Name: characterization of blood outgrowth endothelial cells (boec) from porcine peripheral blood
Uploaded: 2022-01-06T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood at JoVE.com

The authors wish to acknowledge funding from NIH/NHLBI R00 HL129068.

All animal studies were approved by the respective Institutional Animal Care and Use Committees (IACUC) at the Medical College of Wisconsin and Mayo Clinic.
NOTE: In this study, Yorkshire/Landrace/Duroc cross domestic pigs (Sus domesticus), male and female, 40-80 kg, 3-6 months old, were used.
1. Collection of porcine peripheral blood
<ol>
	<li>Prepare materials.
	<ol>
		<li>Dilute heparin solution to 100 U/mL in sterile saline.</li>
		<l...

<ol>
	<li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+II.+Representative+vascular+beds.">Phenotypic heterogeneity of the endothelium: II. Representative vascular beds.</a> Circulation Research. 100 (2), 174-190 (2007).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circulation Research. 100 (2), 158-173 (2007).</li><li>Pober, J. 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(150), e59673 (2019).</li><li>Chang Milbauer, 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=Genetic+endothelial+systems+biology+of+sickle+stroke+risk.">Genetic endothelial systems biology of sickle stroke risk.</a> Blood. 111 (7), 3872-3879 (2008).</li><li>Wei, P., 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=Differential+endothelial+cell+gene+expression+by+African+Americans+versusCaucasian+Americans:+a+possible+contribution+to+health+disparity+in+vascular+disease+and+cancer.">Differential endothelial cell gene expression by African Americans versusCaucasian Americans: a possible contribution to health disparity in vascular disease and cancer.</a> BMC Medicine. 9 (1), 2 (2011).</li><li>Hasstedt, S. 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F., 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=Phenotypic+correction+of+von+Willebrand+disease+type+3+blood-derived+endothelial+cells+with+lentiviral+vectors+expressing+von+Willebrand+factor.">Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor.</a> Blood. 107 (12), 4728-4736 (2006).</li><li>Bodempudi, 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=Blood+outgrowth+endothelial+cell-based+systemic+delivery+of+antiangiogenic+gene+therapy+for+solid+tumors.">Blood outgrowth endothelial cell-based systemic delivery of antiangiogenic gene therapy for solid tumors.</a> Cancer Gene Therapy. 17 (12), 855-863 (2010).</li><li>Dudek, A. Z., 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=Systemic+inhibition+of+tumour+angiogenesis+by+endothelial+cell-based+gene+therapy.">Systemic inhibition of tumour angiogenesis by endothelial cell-based gene therapy.</a> British Journal of Cancer. 97 (4), 513-522 (2007).</li><li>Moubarik, C., 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=Transplanted+late+outgrowth+endothelial+progenitor+cells+as+cell+therapy+product+for+stroke.">Transplanted late outgrowth endothelial progenitor cells as cell therapy product for stroke.</a> Stem Cell Reviews and Reports. 7 (1), 208-220 (2011).</li><li>Pislaru Sorin, 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=Magnetic+forces+enable+rapid+endothelialization+of+synthetic+vascular+grafts.">Magnetic forces enable rapid endothelialization of synthetic vascular grafts.</a> Circulation. 114 (1), 314 (2006).</li><li>Satyananda, 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=New+concepts+of+immune+modulation+in+xenotransplantation.">New concepts of immune modulation in xenotransplantation.</a> Transplantation. 96 (11), 937-945 (2013).</li><li>Klymiuk, N., Aigner, B., Brem, G., Wolf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modification+of+pigs+as+organ+donors+for+xenotransplantation.">Genetic modification of pigs as organ donors for xenotransplantation.</a> Molecular Reproduction and Development. 77 (3), 209-221 (2010).</li><li>Ryczek, N., Hryhorowicz, M., Zeyland, J., Lipi&#324;ski, D., S&#322;omski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas+technology+in+pig-to-human+xenotransplantation+research.">CRISPR/Cas technology in pig-to-human xenotransplantation research.</a> International Journal of Molecular Sciences. 22 (6), 3196 (2021).</li><li>Cooper, D. 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BOECs are a powerful tool that may be used in various scientific and therapeutic approaches7,8,16. BOECs have been used to analyze EC gene expression to elucidate the key factors responsible for the development of vascular diseases and cancer5,19,20,21. BOECs have also been used in therapeutic applicat...

