METHOD OF TREATING CENTRAL NERVOUS SYSTEM DISEASE

 Blood-Brain Barrier (BBB) Permeability Assay

Yes, These are troubled times. With our current focus on eradicating COVID-19, we forget that many of the world's most insidious and costly diseases have limited treatments and no cures.

That is why Neuromics continues to develop its human based 3-D Cell-Based Assays. At the center if our offerings is our BBB Model.

Here's a recent patent application that includes a permeability assay using our model-Sookhee Bang, Jeong Kuen Song, Seung-wook Shin, Kwan Hee Lee, and Ho June Lee. (2020). Method of Treating Central Nervous System Disease. United States Patent Application 20200230218

BBB Permeability Assay of AL04 The in vitro human BBB model (Neuromics, USA) was established using co-cultures of primary Human brain endothelial cells (HBEC), Human brain pericytes (HBPC), and Human brain astrocytes (HBAC). In vitro human BBB model kit has two sides (luminal, blood/abluminal, brain) with 12 transwell inserts (polyester membrane, 0.4 um pore, diameter 12 mm, insert growth area: 1.12 cm2). HBPC were grown on the bottom side of the inserts, HBEC were monolayered on the upper side of the inserts, and HBAC were grown on the bottom of the 12-well culture plate (Neuromics, USA). In vitro BBB model was activated according to the manufacturer's instructions for 4 days. Briefly, the medium from both luminal and abluminal (lower, brain) sides of the transwell insert was changed every other day. Before the transport experiment, the abluminal side was filled with the permeability assay medium. For the permeability assay, purified AL04 or recombinant HSA (Sigma, USA) were added to the luminal side (0.3 ml) of the transwell insert to yield a final concentration of 1 or 10 uM. Incubations were performed on orbital shaker (100 rpm) at 37° C. Samples (150 μl) were collected from the abluminal side (1.2 ml) at 60, 120 and 240 min and immediately replaced with fresh permeability assay medium. The concentrations of transported AL04 or rHSA were measured by Human albumin quantitation ELISA kit (Bethyl laboratory, USA) and analyzed using the standard curve method. Permeability coefficients (Pe, cm s−1) were calculated using the equation: Pe=(VA/(A×c0))×(dQ/dt), where VA is volume of assay buffer in blood side (inside of the insert), A is the surface area of the insert (1.12 cm2), c0 the initial concentration of protein sample added into blood side, dQ/dt the concentration of transported protein sample in brain side in a defined time period. The permeability coefficients (Pe, cm s−1) for the purified AL04 or recombinant HSA were calculated as previously described (Prades, 2015; Nakagawa, 2009).

If you have interest and want to learn more, please email Rose Ludescher, VP, Customer Satisfaction-rose@neuromics.com

Be safe; be healthy.

New 3-D Eye Model

More in-vivo like Model
We see our world in 3-D. Diseases of the eye compromise this ability.

Neuromics' is pleased to announce that we have a 3-D model aimed at accelerating drug discovery for these diseases. Sight is a terrible thing to lose and the faster new drugs can be discovered, fewer people will have to suffer the loss of sight.

Our 3D Human Retinal Microvascular Angiogenesis model is constructed using GFP‐Tagged human Retinal Microvascular Endothelial cells. They are co-cultured with RFP-Tagged human supporting cells. GFP positive human retinal capillary-like tubule formation can be monitored in real time under fluorescence microscope throughout the whole process of the experiment.

HUVECS in 3-D Action

Form Vascular Networks in Microfluidics Model
In this study, the authors developed a 3D functional human microvascular network in a microfluidic device. The established model enables Neuromics GFP-labeled human umbilical vein endothelial cells to form vessel-like microtissues and have physiological functions which are closer to cells in human blood vessels. The perfusable microvasculature allows the delivery of nutrients, and oxygen, as well as flow-induced mechanical stimuli into the luminal space of the endothelium. The microflow effectively mimic the blood flow in human vessels.

