Name: leveraging turbidity and thromboelastography for complementary clot characterization
Uploaded: 2020-06-04T15:00:00
Description: Watch this Scientific Journal Video about Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization at JoVE.com

1. Preparation of phosphate buffer saline (PBS)
NOTE: PBS was used throughout this study as the described assays did not require the addition of calcium. It is important to note that when adding calcium, often utilized to re-calcify citrated blood products, PBS should be avoided as calcium is known to precipitate in phosphate buffers.
<ol>
	<li>Make a 0.01 M, pH 7.4 PBS buffer by mixing 137 mM sodium chloride, 1.8 mM potassium phosphate monobasic, 10 mM sodium phosphate dibasic and 2.7 mM po...

<ol>
	<li>Beckman, M. G., Hooper, W. C., Critchley, S. E., Ortel, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venous+Thromboembolism.+A+Public+Health+Concern.">Venous Thromboembolism. A Public Health Concern.</a> American Journal of Preventive Medicine. 38, 495-501 (2010).</li><li>Goldhaber, S. Z., Bounameaux, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+embolism+and+deep+vein+thrombosis.">Pulmonary embolism and deep vein thrombosis.</a> The Lancet. 379 (9828), 1835-1846 (2012).</li><li>Weisel, J. W., Litvinov, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+fibrin+polymerization+and+clinical+implications.">Mechanisms of fibrin polymerization and clinical implications.</a> Blood. 121 (10), 1712-1719 (2013).</li><li>Weisel, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+assembly.+Lateral+aggregation+and+the+role+of+the+two+pairs+of+fibrinopeptides.">Fibrin assembly. Lateral aggregation and the role of the two pairs of fibrinopeptides.</a> Biophysical Journal. 50 (6), 1079-1093 (1986).</li><li>Tripathi, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+evaluation+of+whole+blood+prothrombin+time+(PT)+and+international+normalized+ratio+(INR)+using+a+Laser+Speckle+Rheology+sensor.">Clinical evaluation of whole blood prothrombin time (PT) and international normalized ratio (INR) using a Laser Speckle Rheology sensor.</a> Scientific Reports. 7 (1), 1-8 (2017).</li><li>Ryan, E. A., Mockros, L. F., Weisel, J. W., Lorand, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+origins+of+fibrin+clot+rheology.">Structural origins of fibrin clot rheology.</a> Biophysical Journal. 77 (5), 2813-2826 (1999).</li><li>Carr, M. E., Hermans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+Density+of+Fibrin+Fibers+from+Turbidity.">Size and Density of Fibrin Fibers from Turbidity.</a> Macromolecules. 11 (1), 46-50 (1978).</li><li>Carr, M. E., Shen, L. L., Hermans, J. A. N., Chapel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass-Length Ratio of Fibrin Fibers from Gel Permeation and Light Scattering. 16, 1-15 (1977).</li><li>Gabriel, D. A., Muga, K., Boothroyd, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Effect of Fibrin Structure on Fibrinolysis. , 24259-24263 (1992).</li><li>Wolberg, A. S., Gabriel, D. A., Hoffman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+fibrin+clot+structure+using+a+microplate+reader.">Analyzing fibrin clot structure using a microplate reader.</a> Blood Coagulation and Fibrinolysis. 13 (6), 533-539 (2002).</li><li>da Luz, L. T., Nascimento, B., Rizoli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombelastography+(TEG):+practical+considerations+on+its+clinical+use+in+trauma+resuscitation.">Thrombelastography (TEG): practical considerations on its clinical use in trauma resuscitation.</a> Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine. 21 (1), 29 (2013).</li><li>Whitten, C. W., Greilich, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thromboelastography:+past,+present,+and+future+.">Thromboelastography: past, present, and future .</a> Anesthesiology: The Journal of the American Society of Anesthesiologists. 92 (5), 1223-1225 (2000).</li><li>Ranucci, M., Laddomada, T., Ranucci, M., Baryshnikova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+viscosity+during+coagulation+at+different+shear+rates.">Blood viscosity during coagulation at different shear rates.</a> Physiological Reports. 2 (7), 1-7 (2014).</li><li>Zeng, Z., Fagnon, M., Nallan Chakravarthula, T., Alves, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+clot+formation+under+diverse+clotting+conditions:+Comparing+turbidimetry+and+thromboelastography.">Fibrin clot formation under diverse clotting conditions: Comparing turbidimetry and thromboelastography.</a> Thrombosis Research. 187, 48-55 (2020).</li></ol>

This protocol demonstrates the utilization of two distinct clot characterization tools testing a simplified fibrinogen/thrombin clot model using commercially available components. Both TEG and turbidity assays are easy to conduct. They not only provide end point clot examinations such as max clot formation (TurbMax and TEGMax) and clot formation times (TurbTime and TEGTime) but also assess the dynamic clot forming process. This makes TEG and turbidity valuable tools for clot ch...

