The work described was supported by the Cornell Center on the Microenvironment &amp; Metastasis through Award Number U54CA143876 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. This work was additionally funded in part by a National Science Foundation Graduate Research Fellowship (A.D.H).

The following protocol is for the production of a single microtube device.
1. Preparation of Halloysite Nanotube Solution
<ol>
	<li>Sonicate (10-13 W (rms)) 250 &mu;L halloysite nanotube solution (6.6 wt% in water) 30 sec. Cool solution with cold water and repeat sonication once. Cool again.</li>
	<li>Draw sonicated solution into a syringe, attach a 0.45 &mu;m syringe filter, and filter solution into clean microfuge tube. Vortex the solution occasionally to maintain homogeneity.</li>
</ol>
2. Coating of Microtube Inner Surface with Halloysite Nanotubes
<ol>
	<li>Obtain 50 cm long section of Micro-Renathane microtubing (300 &mu;m inner diameter, 35.3 &mu;L inner volume). Cut one end of the microtube at a diagonal and insert into a small IDEX adaptor piece. Insert the needle of a 3/10 cc 29G syringe into the other end of the microtube.</li>
	<li>To clean the microtube, place the open end of microtube (the end with the adaptor) into 70% ethanol and draw ~50 &mu;L ethanol into the syringe to fill microtube.</li>
	<li>Rinse the ethanol out of the microtube by drawing a generous volume of distilled water into the syringe.</li>
	<li>Detach the syringe from the microtube, empty the syringe, and then reattach to the microtube.</li>
	<li>Prepare a solution of 0.02% w/v poly-L lysine in water. Draw 50 &mu;L into the microtube and allow to sit for 5 min at room temperature (RT).</li>
	<li>Draw 100 &mu;L filtered nanotube solution into the microtube and allow to incubate for 3 min at RT.</li>
	<li>Draw 100 &mu;L water through the microtube to rinse out the nanotube solution. Allow coated microtube to sit, filled with water, overnight at RT.</li>
</ol>
3. Preparation of Microtubes for Cell Isolation
<ol>
	<li>Prepare a solution of 10 &mu;g/mL protein G in 1X Dulbecco's phosphate buffered saline (PBS, pH 7.0 - 7.2). Draw 50 &mu;L PBS through microtube and then draw 50 &mu;L of the protein-G solution and allow to incubate for 1.5 hr at RT.</li>
	<li>Prepare a solution containing 5 &mu;g/mL E-selectin-IgG and 50 &mu;g/mL antibody (anti-EpCAM for most cancer samples, anti-PSMA for prostate cancer samples) in PBS. Draw 50 &mu;L of the E-selectin and antibody solution into the microtube and allow to incubate for 2 hr at RT.</li>
	<li>Nonspecific cellular adhesion is blocked with 5% milk protein. Prepare a solution of 5% (w/v) milk protein in PBS. Draw 50 &mu;L milk protein solution into the microtube and allow to incubate for 1 hr at RT.</li>
	<li>Draw 50 &mu;L PBS into tube and leave at RT until blood or buffy coat samples are ready for processing.</li>
	<li>10 min prior to using the microtube for isolation, draw 50 &mu;L PBS that has been saturated with Ca2+ ("PBS+") to activate the selectin molecules.</li>
</ol>
4. Preparation of samples for cell isolation
<ol>
	<li>Draw or obtain 10 mL blood from patient into heparinized tube.</li>
	<li>Place 10 mL Ficoll-Paque lymphocyte isolation solution into 50 mL centrifuge tube. Gently layer 10 mL whole blood on top of Ficoll so as not to mix the blood and Ficoll.</li>
	<li>Centrifuge at 2000x g for 15 min at 4 &deg;C with minimal deceleration.</li>
	<li>Remove the buffy coat layer and place in new tube.</li>
	<li>Wash buffy coat with PBS (centrifuge at 230x g for 10 min, discard the supernatant).</li>
	<li>Gently resuspend cells with 1 mL RBC lysis buffer and incubate for 10 min at RT to lyse erythrocytes.</li>
	<li>Add 10 mL PBS, mix gently, and centrifuge at 230x g for 10 min. Discard the supernatant and gently resuspend the pellet in 3 to 4 mL PBS+.</li>
</ol>
5. Cell isolation
<ol>
	<li>Attach one end of the functionalized microtube to a 5 mL syringe using IDEX adaptors.</li>
	<li>Insert the syringe onto a syringe pump.</li>
	<li>Submerge the open end of the functionalized microtube into the cell suspension.</li>
	<li>Process the cell suspension through the microtube at 1 to 4 mL/h.</li>
	<li>Transfer the open end of the microtube to a tube containing PBS+ and draw 300 &mu;L PBS+ into syringe at 0.016 mL/min to remove unbound and loosely bound cells from the microtube.</li>
	<li>Place the open end of the microtube into a clean tube. Disconnect the syringe from the microtube and attach a syringe prefilled with Accutase. Gently perfuse enough Accutase into the microtube to fill (~50 &mu;L) and allow to incubate at RT for 10 min.</li>
	<li>Attach a syringe prefilled with 1 mL of growth media (79% RPMI, 20% FBS, 1% penicillin streptomycin) and perfuse into microtube, collecting effluent into tissue culture treated well plate.</li>
	<li>Culture cells at 37 &deg;C and 5% CO2 under humidified conditions.</li>
</ol>
6. Representative Results
The goal of this technique is to isolate viable cancer cells from the blood of cancer patients. Several methods exist to identify cancer cells in culture; a necessary verification of device success. We have chosen to stain cells in culture with antibodies against epithelial moieties such as EpCAM (epithelial cellular adhesion molecule) or PSMA (prostate specific membrane antigen), in addition to DAPI to identify intact cell nuclei (Fig. 2 and 3). The number of cancer cells captured using this technique is necessarily a function of the number of CTCs in the starting sample, and patient variability can be high. In processing samples taken from patients diagnosed with stage IV cancers, we routinely capture between 100 and 500 cancer cells per tube of blood, at purities &gt;50%. Immediately following isolation, greater numbers of contaminating leukocytes may be present. However these numbers will be depleted following incubation in culture medium for up to 5 days.
<img alt="Figure 1" src="/files/ftp_upload/4248/4248fig1.jpg" /> 
Figure 1. Schematic of functionalized microtube for CTC isolation, showing selectin-mediated rolling followed by antibody-mediated static adhesion.
<img alt="Figure 2" src="/files/ftp_upload/4248/4248fig2.jpg" /> 
Figure 2. Representative data of CTC isolation from blood samples drawn from a breast cancer patient and a lung cancer patient. The number of viable cells positively identified as CTC based on EpCAM staining is represented by the solid bars and pertain to the left ordinate and the percentage of cells that were identified as CTC compared to the total number of captured cells is represented by the open bars and measured on the right ordinate. Results are following five days in culture.
<img alt="Figure 3" src="/files/ftp_upload/4248/4248fig3.jpg" /> 
Figure 3. Representative micrographs from separate donors (A and B) of CTC in culture 5 days subsequent to isolation from a cancer patient blood sample. Cells were fluorescently stained for EpCAM with AlexaFluor 488 (green) and 4',6-diamidino-2-phenylindole (DAPI) to visualize the nucleus (blue).

