This study was funded by grants awarded to R.L.M. from the National Health and Medical Research Council of Australia (grant # 606658).

1. Cell Culture (3-7 Days Prior to BBB Assembly)
1.1. Primary Human Brain Microvascular Endothelial Cells (BECs)
BECs were commercially obtained. The cells were produced by dispase dissociation of normal human brain cortex tissue and provided frozen at passage 3 (&#60;12 population doublings).
<ol>
	<li>Substratum: Coat tissue-culture vessels with &#34;Attachment Factor&#34; as per the manufacturer instructions.</li>
	<li>Cell maintenance: Maintain BECs in serum-containing complete medium. For experimentation, culture BECs in complete serum-free medium. Maintain the cells in a humidified 5% CO2, 21% O2 incubator at 37&#160;&#176;C 
	Note: As an alternative to specialized reagents, 0.1% (w/v) gelatine solution can serve as a coating reagent while DMEM/F-12 supplemented with 15 mM HEPES, 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 50 &#181;g/ml gentamycin, 20 &#181;g/ml heparin and 20 &#181;g/ml endothelial cells growth supplement can be used for BEC maintenance.</li>
	<li>Sub-culturing for experimentation: Split confluent BECs at a ratio of 1:3 to 1:6, depending on the intended time for next use (3-7 days). Change the maintenance medium every 3 days. Use BECs up to 15 passages.</li>
</ol>
1.2. SVG Human Fetal Astroglial Cell-line
SVG human fetal astroglial cells22 were originally derived from primary cultures of human fetal brain transformed with replication-deficient simian virus 40.
<ol>
	<li>Maintenance: Culture SVG cells in minimum essential medium with Earle&#39;s balanced salt solution supplemented with 20% (v/v) FCS (same serum percentage used to maintain their parental primary cells 22), 2 mM L-glutamine, 50 U/ml penicillin and 50 &#181;g/ml streptomycin. Maintain the cells in a humidified 5% CO2, 21% O2 incubator at 37 &#176;C.</li>
	<li>Sub-culturing for experimentation: Split confluent SVGs at a ratio of 1:5 to 1:10, depending on the intended time for next use (3-7 days). Removal of trypsin is not required due to the high content of serum in the medium. Change the medium every 3 days. Use SVGs up to 20 passages.</li>
</ol>
2. Assembly and Coculturing of the Human In vitro BBB Model (Day 0)
Comment: The human in vitro contact BBB model is prepared according to published protocols 13,23, with modifications.
2.1. Coating of Inserts
<ol>
	<li>Place tissue-culture inserts (6.5 mm in diameter with polyester porous membrane, 3 &#181;m pore-size) in the wells of the supplied 24-well plate. Coat the luminal surface of the inserts<s> </s>overnight in a humidified 37 &#176;C incubator with rat collagen I (20 &#181;g/cm2; 50 &#181;l of 132 &#181;g/ml collagen I in 0.02% acetic acid in Milli-Q water (MQH2O)). Wash the inserts once (both from the abluminal and luminal side) with MQH2O to remove the residual acid. 
	Note: Basement membrane of brain capillaries contains no collagen I but mainly laminins, collagen type IV isoforms, nidogens, and heparan sulfate proteoglycans9. For this reason, some researchers utilize coating agents such as collagen IV, fibronectin13, or Matrigel (BD biosciences)16 for a more authentic BBB modeling.</li>
</ol>
2.2. Seeding SVGs on the Underside (Abluminal) Membrane Surface
<ol>
	<li>Invert the insert and gently fit around the rim of the porous membrane a short piece of elastic silicone tubing (&#34;external tubing&#34;; ~10 mm long, 8 mm internal diameter, 1.6 mm wall), creating essentially a new well above the abluminal surface (Figure 2A).</li>
	<li>The underside of the insert needs to be sealed to avoid media leakage. First, prepare a plug (Figure 2A and 2B) made of a silicone tubing (termed &#34;internal tubing&#34;; ~8 mm long, 3.2 mm internal diameter, 1.6 mm wall; Figure 2B), sealed at one end with a plastic sealing cone (~3mm long; prepared by cutting the bottom of a 0.2 ml polymerase chain reaction (PCR) tube; Figure 2B). This cone not only seals the internal tubing lumen, but also widens its edge for tighter fitting into the insert. Next, using a sterile forceps, insert the silicone plug into the luminal cavity and advance it until it reaches up-to ~1-2 mm from the membrane (Figure 2A).</li>
