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).

GLS and SL drafted the manuscript. All authors contributed to the protocol development and edited the manuscript. TM, MT, and WHZ are scientific advisors to myriamed GmbH. WHZ is the founder and shareholder of myriamed GmbH.

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 within each passage and prior to their use in ECT generation (Protocol step 3). For culturing primary human CF-monolayers and&#160;-derived ECT in particular, it is advised to use commercial medium and supplements appropriate for CF (see Table of Materials). Medium supplementation with serum is critical to ensure the expansion of CF in standard 2D cultures. Using serum-free or low serum conditions in 3D cultures, including ECT generation and further culture, can be considered depending on the selected fibroblast type. However, when using CF for ECT generation, it is advised to at least include serum in the casting hydrogel for proper initial tissue compaction.
One limitation in the procedure is associated with CF expansion in 2D culture necessary for ECT generation, which typically leads to a conversion of fibroblasts into myofibroblasts (indicated by enhanced SMA and associated stress fiber formation4). Due to their continuous transdifferentiation, consider that fibroblasts in different passages can give different results when used to generate ECT. In the ECT model, two processes need to be discriminated. After suspension in a collagen hydrogel and ECT formation, cells adapt to their 3D environment and the myofibroblast phenotype may be at least partially reversed. In the following culture phase, the cells might then potentially undergo a switch again in the opposite phenotypic direction, especially by using poles with increasing stiffness or by the addition of pro-fibrotic factors (such as TGF-&#946;1). The possibility to tune the dynamic phenotypic adaptation creates the opportunity to dissect the underlying and biomechanically controlled molecular mechanisms. Such studies may ultimately allow for the modeling of fibrotic conditions and the identification&#160;of pharmacological or gene therapy interventions targeting organ fibrosis. The use of fibroblasts of various origins may further allow for the investigation of processes underlying tissue-specific fibrosis. Application of fibroblasts or other stromal cells not only of different origin but also from different species allows for cross-species studies of mechanisms underlying fibrosis or cell-matrix interactions. Nonetheless, it needs to be noted that by using primary cells from humans, the inter-individual differences between the cells must be taken into consideration. A failure in tissue contraction (see also below) is not necessarily a cause of an experimental error but can result from the intrinsic contractile properties of the individual cell line. Therefore, it is always preferred to use cells from different donors to allow for the discrimination of general mechanisms and donor-dependent differences. Similar to the variability of the obtained results, which could arise from the individual biology of the cell, it is important to mention that all biological material can show significant variability. Therefore, parallel testing of the material from different lots is recommended, at least when it becomes necessary to change the lot.
Moreover, tissues grown on the pole pairs exhibit &#34;arm&#34; and &#34;pole&#34; regions that are structurally and biomechanically dissimilar. It remains to be determined how much the pole region contributes to stretch experiments.
The tissue preparation process must be thoroughly fast to avoid gelation at room temperature. Cell-collagen hydrogel gelation is mainly driven by collagen self-assembly, and largely cell-independent29. It is the first step during tissue formation, and it should occur during the first 15-30 min once placed in a culture incubator. Collagen fibrillogenesis and gelation are impacted by, e.g., hydroxylation of prolines and lysines, and highly dependent on collagen type, ionic strength, pH, and temperature, which affects fiber bundling and pore size of the collagen network42. That could ultimately influence the cell component and, thereupon the structure and mechanical properties of the tissues. When choosing collagen sources and the chemical composition of naturally derived collagen, it is important to identify a reliable high-quality collagen solution for tissue engineering. The use of commercial acid-solubilized bovine type I collagen is recommended at an approximate stock concentration of 6-7 mg/mL. Nonetheless, other collagen solutions with a concentration of &#8805; 4 mg/mL may also be compatible with this. Several other factors such as purity, molecular integrity, solubilizing agent, and shelf-age can influence the incubation time necessary for reconstitution (solidification) of the ECT hydrogel mixture, which should under no circumstances exceed 1 h to avoid cell sedimentation. For optimal results, store and handle collagen-containing solutions at 4 &#177; 2 &#176;C. Collagen integrity can be disrupted if frozen or handled at room temperature and consequently prevent fibrillogenesis and hydrogel gelation. After pH neutralization and cell reconstitution in the collagen hydrogel, pipetting during casting must be gentle as strong shear forces may affect the integrity of the collagen structure and matrix assembly. Variability between batches of collagen or different suppliers can have an impact on ECT formation. It is advisable to test collagen hydrogel to ascertain ideal condensation properties before use in ECT preparation. Moreover, to guarantee appropriate pH neutralization, NaOH volume must be titrated for each individual collagen batch.&#160;In general, additional quality controls are recommended, e.g., SDS-PAGE analysis for investigating collagen integrity and concentration and shear rheology to determine the viscous properties of the collagen solution.
After the initial phase of hydrogel solidification due to collagen self-assembly, the cell component drives matrix compaction further. If ECT do not compact visibly within 24 hours after casting, this may be related to cell viability. A minimum of 80&#160;% cell viability is recommended. Ensure proper cell viability after enzymatic detachment of input cells to obtain proper tissue compaction and functionality. In this protocol, ECT are generated with 0.75 &#215; 106 cells in a final volume of 180&#160;&#181;L per tissue, but different cell numbers may be required depending on the source of cells (e.g., CF donor, vendors). Thus, it is recommended to perform a cell titration experiment in the beginning. Typically, a range of cells from 150,000 to 750,000 can be tested for optimal formation and compaction of the tissues. Generally, this protocol uses 0.3 mg of collagen per ECT corresponding to 1.67 mg/mL collagen in a final volume of 180 &#181;L. If necessary, adjust the ratio between cell number and collagen concentration (collagen concentration from 0.14 to 0.4 mg per tissue can be tested). Moreover, ensure a correct neutralization of acetic acid-solubilized collagen during hydrogel preparation as inadequate pH may be detrimental for cell viability.
As shown in Figure 3 (bottom panel), ECT may not form uniformly. After cell reconstitution in the pH-neutralized collagen hydrogel mixture, the gelation process ensues even at 4 &#176;C and is accelerated at room temperature (once cast into the mold). Ensure that the casting procedure is completed within 15-20 min. Premature gelation will impede proper pipetting of the mixture due to the increased viscosity. When casting the viscous cell-collagen hydrogel, pre-wet pipette tip with hydrogel or use a low retention pipette tip, and follow using the same tip to cast multiple ECT. This practice will reduce variation in hydrogel volume and the formation of bubbles during blow-out (Figure 3D). Ensure to complete the loop within the mold to form a ring-shaped ECT (Figure 3A-B). In addition, make sure that the input cell suspension is homogeneous and free of aggregates at all stages of casting. Mix frequently the cell-collagen hydrogen mixture by swirling the tube while carrying out the casting procedure into the 48-well casting plate. Finally, the gelation during incubation at 37 &#176;C should occur maximally within 15-30 minutes. If this process takes longer, the chance of cell sedimentation increases, producing unevenly populated tissues.
Moreover, unevenly populated tissues and uneven distribution of the cell-collagen hydrogel into the molds can lead to irregular morphology of the ECT, and ECT may not contract uniformly throughout the 48-well casting plate. The ECT position on the flexible poles can also influence the contraction levels and contribute to a similar phenomenon. If the forming ECT detaches from the bottom while adding culture medium, it might float and will compact above the anchorage point of the poles with a defined bending force (Figure 4). This may lead to an overestimated pole deflection and induce variability between tissues/experiments. To avoid this, the hydrogel should be carefully overlaid with the culture medium via the well wall.