<ol>
	<li>Allard, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cells+circulate+in+the+peripheral+blood+of+all+major+carcinomas+but+not+in+healthy+subjects+or+patients+with+nonmalignant+diseases.">Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.</a> Clin. Cancer Res. 10, 6897-6904 (2004).</li><li>Hughes, A. D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobiotechnology+for+the+capture+and+manipulation+of+circulating+tumor+cells.">Nanobiotechnology for the capture and manipulation of circulating tumor cells.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. , (2011).</li><li>Hughes, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtube+Device+for+Selectin-Mediated+Capture+of+Viable+Circulating+Tumor+Cells+from+Blood.">Microtube Device for Selectin-Mediated Capture of Viable Circulating Tumor Cells from Blood.</a> Clinical Chemistry. 58, 846-853 (2012).</li><li>Hughes, A. D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+naturally+occurring+halloysite+nanotubes+for+enhanced+capture+of+flowing+cells.">Use of naturally occurring halloysite nanotubes for enhanced capture of flowing cells.</a> Langmuir. 26, 12155-12164 (2010).</li><li>Al-Mehdi, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat. Med. 6, 100-102 (2000).</li><li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+Building+a+framework.">Cancer metastasis: Building a framework.</a> Cell. 127, 679-695 (2006).</li><li>Lee, D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing+of+P-Selectin+Increases+the+Rate+of+Neutrophil+Recruitment+Under+Shear+Flow.">Microcontact Printing of P-Selectin Increases the Rate of Neutrophil Recruitment Under Shear Flow.</a> Biotechnology Progress. 24, 1052-1059 (2008).</li><li>Gout, S., Tremblay, P. L., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectins+and+selectin+ligands+in+extravasation+of+cancer+cells+and+organ+selectivity+of+metastasis.">Selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis.</a> Clin. Exp. Metastasis. 25, 335-344 (2008).</li><li>Geng, Y., Marshall, J., King, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomechanics+of+the+Metastatic+Cascade:+Tumor+Cell-Endothelial+Cell+Interactions+in+the+Circulation.">Glycomechanics of the Metastatic Cascade: Tumor Cell-Endothelial Cell Interactions in the Circulation.</a> Annals of Biomedical Engineering. , 1-16 .</li><li>Stott, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+circulating+tumor+cells+using+a+microvortex-generating+herringbone-chip.">Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 18392-18397 (2010).</li></ol>

