Name: fibroblast derived human engineered connective tissue for screening applications
Uploaded: 2021-08-20T13:00:00
Description: Watch this Scientific Journal Video about Fibroblast Derived Human Engineered Connective Tissue for Screening Applications at JoVE.com

This work was supported by the German Cardiac Society (DGK Research Fellowship for GLS) and by the German Research Foundation (DFG through the project IRTG 1816 for GLS and AD; DFG 417880571 and DFG TI 956/1-1 for MT; SFB 1002 TP C04 for MT and WHZ; SFB 1002 TP S01 for WHZ; and EXC 2067/1-390729940J for WHZ). WHZ is supported by the German Federal Ministry for Science and Education (BMBF through the project IndiHEART), and the Fondation Leducq (20CVD04). MT, WHZ and SL are supported by the German Center for Cardiovascular Research (DZHK).

All steps must be undertaken in a Class II biosafety hoods installed in laboratories under containment level 1. Depending on local regulations and type of manipulations to be performed, such as viral-mediated gene transfer, the containment level must be increased to the biosafety level 2 or 3. All cultures are maintained at 37 &#176;C in a cell culture incubator with a humidified atmosphere of 5 % CO2 in the air. Note that the volumes (Steps 1 and 2) are provided for a T75 cell culture flask. Adjust the volume...

The presented protocol describes the generation of ECT from primary human CF, which allows studying the mechanical impact of these cells on their extracellular matrix environment and vice-versa.
The fibroblasts need to be expanded to yield sufficient cells for the planned ECT experiments (0.75 x 106 cells/ECT). For the best reproducibility, it is advised to pre-culture frozen or tissue-derived fibroblasts in 2D monolayer culture for a standardized duration up to 80 % confluency with...

