This work was funded by US Army Grant (W81XWH-08-1-0701) and a Fellowship from Carle Foundation Hospital.

Procedure is in full compatibility with IACUC protocol number 13302 and has been approved by the division of animal resources.
1. Instrument Preparation
<ol>
	<li>Autoclave all non-disposable instruments used prior to the surgical procedure to ensure sterility.</li>
	<li>Sterilize instruments used multiple times in a session with a glass bead sterilizer between uses.</li>
</ol>
2. Hydrogel Preparation
<ol>
	<li>Thaw and plate stem cells at least 24 hr prior to creation of the hydrogel construct.</li>
	<li>Prepare patches one day prior to the surgical procedure using a stereolithography apparatus (or similar instrument) which has been described in full detail in previous publications8,18.</li>
	<li>To create the hydrogel, first generate a computer aided design (CAD) model of the desired patch and export the design to stereolithographic format (STL).</li>
	<li>Create a pre-gel solution by dissolving poly(ethylene glycol) dimethacrylate (PEGDMA) in sterile 1x PBS to obtain a 20% w/v solution. Concurrently, dissolve the photoinitiator 1-[4-(2-hydroxy- ethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one in DMSO. Add the photoinitiator to the PEGDMA solution at a final w/v of 0.5%. Directly before crosslinking, add 100 ml of the desired density of cells. Note: A cell density between 2.0 x 105-2.0 x 106 cells/ml&#160;is recommended.</li>
	<li>Pipette the pre-gel solution into a dish at the center of the SLA platform and run the SLA software with the previously uploaded STL formatted design.</li>
	<li>Remove the patches from the apparatus and incubate the hydrogels O/N in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 U/ml&#160;penicillin, and 100 mg/ml&#160;streptomycin to allow for cells to adjust to the environment within the patch.</li>
</ol>
3. Animal Preparation and Oral intubation
<ol>
	<li>Evaluate each mouse prior to the procedure to ensure that the animal is in good health, as determined from their grooming habits and interactions with their cage mates. Note: Female 8-10&#160;week&#160;old C57BL/6J mice were used for this procedure, however, any mouse age or type can be used.</li>
	<li>Place animals into an anesthetic chamber and expose to 5% isofluorane with a 1 L/min O2 support. Monitor the level of anesthesia by toe pinch reflex.</li>
	<li>Once the animal is properly anesthetized, weigh and place them on the intubation stand.</li>
	<li>Direct a light source towards the animal&#8217;s chest cavity and use forceps to retract the laryngeal surface and expose the vocal cords. Carefully guide a 20 G angiocath between the vocal cords, and smoothly insert into the trachea. Detect proper insertion by a mechanical movement of the chest cavity once the catheter is connected to the small animal ventilator.</li>
	<li>Adjust ventilation settings to the weight of the animal based on the manufacturer&#8217;s guidelines.</li>
	<li>Once intubated, place the animal in the supine position on a heating pad to prevent hypothermia. Ensure that the heating pad does not exceed 40 &#176;C. If necessary, place a barrier between the animal and the heating pad to minimize the chance of burns.</li>
	<li>Remove hair from the surgical site through the use of a depilatory cream. Sterilize the site by 3 alternating scrubs of betadine and 75% ethanol to ensure proper sterilization. Place a drape to expose just the surgical site, and place vet ointment on the eyes to prevent dryness during the procedure.</li>
</ol>
4. Surgical Procedure
<ol>
	<li>The thoracotomy is performed on the left side of the animal, therefore, make a skin incision approximately 1 cm to the left of the sternum, that runs the length of the sternum.</li>
	<li>Use forceps to separate the muscle layer from the underlying ribcage in two steps. Observe a delineating line that represents the pectoralis major muscles of the animal. Lift this muscle slightly, separate it from the underlying external oblique muscle and retract medially. Then, free the external oblique muscle from the underlying ribcage in the same manner, and retract laterally, providing an unobscured view of the second, third, and fourth ribs.</li>
	<li>Perform the thoracotomy between the third and fourth ribs. Gently lift the fourth rib and use a cauterizer to open the chest cavity between the third and fourth ribs. Place retractors to further open the cavity and expose the heart.</li>
	<li>Use forceps to rupture the thin pericardium of the heart.</li>
	<li>Ligate the left coronary artery with an 8-0 monofilament nylon suture. Place the suture approximately 4 mm from the apex of the heart, directly below the bottom tip of the left atrium. Determine proper suture placement by a blanching of the ventricular myocardium and an increase in the size of the left atrium after the suture has been tied.</li>
</ol>
5.&#160;Patch Placement and Glue
<ol>
	<li>Keep patches at 37 &#176;C and 5% CO2 conditions until use.</li>
	<li>Gently lift the patch using a flat ended spatula and gently place it on the surface of the heart, at the site of infarction. 
	Note: To prevent slipping off the left ventricle, the patch can be held in place by lightly maintaining contact using either the spatula or the tip of a pair of forceps.</li>
	<li>Prepare the glue by mixing a 4:1 ratio of fibrinogen to thrombin. Mix the solution by repeated pipetting until it begins to thicken, usually within 1 min after the beginning of the preparation.</li>
	<li>Once the solution viscosity reaches the desired level, quickly transfer approximately 10 ml&#160;to the patch surface. Note: The clotting time of the fibrin glue is rapid, providing a small window of opportunity for efficient transfer of the glue.</li>
</ol>
6. Suturing
<ol>
	<li>Perform all closing sutures using a 6-0 monofilament nylon suture.</li>
	<li>Close the rib layer with three to four individual interrupted sutures.</li>
	<li>Before full closure of the intercostal layer, insert a PE-10 cannula into the incision to evacuate the chest cavity after closure is complete and reestablish proper intrapleural pressure.</li>
	<li>Close the pectoral muscles with three to four individual interrupted sutures.</li>
	<li>Seal the skin layer with a continuous suture.</li>
	<li>Following full closure, attach a 1.0 ml syringe to the end of the cannula and use it to evacuate the chest cavity.</li>
	<li>Apply a tissue adhesive to the incision site to reinforce the suture site.</li>
</ol>
7. Post-surgical Treatment
<ol>
	<li>Give mice an injection of buprenorphine (0.05-1.0 mg/kg) and carprofen (2.2 mg/kg) subcutaneously immediately following the coronary ligation and least 20 min&#160;prior to revival. Administer both drugs 6-8 hr after surgery, and then give twice daily for up to three days to control pain and distress.</li>
	<li>Continuously monitor the animals until conscious and every hour for the first 4 hr following surgery. Perform a second checkup the night of the surgery (2-4 hr later) to administer a second dose of analgesic.</li>
	<li>Monitor the animals daily after the second day following the procedure, until animals appear stable.</li>
</ol>
8. Analysis of Heart Function and Histology
<ol>
	<li>Perform echocardiographs on the mice 4 weeks following the procedure to determine the extent of damage following the MI.</li>
	<li>Anesthetize mice with 4-5% isoflurane within an anesthesia chamber for induction of inhalation anesthesia, and then 2% isoflurane via face mask for maintenance anesthesia.</li>
	<li>Clear the imaging area with a depilatory cream and use a small animal ultrasound to obtain a 2D M-mode echocardiography scan of the beating heart and record the functional parameters associated with it.</li>
	<li>Euthanize the mice in a carbon dioxide chamber, then collect the hearts from the animals and place in a 10% formalin solution for 24 hr. After 24 hr, transfer the hearts to 70% ethanol and keep at 4 &#176;C until tissue processing.</li>
	<li>Process the hearts and embed them in paraffin blocks.</li>
	<li>Cut 5 mm sections through the ventricular region of the heart, starting at the apex and ending at the atrium.</li>
	<li>Stain sectioned slices with both Masson&#8217;s trichrome and hematoxylin and eosin stain using standard protocols.</li>
</ol>