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.remotexs.ntu.edu.sg/files/ftp_upload/62700/62700fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><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 &amp;ndash; TrypLE Express</td><td>Gibco</td><td>12604013</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) powder, high glucose</td><td>Gibco</td><td>12100061</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;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&amp;nbsp;Fibroblast Growth Basal Medium</td><td>Lonza</td><td>CC-3131</td><td></td></tr><tr><td>FGM-2 Fibroblast Growth Medium-2 SingleQuots,&amp;nbsp;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 &amp;mu;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 &amp;mu;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&amp;Prime; 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. Typically, biomechanical properties are measured at day 5 of culture, but any time point can be selected. As an example of the applicability of the ECT model, it is shown how this protocol can assist in studying the impact of actin cytoskeleton integrity on the tissue function. ECT were prepared in the 48-well casting plate and treated with the actin polymerization inhibitor Latrunculin A (Lat-A, 7 ng/mL). The treatment reduced the ECT compaction as indicated by the significant increase of 1.7-fold in CSA compared to control (Figure 8A,B). Moreover, the contraction of the tissues was assessed during the 5 days of culture. In the absence of the drug, the contraction gradually increased up to day 5, reaching ~40 % contraction. Lat-A affected tissue contraction, resulting in only ~20 % maximum contraction (Figure 8A,C). Destructive unidirectional stress-strain testing was performed on day 5. From a typical stress-strain curve as the ones obtained for ECT (Figure 7B), several biomechanical parameters can be extracted. Exemplarily, it is shown that inhibition of actin polymerization led to a significant reduction of ~50 % in tissue stiffness over the control (Figure 8D). Taken together, the exemplary data show that the actin cytoskeletal integrity is essential for ECT compaction, contraction, and stiffening.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62700/62700fig08.jpg" /> 
Figure 8: Inhibition of the actin polymerization influences ECT compaction, contraction, and stiffness. ECT generated with human CF and collagen type I were cultured for 5 days around two flexible poles in the presence or absence of 7 ng/mL Latrunculin-A (Lat-A). (A) Representative images of the control and treated ECT placed in flexible poles after 5 days are shown. Scale bar = 2 mm. (B) Cross-sectional areas (CSA) were calculated from macroscopic images (n = 22). (C) Pole deflection was calculated over a period of 5 days. Values are given as means&#177;SEM (n = 22). Significant changes were assessed by 2-way ANOVA with Dunnett&#39;s (*p&#60;0.05 vs. Control) post hoc tests for multiple comparisons. (D) Tissues were subjected to rheological destructive tensile measurements and the Young&#39;s moduli were retrieved from the stress-strain analyses (n = 16). (B and D) Boxes indicate the lower and upper quartile. Horizontal line in each box represents the median CSA and stiffness, respectively. The means for each group are indicated by a +. Vertical lines extending from each box represent the minimum and maximum values measured. Significant changes in B and D were assessed by unpaired, two-tailed Student&#39;s t-test (*p&#60;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62700/62700fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.3K Views.  University Medical Center Goettingen. Met dit protocol kunnen de fibroblasten hun eigen omgeving bouwen en organiseren onder gedefinieerde mechanische omstandigheden. Dit resulteert in anisotrope weefsels met stijfheid en matrixsamenstellingen die vergelijkbaar zijn met de natuurlijke omgeving van de cel. Deze methode maakt het mogelijk om tot 48 omstandigheden parallel te vergelijken.Het is flexibel met betrekking tot het gebruikte celtype en de kweekomstandigheden. Door slechts enkele goed gedefinieerde componenten te ge...

