Name: lucifer yellow - a robust paracellular permeability marker in a cell model of the human blood-brain barrier
Uploaded: 2019-08-19T13:00:00
Description: Watch this Scientific Journal Video about Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier at JoVE.com

The authors are thankful for the financial support from the 2017 New Investigator Award from the American Association of Pharmacy, a Hunkele Dreaded Disease award from Duquesne University and the School of Pharmacy start-up funds for the Manickam laboratory. We would like to thank the Leak laboratory (Duquesne University) for western blotting assistance and allowing use of their Odyssey 16-bit imager. We would also like to include a special note of appreciation for Kandarp Dave (Manickam laboratory) for help with western blotting.

1.General hCMEC/D3 cell culture
<ol>
	<li>Resuscitation of frozen cells 
	NOTE: All cell culture maintenance and experiments were performed inside a sterile biosafety hood. Culture media, supplements and reagents were either purchased as sterile products or sterilized via filtration using a 0.22 &#181;m membrane filter to prevent microbial contamination.
	<ol>
		<li>Add 8.5 mL of collagen solution (0.15mg/mL) in a tissue culture flask (75 cm2&#160;growth area; referred henceforth as T75...

<ol>
	<li>Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+blood-brain+barrier.">Structure and function of the blood-brain barrier.</a> Neurobiology Of Disease. 37 (1), 13-25 (2010).</li><li>Griep, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BBB+on+chip:+microfluidic+platform+to+mechanically+and+biochemically+modulate+blood-brain+barrier+function.">BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function.</a> Biomedical Microdevices. 15 (1), 145-150 (2013).</li><li>Camos, S., Mallolas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+for+assaying+microvascular+endothelial+cell+pathophysiology+in+stroke.">Experimental models for assaying microvascular endothelial cell pathophysiology in stroke.</a> Molecules. 15 (12), 9104-9134 (2010).</li><li>Cucullo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+human+brain+endothelial+cells+and+flow-based+vascular+modeling:+a+marriage+of+convenience+for+rational+neurovascular+studies.">Immortalized human brain endothelial cells and flow-based vascular modeling: a marriage of convenience for rational neurovascular studies.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 28 (2), 312-328 (2008).</li><li>Wolff, A., Antfolk, M., Brodin, B., Tenje, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Blood-Brain+Barrier+Models-An+Overview+of+Established+Models+and+New+Microfluidic+Approaches.">In vitro Blood-Brain Barrier Models-An Overview of Established Models and New Microfluidic Approaches.</a> Journal of Pharmaceutical Sciences. 104 (9), 2727-2746 (2015).</li><li>Weksler, B., Romero, I. 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A., Couraud, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hCMEC/D3+cell+line+as+a+model+of+the+human+blood+brain+barrier.">The hCMEC/D3 cell line as a model of the human blood brain barrier.</a> Fluids and Barriers of the CNS. 10 (1), 16 (2013).</li><li>Llombart, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+secretomes+from+a+human+blood+brain+barrier+endothelial+cells+in-vitro+model+after+ischemia+by+stable+isotope+labeling+with+aminoacids+in+cell+culture+(SILAC).">Characterization of secretomes from a human blood brain barrier endothelial cells in-vitro model after ischemia by stable isotope labeling with aminoacids in cell culture (SILAC).</a> Journal of Proteomics. 133, 100-112 (2016).</li><li>Weksler, B. 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J., Abbott, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Blood-Brain Barrier. , 307-324 (2003).</li><li>Deli, M. A., &#193;brah&#225;m, C. S., Kataoka, Y., Niwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+Studies+on+In+vitro+Blood&#8211;Brain+Barrier+Models:+Physiology,+Pathology,+and+Pharmacology.">Permeability Studies on In vitro Blood&#8211;Brain Barrier Models: Physiology, Pathology, and Pharmacology.</a> Cellular and Molecular Neurobiology. 25 (1), 59-127 (2005).</li><li>Ren, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+General+Method+To+Increase+Stokes+Shift+by+Introducing+Alternating+Vibronic+Structures.">A General Method To Increase Stokes Shift by Introducing Alternating Vibronic Structures.</a> Journal of the American Chemical Society. 140 (24), 7716-7722 (2018).</li><li>Pack, D. W., Hoffman, A. S., Pun, S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+development+of+polymers+for+gene+delivery.">Design and development of polymers for gene delivery.</a> Nature Reviews Drug Discovery. 4 (7), 581-593 (2005).</li><li>Oupick&#253;, D., Kon&#225;k, C., Ulbrich, K., Wolfert, M. A., Seymour, L. 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E., Artursson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+drug+permeability+and+prediction+of+drug+absorption+in+Caco-2+monolayers.">Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers.</a> Nature Protocols. 2 (9), 2111-2119 (2007).</li><li>Balda, M. S., Anderson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+classes+of+tight+junctions+are+revealed+by+ZO-1+isoforms.">Two classes of tight junctions are revealed by ZO-1 isoforms.</a> The American Physiological Society. , (1992).</li><li>Brown, R. C., Davis, T. 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A key role of the BBB is to prevent the exchange of non-essential ions and toxic substances between the systemic circulation and the brain to maintain hemostasis of neural microenvironment. One of the characteristic features of the BBB is the ability of the capillary endothelial cells to form tight junctions (TJs) that effectively seal the paracellular route of transport. We demonstrated a LY Papp assay as a quantitative method to determine the apparent kinetics of TJ barrier formation in cultured hCMEC/D3 mon...