	<li>Next, seed 4 x 104 SVG cells in 200 &#181;l SVG maintenance medium (section 1.2.2 above) directly into the silicone well above the abluminal membrane surface (Figure 2A). Allow SVGs to adhere for at least 4 hr in the incubator. 
	Note: To maintain sterility transport the assembled inserts in between two 6-well plates (one plate inverted over the other; Figure 2C) to minimize exposure to unfiltered air during the procedure. Silicone tubes and plugs are washed in ethanol and autoclaved for later use. 
	Note: Use plugs (section 2.2.2) only for inserts with membrane pore sizes larger than 1 &#181;m. Membranes with pore diameter &#8804;1 &#181;m rarely leak and require only the fitting of the external silicone tubing (section 2.2.1). In addition, some coating agents other than collagen I (see note to section 2.1) may prevent leakiness even through 3 &#181;m pores. Determine this empirically.</li>
	<li>In order to monitor the adherence state of your astrocytes, seed in parallel into a standard 96-well an identical volume of the astrocyte cell-suspension. This well has the same surface area as the 6.5 mm insert (0.33 cm2) and permits easier visualization of cells by phase-contrast microscopy than the insert membrane. When you observe sufficient adherence of the astrocyte in the control 96-well, the astrocytes have adequately adhered also to the insert membrane.</li>
	<li>When astrocytes have sufficiently adhered in the control 96-well, transfer the insert assemblies to the tissue culture hood (see note above) and gently remove the external silicone tubing and plugs. Return inserts to the normal (i.e. upright) orientation into the supplied wells containing 800 &#181;l/well of BEC maintenance medium (section 1.1.2 above).</li>
</ol>
2.3. Seeding of BECs into the Luminal Chamber above the Astrocytes
Seed 2x104 BECs in 200 &#956;l onto the collagen-coated luminal surface and return the inserts into the incubator.
2.4. Coculturing
Coculture the SVG/BEC-seeded inserts for 3 days in BEC medium without media change before experimentation.
3. Experimentation with the Human BBB Model (Day 3)
<ol>
	<li>To stimulate the in vitro BBB first wash cells by replacing the abluminal and luminal media with 600 &#181;l and 100 &#181;l of serum-free BECs medium, respectively.</li>
	<li>Aspirate the luminal medium again and replace with 100 &#181;l of serum-free medium with stimulating agents. 
	Note: If stimulation of the abluminal chamber is required (to activate the in vitro BBB from the astrocytic compartment), aspirate the abluminal medium and replace with 600 ul of serum free medium with stimulating agents.</li>
	<li>Test each experimental group in triplicate (hence, if luminal stimulation is performed, which requires 3 x 100 &#181;l, we recommend diluting the reagents to their final concentrations in 350 &#181;l of serum-free medium per group to minimize pipetting errors between wells).</li>
	<li>Perform standard paracellular and/or transcellular permeability assays of labeled tracers 16,24 (for the latter we use fluorescein isothiocyanate (FITC)-conjugated albumin) and/or measurement of TEER 13,16,25 as previously described.</li>
</ol>
To account for fluctuations in baseline permeability between experiments, analyze changes in permeability relative to a coated insert without cells (which serves as a reference for maximal permeability) and to the vehicle-treated group using the formula: permeability (% of max) = (permeability value of experimental insert - average permeability value of the vehicle group) / (permeability value of the blank insert - average permeability value of the vehicle group) X 100.
4. Visualization of Cells on Porous Membranes
Comment: Unstained cells on insert membranes are difficult to observe by phase contrast or differential interference contrast (DIC) microscopy since the membrane is quite opaque and interferes with light transmission. To deal with this issue we developed various staining procedures of cells on inserts, which greatly enhance cell appearance on the membrane. Generally, stain the insert membranes while still attached to the insert, in the wells. The membranes should be removed only at the end of the staining procedure for mounting purposes.
4.1. Hematoxylin Staining
