The authors thank Prof Hendrik Tevaearai and the Department of Cardiovascular Surgery for financial support for the initial part of the study.

NOTE: Lewis Male and Female rats, 200-220 g were housed under standard laboratory conditions (12 hr light and dark cycle, ad libitum water and food, IVC cage). All animals were treated in compliance with the recommendations of the FELASA and the Swiss Law on animal protection.
1. Cell Preparation: Mesenchymal Stem Cell Isolation from Bone Marrow
<ol>
	<li>Anesthetize the rats with isoflurane 5% and O2 5 L/min in an induction chamber for 5 min. Place the animal&#8217;s snout into a nose cone connected to the anesthesia system. Perform a toe or tail pinch to confirm sedation.</li>
	<li>Remove the skin from the leg with scissors by cutting the skin from ankle to the hip. Remove the muscles and cut the femoral artery for animal exsanguination. Expose the hip joint and dislocate the femur head. It is important not to damage the femur head. Place the bones in sterile PBS in a 50 ml plastic tube.</li>
	<li>Remove all muscles and ligaments from the bones without breaking them. NOTE: It is important that the bone remains intact to avoid linkage of flushing solution or contamination with ethanol. Rinse the peeled bones in 70% ethanol for 5 min.</li>
	<li>Under the sterile laminar flow hood, cut both tips of the bones with scissors. Flush the bone marrow by injecting sterile PBS from the bone extremity. Collect marrow into a 15 ml tube. Centrifuge 7 min at 300 x g.</li>
	<li>Remove the supernatant. Suspend the pellet in 3 ml of Red Blood Cell Lysis buffer. Incubate the suspension for 1 min at room temperature before spinning again for 7 min at 300 x g. In a 150 ml culture flask, add sterile cell culture medium (395 ml IMDM medium, 5 ml pen/strep, 100 ml FBS) and seed the cells. </li>
	<li>Change medium the second day to remove nonadherent cells.
	<ol>
		<li>Perform medium changes every second day for 2-3 weeks until desired cell quantity is obtained.</li>
		<li>Alternatively, prepare the biograft by seeding the cells onto the solid matrix as needed and described elsewhere3.</li>
	</ol>
	</li>
	<li>The day of cardiac implantation, prepare the cells or harvest the biograft just before epicardial application.
	<ol>
		<li>Collect the cells using Accutase cell detachment solution. Count the cells. Fill a 1.5 ml centrifuge tube with the volume of cell suspension calculated to obtain desired cell quantity. Centrifuge for 7 min at 300 x g and remove the supernatant.</li>
		<li>Alternatively, harvest the biograft from cell culture medium, rinse in sterile PBS, and keep in fresh culture medium without serum.</li>
	</ol>
	</li>
</ol>
2. First Thoracotomy and LAD Ligation
<ol>
	<li>Weigh the rat. Turn on the heating pad at 37 &#176;C. Anesthetize the rat with 5% isoflurane and 5 L/min 100% O2 in an induction chamber for 5-7 min. Perform a toe or tail pinch to confirm sedation. Place the rat on the heating pad.</li>
	<li>Intubate the animal with a 14 G i.v. catheter. Connect the intubation catheter to a rodent ventilator programmed for 2.5 L/min oxygen, 2.5% isoflurane, tidal volume of 2 ml, and a breathing frequency of 90 breaths/min.</li>
	<li>Prepare a buprenorphine solution at 0.1 mg/kg. Inject subcutaneously one third of the solution. Shave the left part of the thorax. Disinfect with a 1% Betadine solution.</li>
	<li>Incise the skin perpendicular to the sternum at the fourth intercostal space. Separate the 3 layers of thoracic muscle (pectoralis major, ascending pectoralis, and external oblique pectoralis). Open the fourth intercostal space (between ribs 4 and 5).</li>
	<li>Use a small retractor to spread the ribs and to expose the heart. Open the pericardium carefully. Locate and ligate the left anterior descending coronary artery (LAD) with a 7.0 suture 4 mm below the atrium.</li>
	<li>Close the intercostal space with two stitches using 3.0 sutures. Position the two sutures proximally and distally from the sternum. First, tighten the distal suture. Clamp the ventilator exhaust tubing for 2 sec in order to inflate the chest and avoid any pneumothorax. Tighten the second suture. Position the muscles layers back in place (no sutures are needed). Close the skin with a 5.0 suture.</li>
	<li>Disinfect the suture with 1% Betadine solution. Inject the rest of the buprenorphine solution. Turn off the anesthesia system. Remove intubation catheter.</li>
	<li>Keep the rat in the cage under a warm Lamp for 1-2 hr. Keep a thermometer in the cage and control the distance between the warm lamp and the cage in order to avoid over heating. Return the rat in the IVC unit. Inject subcutaneously Buprenorphine 0.05-0.1 mg/kg post &#8211;operatively every 6-12 hr for 48 hr.</li>
</ol>
3. Epicardial Administration of the Treatment Via a Second Thoracotomy
<ol>
	<li>Repeat steps 2.1-2.3.</li>
	<li>Incise the skin perpendicular to the sternum at the fifth intercostal space. Separate the 3 layers of thoracic muscle (pectoralis major, ascending pectoralis and external oblique pectoralis). Open the fifth intercostal space (between ribs 5 and 6).</li>
	<li>Use a small retractor to spread the ribs and to expose the heart. If there is some adherence, carefully remove them with fine forceps.</li>
	<li>Locate the infarct area that appears as a pale area below the ligature. NOTE: When necessary, use a 10 cm piece of suture (7.0) inserted at the apex to better visualize the left ventricle. Pull gently to the suture maintained loosely by a clamp to expose the heart.</li>
	<li>Apply one of the following treatments:
	<ol>
		<li>Position the biograft at the surface of the heart. Use the prefilled fibrin sealant syringe prewarmed at room temperature for 10 min. Apply a drop (50-100 &#956;l) of fibrin sealant under the biograft using a 25 G Luer needle. Verify that the biograft is perfectly sealed.</li>
		<li>Alternatively, apply the cell pellet collected with a pipette tip at the surface of the heart onto the pale area below the ligature. Apply a drop (50-100 &#956;l) of fibrin sealant onto the cell pellet.</li>
	</ol>
	</li>
	<li>Remove the piece of suture from the apex.</li>
	<li>Repeat steps 2.6-2.8.</li>
</ol>