Blood-brain barrier (BBB) is the protective barrier limiting the influx of plasma components into the brain tissue and consists of brain endothelial cells along with supporting cells such as pericytes. The major role of BBB is to serve as a barrier that seals the space between peripheral blood and central nervous system (CNS) to maintain hemostasis of the neural microenvironment1,2. The brain capillary endothelial cells effectively seal the paracellular pathway via formation of intercellular tight junctions (TJs)1. This protective barrier allows glucose and selected nutrients to enter the brain while it prevents the majority of ions, toxic substances and drugs from passing through this tight barrier. Apart from its protective role, the natural barrier function of the BBB poses a severe challenge in the development of drug delivery systems targeting the CNS.
In vitro cell culture models of the BBB are helpful tools to study its biology and to understand the effects of drug treatment on TJ barrier integrity. We used the human cerebral microvascular endothelial cell line (hCMEC/D3) as an in vitro model since it is an accepted model of human brain endothelium3 and recapitulates many functions of the human BBB. hCMEC/D3is one of the most commonly used cell lines for modeling the BBB in vitro4,5,6,7,8,9. Despite its comparatively low values of transendothelial electrical resistance (TEER), a measure of barrier tightness, this cell line retains most of the morphological and functional properties of brain endothelial cells, even as a monoculture in the absence of cocultured glial cells6,7. The hCMEC/D3 cell line expresses multiple BBB markers including active transporters and receptors until approximately passage 35 without undergoing dedifferentiation to unstable phenotypes6,7,9,10,11. The most striking characteristic of hCMEC/D3 cell line as an in vitro BBB model is its ability to form TJs5,9,11,12. It should be noted that although stem cell-derived BBB models showed higher permeability in many studies compared with hCMEC/D3 cell line and they do express some BBB markers, they are yet to evolve as the most common BBB cell model13. Importantly, stem-cell derived BBB models remain to be characterized with respect to maximum passage numbers that allow the cells to maintain stable BBB phenotypes14.
Three primary methods are commonly used to determine the TJ barrier integrity, including the measurement of TEER, measurement of apparent permeability coefficient (Papp) of small hydrophilic tracer molecules such as sucrose, inulin, Lucifer Yellow, etc. and immunostaining of known molecular markers of TJs such as claudin-5, ZO-1, occludin, etc.5. TEER is a relatively simple and quantitative method that measures the electrical resistance across the cell monolayers cultured on a porous membrane substrate5. However, TEER values can be influenced by experimental variables such as composition of the culture medium and the type of measurement instrument. A likely combination of these factors leads to a broad distribution of TEER values ranging from 2 to 1150 &#937; cm2 in the hCMEC/D3 cell line cultured for 2-21 days13. Immunostaining is a visual method to determine the presence of TJ proteins by labelling the targeted protein using antibodies. However, immunostaining involves a series of experimental steps, including the need to fix/permeabilize cells that may result in experimental artifacts and the fluorescent signals may fade over time. The above factors may lead to subjective errors affecting data quality.
The primary focus of this work is to present a LY-based apparent permeability assay determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells. Although other advanced in vitro BBB systems, such as co-culture systems, microfluidic systems, are physiologically more relevant mimics with significantly improved barrier function15,16,17, the hCMEC/D3 transwell setup is a simple and reliable model to estimate the kinetics of TJ formation and rapidly screen the effect of different drug formulations on barrier function. In general, Papp values are consistent for various hydrophilic solutes in hCMEC/D3 monolayers. For example, the reported Papp values for various low molecular mass solutes (such as sucrose, mannitol, LY, etc.) in different in vitro&#160;BBB models are in the order of 10-4 cm/min5,18,19,20. In our experimental setup, the brain endothelial cells are seeded on a collagen-coated microporous membrane for cell attachment and monolayer formation to mimic the in vivo barrier. The LY added in the apical side is expected to traverse the intercellular tight junctions and accumulate in the basolateral side. Greater concentrations of LY in the basolateral side indicate an immature, not-fully functional barrier while lower concentrations reflect restricted transport due to the presence of functional TJs resulting in a mature barrier.
LY is a hydrophilic dye with distinct excitation/emission peaks and avoids the need to radiolabel tracer molecules such as sucrose, mannitol or inulin. Thus, the fluorescence values of LY can be used to directly calculate its paracellular permeability across the BBB monolayers. Also, compared to many commercially available dyes used in biomedical fields that suffer from small Stokes shifts such as fluorescein21, the Stokes shift of LY is about 108 nm with sufficient spectral separation, thus allowing LY fluorescence data as a robust readout to determine paracellular permeability. We used Western blotting as an orthogonal technique to demonstrate changes in expression of the tight junction marker protein, ZO-1, over culture time. ZO-1 expression detected via Western blotting is used to supplement the LY Papp data and in combination, these data suggest that the observed changes in LY Papp values is reflective of the formation of a monolayer with gradual increase in expression of the tight junction marker, ZO-1.
As pointed out earlier, the central focus of this work is to demonstrate a LY assay as a simple technique to monitor the formation of a confluent monolayer with functional tight junctions. However, to demonstrate an additional utility of the developed assay, we measured the LY Papp in DNA nanoparticle-transfected hCMEC/D3 monolayers. Nucleic acids can be condensed into polyelectrolyte nanoparticles with a diameter of 100-200 nm via electrostatic interaction between the positively charged groups of polymers and the negatively charged phosphate groups of nucleic acids22,23. We refer to these complexes as DNA nanoparticles (DNA NPs) in our work. While our intention is to transfect cells and express the desired protein, we must ensure that the barrier properties of the hCMEC/D3 monolayers are not compromised. Our data suggests that a standard 4 h luciferase gene transfection regime does not measurably change the LY permeability demonstrating the utility of the LY Papp assay to determine changes in TJ barrier integrity.