A major obstacle in the study of fibrotic diseases is the lack of representative human 3D tissue models that provide insight into the behavior of fibroblasts and their pathological derivatives. To study fibrotic processes, standard 2D culture systems are sub-optimal since isolated fibroblasts transdifferentiate rapidly into &#945;-smooth muscle actin (SMA)-expressing myofibroblasts when cultured on non-compliant 2D substrates1,2,3. Thus, fibroblasts in the standard 2D culture do not reflect a regular &#34;healthy&#34; tissue phenotype3,4,5,6. Cultures on pliable substrates have been introduced to simulate non-fibrotic (10 kPa) and fibrotic (35 kPa) tissue environments7, but these lack the third dimension, which is very important with respect to pathophysiology. Tissue engineering provides the opportunity to overcome this limitation by allowing fibroblast culture in a defined and experimentally tunable extracellular matrix (ECM)-context, for example, by alterations in the cellularity, ECM composition, and ECM concentration, all of which can determine the tissue biomechanics.
Various 3D models have been generated using fibroblasts. Floating discs and microspheres were among the first and demonstrate that collagen is remodeled and compacted in a time-dependent manner. Fibroblasts exert traction forces on collagen fibrils, a process which can be facilitated by the addition of pro-fibrotic agents such as transforming growth factor-beta 1 (TGF-&#946;1)8,9,10,11,12,13,14,15,16. However, freely floating cultures do not allow for the controlled external loading and, therefore, constitute continuously shrinking or compacting models. Sheet-like engineered tissues opened the possibility of studying homeostatic regulation of biomechanical properties of tissues, namely through uni, bi, multiaxial, or cyclic strain testing17,18,19,20. These models have been used, e.g., to demonstrate the influence of the cell number on the tissue stiffness, which was found to correlate positively with cytoskeleton integrity and actomyosin cytoskeleton contractility18,19. However, it is important to note that force-to-strain conversions are complicated by the non-uniform tissue deformation around clamp points of force transducers and anchor points. This inherent limitation can be bypassed by dog-bone or ring-shaped tissues, offering some tissue enforcement at anchor-points21,22,23. Ring-shaped tissues can be prepared by distributing a cell-collagen hydrogel into ring-shaped molds. As the hydrogel compacts, a tissue forms around the uncompressible inner rod of the mold, which offers resistance for further tissue contraction24,25,26,27. After initial and typically maximal compaction, tissues may also be transferred to adjustable spacers to further restrain circular ECT at a defined tissue length3,24,25,26,27,28,29,30. Biophysical properties can be assessed in standard horizontal or vertical strain-stress devices with appropriate load cells under unidirectional or dynamic strain3. As the tissues have a largely uniform circular structure and can be held on bars/hooks (anchorage points and/or force transducers), although these may still enclose compression areas around the loading bars, this format allows a more uniform strain variation as compared to clamping3. Furthermore, anchored tissues elicit a bipolar cell shape, and cells adapt to the tissue forces by elongation along force lines promoting anisotropic traction31,32,33,34,35,36. We have previously applied ring-shaped ECT from rat and human cardiac fibroblasts (CF) around a single stiff pole in functional stress-strain experiments and performed gain and loss of function studies by using virally transduced fibroblasts24,25,26 and pharmacological studies37. Further, we could identify sex differences in CF-mediated fibrosis in the ECT model27.
The following protocol for the generation of human ECT, exemplified with primary human CF obtained as cryopreserved CF from commercial vendors (see Table of Materials), combines the advantages of ring-shaped tissues with an easy and fast way of producing macroscopic tissues for a 48-well platform designed for parallel high-content testing.
Importantly, the ECT model is not restricted to a specific fibroblast type, with the documented use in the investigation of other fibroblasts, e.g., skin fibroblasts38,39. Moreover, fibroblasts from patient&#39;s biopsies work equally well, and the choice of fibroblasts ultimately depends on the scientific question to be addressed.
The platform used for the generation of ECT described in this protocol is a commercially available 48-well 3D cell/tissue culture plate (Figure 1A). The methods for the preparation, culturing, and monitoring ECT formation and function under a defined geometry and mechanical load with the help of the 48-well plate are described. The formed ECT are held by integrated flexible poles and the mechanical load can be fine-tuned according to the final purpose by using poles with different hardness (Shore A value 36-89), influencing their bending stiffnesses. Poles with a shore A value of 46 are recommended. The protocol is, in addition, compatible with a previously described custom circular mold, where the ECT is held around a single stiff rod37. The dimensions of this mold are given in Figure 1B.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62700/62700fig01.jpg" /> 