Thrombosis is the pathological formation of a blood clot in the body that blocks blood circulation leading to high morbidity and mortality worldwide. There are 1 to 2 cases of venous thromboembolism and 2 to 3 cases of thrombosis-induced vascular diseases per 1000 people annually1,2. Presented here is a method leveraging thromboelastography (TEG)&#160;and turbidity to monitor clot formation under various clotting conditions. Fibrin(ogen) is the primary protein that is responsible for clot formation in the body. In the final steps of the coagulation cascade, fibrinopeptides are cleaved from fibrinogen by thrombin initiating the polymerization of insoluble fibrin monomers as the clot develops3,4. To understand clot formation in pathological thrombosis, it is necessary to characterize fibrin formation under diverse clotting circumstances. Multiple clot monitoring assays have been utilized to study fibrin clot formation in vitro. Prothrombin time (PT/INR) and activated partial thromboplastin time (aPTT) are two common clinical assays that measure the integrity of a specific coagulation pathway. However, they use time as the only variable that gives no indication of physical clot properties5. Electron microscopy allows visualization of the micro-structure of a completely formed fibrin clot but provides no information about the clot forming process itself6. Among all assays, turbidity assays and TEG offer the ability to track clot characteristics dynamically over time. These techniques enable the measure of comprehensive clotting profiles and therefore, provide some benefit over other fibrin clot characterization tools.
Specifically, turbidity assays (or clot turbidimetry) is widely used for research and clinical applications due to its simplistic implementation and the wide accessibility of spectrometers in research laboratories. This assay allows a dynamic measurement of light transmittance through a forming clot by taking individual repetitive readings at a defined wavelength (most commonly at a wavelength in the range of 350 &#8211; 700 nm)7. Temperature in the reading chamber can also be adjusted. As fibrin gel forms, the amount of light that travels through the protein network is reduced causing an increase in absorbance over time. Similarly, absorbance decreases when the clot network degrades. Turbidity assays can easily be multiplexed using a multi-well plate format to allow for high throughput sample screening in both 96- and 384-well plates. Several clot characteristics can be derived from a turbidity tracing curve (absorbance over time measurement) that include: maximum turbidity, time to maximum turbidity, time to clot onset, and clot formation rate (Vmax). A fibrin fiber mass/length ratio can also be derived from raw turbidity data to estimate fibrin fiber thickness8,9,10.
TEG is primarily utilized in the clinical setting to assess patients&#8217; hemostasis and clot lysis. It is also commonly used in surgical applications to determine when anti-fibrinolytic drugs or hemostatic blood products should be administered11,12. Clot formation occurs inside a TEG cup with all the clotting components being added to the cup prior to the initiation of the assay. The cup, with evolving clot, physically rotates against a pin that is inserted into its center and an electromechanical torsion sensor measures the increasing viscoelastic strength of the clot. This assay is typically carried out at the physiological temperature of 37 &#176;C; however, the temperature can be manually adjusted on the instrument. Maximum amplitude (MA), reaction rate (R), kinetics time (K), &#945;-angle (Angle), and time to maximum amplitude (TMA) are extracted by the TEG software from the dynamic TEG tracing. These values are typically compared with clinical normal ranges to assess a patient&#8217;s coagulation state. While TEG is not precisely a viscometer, as it measures clot strength in millimeter units, it does provide important viscoelastic clot data and functions as a valuable clinical decision making tool for physicians to decide to administer specific blood products and adjust therapeutic dosing13. When both TEG and turbidity assays are utilized together, they&#160;provide&#160;complementary clot characterization information as clot strength and kinetics are easily extracted from TEG and fibrin fiber thickness can be accessed by optical turbidity measurements.
As fibrin is a critical component of a blood clot, fibrin clot characterization under diverse clot formation conditions can provide valuable insight into how a specific variable contributes to the clot formation process and ultimate clot properties. Understanding this can provide guidance for thrombosis diagnosis and the development of therapeutics. To obtain a more representative fibrin clot characterization, plasma can be substituted to monitor clot formation as it resembles in vivo clotting conditions more closely than a simplified fibrinogen/thrombin model system. However, due to the intricate nature of the coagulation cascade, clot formation using plasma adds to the complexity, making it more difficult to isolate the impact of individual factors. Utilizing a simplified fibrinogen/thrombin model prevents the need to initiate the entire clotting cascade allowing for isolation of the final fibrin formation step. By including two major fibrin forming components (fibrinogen and thrombin), this setup creates a highly controlled clot formation condition. It is also important to note that while the simplified clot model is used here, this protocol can also be utilized to characterize more complex clots by including additional clotting factors. In this study, fibrin clot characterization using turbidity and TEG are carried out by varying fibrinogen and thrombin concentrations, ionic strength, pH, and total protein concentration in the clotting solution to mimic different in vivo clotting circumstances14. Details regarding these variations to the protocol have been included in Section 5.