<table border="0" cellpadding="0" cellspacing="0" style="width:1035px;" width="1033">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">hCMEC/D3 cell line</td>
			<td style="width:195px;">Cedarlane Laboratories</td>
			<td style="width:123px;">102114.3C-P25</td>
			<td style="width:172px;">human cerebral microvascular endothelial cell line&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gWizLuc</td>
			<td>Aldevron&#160;</td>
			<td>5000-5001</td>
			<td>Plasmid DNA encoding luciferase gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lucifer yellow CH dilithium salt&#160;</td>
			<td>Invitrogen</td>
			<td>155267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell inserts with polyethylene terephthalate (PET) track-etched membranes</td>
			<td>Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture flask&#160;</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Flat Bottom&#160;</td>
			<td>Olympus Plastics</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Black 96-Well Immuno Plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>437111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S-MEM 1X</td>
			<td>Gibco</td>
			<td>1951695</td>
			<td>Spinner-minimum essential medium (S-MEM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EBM-2</td>
			<td>Clonetics</td>
			<td>CC-3156</td>
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			<td height="42" style="height:42px;width:545px;">Alexa Fluor680-conjugated AffiniPure Donkey Anti-Mouse LgG(H+L)</td>
			<td style="width:195px;">Jackson ImmunoResearch Inc</td>
			<td style="width:123px;">128817</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:545px;">12-well, Flat Bottom</td>
			<td style="width:195px;">Olympus Plastics</td>
			<td style="width:123px;">25-106</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:545px;">RIPA buffer (5X)</td>
			<td style="width:195px;">Alfa Aesar</td>
			<td style="width:123px;">J62524</td>
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			<td height="21" style="height:21px;width:545px;">Aprotinin</td>
			<td style="width:195px;">Fisher BioReagents</td>
			<td style="width:123px;">BP2503-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:545px;">Odyssey CLx imager</td>
			<td style="width:195px;">LI-COR Biosciences</td>
			<td style="width:123px;"></td>
			<td>for scanning western blot membranes</td>
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	</tbody>
</table>

lucifer yellow - a robust paracellular permeability marker in a cell model of the human blood-brain barrier

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

First, we determined the effect of culturing time on LY permeability to determine the apparent kinetics of TJ formation. The mean LY Papp values from day 1 to 10-post seeding are shown in Figure 2a. On day 1, the mean Papp was 4.25 x 10-4&#160;cm/min and slightly dropped to 3.32 x 10-4&#160;cm/min on day 2. The mean Papp value slightly increased to 3.93 x 10-4&#160;cm/min on day 3 and fluctuate...