The endothelium is a highly complex, dynamic structure and a vital component of the vascular wall. It lines the inner surface of blood vessels to provide a physical interface between circulating blood and surrounding tissues. This heterogeneous structure is known to perform various functions such as angiogenesis, inflammation, vasoregulation, and hemostasis1,2,3,4. Human umbilical vein endothelial cells are a widely studied cell type to assess the functionality of endothelial cells. However, the patient-specific batch variability, inconsistent phenotype, and minimum splitting efficiency suggest a need to determine a cell source that could improve upon all of these features5.
Obtaining a homogenous population of primary endothelial cells can be technically challenging, and primary endothelial cells do not possess high proliferative capacity6.&#160;Hence, to study vascular regeneration and evaluate pathophysiological processes, various groups have tried to obtain and assess different types of endothelial cells derived from peripheral blood, e.g., endothelial progenitor cells (EPCs) or blood outgrowth endothelial cells (BOECs)6,7,8,9. The spindle-shaped early EPCs originate from hematopoietic stem cells (HSCs) and have limited growth potency and limited angiogenic ability to produce mature endothelial cells. Furthermore, they closely resemble inflammatory monocytes. In addition, their capacity to further differentiate into functional, proliferating, mature endothelial cells is still debatable6,7,9,10.&#160;The continuous culture of peripheral blood mononuclear cells (PBMCs) can give rise to a secondary population of cells known as late-outgrowth EPCs, BOECs, or endothelial colony-forming cells (ECFCs)6,7,9,10. Medina et al. in 2018, acknowledged the limitations of EPCs, the ambiguity of their nomenclature, along with a general lack of concordance with many distinct cell types continuously grouped under EPCs11. In contrast, BOECs have become recognized for their role in vascular repair, health and disease, and cell therapy. Further study and therapeutic use of these cells will rely on protocols to consistently derive these cell types from circulating progenitor cells.
Primary cells such as BOECs can be used as a surrogate to obtain highly proliferative mature endothelial cells6. BOECs are phenotypically distinct from early EPCs and exhibit typical endothelial features such as cobblestone morphology and expression of adherens junctions and caveolae12. Gene profiling by Hebbel et al.13,14,15 found that BOECs or ECFCs are the true endothelial cells as they promote microvascular and large vessel formation. Thus, BOECs can be used as a tool to evaluate pathophysiological processes and genetic variation16. They are also considered an excellent cell source for cell therapy for vascular regeneration17. Hence, a standardized protocol to consistently derive these highly proliferative cells is essential.
While BOECs provide a powerful tool for studying human pathophysiological and genetic variation, a more homogenous source of BOECs may provide more robust and reliable experimental and therapeutic outcomes. Superior homogeneity can be achieved by using xenogeneic cell sources derived from genetically similar animals raised in similar conditions18. While xenogeneic cell sources are prone to eliciting a host immune response, immunomodulation strategies are being developed with the goal of generating immunocompatible animals and animal products, including cells. Pigs, in particular, are an abundant source of peripheral blood and are commonly used to study medical devices and other therapies due to anatomical and physiological similarities to humans. Hence, this study refines the protocol for the isolation and expansion of highly proliferative BOECs from porcine peripheral blood.The protocol detailed below is a straightforward and reliable method to obtain a large number of BOECs from a relatively small volume of blood. The cultures can be expanded through several passages to generate millions of cells from a single blood sample.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>19 G needle</td>
			<td>Covidien</td>
			<td>1188818112</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL syringes</td>
			<td>Covidien</td>
			<td>8881560125</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride solution (0.8%)</td>
			<td>Stemcell Technologies</td>
			<td>07850</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic solution (100x)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-253-839</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 culture medium</td>
			<td>Lonza Walkersville</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Extension tube</td>
			<td>Hanna Pharmaceutical Supply Co.</td>
			<td>03382C6227</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlas Biologicals</td>
			<td>F-0500-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque 1077</td>
			<td>Cytiva</td>
			<td>17144003</td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>Heparin sodium injection (1,000 units/mL)</td>
			<td>Pfizer</td>
			<td>00069-0058-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Gibco</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Eppendorf</td>
			<td>2231300004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Gibco</td>
			<td>15230-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin pipette</td>
			<td>Celltreat Scientific</td>
			<td>229280</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of blood outgrowth endothelial cells (boec) from porcine peripheral blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Morphology of the cultured cells was observed from the start of the culture until BOEC colonies were observed (Figure 1). A smaller population of adherent cells started to attach to the culture dishes and grow, while non-adherent cells were removed with culture medium changes (Figure 1B). Colonies first appeared on day 6 as a collection of endothelial-like cells proliferating radially outward from a central point (Figure 1D). As the...