This in-vivo like model is then used for toxicity assays-Yan Li, Qing-Meng Pi, Peng-Cheng Wang, Lie-Ju Liu, Zheng-Gang Han,Yang Shao, Ying Zhai, Zheng-Yu Zuo, Zhi-Yong Gong, Xu Yang and Yang Wu. Functional human 3D microvascular networks on a chip to study the procoagulant effects of ambient fine particulate matter. : RSC Adv., 2017, 7, 56108

Images: Microvascular network formation based on microfluidic 3D HUVEC culture. (A) Schematic diagram of a microfluidic device. (B) Schematic diagram of microvascular network formation based on microfluidic 3D HUVEC culture. (C) Schematic diagram of loading microparticles in microvascular networks. (D) Microscope image of HUVECs seeding in fibrin hydrogel. (E) Confocal microscope image of fluorescent microvascular networks.

Our human primary and stem cells are widely used and frequently published. We will continue to post relevant results from researchers using the cells here.

3D Printing the Way to Artificial Muscles

Muscles Contract Under the Control Motor Neurons

The methods used to develop the artificial muscles included use of our GDNF Protein to maintain motor neurons in the 3-D Culture-Caroline Cvetkovic, Max H. Rich, Ritu Raman, Hyunjoon Kong , Rashid Bashir. A 3D-printed platform for modular neuromuscular motor units. Microsystems; Nanoengineering 3, Article number: 17015 (2017) doi:10.1038/micronano.2017.15

Skeletal muscle cells and motor neurons were combined into a fabricated 3D co-culture system. C2C12 myoblasts were differentiated into multinucleated myotubes (a) and combined with extracellular matrix (ECM) proteins to create an engineered muscle ring tissue (b). In parallel, mouse embryonic stem cells (HBG3 mESCs) were differentiated into motor neurons (MNs) through the formation of embryoid bodies (EBs) (c and d) and then combined with the engineered muscle tissue and ECM proteins (e) on 3D-printed hydrogel devices (f and g). Once the multi-layered rings sequentially compacted and fused together, they were then placed on a stationary hydrogel skeleton (h). Scale bars, 50 μm (b and d), 500 μm (c), and 10 μm (d, inset).

Immuno-fluorescence in 3-D

Potent Markers!

We are seeing more 3-D Cell Based Assays being used for Neuroscience Research. It is important researchers have the Neuron-Astroglial-Progenitor Markers required for staining in 3-D. This helps researchers determine the cell types present in their assays.

I have posted here published results. I would like to share the latest: Yiting Liu, Katherine S. Given, Danielle E. Harlow, Adeline M. Matschulat, Wendy B. Macklin, Jeffrey L. Bennett and Gregory P. Owens. Myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination. Acta Neuropathologica Communications Neuroscience of Disease 20175:25
DOI: 10.1186/s40478-017-0428-6

3D movie reconstructed by super-resolution structured illumination microscopy (SIM) imaging of live organotypic mouse cerebellar slices stained with MS#30 (red), then fixed and stained for MAG (blue) and NF-H (purple). MS#30 reactivity was on oligodendrocyte processes, including those contacting adjacent axons, and on myelinated MAG+ axons, outside of MAG layer. Scale bar: 2 μm. (MPG 90340 kb).

We will continue to post cool applications for our antibodies here.

Check Out Our 3D Human Skin Tissue Model

In vivo Like Model for Studying Skin Diseases/Irritants

I am pleased to announce this new addition to our Cell Based Assay offerings.

3D Human Skin Tissue Model

Cross section of Human Skin Model

The model includes precultured tissue in our engineered and tissue/cell specific Collagel-Hydrogels.

Our 3D human skin model is manufactured in a GMP lab. The model is engineered from multilayered, differentiated epidermal cells. The tissue is provided cultured in inserts using serum free media. The model mimics human skin. It provides an in vivo like environment for studying dermal related diseases and cancers. It is also excellent for skin toxicology and irritancy assays.

