The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.

This protocol follows the LUMC and AMC guidelines of human research ethics committee.
1. Surgical Procedure and Tissue Collection
Note: Although various techniques for surgical excision of Dupuytren&#8217;s contracture exist, the current gold standard is the partial fasciectomy 15. Most patients are treated in the day-surgery clinic.
<ol>
	<li>Perform surgery under general or regional anesthesia according to patient&#39;s and anaesthesiologist&#39;s preferences and to patient&#8217;s co-morbidities. These considerations are beyond the scope of this paper. Prior to incision, touch the skin with a sharp object to verify that anesthesia is satisfactory. Use a tourniquet to facilitate surgical visualization in a bloodless field.</li>
	<li>After sterile prepping and draping, mark out the anticipated incision with a pen. Incise the skin with a scalpel under loupe magnification either longitudinally or using zigzag incisions to prevent additional contractures and also to allow for sufficient exposure.</li>
	<li>Using scissor dissection, free the skin and subcutaneous layer from the underlying contracted fascia palmaris. Identify and protect the neurovascular bundle of the finger because the diseased cord can displace it toward the midline. Once the fibrotic tissue (nodule and/or cord) is outlined, excise it sharply and regionally (subtotal) using a scalpel. Perform meticulous hemostasis under tourniquet control at the end of the procedure to prevent hematoma formation.</li>
	<li>To close the skin, use additional Z-plasties to obtain partial lengthening of the skin.</li>
	<li>For these studies use only the nodule part of the fibrotic cord, the most active and cellular part of the disease. Have the surgeon perform macroscopic identification. The isolated nodules in the palm of the hand are often precursors or a cord but are hardly ever resected, as they usually do not cause symptoms. The ones we resected were the nodules that were part of a cord of the contracted finger. Identify these nodules by the hard thick part of the cords in the handpalm (Figure 1A).</li>
	<li>Once the surgeon removes the tissue, immediately transfer it using sterile forceps to a 50 ml tube containing DMEM medium supplemented with 10% FCS and 1% (P/S). Keep the tissue for a maximum of 2 hr in this medium on a box of wet ice (4 &#176;C) (e.g., transportation of the tissue from the operation room to the cell culture facility).</li>
	<li>Keep the samples on wet ice (4 &#176;C) while preparing for the next step. Preparation of the materials and cell culture chamber requires 10-20 min.</li>
</ol>
2. Preparation of Instruments, Culture Medium and Culture Inserts
Note: All procedures are performed at RT&#160;unless specifically stated.
<ol>
	<li>Prepare 500 ml of DMEM supplemented with 10% FCS and 1% (P/S) and filter sterilize. Prewarm the medium at 37 &#176;C.</li>
	<li>Autoclave forceps, a pair of curved-bladed Mayo scissors (150-170 mm in length) and a pair of Metzenbaum scissors and store in a sterile container.</li>
	<li>Under laminar flow, open a sterile 12-well culture plate. Place one culture insert (0.45 &#181;m pore size, 12 mm diameter) in each well of the 12-well plate (Figure 1B, lower panel).</li>
	<li>Fill the 12-well plate with 600 &#181;l of prewarmed (37 &#176;C) culture media by pipetting on the well and not directly on the membrane of the insert. Place the plate into the incubator (37 &#176;C, 5% CO2) (Figure 1B, upper panel).
	<ol>
		<li>Alternatively, use a 24-well plate and add 300 &#181;l of medium per well. The volume of medium per well is optimal when a meniscus layer is formed between the liquid and the lower part of the membrane insert. 
		Note: the medium at the bottom of the well diffuses through the nitrocellulose membrane forming the meniscus layer as depicted in schemes of Figure 1B (lower panel).</li>
	</ol>
	</li>
</ol>
3. Tissue Preparation and Ex Vivo Tissue Culture Setup
Note: All procedures are performed at RT unless specifically stated.
<ol>
	<li>Transfer the nodule part of the cord from the 50 ml tube on a 100 mm Petri dish and add 10 ml of prewarmed (37 &#176;C) medium. Ensure that the tissue is in contact with the liquid during the entire procedure.
	<ol>
		<li>Remove the perinodular fat layer using the Metzenbaum scissors. Discard the fat tissue. With the use of forceps and the curved-bladed scissors, cut transversally the tissue into smaller pieces (maximum thickness of 200 &#181;m).</li>