<table>
	<tbody>
		<tr>
			<td>96-Well Clear Flat Bottom UV-Transparent Microplate</td>
			<td>Corning</td>
			<td>3635</td>
			<td>Non-treated acrylic copolymer, non-sterile</td>
		</tr>
		<tr>
			<td>Albumin from human serum</td>
			<td>Millipore Sigma</td>
			<td>A1653</td>
			<td>&#8805;96%, lyophilized powder</td>
		</tr>
		<tr>
			<td>Arium Mini Plus Ultrapure Water System</td>
			<td>Sartorius</td>
			<td>NA</td>
			<td>DI water source</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td>&#8805;96%, lyophilized powder</td>
		</tr>
		<tr>
			<td>Disposable Cups and Pins for TEG 5000 (Clear)</td>
			<td>Haemonetics</td>
			<td>REF 6211</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen, Bovine Plasma</td>
			<td>Millipore Sigma</td>
			<td>341573</td>
			<td>contains more than 95% clottable protein</td>
		</tr>
		<tr>
			<td>Fibrinogen, Plasmingogen-Depleted, Human Plasma</td>
			<td>Millipore Sigma</td>
			<td>341578</td>
			<td>Contains &#8805; 95% clottable proteins.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Millipore Sigma</td>
			<td>P3813</td>
			<td>Powder, pH 7.4, for preparing 1 L solutions</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Millipore Sigma</td>
			<td>60130</td>
			<td>&#8805;99.5% purity</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Millipore Sigma</td>
			<td>P9791</td>
			<td>&#8805;98% purity</td>
		</tr>
		<tr>
			<td>SevenEasy pH Meter</td>
			<td>Mettler Toledo</td>
			<td>S20</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>71378</td>
			<td>&#8805;99.5% purity</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Millipore Sigma</td>
			<td>71636</td>
			<td>&#8805;99.5% purity</td>
		</tr>
		<tr>
			<td>SpectraMax M5 multi-detection microplate reader system</td>
			<td>Molecular Devices</td>
			<td>M5</td>
			<td></td>
		</tr>
		<tr>
			<td>TEG 5000 Thrombelastograph Hemostasis analyzer system</td>
			<td>Haemonetics</td>
			<td>07-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin, Bovine</td>
			<td>Millipore Sigma</td>
			<td>605157</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin, Human Plasma, High Activity</td>
			<td>Millipore Sigma</td>
			<td>605195</td>
			<td></td>
		</tr>
	</tbody>
</table>

leveraging turbidity and thromboelastography for complementary clot characterization

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, characterizing fibrin clot formation is beneficial to the study of thrombosis. Turbidity and thromboelastography (TEG) are both widely utilized in vitro assays for monitoring clot formation. Turbidity dynamically measures the light transmittance through a fibrin clot structure via a spectrometer and is often used in research laboratories. TEG is a specialized viscoelastic technique that directly measures blood clot strength and is primarily utilized in clinical settings to assess patients&#39; hemostasis. With the help of these two tools, this study describes a method for characterizing an in vitro fibrin clot using a simplified fibrinogen/thrombin clot model. Data trends across both techniques were compared under various clotting conditions. Human and bovine fibrin clots were formed side-by-side in this study as bovine clotting factors are often used as substitutes to human clotting factors in clinical and research settings. Results demonstrate that TEG and turbidity track clot formation via two distinct methods and when utilized together provide complementary clot strength and fiber structural information across diverse clotting conditions.