<ol>
	<li>Fix inserts with ice-cold 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS; 130 mM NaCl, 10 mM Na2HPO4, 10 mM NaH2PO4, pH 7.2) for 20 min. Wash once in PBS.</li>
	<li>Submerge the insert in 0.1% Mayer&#39;s hematoxylin solution for 2 min.</li>
	<li>Gently wash the insert with tap water (by submerging the insert in a beaker containing tap water and gently pipetting the excess water out). Transfer the insert to Scott&#39;s tap water solution (2 g/L NaHCO3, 20 g/L MgSO4) for ~20 sec until the desired blue color develops. Wash the insert again with tap water (section 4.1.3). An optional eosin stain can be introduced here by dipping the insert in 1% eosin solution for 10 sec followed by a tap water wash.</li>
	<li>Air-dry the insert membrane. Using a scalpel (cutting around the membrane edge) remove the membrane and mount it on a glass slide for bright-field imaging 
	Note: If di-N-butylphthalateinxylene (DPX) mounting medium is utilized wash the insert with 100% ethanol followed by xylene before removing and mounting the membrane.</li>
</ol>
4.2. Scanning Electron Microscope (SEM)
<ol>
	<li>Fix inserts with ice-cold 4% (w/v) PFA in PBS for 20min. Wash three times in PBS.</li>
	<li>Post-fix the membranes for 30 min in 1% (w/v) osmium tetroxide in MQH2O.</li>
	<li>Wash inserts in water (section 4.1.3) and dehydrate in an escalating ethanol gradient (50%, 70%, 90% for 5 min each, then 3x with 100% ethanol for 10 min per interval).</li>
	<li>Treat dehydrated inserts with a mixture of hexamethyldisilazane (HMDS):ethanol (1:2 then 2:1 for 5 min).</li>
	<li>Treat inserts with 100% HMDS for 5 min, air-dry and store under desiccated conditions.</li>
	<li>Remove the membrane using a scalpel (cutting around the membrane edge). Examine membranes by scanning electron microscopy.</li>
</ol>
4.3. Immunofluorescence
<ol>
	<li>Fix inserts with ice-cold 4% (w/v) PFA in PBS for 20 min, wash once in Tris-buffered saline (TBS; 50 mM Tris-HCl pH 7.6, 154 mM NaCl, 0.22 &#181;m filtered ).</li>
	<li>Perform immunostaining of the cells on the insert membrane as previously described26.</li>
	<li>Remove the membrane using a scalpel (cutting around the membrane edge) and mount it on a glass slide with fluorescence mounting medium. Examine membrane by fluorescent microscopy.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+of+alteplase+in+the+treatment+of+ischaemic+stroke.">Pharmacokinetics of alteplase in the treatment of ischaemic stroke.</a> Expert Opin. Drug Metab. Toxicol. 8, 271-281 (2012).</li><li>Derex, L., Nighoghossian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+haemorrhage+after+thrombolysis+for+acute+ischaemic+stroke:+an+update.">Intracerebral haemorrhage after thrombolysis for acute ischaemic stroke: an update.</a> J. Neurol. Neurosurg. Psychiatry. 79, 1093-1099 (2008).</li><li>Garber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke+treatment--light+at+the+end+of+the+tunnel.">Stroke treatment--light at the end of the tunnel.</a> Nat. Biotechnol. 25, 838-840 (2007).</li><li>Liebeskind, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+Stroke+in+the+2010s.">Reversing Stroke in the 2010s.</a> Stroke. 40, 3156-3158 (2009).</li><li>Weksler, B. 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=Blood-brain+barrier-specific+properties+of+a+human+adult+brain+endothelial+cell+line.">Blood-brain barrier-specific properties of a human adult brain endothelial cell line.</a> FASEB J. 19, 1872-1874 (2005).</li><li>Song, L., Pachter, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+murine+brain+microvascular+endothelial+cells+that+maintain+expression+and+cytoskeletal+association+of+tight+junction-associated+proteins.">Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins.</a> In Vitro Cell. Dev. Biol. Anim. 39 (2003), 313-320 (2003).</li><li>Steiner, O., Coisne, C., Engelhardt, B., Lyck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+immortalized+bEnd5+and+primary+mouse+brain+microvascular+endothelial+cells+as+in+vitro+blood-brain+barrier+models+for+the+study+of+T+cell+extravasation.">Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood-brain barrier models for the study of T cell extravasation.</a> J. Cereb. Blood Flow Metab. 31, 315-327 (2011).</li></ol>