Watch this Scientific Journal Video about Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood at JoVE.com

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

The protocol provides highly proliferative blood outgrowth endothelial cells that can be used for various tissue engineering, cell therapy, and disease modeling investigations. This technique consistently yields many cells from a single blood draw. For this technique to be successful, it is crucial to quickly get the cells into the culture medium and incubator environment as soon as possible.First, dilute heparin solution to 100 units per milliliter in sterile saline. Add three to four milliliters of heparin solution to each of two 50-milliliter conical tubes and two 60-milliliter syringes. Then, draw undiluted 1, 000 units per milliliter heparin solution into an extension tube.Connect a 19-gauge needle to one end of the heparin-filled extension tube, and a heparin-containing 60 milliliter syringe to the other end of the extension tube. Next, clean the femoral groin area of an anesthetized pig with a Betadine scrub solution. Puncture the femoral vein or artery with a 19-gauge needle and at one to two milliliters per second, slowly draw 50 milliliters of blood into the syringe.Leaving the needle in place, immediately kink the extension tube and disconnect the 60 milliliter syringe. Slowly transfer the blood to a heparin-containing 50-milliliter conical tube. Tightly cap the conical tube, gently invert the tube a few times to mix, and place it on ice.Connect the remaining heparin-containing 60-milliliter syringe to the extension tube. Unkink the extension tube, and slowly draw additional 50 milliliter blood into the syringe. Immediately remove the needle from the artery or vein and slowly draw the remaining blood from the extension tube into the syringe.Disconnect the 60-milliliter syringe from the extension tube and slowly transfer the blood to the remaining heparin-containing 50-milliliter conical tube. Tightly cap the 50-milliliter conical tube, gently invert a few times to mix, and place on ice. Apply pressure hemostasis to the pig's femoral groin area.Dilute the blood one-to-one with PBS in four 50-milliliter conical tubes. Add 25 milliliters of room temperature, ready-to-use density gradient solution to eight 50-milliliter conical tubes. Slowly pipette 25 milliliters of blood saline solution inside each 50-milliliter conical tube containing density gradient solution by holding the tube at a sharp angle to the pipette tip.Centrifuge the tubes for 30 minutes at 560 g and room temperature. Carefully collect the buffy coat, which is the cloudy layer of mononuclear cells above the density gradient solution and below the clear plasma, from each tube with a thin pipette and distribute it evenly into two new 50-milliliter conical tubes. Discard the density gradient solution containing tubes.To coat a six-well plate with fibronectin, make a stock solution of human plasma fibronectin by diluting it to one milligram per milliliter in sterile water. Make 3.6 milliliters of a working solution of fibronectin by diluting 600 microliters of fibronectin stock solution in three milliliters of PBS. Transfer 600 microliters of the fibronectin working solution into each well of a six-well plate and gently rock until evenly coated.And gently aspirate the solution out of each well. To wash the mononuclear cells while waiting for the fibronectin to coat, top up the tubes with up to 45 milliliters of PBS, centrifuge for five minutes at 560 X g and four degrees Celsius with low break. Aspirate the supernatant from both tubes.Resuspend each cell pellet in 25 milliliters of PBS. Resuspend each cell pellet in five milliliters of PBS and add 15 milliliters of 0.8%ammonium chloride solution to each tube. Wash the cells as before by adding PBS.Aspirate the supernatant from both tubes. Resuspend each cell pellet in 25 milliliters of PBS. Resuspend each cell pellet in six milliliters of EGM-2 culture medium supplemented with 10%FBS and 1X antibiotic-antimycotic solution.Gently transfer two milliliters of the cell suspension to each well of the fibronectin coated six-well plate and gently rock until evenly coated. 24 hours after seeding the cells, gently add one milliliter of fresh culture medium to each well. The following day, gently aspirate 1.5 milliliters of culture medium from each well, and replace it with 1.5 milliliters of fresh culture medium.For the next five days, gently change the culture medium with two milliliters of fresh culture medium per well. Regularly visualize the cell cultures under low power 4X objective light microscopy. Morphology of the cultured cells was observed from the start of the culture until BOEC colonies were observed.A smaller population of adherent cells started to attach to the culture dishes and grow, while non-adherent cells were removed with culture medium changes. Colonies first appeared on day six as a collection of endothelial-like cells proliferating radially outward from a central point. As the culture progressed, cell colonies became more dense and displayed a cobblestone morphology similar to mature endothelial cells.Two-dimensional phase contrast microscopy was used to image tube formation in serum-free medium and complete growth medium. Cells in both media conditions underwent morphological differentiation and rapidly organized into extensive networks of capillary tube-like structures. BOECs were further characterized by the expression of mature endothelial cell marker CD31 or platelet endothelial cell adhesion molecule one.BOECs showed uniform expression of CD31. Furthermore, flow cytometry analysis of BOECs with CD14 AF 700 antibody compared to positive control confirmed the absence of EPCs, as the cell stained negative for monocyte marker CD14 compared to the positive control group of PBMCs. Positive CD14 staining shown in blue, and unstained cells shown in red.During blood collection, it is essential to collect as much of the buffy coat layer as possible while collecting as little of the adjacent layers as possible. This technique has enabled researchers to study new strategies for endothelializing implantable cardiovascular devices using a pig model with autologous blood outgrowth endothelial cells.

characterization-blood-outgrowth-endothelial-cells-boec-from-porcine

Research

JoVE Journal

Bioengineering

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

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.

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 ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...