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.

Human Primary Cells Trifecta

Tri-culture of Neuromics' Human Primary Cells in 3-D BBB Model

I mentioned in an earlier post that we take Researchers questioning the validity of our Human Primary Cells very seriously. We follow up with our clients to make sure our cells are working as expected. If they are not 100% happy, we offer a free replacement or full refund.

...So it is encouraging to see Researchers using our Human Pericytes,  Brain Microvascular Endothelial Cells (HBMES) and Astrocytes to build a static 3-D Blood-Brain Barrier Model.

Ece Bayir, M. Mert Celtikoglu, Aylin Sendemira. The use of bacterial cellulose as a basement membrane improves the plausibility of the static in vitro blood-brain barrier model. https://doi.org/10.1016/j.ijbiomac.2018.12.257.

Image: Pericytes, HBMECS, and Astrocytes 5 days after culturing. Live cells (green) and dead cells (red).

The investigators concluded, "Caffeine and sucrose permeability values obtained from all models were close to literature data and physiological values."

Primary Human Brain Pericytes

Pure and Potent
The authors of a chapter on pericytes published in SpringerLink referred to our human primary pericytes as "pericyte-like", but chose not to characterize them because of the "exorbitant cost". Dore-Duffy P., Esen N. (2018) The Microvascular Pericyte: Approaches to Isolation, Characterization, and Cultivation. In: Birbrair A. (eds) Pericyte Biology - Novel Concepts. Advances in Experimental Medicine and Biology, vol 1109. Springer, Cham

Given that these pericytes are also part of our hot selling 3-D Human BBB Model, we are in the process of challenging these claims. We will always take inaccurate claims regarding our solutions seriously, and we take immediate action.

• Characterization of cells-we validated several key markers by immunofluorescence.

Staining of Desmin (dilution 1:100). Secondary antibody conjugated to Alexa 594 (red) and counterstained with DAPI (blue). Cells were mounted using iBrite mounting media.

Staining of Actin (dilution 25 ug/mL). Secondary antibody conjugated to Alexa 594 (red) and counterstained with DAPI (blue). Cells were mounted using iBrite mounting media.

We also plan on doing a phenotypic analysis of these cells and will post here when completed.

• Cost of cells-isolating pericytes from human donors, and expanding to the required number of cells involves much time and effort. We know! Further differentiating stem cells into pericytes is hard and time-consuming. Against this backdrop, we consider our pricing to be inexpensive. Though they are more difficult to derive, they are priced equivalently with our other human primary cells ($789/500,000 USD Cells).

As always, we will post new data here.

Human Brain Microvascular Endothelial Cell in Action

Recent Pubs
Our Human Brain Microvascular Endothelial Cells (HBMECs) are known for the performance in drug discovery and tox assays. These 21-CFR compliant cells are used in the "blood side" of our 3-D BBB Models.

1. Shavali Shaik, Bridget Kennis, Shinji Maegawa, Keri Schadler, Yang Yanwen, Keri Callegari, Rishi R. Lulla, Stewart Goldman, Javad Nazarian, Veena Rajaram, Jason Fangusaro, and Vidya Gopalakrishnan. (2018). REST upregulates gremlin to modulate diffuse intrinsic pontine glioma vasculature. Oncotarget. 2018 Jan 12; 9(4): 5233–5250. doi: 10.18632/oncotarget.23750 
2. Hu et al. (2016). Epigenetic Activation of WNT5A Drives Glioblastoma Stem Cell Differentiation and Invasive Growth. Cell. 167, 1281–1295. http://dx.doi.org/10.1016/j.cell.2016.10.039,
   