Authors have nothing to disclose.

Medical research into brain pathologies suffers much translational difficulty. In the area of acute ischemic stroke, for example, many drugs which showed great promise in animal models failed at the clinic38,39. The reasons for these disappointing results are diverse and include infidelity of the preclinical test systems to the human stroke scenario and overstatement of results obtained from animal studies21. One strategy to improve the predictive value of preclinical findings to the clinical phase ...

The BBB is a specialized interface between the peripheral blood circulation and the central nervous system, crucially responsible for the maintenance of brain hemostasis. It comprises distinct brain microvascular endothelial cells (BECs) which are functionally influenced by few cellular and acellular components (below) to form a tight and dynamic gateway into the brain. Under physiological conditions the BBB restricts the passage of blood cells, plasma components and harmful substances, all potentially neurotoxic, into the brain. In parallel, the BBB selectively exchanges key ions and nutrients (glucose and amino-acids) and metabolic waste products between the brain and the circulation to precisely maintain the brain environment1,2. In recent years it is becoming evident that failure of the BBB occurs in a variety of chronic brain pathologies, such as neurodegenerative or inflammatory-related diseases (e.g. Alzheimer&#39;s disease and multiple sclerosis, respectively)3, as well as in acute conditions like ischemic stroke4.
The unique BBB properties of brain endothelial cells (BECs) are largely induced by their cerebral environment5, and in particular by astrocytes6,7. There is a growing understanding that other cell-types, such as pericytes8, neurons and microglia1,3, as well as the basement membrane9, support BECs and form together a functional unit termed the &#34;neurovascular unit&#34; (NVU) which simultaneously couples neuronal metabolic demands to their supplying capillaries10.
The involvement of the BBB in pathological situations underlies numerous attempts to develop in vitro BBB models to assist in BBB-related research11,12. These models aim to mimic as close as possible in vivo BBB characteristics according to the NVU principle. In vitro BBB models generally rely on a monolayer of tight-junction-forming BECs (mainly from bovine13, human14, rat15, mouse16, and porcine17,18 origins), cultured on a porous membrane together with supporting astrocytes (extensively reviewed by Deli et al. 200511).
Astrocytes can be grown in non-contact conditions on the bottom of a tissue culture well, separated from BECs (cultivated on the upper surface of the membrane) by the culture medium yet communicating with BECs via soluble factors16. In more advanced models which better resemble the anatomical structure of the BBB in vivo, astrocytes are maintained in contact conditions and cultured directly on the opposite side of the membrane in close proximity to BECs13,15,17 (Figure 1). This configuration enables physical contact between BECs and astrocytes, established when astrocytes project their processes through the porous membrane. Importantly, for a true contact to occur the pores should be &#8805;1&#181;m in diameter, since astrocytic end-feet cannot pass through smaller pore sizes (i.e. 0.4 &#181;m)14,15. Notably, contact BBB systems are demonstrated in some studies to be superior to their non-contact counterparts regarding their trans-endothelial electrical resistance (TEER) and endothelial permeability values of various tracers13,17,18. An additional dimension of media flow was recently added in a number of in vitro BBB models to apply shear forces to the endothelium for closer simulation of the brain vasculature12,19.
One technical obstacle to overcome when generating a contact BBB model is the seeding of astrocytes against gravity on the abluminal surface of the porous membrane. Previous protocols13,20, where astrocytes were simply seeded in a drop of media on top of an inverted insert, allowed only short seeding times (i.e. 10 min13 or 2 hr20) which were found in our hands to be insufficient for proper cell attachment. Using this basic method, a longer astrocyte attachment period requires constant monitoring of the inserts by frequent opening of the incubator (causing fluctuations in temperature, pH and humidity) and is also prone to uneven cell seeding due to leakage of media through the pores, especially if pores larger than 1 &#181;m are employed.