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The authors declare that they have no competing financial interests.

With this approach to the murine MI model, we have developed a system that minimizes the damage to non-myocardium areas that are associated with other murine MI techniques. These areas include damage caused by a tracheostomy, the cutting of the muscular layer, and the breakage of ribs to expose the chest cavity. We believe that these changes improve the overall surgical outcome thanks to the care taken to keep as much of the major structures, including the ribs and muscle layers, intact during the surgical procedure.
It should be noted that there are some limitations to this technique, with the largest being its time-consuming nature, resulting in only 8-10 full surgical procedures in one day. To overcome this, a second ventilator can be used, allowing for the procedure to be performed on two animals concurrently. Additionally, major vessels, such as the intercostal vessels and interior thoracic artery, should be avoided when performing the thoracotomy. If near one of these vessels, a cauterizer should be used to prevent bleeding out due to a ruptured artery.
Fibrin glue is known in both the laboratory and clinical settings thanks to its ability to quickly aid in clot formation, as well as its potential to deliver and host MSCs14-17,19. For our purposes, fibrin glue served as a biocompatible approach to adhering a water-based hydrogel construct to the surface of the heart. The use of a fibrin-based glue has proven successful in keeping the hydrogel construct at the damaged site for up to 8 weeks, with the possibility for tissue contact of a longer amount of time. The use of this adhesion system allowed control for placement of the delicate construct without the need for additional sutures that harm the myocardium as well as the construct itself. We have found that the glue itself is non-toxic to the encapsulated cells and does not negatively affect the underlying tissue. The hydrogel and fibrin-based glue system can be applied to a variety of other tissue engineering purposes that desire cell or therapeutic contact for an extended period of time. Furthermore, with the use of the SLA fabrication method, designs and structures may be incorporated within the patch to create a more complex system for cell delivery18.
One setback to the use of the fibrin-based glue is the fact that more than one researcher must be present in the surgical room during patch placement and glue addition. It is the job of the first researcher to make sure the patch remains in place and does not get lost in the chest cavity, while the second researcher mixes the solution and applies it to the patch after proper viscosity is reached. To optimize the timing of the glue delivery, it is suggested that the second researcher also tests and times the gelation of the glue prior to patch placement, since proper gelation of the glue is critical for patch adhesion.
There are a number of critical steps and error prone areas within the protocol, with the first being the induction of the myocardial infarction. It is imperative to check for signs, such as the blanching of the left ventricular wall and the enlargement of the left atrium, to ensure occlusion of the left coronary artery. This can be done prior to the addition of the stitch by simply crossing and tightening the end of the suture strings. If it is determined that the left coronary artery is not enclosed by the suture, the string can be taken out and re-applied. A second critical measure is to continue to monitor the intubation tube throughout the procedure, to ensure that it remains within the trachea and does not get pulled out mid-surgery. Finally, throughout the surgery, all attempts should be made to avoid contact with the lungs, as any contact has the potential to lead to a collapsed lung. If, during the surgery, lung collapsing occurs, simply block the outflow tube of the intubator for up to 3 sec in an attempt to re-inflate troubled regions of the lungs.