Watch this Scientific Journal Video about Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier at JoVE.com

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

This protocol is a robust technique to determine the functional integrity of intercellular tight junctions in a cell model of the human blood-brain barrier. This is a simple and fairly inexpensive protocol that allows calculating the apparent permeability in a shorter time period instead of running a longer kinetic assay. This method can also be used to determine the functional integrity of tight junctions in brain endothelial cells transfected with polymer DNA nanoparticles.To begin cell plating, place tissue culture inserts with microporous membranes into a 24-well culture plate. Add 400 microliters of 0.15-milligrams-per-milliliter collagen type one in each tissue culture insert. Then incubate for one hour in a 37 degree Celsius, 5%carbon dioxide incubator.Rock the 24-well plate gently to allow even spreading of the collagen solution over the microporous membrane in the tissue culture inserts. After an hour, remove the collagen solution and gently wash the microporous membrane with 0.4 milliliters of 1X PBS buffer. Now, plate hCMEC/D3 cells with a density of 50, 000 cells per square centimeter in the cell inserts.Place the 24-well plate with tissue culture setup in the incubator to allow cell attachment and proliferation. Incubate the plate for seven days to allow the cells to reach 100%confluency. Remove the growth medium every other day and transfer 0.5 milliliters of pre-warmed fresh media into the tissue culture inserts.Repeat the plating procedure for Western blotting to determine changes in ZO-1 expression for DNA nanoparticle transfection, and for the ATP assay to determine cell viability in transfected cells. For determining Lucifer Yellow apparent permeability, on each day post-seeding, remove the growth medium and add 1.5 milliliters of pre-warmed transport buffer to the basolateral side of the well. Now add 58.3 microliters of 20-micromolar Lucifer Yellow solution to the apical side of each trans-well insert.Save 50 microliters of the solution for fluorescence measurements. After completely removing residual PBS buffer from the apical side, add Lucifer Yellow solution as quickly as possible to avoid drying the cells. Ensure accurate volumes of Lucifer Yellow solution in the apical side.Make sure to add the same and accurate volume of Lucifer Yellow solution in all inserts. Work quickly to avoid drying the cells. Incubate the cells in a rotary plate shaker at 100 RPM and 37 degrees Celsius for 60 minutes.Then remove 30 microliters of the Lucifer Yellow sample from each apical compartment. Transfer the 20-micromolar Lucifer Yellow solution and the apical side samples to pre-labeled tubes. Now dilute the sample tenfold using transport buffer.Remove 500 microliters from each basolateral compartment, and transfer the sample to pre-labeled tubes. Prepare a series of Lucifer Yellow standards for the standard curve. Add 100 microliters of each standard apical and basolateral sample to each well of a black 96-well plate in duplicate.Use a fluorescence microplate reader to measure the Lucifer Yellow fluorescence intensity and calculate the apparent permeability, as described in the manuscript text. The effect of culturing time on Lucifer Yellow permeability was used to determine the apparent kinetics of tight junction formation. The apparent permeability values significantly decreased on day seven compared with day one, suggesting that the barrier became tighter.The apparent permeability values then stabilized until day 10. This implied the barrier formation was complete and functional, resulting in decreased Lucifer Yellow paracellular transport. Western blotting was used to detect changes in the expression of the tight junction protein ZO-1 over time.The two bands represent the two ZO-1 isoforms. Densitometry analysis revealed that the pixel value of ZO-1 increased from days three through seven post-seeding, suggesting that the tight junction protein ZO-1 formed continually from days three through seven. After calcium depletion treatment on day seven post-seeding, the band of ZO-1 was almost undetectable, indicating that ZO-1 was unable to form in the absence of calcium ions.As shown in the results, Western blotting of ZO-1 protein can be performed to determine changes in the expression level of tight junction proteins, complementing the Lucifer Yellow-based functional activity data. We also discovered that the functional activity of tight junctions in human brain endothelial cells was not affected by DNA nanoparticle transfection.

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