Figure 1:&#160;Schematic representation of casting molds. (A) Technical drawing and dimensions of a casting mold with two flexible poles. The mold comprises an inner circumference delimited by a short wall that holds double retaining poles at the mold&#39;s main body. The flexible poles have a free horizontal distance to one another and are connected at the base. The mold allows for 180 &#181;L casting volume. The well of each mold allows a volume capacity of at least 600 &#181;L of culture media. Different material compositions can be used to produce poles with specific stiffnesses (e.g., TM5MED-TM9MED). (B) Technical drawing and dimensions of a ring-shaped mold with a single stiff rod. This is an alternative mold with distinct geometry and mechanical environment, which can be used with the ECT casting protocol37. The ring-shaped mold assembly method was adapted from published bigger formats28,41. In brief, the method includes (1) imprinting polytetrafluoroethylene (PTFE) molding spacers (8 mm diameter) in polydimethylsiloxane (PDMS, silicone) poured into glass dishes (diameter 60 mm), and (2) fixing a PDMS pole holder (1.5 mm diameter) concentrically inside of the formed hollow cavity, which serves to (3) hold a removable pole (4 mm diameter silicone tube). The hollow space resultant allows for 180 &#181;L of casting volume. Each glass dish can comport multiple imprinted molds (exemplarily shown with 5 molds) and has the capacity for up to 5 mL of culture medium. <a href="https://www.jove.com/files/ftp_upload/62700/62700fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture reagents:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase Solution</td>
			<td>Merk Millipore</td>
			<td>SCR005</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation reagent &#8211; TrypLE Express</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) powder, high glucose</td>
			<td>Gibco</td>
			<td>12100061</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate buffered saline (DPBS), pH 7.2, -Ca2+, -Mg2+</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>FGM-2 Fibroblast Growth Medium-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3132</td>
			<td></td>
		</tr>
		<tr>
			<td>FBM&#160;Fibroblast Growth Basal Medium</td>
			<td>Lonza</td>
			<td>CC-3131</td>
			<td></td>
		</tr>
		<tr>
			<td>FGM-2 Fibroblast Growth Medium-2 SingleQuots,&#160;Supplements and Growth Factors</td>
			<td>Lonza</td>
			<td>CC-4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Medium 3 KIT</td>
			<td>PromoCell</td>
			<td>C-23130</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Basal Medium 3</td>
			<td>PromoCell</td>
			<td>C-23230</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Medium 3 SupplementPack</td>
			<td>PromoCell</td>
			<td>C-39350</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin (10000 U/mL)/ Streptomycin (10000 &#956;L/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution (NaOH) 1.0 N</td>
			<td>Sigma-Aldrich</td>
			<td>S2770-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sources:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal human cardiac fibroblasts from the ventricle (NHCF-V)</td>
			<td>Lonza</td>
			<td>CC-2904</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cardiac Fibroblasts (HCF-c)</td>
			<td>PromoCell</td>
			<td>C-12375</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Cardiac Fibroblasts (HCF-p)</td>
			<td>PromoCell</td>
			<td>C-12377</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human foreskin fibroblasts-1 (HFF-1)</td>
			<td>ATCC</td>
			<td>SCRC- 1041</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen sourses:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I (bovine) in 0.01 M HCl</td>
			<td>LLC Collagen Solutions</td>
			<td>FS22024</td>
			<td>6-7 mg/mL</td>
		</tr>
		<tr>
			<td>Collagen Type I (rat tail) in 0.02 M HCl</td>
			<td>Corning</td>
			<td>354236</td>
			<td>~4 mg/mL</td>
		</tr>
		<tr>
			<td>Drugs:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latrunculin-A (Lat-A)</td>
			<td>Enzo Life Sciences</td>
			<td>BML-T119-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic ware:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plastic ware</td>
			<td>Sarstedt and Starlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh cell strainer (Nylon, pore size 40 &#956;m)</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>myrPlate-uniform</td>
			<td>myriamed GmbH</td>
			<td>TM5 med</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes wide opening, sterile (10 mL)</td>
			<td>Corning</td>
			<td>07-200-619</td>
			<td></td>
		</tr>
		<tr>
			<td>Specific instruments:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bi-telecentric CORE lens for 1/2&#8243; detectors</td>
			<td>OptoEngineering</td>
			<td>TCCR12096</td>
			<td></td>
		</tr>
		<tr>
			<td>Area scan camera Basler ace acA4024</td>
			<td>Basler</td>
			<td>107404</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