M.R.K. is a scientific advisor of CellTraffix, Inc. (Rochester, NY).

It is often the case that the early steps in the discovery of new cancer therapeutics utilize cancer cell lines, which bear questionable resemblance to primary cancer cells yet remain in use due to their ease of use in the laboratory. Research into the development of new cancer therapies would be expedited if primary human cancer cells were utilized early in novel research. CTC are the most easily accessible type of cancer cell, due to their presence in blood and the ease of a standard blood draw. In addition, circulating tumor cells represent a necessary step in the process of metastasis5,6, so their relevance to the disease and utility for targeting new drugs is clear. Isolation of CTC from blood in vitro is complicated by their low concentrations: on the order of one per million leukocytes or one per billion erythrocytes7. The majority of current methods for detecting CTC in blood, including the only FDA-approved technique CellSearch(Veridex), damage or destroy cells in the detection process, precluding use beyond enumeration. The method described above does not compromise cell viability and thus opens the door for future clinical research on cancer. As the recovery of intact cells is the goal of this technique, it is imperative that cells be handled with care, especially during blood separation steps.
There are a number of tunable parameters in this system that could be altered to achieve improved yield depending on critical characteristics such as cancer type. In the steps described above, we had chosen to use EpCAM as a CTC-specific antibody for all cancer types except when processing prostate cancer samples, wherein anti-PSMA was used. Further substitutions of cancer-specific antibodies can certainly be used to improve performance for individual patients. Furthermore, selectin and antibody concentrations can be altered to enhance capture8.
An essential feature of the device is the incorporation of E-selectin molecules onto the surface. E-selectin is normally expressed on the luminal surface of endothelial cells and function to recruit fast moving leukocytes to sites of inflammation. Flowing leukocytes bind transiently to selectin molecules, resulting in a slower, rolling behavior that facilitates slower and stronger binding of the cell to the endothelium by integrins. Convincing experimental evidence exists that makes the case that CTC can extravasate by a similar mechanism9,10. The inclusion of selectin also allows the device to be operated at greater flow rates, rates that would otherwise prevent cells from binding to antibodies11.Thus our device physiologically mimics a venule to biomimetically capture flowing CTC without inflicting cellular injury.
Enhanced device performance can be attributed to the addition of the halloysite nanotube coating to the luminal surface of the device. Previous studies have shown that there are three major components of the nanotube coating that allows for improved functionality. First, the nanotube coating provides increased surface area, allowing for greater protein deposition onto the surface4. Second, in performing atomic force microscopy on the nanotube coating we have determined that individual nanotubes protrude from the surface into the flow. This allows selectin molecules to be presented as much as one micron above the surface so that cells can be captured and recruited to the surface earlier in their trajectory through the tube4. Finally, the halloysite nanotube coating is able to prevent leukocyte adhesion and spreading on the surface, allowing for a reduced number of leukocytes captured along with the CTC and thus greater subsequent CTC purities3.