Here, we describe a general protocol for preparation of a contact BBB model. Our procedure includes an alternative method for cell-seeding on inverted inserts, which addresses the above mentioned limitations. The method permits undisturbed adherence of astrocytes onto the abluminal membrane surface, in an equilibrated incubator, for an extended period of time. As a result, a uniform seeding of astrocytes is achieved which increases barrier quality and minimizes basal permeability variations between inserts.
As the use of human cells is important for human-relevant research21, we additionally demonstrate in this article the specific utilization of a novel combination of primary human BECs and immortalized human astrocytes for establishment of a contact human BBB model with a high throughput screening capacity. Since viewing of cells on porous membranes can be difficult, we also detail staining techniques which can assist in determination of confluence and cell morphology on the porous membranes. Finally, we exemplify how our human BBB model can be utilized to examine the effect of tissue-type plasminogen activator (t-PA) - a clot busting enzyme which serves as a sole treatment option for acute ischemic stroke - on the BBB.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Attachment Factor</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-210</td>
			<td style="width:243px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Brain microvascular endothelial cells (BECs), primary, human</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Complete medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC JetFuel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Complete serum-free medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">SF-4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC RocketFue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DMEM/F-12 with 15 mM HEPES</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Endothelial cells growth supplement (from bovine origin)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">External silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A080.016</td>
			<td style="width:243px;">Pumpsil brand, 8 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Foetal calf serum</td>
			<td style="width:193px;">Lonza</td>
			<td style="width:171px;">14-501F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Gentamycin sulfate</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Heparin sodium</td>
			<td style="width:193px;">Pfizer</td>
			<td style="width:171px;"></td>
			<td style="width:243px;">1,000 U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Hexamethyldisilazane (HMDS)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">H4875</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Human brain microvascular endothelial cells</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Internal silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A032.016</td>
			<td style="width:243px;">Pumpsil brand, 3.2 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">L-Glutamine</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Mayer&#8217;s hematoxylin solution</td>
			<td style="width:193px;">Amber Scientific</td>
			<td style="width:171px;">MH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Minimum essential medium with Earle&#8217;s balanced salt solution</td>
			<td style="width:193px;">HyClone Laboratories</td>
			<td style="width:171px;">SH30244.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rat collagen I</td>
			<td style="width:193px;">Trevigen</td>
			<td style="width:171px;">3440-100-01</td>
			<td style="width:243px;">Cultrex brand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Tissue culture inserts</td>
			<td style="width:193px;">Corning Life Sciences</td>
			<td style="width:171px;">3472</td>
			<td style="width:243px;">Transwell brand, 6.5 mm in diameter, with polyester porous membrane, 3 &#181;m pore-size</td>
		</tr>
	</tbody>
</table>

improved method for the preparation of a human cell-based, contact model of the blood-brain barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

In order to establish a human, contact BBB model we had to cultivate SVGs and BECs on porous membranes with a 3 &#181;m pore-size, shown to permit passage of astrocyte end-feet for contact with endothelial cells14,15,27,28. A schematic representation of the complete contact model is illustrated in Figure 1 (left illustration). The main technical challenge presented by a contact system is the need to seed astrocytes against gravity on the abluminal surface of the membrane. We have succeeded in ...

