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 ...

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