	</ol>
	</li>
	<li>Transfer the 12-well plate from the incubator into the laminar hood. Check that the membrane of the insert has turned transparent (wet) and therefore is in contact with the medium.</li>
	<li>Using forceps carefully lift a tissue part by touching the outer layer (do not apply tension on the tissue). Place one tissue part onto the membrane of the cell culture insert so that the tissue remains in the air-medium interphase. Culture a maximum of two tissue parts on the same insert/ well.</li>
	<li>Ensure that the tissue is placed flat on the membrane, in longitudinal contact with the medium such that the tissue neither floats off the membrane nor is fully covered by medium. Avoid air bubbles. 
	Note: At this stage, it is possible to add growth factors or inhibitors of interest to the media; for instance, test compounds (A, B, C, D, E) in replicates and/or in concentration curve. Include at least 2 control (untreated) pieces of tissue under normal growth conditions to ensure that the experiment has been set up properly. Incubate tissues for 3-7 days in a tissue culture incubator.</li>
	<li>Infect the tissue with either lentiviral or adenoviral construct if overexpression or knock down experiments of a gene-of-interest need to be performed (Figure 2). Transfer a tissue part from the 12-well plate into a 35 mm dish using forceps.
	<ol>
		<li>Use a maximum volume of 10 &#181;l of high titer virus.</li>
		<li>Perform the injection under a dissection microscope with an insulin syringe loaded with 50 &#181;l PBS containing 10 &#181;l of the selected virus. Place the needle perpendicular to the center of the tissue.</li>
		<li>Slowly inject the content of the syringe. Do not retract the needle directly. When puncturing the tissue, do not let the needle go through the other side but maintain the needle within the tissue itself (around the center of the tissue).</li>
		<li>Transfer the tissue back into the 12-well plate, close the culture plate and place it into the incubator (37 &#176;C, 5% CO2).</li>
	</ol>
	</li>
</ol>
4. Whole-mount Immunofluorescence Staining and 3D Reconstruction
Note: All procedures are performed at RT unless specifically stated. The protocol for whole mount immunostaining and imaging described below is an adaptation from previously reported methods used for other tissues 16-19. Buffers and materials are described in Table 1 and Materials List.
<ol>
	<li>Transfer tissues from the culture plate to 2 ml microcentrifuge tubes containing PBS. Wash 2x for 5 min in PBS solution on a rotating platform. Fix tissues in 4% buffered PFA (1.5 ml solution per tissue in 2 ml microcentrifuge tubes), O/N at 4 &#176;C on a rotating platform.</li>
	<li>Remove PFA and wash 3x in PBS for 5 min each.</li>
	<li>Dehydrate tissues by immersion in a 25% methanol solution for 2 hr at RT on a rotating platform. Increase the methanol percentage gradually (50%, 75%, 100%) with the tissues immersed at each solution for 2 hr at RT on a rotating platform.</li>
	<li>Transfer tissues in DMSO/methanol solution (permeabilization step) for 3 weeks at 4 &#176;C, as described previously 18. 
	Note: At this stage, tissues can be stored long term in 100% methanol at -20 &#176;C.</li>
	<li>Proceed with staining procedure 16, 17; gradually rehydrate tissues (75%-50%-25%-0% methanol series). Perform each rehydration step for 2 hr at RT or O/N at 4 &#176;C, under constant agitation.</li>
	<li>Incubate samples with primary antibodies in PBS containing 1% bovine serum albumin (BSA) and 20% DMSO O/N at 4 &#176;C. Use antibodies at the following ratios using the commercial stocks in PBS: anti-&#945; smooth muscle actin (1:500, mouse), anti-collagen type I (1:500, goat), anti-collagen type III (1:500, goat).</li>
	<li>Wash extensively in PBS for 48 hr at 4 &#176;C (change PBS solution 3 to 4 times).</li>
	<li>Dilute appropriate secondary antibodies (Alexa 555 anti-mouse, Alexa 488 anti-goat) at 1:250 dilution in PBS containing 1% (BSA) and 20% DMSO. Incubate O/N at 4 &#176;C.</li>
	<li>Wash extensively in PBS for 48 hr at 4 &#176;C and incubate with nuclear counterstain, TO-PRO-3 diluted 1:500 in PBS at the final washing step. 
	Note: TO-PRO-3 is a far-red emitting DNA dye (with a He-Ne 633 nm laser line) and is combined for simultaneous imaging with other channels; in this case collagen type I / collagen type III (488 nm), &#945; smooth muscle actin (555 nm), TO-PRO-3 (647 nm).</li>
	<li>Transfer the tissues to a methanol series (25%-50%-75%) to slowly dehydrate the tissue; perform each dehydration step for 2 hr at RT or O/N at 4 &#176;C under constant agitation.
	<ol>
		<li>Transfer tissues in polypropylene tubes. Clear tissues in methylsalicylate (handle under chemical hood) 18 or as an alternative use BABB solution 19. Incubate at RT under constant agitation until the tissues become transparent. Mount between a slide and a coverslip sealed with hard set mounting medium (Figure 1C, upper panel).</li>
	</ol>
	</li>
	<li>Image samples on an inverted confocal microscope equipped with high NA 63X and/or 100X oil immersion objectives (Figure 1C).
	<ol>
		<li>Clean the lens with cleaning solution provided by the microscope manufacturer.</li>
		<li>Apply one drop of the oil on the objective lens. Place the slide with the specimen on the microscope stage and focus on the sample.</li>
	</ol>
	</li>
	<li>Set the configurations on the microscope for simultaneous imaging of three-channel fluorescence with laser lines 488 nm, 514 nm and 633 nm.</li>
	<li>Acquire single XY images or multiple focal plane images (Z stack) (Figure 3A). Use increased width of Z stack for performing 3D reconstruction. To obtain a high-resolution image use low scanning speed, number of average scans (&#8805; 3) and acquire 16-bit images of 1,024 x 1,024 pixel resolution.</li>
	<li>Analyze the Z stack acquired images: first rename and save image file (xyz). Using the software program of the confocal microscope, configure a maximum intensity projection of the Z stack images into a 3D reconstructed image.
	<ol>
		<li>Select the xyz file, in the &#8220;Process Tools&#8221;, click under &#8220;Visualisation&#8221; and &#8220;3D Projection&#8221;. Enter &#8220;Maximum&#8221; in the method of projection (or &#8220;Average&#8221; if the fluorescent signal is saturated). For a single projection do not change the X, Y, and Z planes, and apply the projection tool (Figure 3B, r1).</li>
	</ol>
	</li>
	<li>Use the Z stack images to create a 3D projection with animation (confocal software) for viewing purposes. Select the xyz file, in the Process Tools, click under &#8220;Visualisation&#8221; and &#8220;3D Projection&#8221;.
	<ol>
		<li>Click on &#8220;Create Movie&#8221;. Enter the Start Rotation angle in degrees (e.g., 0&#176; as starting point of the movie, Y axis) and click on &#8220;Set Start&#8221;. Enter the End Rotation angle in degrees (Y axis) as end point of the movie (e.g., 180&#176; or 360&#176; for a complete rotation) and click on &#8220;Set End&#8221;.</li>
		<li>Select &#8220;Maximum&#8221; as a method of projection (or &#8220;Average&#8221; if the fluorescent signal is saturated). Enter the &#8221;Number of Frames&#8221; for the rotation of the 3D reconstruction (e.g.,&#160;20 rotations or higher to reduce the speed of rotation). Click on &#8220;Apply&#8221;. Export the movie as visual file (e.g., &#8220;avi&#8221;) (Supplementary&#160;Movie 1) or export as &#8220;tiff&#8221; file which creates an image file for each rotation angle (Figure 3B; rotation r4, r10, r18).</li>
	</ol>
	</li>
</ol>
5. Combined Second Harmonic Generation (Collagen) and Two-photon Excited Fluorescence (Elastin) Imaging on Ex Vivo Tissue during Culture
Note: All procedures are performed at RT unless specifically stated.
<ol>
	<li>Remove transwell plates from incubator and place a single (unfixed) tissue part in a 35 mm cell culture plate containing 500 &#181;l of medium.</li>
	<li>Position the tissue so that the long axis is flat on the plate. Keep tissue wet at all times however, not floating.</li>
	<li>Place water-immersed objective of the multiphoton microscope directly in contact with the tissue (Figure 1C).</li>
	<li>Obtain images with an excitation wavelength of 800 nm and collect emitted light between 371-425 nm (SHG, collagen) and 474-532 nm (autofluorescence, elastin) (Figure 3C). Perform two-photon microscopy in an upright confocal microscope equipped with a femtosecond laser using a 20X/1.0 NA water-immersion objective. Process confocal stacks with manufacturer&#8217;s software. 
	Note: Excitation with a near infrared laser of collagen molecules creates high contrast imaging of native collagen structures in different depths.</li>
</ol>

