS may represent a new and more accessible target for limiting sensory-autonomic dysfunction following SCI.

I will keep you posted as more progress is made towards finding therapeutic targets AD.