The experiments shown in Figure 1 are representative turbidity tracing curves of human and bovine fibrin clots at different fibrinogen levels. Representative TEG tracing curves for fibrin clot formation at different fibrinogen levels are shown in Figure 2. Both tracing curves demonstrate that after a lag period following clot initiation, clot turbidity or clot amplitude increases over time and levels off at the end of clot formation. An endpoint value of maximum...

Watch this Scientific Journal Video about Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization at JoVE.com

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Thrombosis is a leading cause of death worldwide. Fibrin(ogen) is the protein primarily responsible for clot formation or&#160;thrombosis. Therefore, ...

Thromboelastography and turbidity assays are two simple and distinctive methods for clot characterization. These techniques together offer a more comprehensive understanding of how clotting variables impact clot features. Both of this assays, not only offer endpoint clot analysis but also attract fibrin clot formation over time, unlike many other clot characterization tools These techniques can help develop a physiologically relevant synthetic clot platform that can provide reliable diagnosis of the fibrinolytic state of thrombosis patients To monitor clot turbidity over time, use any commercially available spectrometer that has an absorbance range of 350 to 700 nanometers.Turn on the spectrometer and open the corresponding analysis software. Then select plate one and open the plate settings tab. Click ABS and kinetic to monitor a dynamic absorbance over time.Select 550 nanometers in the wavelength tab, then switch to the timing tab and adjust the total runtime to 60 minutes with an interval of 30 seconds, select the wells of interest by highlighting them. Pipette 140 microliters of PBS into one well of a UV transparent 96 well plate. Then add 10 microliters of thrombin and mix, immediately initiate clotting by adding 50 microliters of fibrinogen to the well and pipette up and down five times using only the first stop of the pipette taking care to avoid creating bubbles.Place the plate in the plate holder and click read in the software to start the turbidity reading. When the read has finished retrieve the turbidity data and obtain a turbidity tracing curve by plotting the absorbents change over time, derive maximum turbidity by taking the max absorbance value of the curve over time, then calculate 90%maximum turbidity by multiplying maximum turbidity by 0.9. Derive the time to maximum turbidity by computing the time from clot initiation to 90%maximum turbidity.Turn on the thromboelastography or TEG analyzer, and wait for the temperature to stabilize at 37 degrees Celsius, then open the TEG software and create an experiment name under the ID section. Click the TEG tab and follow the onscreen prompts to conduct an eTest test for all channels. Then place the lever back to the load position once all checks are complete.Click done in the information page and input sample information for channels that will be used. Place a clear uncoated TEG cup in its corresponding channel. Slide the carrier up to the top and press the cup bottom five times to a fix the pin to the torsion rod then lower the carrier and press the cup downward into the base until it clicks.Pipette 20 microliters of thrombin solution into the TEG cup, then initiate clotting by adding 340 microliters of fibrinogen into the cup to obtain a 360 microliter clotting solution. Mix the contents of the cup by pipetting up and down five times. Slide the cup loaded carrier up move the lever to the read position and click start in the software to initiate the TEG reading.Once the reading is completed retrieve the parameters and obtain a TEG tracing curve by plotting amplitude over time. Elect MA as maximum amplitude, which is indicative of clot strain and TMA as time to maximum amplitude from the software. Representative turbidity and a TEG tracing curves of human and bovine fibrin clots at different fibrinogen levels are shown here.The tracing curves demonstrate that after a lag period following clot initiation, clot turbidity or clot amplitude increases over time and levels off at the end of clot formation. Maximum turbidity and time to maximum turbidity are the two parameters derived from turbidity, while maximum amplitude and time to maximum amplitude are derived from TEG. At a higher level of fibrinogen in the clotting solution all four values increase.Maximum turbidity is an optical measure of clot structure, which is indicative of fibrin fiber thickness and fibrin network density. While maximum amplitude is a mechanical measure that reflects absolute clot strain, taken together the two values provide complimentary insight about the clot micro structures. This procedure demonstrates a simplified clotting model to examine variables that mainly affect fibrin polymerization.This model can be customized by inclusion of additional clotting factors to study other parameters. The clotting model can be easily modified using clinical plasma samples to monitor clot formation and disillusion in the presence of antithrombotic drugs. Results could guide therapeutic management in patients.

leveraging-turbidity-thromboelastography-for-complementary-clot

Research

JoVE Journal

Medicine

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...