GREM-1 is required for tube formation in vitro (A) Q-RT-PCR analysis of GREM-1 gene expression in SU-DIPG-IV cells stably expressing either control shRNA or GREM-1-specific shRNA. Lentiviral constructs expressing two different GREM-1 shRNAs (GREM-1.1 and GREM-1.2) were used to knockdown GREM-1. Efficiency of GREM-1 knockdown was determined by Q-RT-PCR and expression was normalized to 18s RNA. Significance is as shown (**less than.01). (B-C) HUVEC or human brain microvascular endothelial cells (HBMEC) were cultured in endothelial cell medium and or conditioned medium from either control shRNA or shGREM-1.2 transfected SU-DIPG-IV cells. Tube formation in matrigel was measured after 16h and images were obtained. The ability of GREM-1 to rescue loss of tube formation upon REST knockdown was determined by addition of human-recombinant GREM-1 (rGREM-1) to conditioned media-endothelial media mix. Scale bars, 100μm. (C) Quantification of tubes in matrigel shown in Figure B (right panels). Data shown is mean +/- SD, ***p less than .001, n=3. (D) Western blot analysis to assess VEGFR2 levels in SU-DIPG-IV, -VI and –XIII cells, HUVECs and HBMECs was done using anti-VEGFR2 antibodies. Tubulin served as a loading control. (E) Western blot analysis was performed to assess AKT signaling downstream of GREM-1 interaction with its potential receptor VEGFR2 in HUVEC and HBMEC. Anti-pAKT (S473), anti-pAKT (T308), total AKT, and anti-actin were employed.

If these cells fit your assay requirements and need more data/info, do not hesitate to contact me, Pete Shuster, CEO at pshuster@neuromics.com or cell: 612-801-1007.

Neuromics' BBB Model and Permeability Assays

Crossing the Blood-Brain Barrier
Our BBB Model is being increasingly used by Bio-Pharma for drug permeability studies. Customers include Amgen, Genentech, Boehringer Ingelheim, and Merck.

These assays are key for making sure molecules/compounds of interest will cross the barrier into the brain and at what rate. This data, in part, add clarity on best candidates to move into in-vivo testing.

Human Blood Brain Barrier Model	3D45002	12 well
Human Blood Brain Barrier Model	3D45002	24 well
Human Blood Brain Barrier Model	3D45002	6 wells

I you have interest in using our models, Rose Ludescher, Manager of Customer Satisfaction, can provide information aligned with your assay requiremenst. rose@neuromics.com or 1-866-350-1500.

3-D BBB Penetration Assays

Making 2017 the Year of Tissue-Like, 3-D Models

There is growing interest in our 3-D BBB Model for testing penetration of compounds, small molecules, peptides and oligos through the BBB into our tissue like brain side.

 I would like to present related data:

We plan on adding a pancreatic model in January so stay tuned.

Neuromics Cornerstone

Human Primary + Stem Cells and 3-D Tissue Like Models

We are pleased by growing demand for our "hard to find" primary cells and 3-D BBB model. These are proving to be engines for drug discovery and toxicology assays.

We have have the capabilities isolate cells from healthy human donors. We then grow and characterize the cells and cryo-preserve in vials of 500,000 cells. They are now ready to culture.

We are increasingly determining types of cells to add to our growing catalog by meeting the specific demand of our clients. For example, our addition of primary human schwann cells and microglia happened, because they were needed for a specific client study.

Human Schwann Cells

Our 3-D Blood Brain Barrier Model is also working well in the hands of our clients. We are receiving positive input and this is supported by repeat orders. It is designed for users to test the ability of compounds, molecules and oligos to pass the BBB and penetrate into the brain.

Our cornerstone: hard to find human primary/stem cells + 3-D tissue like assays.

We can say, "Need cells or 3-D tissue like models?  Just ask us!". Pete Shuster, CEO and Owner-direct phone: 612-801-1007 or pshuster@neuromics.com

Human Astroglia and Schwann Cells-BBB Model

Broadening our Capabilities

At the core of our solutions are many options for primary human cells. We are especially pleased that we have growing capabilities to provide new cells to researchers studying autoimmune neuro-degenerative diseases like ALS and MS with the addition of:
Schwann Cells

Human Schwann Cells (HSwC) are isolated from human spinal nerve. HSwC are cryopreserved at passage one and delivered frozen. Each vial contains >5 x 10^5 cells in 1 ml volume. HSwC are characterized by immunofluorescence with antibodies specific to S100, GFAP, and CD90. HSwC are negative for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast, and fungi. HSwC can to further expand for 10 population doublings in our Schwann Growth medium (cat # SGM001).
Human Astrocytes

 Human Brain Astrocytes cultured with AlphaBioCoat.
Human Blood Brain Barrier Model
I will continue to post updates here.