Check all our  3D Models:

Name	Applications	Size	Price
Human Blood Brain Barrier Model	In vivo Like Assays	6 wells 12 well 24 well	$1,780 $2,600 $3,625
3D HUVEC Model	In vivo Like Assays	6 well 12 well 24 Well	$1,269 $1,399 $1,539
3-D Human Skin Tissue Model	In vivo Like Assays	6 well 12 well 24 Well	$1,269 $1,399 $1,539

Questions? Contact me directly-Pete Shuster, CEO and Owner, 612-801-1007 or pshuster@neuromics.com.

Why You Should Consider Culturing Your Cells in 3-D

2-D versus 3-D and meaningful outcomes

We have seen recent growth in our customers' demand for 3-D cell based assay solutions. This could be due to more the fact that these assays our proving to generate more in vivo like data.

Breast cancer cells, for example, grown in 2-D can easily be killed by low doses of chemotherapeutic drugs or low doses of radiation. If those same cells are grown in 3-D, they are resistant to the same doses of chemotherapeutic drugs or radiation, just like cancer as its found in the body. In this way, cells grown in 3D are more valid targets for testing and discovering new drugs to treat cancer. Another benefit of testing drugs in three dimensional cell culture versus two dimensional cell culture is that cells in 3D form multi-layers of cells whereas cells grown in 2-D form a monolayer of cells that is spread very thin on a plastic surface. When testing a drug in 2-D, it needs only to diffuse a short distance across the cell membrane to reach its intended target. In 3-D, the situation is more realistic and a drug needs to diffuse across multi-layers of cells to reach the cells on the inside of a microtissue.

Neuromics has a variety of options that will enable you to choose the best option(s) for your 3-D assays. These include:

• Collagel Hydrogels-New-Formulated using Type 1 Bovine, Human or Rat Collagen
• 3-D Nanofiber Solutions-Aligned Scaffolds, Randomly Oriented Scaffolds and Interactive Microenvironment System (IMEMS). 
• AlphaMAX3D-Potent three dimensional environment for cell culturing 
• AlphaGEL3D-Novel synthetic peptide hydrogel 
• Extracellular Matrix (ECM) Proteins-ECM proteins for cell culturing

Look for more posting on these important solutions.

Staining Neurons and 3-D

Tuj-1 or Beta Tubulin Antibody in Action!

Our potent Neuron-Glia Markers are widely used and frequently published. We are also pleased with the many positive reviews.

In this study researchers use our Tuj-1/Beta Tubulin Antibody to stain neurons in 3-D Cultures: Gaoying Sun, Wenwen Liu, Zhaomin Fan, Daogong Zhang, Yuechen Han, Lei Xu1, Jieyu Qi, Shasha Zhang, Bradley T. Gao, Xiaohui Bai,Jianfeng Li,Renjie Chai, Haibo Wang. The three-dimensional culture system with matrigel and neurotrophic factors preserves the structure and function of spiral ganglion neuron in vitro...Tuj-1-β-tubulin (1:1000, Neuromics, USA)...

Images: Morphology of the SGNs growth cone cultured in 2D and 3D systems. Phalloidin, green; β-tubulin, red.

I will continue to post positive developments concerning use of our Neuron-Glia Markers.

Reconstructing 3D Living Biomaterials

Hydrogel Solutions for Tissue Reconstruction and Repair

Alphabioregen/Neuromics are pleased to announce the use of our Hydrogels for reconstructing living biomaterials for regenerative biology and medicine: Suwan N. Jayasinghe,Jensen Auguste, Chris J. Scotton. Platform Technologies for Directly Reconstructing 3D Living Biomaterials. DOI: 10.1002/adma.201503001.

In this study, researchers use our  Collagen Hydrogel, Collagen Hydrogel Soft and Collagen Hydrogel Soft+  to describe  in vivo applications using a murine model to interrogate biocompatibility and cellular behavior post-transfer.

Luc macrophages (CC or BES) were then incorporated into a fl uid mixture containing one of three alternative collagen-based hydrogels, namely, Collagen Hydrogel, Collagen Hydrogel Soft, or Collagen Hydrogel Soft+ (resulting in six possible combinations of macrophages and biopolymer).

Overview of Results

Figure: Bioluminescent imaging of implanted macrophage–biopolymer mixes. IC-21-Luc macrophages (CC or BES) were mixed with biopolymer (Hydrogel, Soft, or Soft+) and subcutaneously injected into the dorsal fl anks of C57Bl/6 mice (three mice per hydrogel, with CC on the left fl ank and BES on the right). Following intraperitoneal injection of delta-luciferin, macrophage bioluminescence was detected using an IVIS Lumina II imaging system. A representative image from day 1 post-implantation (and 25 min post luciferin injection) is shown, indicating the peak detectable radiance (photons s −1 cm −2) in identically sized regions of interest. The CE results were very similar to the BES implants.

Figure:  Histological analysis of the macrophage–biopolymer implantation site (MSB staining). At day 4 post-implantation, skin was harvested from each dorsal fl ank, and processed for histology. Where possible, serial sections directly adjacent to those shown in Figure 4 were stained with a modifi ed trichrome stain. Mature fi brillar collagen within the dermis appeared dark blue, while the collagen within the hydrogel was generally lighter blue in appearance, indicating a less highly cross-linked or fi brillar form of collagen. Scale bar: 200 µm.

These Collagen Hydrogels are proving easy to use and effective in the hands of our customers. If you are looking for solutions for tissue reconstruction/repair or 3-D in vivo like cell based assays, do not hesitate to contact me @ direct phone: 612-801-1007 or pshuster@neuromics.com. Thank you, Pete Shuster, CEO and Owner, Neuromics.

Neurons in 3-D

In Vivo Like Neurite Outgrowth Cultures

Culturing in 3-D requires potent Primary Neurons. Here researchers use our e18 Rat Cortical Neurons in developing their 3-D assays: Chandrasekhar R. Kothapallia, Peyman Honarmandib. Theoretical and experimental quantification of the role of diffusive chemogradients on neuritogenesis within 3D collagen scaffolds. Acta Biomaterialia. Available online 14 May 2014. http://dx.doi.org/10.1016/j.actbio.2014.05.009

Abstract: A critical challenge to regenerating close mimics of native axonal pathways under chronic neurodegenerative disease or injury conditions is the inability to stimulate, sustain and steer neurite outgrowth over a long distance, till they reach their intended targets. Understanding neurite outgrowth necessitates quantitative determination of the role of molecular gradients on growth cone navigation under dynamic physiological conditions. High-fidelity biomimetic platforms are needed to computationally and experimentally acquire and analyze spatio-temporal molecular gradient evolution and the growth cone response across multiple conditions along this gradient pathway. In this study, we utilized a simple microfluidic platform in which diffusive gradients were generated within a 3D porous scaffold in a defined and reproducible manner, and its characteristics (spatio-temporal gradient, steepness, diffusion time, etc.) precisely quantified at every specific location within the scaffold. Using this platform, we show that the cortical neurite response within 3D collagen scaffolds, at both the cellular and molecular level, is extremely sensitive to subtle changes in localized concentration and gradient steepness of IGF-1 within that region. This platform could also be used to study other biological processes such as morphogenesis and cancer metastasis, where chemogradients are expected to significantly regulate the outcomes. Results from this study might be of tremendous use in designing biomaterial scaffolds for neural tissue engineering, axonal pathway regeneration under injury or disease, and in formulating targeted drug delivery strategies.

Image: Neurons in 3-D Assay

Neuromics' provides many Stem and Primary Cell Assay Solutions including tools for 3-D Cultures. We also offer services studying the effects of small molecules and compounds on Stem Cell expansion, differentiation and migration. To learn more contact me at 612-801-1007 or pshuster@neuromics.com.

Extracellular Matrix Environment and Chemotherapeutics

Substrate Matters!

Neuromics' has been promoting a variety of 3-D Cell Based Assay Solutions for the past several years. These include: Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins. We have found that the adoption rate for these as standard tools for drug discovery is slower that we anticipated.