Watch this Scientific Journal Video about Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier at JoVE.com

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

improved-method-for-preparation-human-cell-based-contact-model-blood

Research

JoVE Journal

Bioengineering

16.1K Views.  Monash University. Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Video: Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

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.

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 ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Preparation of the Neonatal Rat Cardiomyocyte Balls for the Slow Injection Method

Begin by measuring the neonatal Rat Ventricular Cardiomyocytes concentration using a hemocytometer. Dilute the cell suspension solution to 3, 000 cells per milliliter, with alpha MEM containing 10%heat inactivated FBS. Distribute 100 microliters of the diluted ventricular cardiomyocyte in each well of the cell's non-adhesive 96 well plates.Centrifuge the cells for five minutes at 100 G.And culture them for two to three days in the cell culture incubator, with 5%carbon dioxide at 37 degrees Celsius. After incubation, aspirate the culture medium from the Rat Cardiomyocyte balls seeded in the non-adhesive 96 well plate. Then, transfer the cardiomyocyte balls from each well to a 15 milliliter tube.Centrifuge the cardiomyocyte balls and aspirate the supernatant. Then, wash them using an ADS buffer. To facilitate the tracking of the cardiomyocyte cells in the Rat after engraftment, stain the cells with PKH26.Before centrifuging the cells, track the development of bidim cardiomyocyte balls by observing them under the microscope regularly after seeding. Begin preparing the main injection apparatus for a neonatal cardiomyocyte ball injection using a 29 gauge, 50 millimeter long needle equipped with a syringe. Connect an 18 gauge one milliliter power transfer syringe needle to a thin polypropylene tube with low lumen expandability.Aspirate the water using a syringe, and remove all the bubbles from the syringe and the connected tubes. Repeat it for another 18 gauge needle. Then, connect the other side of the power transfer tube to the 18 gauge needle of the same one milliliter syringe.Confirm the successful power transition by pushing one syringe plunger down, and another syringe plunger up. Once done, set the syringe in a syringe pump. Next, set up the cooling system by attaching the tightly coiled copper tube to the cell containing part of the cell injection syringe.Leaving 10 millimeter of excess pipe at both ends. Connect the copper tube to the flexible plastic tubing before connecting the other ends of the plastic tubing to an external pump. Fill the line with cooling water, and immerse the excess copper tube into crushed ice to cool the water.This cooling system maintains the cell suspension solution in the cylinder at approximately 2 degrees Celsius.

Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method

Prepare the 10%gelatin stock solution by weighing and dissolving the gelatin in the ADS buffer, and autoclaving the solution. Rewarm the 10%gelatin stock at 37 degrees Celsius, until it becomes liquid. Then dilute the gelatin stock, using a prewarmed ADS buffer, to obtain the final injectable 5%gelatin solution.Suspend 96 cardiomyocyte balls in 100 microliters of prewarmed injection solution. Load the cardiomyocyte ball suspension into the cell injection syringe without aspirating the excess air. Next, hold the syringe vertically, with the needle facing upward, and tap the syringe to collect the bubbles at the upper ridge of the syringe.Also, observe the cardiomyocyte balls gradually settling onto the rubber seal of the plunger. Maintain the vertical position of the syringe, and slowly push the plunger to discard the bubbles and excess cell suspension solution, until 20 microliters of the cell suspension remain in the syringe, and the cardiomyocyte ball remains resting on the rubber seal of the plunger. Once done, immerse the capped syringe in an ice bath for five minutes, and fix the injection syringe apparatus on the animal stage before activating the main syringe pump during percutaneous cell transplantation in an anesthetized rat.