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The authors have filed a Patent Application for Dupuytren&#8217;s disease: 3D organ culture. # GB1307200. Commercial license and service contracts are available.

The most critical steps of culturing ex vivo human connective tissue are the immediate use of the tissue after surgical removal to ensure viability; the tissue should remain in medium or saline solution at all times; maintain a sterile culture; transverse sections of tissue should have maximum 200 &#181;m thickness; set up of the ex vivo culture system is optimal when tissue is in contact with the medium but not fully submerged. Medium should be added only on the outside chamber of the transwell in a sm...

Dupuytren&#39;s disease (DD), a benign fibroproliferative disease causes permanent flexion of the fingers due to the formation of nodules and cords in the palm of the hand. Although the disease spread is particularly high among Caucasians of Northern Europe, the underlying genetic etiology of the disease remains unknown 1. The main characteristic of DD is the excess production of extracellular matrix (ECM) proteins (e.g., collagen), which form a tough fibrous tissue occupying the space between the tendons and skin of the palm of the hand and fingers, permanently disrupting the fine movements of the hand 2, 3. The recurrence of the disease suggests underlying genetic alterations as a cause of fibrosis 1, 4. An effective treatment could be to target directly the uncontrollable fibrotic mechanisms at the cellular and molecular level.
Our recent work on fibrosis has led us to the development of a novel 3D culture system that allows short-term culture of human fibrotic tissue with the potential of drug testing. This system has helped to overcome the limiting approach of 2D fibroblast cultures and to define a role for the partial down regulation, achieved by exon skipping, of TGF&#946; pathway activation in mediating fibrosis 5.
We have developed a method to culture ex vivo human resection specimens from DD patients to study the interaction between myofibroblasts and the surrounding ECM 5, 6. The study of DD connective tissue fibrosis as well as other fibrotic diseases relies on histopathological analysis of the excised surgical specimens, isolation of fibroblasts from the tissue and establishment of primary cultures or cell sorting procedures. These approaches are quite static since they do not permit exogenous manipulation of the disease properties or therapeutic intervention by the experimenter. In addition, primary cell cultures tend to adapt to the culture conditions and their gene expression properties differ essentially from the in vivo situation upon every passage, even during early passages (among passage 3 and 6) 7, 8. We have managed to maintain the waste surgical material in ex vivo culture conditions for a time period that allows study of the patient-specific characteristics and screening of anti-fibrotic or anti-inflammatory drug compounds.
The system is based on a nitrocellulose membrane that permits contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue upon attachment, as previously observed when culturing DD fibroblasts as well as other cell types 9. No collagen gel or other ECM protein substrate is required, since the DD tissue itself produces large amounts of these proteins. This is advantageous for the maintenance of native ECM microenvironment and turnover since matrix substrates are important regulator of tissue architecture and function 10, 11. For instance, ECM proteins such as fibronectin, laminin and collagen, may influence front-rear polarity of fibroblasts as similarly shown for apical-basal polarity in epithelial cells 12, 13. Polarized cells have asymmetrical distribution of extracellular molecules which determines cell migration and gene expression, e.g., &#945;1&#946;1 integrin accessibility on the membrane affects cell adhesion to type I collagen14. Since a primary goal of this 3D model was to preserve the native microenvironment, no artificial ECM matrix substrate was used.