A myocardial infarction (MI) is defined as the interruption of blood to a region of the heart caused by the occlusion of a major coronary artery. The damage resulting from an MI is due to the remodeling of the viable heart tissue into non-functional scar tissue, which decreases the ability of the heart or, more specifically, the left ventricle, to beat properly. This results in a decrease in the volume of blood that can be delivered to the body with every heartbeat, known as the stroke volume, and the percentage of blood that is pumped out of the heart with each heartbeat, known as the ejection fraction6. These, along with other diminished functions, increases the strain on the rest of the heart to maintain adequate function. Often, this increased strain can become so severe that it causes a second heart attack, a phenomenon seen in approximately 10% of individuals7.
While medical practices have evolved to treat the immediate aftermath of an MI, no technique has been developed to halt, slow, or reverse the negative side effects of tissue remodeling. Stem cell therapies have emerged as a possible avenue for such a treatment, however, despite their promising potential, stem cells have not proven successful in the clinical setting. One theory for their shortcomings is the inability to ensure the beneficial cells remain at the site of infarction long enough to generate favorable results5. It has been shown that no more than 24% of cells that are simply injected into the site of infarction survived and remained at the damaged site 1 day post-delivery2. A possible prospect for addressing this issue of cell retention is to develop biocompatible hydrogel systems that encapsulate either cells or therapeutics, which can be delivered to the damaged site. The hydrogel of choice in this protocol is a poly(ethylene glycol) dimethacrylate due to its previous use in cell encapsulation procedures, however, any hydrogel capable of encapsulation may be used8. The delivery of the patch directly to the site of injury ensures cell-to-tissue contact over an extended period of time, increasing the length of time the cells can provide beneficial factors to the underlying myocardium.
A bottleneck to the patch approach is the difficulty of adhering the patch to the heart surface. Many groups have overcome this through a variety of techniques, the most prevalent being a simple suture to tie the construct to the heart surface9,10. This has proven successful in a number of cases in which the construct is made of a stiffer material, but fails when attempted on a hydrogel system, due to the high water concentration and delicate nature of the patch construct. To overcome this, we have utilized a fibrin glue external adhesive system that mimics the chemistry of clot formation. Fibrin glue has been used in numerous medical surgeries, including dura tears, bronchial fistulas, and corneal transplantation, highlighting the biocompatibility of the product as a wound sealant11-13. Additionally, fibrin has been used for a variety of cardiac purposes, including surgical treatment of left ventricular ruptures and coronary artery bypass surgeries, however, its use as an adhesion glue for a cardiac patch is not commonly used14-17. A simple formulation of thrombin and fibrinogen results in a biocompatible glue that can be placed directly on the outside of an external cardiac patch, providing a viable adhesion system to ensure patch to heart interaction.