Human Astrocytes

Cortex Derived Astrocytes

Neuromics is pleased to be offering yet another option for culturing Human Astrocytes. They can be passaged up to 10X-Only 749 USD/500,000 Cells

They are deigned to be easy to culture and grow.

 Image: Human Brain Astrocytes cultured with our AlphaBioCoat.

Check out our large offering of Neurons, Astroglia, Progenitors, Brain Endothelial Cells/Pericytes and Blood Brain Barrier (BBB) Model. Should you have questions on these or any of our offerings, I can be reached directly at 612-801-1007 or pshuster@neuromics.com. Thank you, Pete Shuster-CEO and Owner.

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.

3-D Cell Based Assays Are Evolving

3-D Multicell Models and Gels

There is a growing body of publications of why 3-D matters in drug discovery and toxicology assays. This table shows important distinctions for single cell assays.

Cellular characteristics	2D	3D	Refs.
Morphology	Sheet-like flat and stretched cells in monolayer	Natural shape in spheroid/aggregate structures	20,24,50
Proliferation	Often proliferate at a faster rate than in vivo	May proliferate at a faster/slower rate compared to 2D-cultured cells depending on cell type and/or type of 3D model system	17,51
Exposure to medium/drugs	Cells in monolayer are equally exposed to nutrients/growth factors/drugs that are distributed in growth medium	Nutrients and growth factors or drugs may not be able to fully penetrate the spheroid, reaching cells near the core	24,52
Stage of cell cycle	More cells are likely to be in the same stage of cell cycle due to being equally exposed to medium	Spheroids contain proliferating, quiescent, hypoxic and necrotic cells	18,24,53
Gene/protein expression	Often display differential gene and protein expression levels compared to in vivo models	Cells often exhibit gene/protein expression profiles more similar to those in vivo tissue origins	17,40,54
Drug sensitivity	Cells often succumb to treatment and drugs appear to be very effective	Cells are often more resistant to treatment compared to those in 2D culture system, often being better predictors of in vivo drug responses	17,33

Table: Key Differences in Cellular Characteristics and Processes in Two-Dimensional and Three-Dimensional Culture Systems

Assay Drug Dev Technol. 2014 May 1; 12(4): 207–218.

doi:  10.1089/adt.2014.573

From the table, we see, for the most part, the overall advantages of cells in 3-D for generating more in vivo like data. We also see potential issues like: "Nutrients and growth factors or drugs may not be able to fully penetrate the spheroid, reaching cells near the core".  

We have resolved penetration issues with our engineered Collagel Hydrogels. We have further enhanced the assays by including multicell assays that even more closely mimic the in vivo environment.

Our Blood Brain Barrier (BBB) Model, for example, includes our:

• Human Brain Microvascular Endothelial Cells
• Human Brain Microvascular Pericytes (HBMVPCs)
• Human Brain Astrocytes (HBA)

These cells are pre-cultured in the engineered gels and cost a fraction of the individual components (see: culturing and assay protocol).

Name	Catalog #			Size	Price
Human Blood Brain Barrier Model	3D45002-6			6 wells 12 well 24 well	$1,780 $2,600 $3,625

Our intention is for you to generate the most in vivo like data possible.

Solutions for Blood Brain Barrier Research

Neuromics Has the Key Components!

We are pleased to add Human Brain Microvascular Pericytes (HBMVPCs) to our solution set for Blood Brain Barrier (BBB) Researchers. These join our Human Brain Microvascular Endothelial Cells and hAstroPro Human Astrocyte+Astroglial-Neurons Co-Culturing Kits to round out our offerings of cells involved in BBB regulation.

 

Image: Human Brain Pericytes cultured with AlphaBioCoat (AC001).

Pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels (see: http://www.nature.com/nature/journal/v468/n7323/full/nature09522.html).

Questions? Do not hesitate to call me or e-mail me-612-801-1007 or pshuster@neuromics.com). Pete Shuster CEO and Owner.