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>300 um ID tubing</td>
 <td>Braintree Scientific</td>
 <td>MRE025</td>
 </tr>
 <tr>
 <td>Larger tubing for connection to syringe</td>
 <td>IDEX Health &amp; Science</td>
 <td>1507L</td>
 </tr>
 <tr>
 <td>5mL syringe for pump</td>
 <td>BD</td>
 <td>309603</td>
 </tr>
 <tr>
 <td>Connector small to large tubing</td>
 <td>IDEX Health &amp; Science</td>
 <td>P-770</td>
 </tr>
 <tr>
 <td>Connector large tubing to syringe</td>
 <td>IDEX Health &amp; Science</td>
 <td>F-120 and P-659</td>
 </tr>
 <tr>
 <td>Syringe for microtube (3/10cc Insulin Syringe U-100 29G 1/2')</td>
 <td>BD</td>
 <td>309301</td>
 </tr>
 <tr>
 <td>Accutase</td>
 <td>MP Biomedicals LLC</td>
 <td>1000449</td>
 </tr>
 <tr>
 <td>Ficol-Paque Plus</td>
 <td>GE Healthcare Bio-Sciences AB</td>
 <td>17-1440-03</td>
 </tr>
 <tr>
 <td>RBC Lysis Buffer (Buffer EL, 1000mL)</td>
 <td>Qiagen</td>
 <td>1014614</td>
 </tr>
 <tr>
 <td>Protein G, 5 mg</td>
 <td>CalBiochem (calbiochem.com)</td>
 <td>539303</td>
 </tr>
 <tr>
 <td>Human EpCAM/TROP-1 Antibody</td>
 <td>R&amp;D Systems</td>
 <td>MAB960</td>
 </tr>
 <tr>
 <td>PSMA Antibody (GCP-04)</td>
 <td>abcam</td>
 <td>ab66911</td>
 </tr>
 <tr>
 <td>96 well plates (Microtest 96)</td>
 <td>BD Falcon</td>
 <td>35-3072</td>
 </tr>
 <tr>
 <td>Blotting Grade Blocker Non-Fat Dry Milk, 300g</td>
 <td>Bio-Rad Laboratories</td>
 <td>216005508</td>
 </tr>
 <tr>
 <td>Halloysite Nanotubes</td>
 <td>NaturalNano</td>
 <td>NN-HNT200</td>
 </tr>
 <tr>
 <td>Calcium Carbonate, 5g</td>
 <td>Aldrich Chemistry</td>
 <td>481807</td>
 </tr>
 <tr>
 <td>rhE-Selectin/Fc Chimera, 100 ug</td>
 <td>R&amp;D Systems</td>
 <td>724-ES</td>
 </tr>
 <tr>
 <td>RPMI Medium 1640</td>
 <td>Gibco</td>
 <td>22400-089</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Gibco</td>
 <td>14190-095</td>
 </tr>
 <tr>
 <td>Poly-L lysine 0.1% w/v in water</td>
 <td>Sigma-Aldrich</td>
 <td>P8920-100ML</td>
 </tr>
 <tr>
 <td>Sonic Dismembrator</td>
 <td>Fisher Scientific</td>
 <td>Model 100</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Atlanta Biochemicals</td>
 <td>S11056H</td>
 </tr>
 <tr>
 <td>Penicillin Streptomycin</td>
 <td>Sigma Life Siiences</td>
 <td>P0781</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples at JoVE.com

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

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 ...

rapid-isolation-viable-circulating-tumor-cells-from-patient-blood

Research

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Bioengineering

26.4K ビュー.  Cornell University. 循環腫瘍細胞は細胞のダメージを与えることなく癌患者の血液から分離されています。腫瘍細胞の単離は上皮マーカーに対する抗体に加えて、E-セレクチンの二分子表面を使用して行われます。ナノチューブのコーティングは、特に高い捕捉純度で得られた癌細胞の接着を促進する。

ビデオ: Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

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...

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Capture and Release of Viable Circulating Tumor Cells from Blood

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Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic...

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

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 ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

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 ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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.

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, ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...