ECT reach around 95 % compaction compared to the initial cell-collagen hydrogel volume within the first 24 h. Tissue compaction and contraction under control conditions and in the presence of FCS ensues a few hours after casting and notably increases up to day 5 (Figure 5A). Pole deflection may further increase during the following 15 days (20 days was the longest time tested). The magnitude of pole deflection depends on cell type, cell state, and cell and tissue culture conditions. Typicall...

Watch this Scientific Journal Video about Fibroblast Derived Human Engineered Connective Tissue for Screening Applications at JoVE.com

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

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 ...

This protocol allows the fibroblast build and organize their own environment under defined mechanical conditions. These results in anisotropic tissues with stiffness and matrix compositions comparable to the cell's natural environment. This method allows to compare up to 48 conditions in parallel.It is flexible with respect to the used cell type and culture conditions. By using only a few well-defined components, it is reproducible between laboratories. By applying pro-fibrotic factors or antifibrotic drugs, this protocol can be used to study the underlying processes and therapies of fibrotic diseases of any kind.Demonstrating the procedure will be Alisa Degrave, a PhD student from our laboratory, and myself. To begin with, thaw the cryo-preserved cardiac fibroblasts in a water bath at 37 degrees Celsius for approximately two minutes until the vial is left with only a small amount of ice. Next, transfer the cell suspension dropwise using a two milliliter serological pipette into an appropriate sterile centrifuge tube containing 10 milliliters of fibroblast growth medium.For optimal cell retrieval, rinse the cryo-vial with one milliliter of FGM and transfer it to the centrifuge tube. Gently resuspend the cells and transfer into a cell culture flask. Replenish FGM every other day for five days or until the cells have reached 80%confluency.After aspirating the medium from the cultured cells, wash cells with six milliliters of PBS and aspirate, then add six milliliters of the cell dissociation reagent to the cells and incubate until the cell detachment is visible. Neutralize enzymatic activity by adding six to 12 milliliters of FGM to the dislodged cells in the cell association reagent. Gently pipette up and down four to eight times using a 10 milliliter serological pipette to ensure a single-cell suspension and transfer the cells into a fresh 50 milliliter collection tube.Verify the yield with the help of an automated cell counter as per the manufacturer's instructions and pellet down the cells. After centrifugation, aspirate the supernatant, flick the tube to dislodge the pellet and resuspend the cells in FGM. Next, strain the cell suspension through a 40 micrometer mesh cell strainer, then using an automated cell counter, assess cell number and viability by electric current exclusion to ensure reliable cell numbers before proceeding with engineered connective tissues or ECT preparation.To prepare for ECT, adjust the cell suspension to the 8.9 million cells per milliliter at 20 to 25 degrees Celsius by adding FGM and keep the cells on ice. Transfer all the other tubes containing the individual components of ECT hydrogel mixture onto ice and pre-chill an empty 50 milliliter centrifuge tube to prepare the mixture. Start preparing the ECT hydrogel mixture by adding components described in the text to the pre-chilled 50 milliliter centrifuge tube avoiding air bubble formation.Firstly, pipette the acid soluble collagen type I hydrogel into a serological pipette with a wide bore tip. Adjust the salt content of the collagen solution by adding the DMEM while gently swirling the tube for proper mixing. To neutralize the pH, add 0.2 molar sodium hydroxide while swirling the tube and confirm the neutralization by observing the red color of the phenol red indicator.Then add the cell suspension dropwise while gently swirling the tube. Mix the suspension by gently piping up and down only once using a serological pipette with a wide bore tip to avoid bubble formation and minimize the shear stress. Gently swirl the tube 10 times for thorough mixing.Keep the centrifuge tube containing ECT hydrogel mixture on ice throughout the casting process. Wet a one milliliter pipette tip in ECT hydrogel mixture, then distribute 180 microliters of hydrogel mixture evenly into each mold of the 48-well casting plate avoiding excessive shear forces that may affect the integrity of the collagen matrix assembly and ensuring that the entire plate finishes in 15 to 20 minutes. Ensure that a complete loop forms within the mold as discontinuous distribution of ECT mixture will prevent a complete ECT ring formation.Avoid pipetting into the inner well and forming bubbles during pipetting to ensure a uniform ECT hydrogel casting for homogeneous and functional tissue formation. Carefully place the 48-well casting plate inside the cell culture incubator to reconstitute the ECT hydrogel mixture for 15 to 30 minutes. After incubation, it appears gel-like and opaque.Add 600 microliters of 37 degrees Celsius warm FGM per well along the wall gently to avoid ECT detachment from the bottom. Replace the medium every day with 500 microliters of FGM until analysis. At the desired time points, use a stereo microscope to record microscopic images of the top and side views of the ECT.Use an image processing program to perform a line scan analysis. Set a scale and use the straight line tool to trace and measure the ECT diameters at a minimum of six positions per arm in each imaging plane. Image the 48-well casting plate under a recording device with an integrated area scan camera placed at a fixed distance equipped with a high-resolution monochrome image sensor and a near-UV light source.Perform automated detection of the poles'tips containing fluorescent dye for maximizing the contrast. Measure the distance between the poles from daily records using an automated analysis by running the recorded images on software to detect high-contrast bright pixels on a dark background or an image processing program. Remove the ECT from retaining poles and insert a transferring hook and ECT loop.Then transfer the ECT onto two hooks clamped to the stationary arm and the transducer arm of an extensional dynamic mechanical riometer equipped with a 37 degree Celsius tempered organ bath filled with PBS. Apply uniaxial tension at a constant linear rate by setting the riometer at approximately 1%of the initial distance between the hooks per second. A constant stretching rate of 0.03 millimeters per second can be used with typical ECT dimensions.Tear the force of the transducer and initiate the stretch. Keep recording until the point of ECT rupture after normalizing the measured force by cross-sectional area and plot the stress-strain curve determining different biomechanical parameters. Just before the tissue starts micro fracturing, the upper limit of the elastic region corresponds to the yield point and its strain is a measure of tissue elasticity.The plastic region is in between the yield point and the failure point. The failure point corresponds to a sudden drop in the stress due to rupture of the tissue, defining the ultimate strain which is a measure of tissue extensibility. The third measuring point corresponds to the maximum strength defined by the highest stress that the tissue can bear without breaking during the stretch.The resilience and toughness given by the area under the curve corresponds to the energy absorbed by the tissue up to the yield point and failure point respectively. For each obtained curve, the slope of the linear part of the elastic region corresponds to Young's modulus, also known as elastic modulus. It is a mechanical property that measures the stiffness of the tissue.Using this protocol, ECT compaction and contraction under control conditions and in the presence of FCS ensued a few hours after casting and notably increased up to day five. When ECT was treated with the actin polymerization inhibitor Latrunculin A, the ECT compaction was reduced as indicated by the significantly higher cross-section area compared to control. The contraction of the tissues was assessed during the five days of culture.In the absence of Latrunculin A, the contraction gradually increased up to day five reaching about 40%contraction. However, Latrunculin A presence affected the tissue contraction resulting in only about 20%maximum contraction. Actin polymerization inhibition led to a significant reduction of about 50%in the tissue stiffness over the control.These results demonstrate that the actin cytoskeletal integrity is essential for ECT compaction, contraction, and stiffening. It is important to select a reliable, high-quality collagen solution and ensure that a single-cell suspension with high viability is used to reconstitute engineered connective tissues.

fibroblast-derived-human-engineered-connective-tissue-for-screening

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...