We believe substrate matters so I am pleased to share a recent publication that references use of our Collagen IV and other ECM proteins. This confirms the importance of using a more in vivo like environment in testing chemotherapeutics: Thuy V. Nguyen,Marianne Sleiman,Timothy Moriarty,William G. Herrick,Shelly R. Peyton. Sorafenib resistance and JNK signaling in carcinoma during extracellular matrix stiffening. Publication: Biomaterials. Elsevier. 13 April 2014. http://dx.doi.org/10.1016/j.biomaterials.2014.03.058.

Abstract: Tumor progression is coincident with mechanochemical changes in the extracellular matrix (ECM). We hypothesized that tumor stroma stiffening, alongside a shift in the ECM composition from a basement membrane-like microenvironment toward a dense network of collagen-rich fibers during tumorigenesis, confers resistance to otherwise powerful chemotherapeutics. To test this hypothesis, we created a high-throughput drug screening platform based on our poly(ethylene glycol)-phosphorylcholine (PEG-PC) hydrogel system, and customized it to capture the stiffness and integrin-binding profile of in vivo tumors. We report that the efficacy of a Raf kinase inhibitor, sorafenib, is reduced on stiff, collagen-rich microenvironments, independent of ROCK activity. Instead, sustained activation of JNK mediated this resistance, and combining a JNK inhibitor with sorafenib eliminated stiffness-mediated resistance in triple negative breast cancer cells. Surprisingly, neither ERK nor p38 appears to mediate sorafenib resistance, and instead, either ERK or p38 inhibition rescued sorafenib resistance during JNK inhibition, suggesting negative crosstalk between these signaling pathways on stiff, collagen-rich environments. Overall, we discovered that β1 integrin and its downstream effector JNK mediate sorafenib resistance during tumor stiffening. These results also highlight the need for more advanced cell culture platforms, such as our high-throughput PEG-PC system, with which to screen chemotherapeutics.

Figure: High-throughput biomaterial platform for drug screening. (A) The high-throughput platform consists of a black-walled, glass bottom plate, with PEG-PC gels cast in each of the inner 6x10 wells. (B) Gels can be functionalized with any protein or peptide of interest, and they support the adhesion and growth of carcinoma cells. We used this platform to test carcinoma cell response to a kinase inhibitor (sorafenib) as a function of underlying gel stiffness and ECM adhesive protein cocktail. (C) A representative graph of SkBr3 proliferation (y-axis) in response to sorafenib (x-axis) across a range of gel stiffness (colors) demonstrates the IC-50 calculation.

This confirms the importance of considering your substrate environment when developing your in vitro assays for High Content and High Throughput Drug Discovery. We will continue to provide updates.

From Zero to 3-D Cell Based Assays in 15 Minutes

Collagel Hydrogels are Designed to Match Your Cell Types.

We are pleased to add Collagel Hydrogels to our 3-D Cell Based Assay Solutions.

The are 3 different gel types that mimic the different in vivo extracellular environment that your cells experience. You will be able to get really nice adipogenesis with MSCs using our CollaGel Hydrogel Standard. CollaGel Hydrogel Soft is an ideal matrix for growing fibroblast; primary hepatocyte cultures and great for growing smooth muscle cells. CollaGel Hydrogel Soft+ is an ideal matrix for growing nervous cells, veins cells and Hydroxyapatite crystals.

Image: Primary hepatocyte culture in 3D model using our CollaGel Hydrogel. 

Using these gels, you can now form small tube-like structures in combination with Human Umbilical Vein Endothelial Cells (HUVEC) in a 3D model. Our CollaGel Hydrogel can also be used for 3D printing without worrying about the needles in your machine been broken due to the fast gelling process. Collagel Hyrogels can be used for: Stem Cell Behavior Studies Fibrosis Studies,  Cosmetic Toxicology, Hepatocyte Assays, Neuronal Branching, Wound Healing Assays, Cell Invasion Assays, Migration Assays, Cancer Cell Phenotyping and  Bioprinting.

I will be posting Collagel Hydrogel related data and images provided by our customers.