Video: Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Developmental Biology

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

Cancer Research

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Engineering

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Medicine

Databases to Efficiently Manage Medium Sized, Low Velocity, Multidimensional Data in Tissue Engineering

Science relies on increasingly complex data sets for progress, but common data management methods such as spreadsheet programs are inadequate for the growing scale and complexity of this information. While database management systems have the potential to rectify these issues, they are not commonly utilized outside of business and informatics fields. Yet, many research labs already generate "medium sized", low velocity, multi-dimensional data that could greatly benefit from implementing similar systems. In this article, we provide a conceptual overview explaining how databases function and the advantages they provide in tissue engineering applications. Structural fibroblast data from individuals with a lamin A/C mutation was used to illustrate examples within a specific experimental context. Examples include visualizing multidimensional data, linking tables in a relational database structure, mapping a semi-automated data pipeline to convert raw data into structured formats, and explaining the underlying syntax of a query. Outcomes from analyzing the data were used to create plots of various arrangements and significance was demonstrated in cell organization in aligned environments between the positive control of Hutchinson-Gilford progeria, a well-known laminopathy, and all other experimental groups. In comparison to spreadsheets, database methods were enormously time efficient, simple to use once set up, allowed for immediate access of original file locations, and increased data rigor. In response to the National Institutes of Health (NIH) emphasis on experimental rigor, it is likely that many scientific fields will eventually adopt databases as common practice due to their strong capability to effectively organize complex data.

Many researchers generate "medium-sized", low-velocity, and multi-dimensional data, which can be managed more efficiently with databases rather than spreadsheets. Here we provide a conceptual overview of databases including visualizing multi-dimensional data, linking tables in relational database structures, mapping semi-automated data pipelines, and using the database to elucidate data meaning.

Science relies on increasingly complex data sets for progress, but common data management methods such as spreadsheet programs are inadequate for the ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

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