In brief: resection specimens are equally cut in a sterile environment and placed on nitrocellular membranes. If treatment administrated via injection is required the tissues are injected after they have been placed on the membrane. If treatment does not require to be administrated via injection then the compound is added to the culture media (Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM), with 1% fetal calf serum (FCS), 1% penicillin-streptomycin (P/S)). The cultures are maintained for a maximum of ten days after which the tissue is fixed in 4% paraformaldehyde (PFA), processed through 30% sucrose solution, embedded in O.C.T. compound and stored at -80 &#176;C, as previously described 5.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium</td><td>Invitrogen</td><td>11965-084</td><td></td></tr><tr><td>fetal calf serum (FCS)</td><td>Gibco</td><td></td><td>high glucose, heat inactivated</td></tr><tr><td>penicillin-streptomycin</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Cell culture inserts</td><td>Millicell</td><td>PIHA01250</td><td>0.45 &amp;#956;m pore size, 12&amp;nbsp;mm diameter</td></tr><tr><td>anti-collagen type I</td><td>Southern Biotech</td><td>1310-08</td><td></td></tr><tr><td>anti-&amp;alpha; smooth muscle actin</td><td>Sigma</td><td>A2547</td><td></td></tr><tr><td>anti-collagen type III</td><td>Southern Biotech</td><td>1330-01</td><td></td></tr><tr><td>Alexa Fluor555 Donkey Anti-Mouse IgG (H+L)</td><td>Invitrogen</td><td>A-31570</td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody</td><td>Invitrogen</td><td>A-11055</td><td></td></tr><tr><td>TOPRO-3</td><td>Invitrogen</td><td>T3605</td><td></td></tr><tr><td>methylsalicylate</td><td>Sigma</td><td>M6752</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Tissue Tek OCT</td><td>Sakura</td><td>25608-930</td><td></td></tr><tr><td>Microsccope glass coverslips</td><td>Menzel-Glaser</td><td>BB024060A1</td><td>24 x 60 mm</td></tr><tr><td>Microscope SuperFrost slides</td><td>Menzel-Glaser</td><td>AA00000102E</td><td>76 x 26 mm</td></tr><tr><td>VECTASHIELD HardSet Mounting Medium</td><td>Vector laboratories</td><td>H-1400</td><td></td></tr><tr><td>Leica TCS SP5 II confocal microscope</td><td>Leica Microsystems</td><td></td><td>Argon-488, 514 nm and HeNe-633 nm laser lines</td></tr><tr><td>Zeiss 710 NLO upright confocal microscope</td><td>Jena, Germany</td><td></td><td>Equipped with femtosecond Spectra - Physics Deep See MP laser (Santa Clara, United States) using a Plan-Apochromat 20X/1.0 NA water-immersion objective.</td></tr></tbody></table>

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.

The method of ex vivo, 3D culture of connective tissue is an easy and robust set up system to understand the relation between ECM and other cell components constituting the DD tissue and potentially other types of fibrosis as well. Moreover this system allows a reliable method to test the effects of compounds on different cell types and their effects on the fibrotic load 20.
The steps from collection of surgical waste material derived from Dupuytren&#8217;s fibrosis, the as...

Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

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.

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 ...

human-dupuytren-s-ex-vivo-culture-for-study-myofibroblasts

Research

JoVE Journal

Medicine

10.4K Views.  Leiden University Medical Center.  De ziekte van Dupuytren (DD) is een fibroproliferatieve ziekte van de palm van de hand. Hier presenteren we een protocol om de cultuur resectiepreparaten van DD in een driedimensionale (3D) cultuur systeem. Een dergelijke tijdelijke kweeksysteem maakt behoud van de 3D structuur en moleculaire eigenschappen van het fibrotische weefsel. 

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

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Genetics

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Bioengineering

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.

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Biology

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.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

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

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