<table><tbody><tr><td>Harvard Model 687 Mouse Ventilator</td><td>Harvard Apparatus</td><td>55-0001</td><td></td></tr><tr><td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td><td>Fisher Scientific</td><td>NC9531961</td><td></td></tr><tr><td>Leica MZ6 surgical microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Cautery Kit</td><td>Gemini</td><td>GEM 5917</td><td></td></tr><tr><td>Delicate Forceps - 0.4 mm Tips Angled</td><td>Fine Science Tools</td><td>11063-07</td><td></td></tr><tr><td>Agricola Retractor - 3.5 cm Spread</td><td>Fine Science Tools</td><td>17005-04</td><td></td></tr><tr><td>Spring Scissors - 2.5 mm Blades Straight</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Iris Scissors - Delicate Straight 10.5 cm</td><td>Fine Science Tools</td><td>14060-10</td><td></td></tr><tr><td>8-0 monofilament suture</td><td>Ethicon</td><td>8730P</td><td></td></tr><tr><td>6-0 Silk suture</td><td>Ethicon</td><td>639G</td><td></td></tr><tr><td>Thrombin</td><td>Sigma</td><td>T7009</td><td></td></tr><tr><td>Fibrinogen</td><td>Sigma</td><td>F3879</td><td></td></tr><tr><td>Vetbond Tissue Adhesive</td><td>3M</td><td>1469SB</td><td></td></tr></tbody></table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

During the surgical procedure, ligation of the left coronary artery can be identified by a marked blanching downstream of the occluded artery. As a test before tying the knot, the suture can be tightened briefly to check if it is in the appropriate place. Additionally, since occlusion of the artery results in almost instantaneous decrease in the ability of the left atrium to properly beat, the left atrium will enlarge in response to a backflow of blood in the system.
M-mode echocardiography measurements taken as early as 2 days post-infarction show a cessation of left wall movement, indicative of the muscle reconstruction. Qualitative calculations made from the data show a decrease in ejection fraction and stroke volume in the infarcted hearts. At the termination of the experiment, when the hearts are collected for histological purposes, a clear dilation of the left ventricle can be seen, along with a thinning of the left ventricular wall, and the deposition of collagen that denotes scar tissue deposition in place of functioning cardiomyocytes (Figure 1).
Also seen upon histological analysis is the presence of the hydrogel construct thanks to the administration of the fibrin gel (Figure 2). For our purposes, the adhesive system must be viscous enough to allow precise placement control and minimize subsequent runoff into surrounding organs, but malleable enough not to interfere with heart function. Preliminary tests were performed to calculate the viscosity, gelation time, and stiffness of various fibrinogen/thrombin ratios, in order to determine the proper combination that suits our needs. In vivo analysis was performed to test the ability of the fibrin glue to maintain patch-to-tissue adhesion while still allowing for full heart function (data not shown). It should be noted that the fibrin glue did not harm the myocardium, as evidenced by the lack of tissue remodeling or ventricular thinning at the site (Figure 2). The cardiomyocytes remain intact despite the addition of both a cardiac patch and its accompanying glue. Additionally, viability testing confirmed that that the administration of fibrin glue to the external surface of a cell-encapsulating hydrogel patch did not affect cell survival within the patch (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/52562/52562fig1.jpg" /> 
Figure 1: Left Coronary Artery Ligation Results in a Decrease in Cardiac Function as Measured by Echocardiography. M-mode echocardiography of healthy (a) and infarcted (b) hearts. Infarcted hearts show a clear lack of wall movement where viable tissue has been replaced with scar tissue. This correlates with a measureable decrease in left ventricular ejection fraction (EF), cardiac stroke volume (SV), and cardiac output (CO). Histological analysis of healthy (c) and infarcted (d) hearts shows dilation of the left ventricle and thinning of the left ventricular wall of the infarcted myocardium, both signs of tissue remodeling and scar tissue deposition. Histological images are cross sectional slices capturing the left (larger chamber) and right (smaller chamber) ventricles.
<img alt="Figure 2" src="/files/ftp_upload/52562/52562fig2.jpg" /> 
Figure 2: Application of Fibrin Flue Ensures Patch Adhesion to the Heart. The patch is placed on the surface of the heart while the chest cavity is exposed during the procedure (a). Once placed, a fibrin-based glue is added on top of the heart and allowed to dry. The patch can clearly be seen on histological sections of heart tissue that were collected at 1 week post-procedure (b). Dissociation of the patch from the heart surface in histological images is an artifact of the collection and sectioning process. Results are consistent for up to 8 weeks of adhesion.
<img alt="Figure 3" src="/files/ftp_upload/52562/52562fig3.jpg" /> 
Figure 3: Fibrin-based Glue Does Not Harm Encapsulated Tissues. Cell viability was measured via MTT analysis 1 week after the administration of a fibrin-based glue using protocols described in detail in previous publications8. The viability of cells within the patch was not affected by the addition of the glue to the surface of the construct.

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

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Universidad San Sebastian

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JoVE Journal

Medicine

8.6K Views.  University of Illinois at Urbana-Champaign. This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Video: A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Bioengineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering 

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented.&#160;Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area.&#160;LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10%&#160;post-operative mortality.&#160;

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

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Rozdziały w tym wideo

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

LAD-Ligation: A Murine Model of Myocardial Infarction

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

3D Whole-heart Myocardial Tissue Analysis

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells