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

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JoVE Journal

Bioengineering

3.2K visualizações.  University Medical Center Goettingen. Este protocolo permite que o fibroblasto construa e organize seu próprio ambiente em condições mecânicas definidas. Estes resultam em tecidos anisotrópicos com rigidez e composições matriciais comparáveis ao ambiente natural da célula. Este método permite comparar até 48 condições em paralelo.É flexível em relação ao tipo de célula usada e às condições de cultura. Usando apenas alguns componentes bem definidos, é reprodutível entre laboratórios. Aplicando fatore...