<ol>
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The authors have nothing to disclose.

The permanent ligation of LAD causes irreversible myocardial injury. The first animal model was described in 19604. Since then, it has been considered as a standard and suitable model for chronic MI. Its stability and reproducibility allowed experimental evaluation of therapies for MI5. Improved procedures following the initial description reported an operative mortality rate of 35-13%6.
As expected, LAD ligation induced impaired heart function observed within ...

Since the end of the 19th century, cardiovascular disease has remained the number one serial killer in industrialized countries. Among them, coronary artery disease represents the main etiology. The acute phase results in myocardial infarction (MI) and is followed by maladaptive remodeling that progressively develops toward a chronic phase and severe heart failure. Despite recent significant technological and therapeutic advances, the morbidity and mortality due to the progression of heart failure is still growing1. In this context, cell therapy has gained increasing interest as a new therapeutic option to stop the progression of the disease toward heart failure and to stimulate the recently identified regenerative capacity of the myocardium. Experimental and clinical investigations have provided compelling evidence of the beneficial effects obtained after cardiac transplantation of various cell types. Major outcomes included improved cardiac contractile function, decreased left ventricular remodeling, reduced infarct size, and increased vascular density in the infarcted area. However, the low cell number retention after cell injection remained an important drawback. Association of cells with a biomatrix to improve of cell delivery2 have recently fostered researcher and clinical interests.
Ligation of the left anterior descending coronary artery (LAD) is a reference method for MI in small animal model that results in transmural infarction and a mature scar. Cell therapy applied in the chronic phase of MI requires a second surgical intervention. A median sternotomy is usually performed to allow intramyocardial injection of the cells or epicardial implantation of biografts. Such invasive surgical procedures increase the mortality rate, post-surgery recovery time, pain, and risk of infection. The minimally invasive approach presented here not only prevents such bias but also provides optimal accessibility of the heart for treatment application. MI and epicardial implantation of cells associated with a gel type biomatrix are performed on a beating heart via left intercostal thoracotomies.

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:104px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 3-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">POLYSORB 5-0 suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">UL 202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SURGIPRO II 7-0 Suture</td>
			<td style="width:104px;">COVIDIEN</td>
			<td style="width:119px;">VP904X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CATHETER INSYTE 14G</td>
			<td style="width:104px;">BD</td>
			<td style="width:119px;">381267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">557058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
			<td style="width:104px;">FST Germany</td>
			<td style="width:119px;">11274-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibrin sealant</td>
			<td style="width:104px;">BAXTER</td>
			<td style="width:119px;">1501441</td>
			<td>TISSEEL Glue, Frozen -20&#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CASTROVIEJO EYE SPECULA</td>
			<td style="width:104px;">HARVARD APPARATUS</td>
			<td style="width:119px;">72-8925</td>
			<td>use as retractor for the ribs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMDM Glutamax</td>
			<td>Gibco</td>
			<td>31980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>PAA Clone</td>
			<td>&#160;&#160;&#160;&#160;&#160; A15-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">bone scissors</td>
			<td style="width:104px;">Fine Science Tols</td>
			<td style="width:119px;">16044-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Blood Cell Lysis Solution</td>
			<td>Gentra Systems</td>
			<td>D-50k1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
			<td style="width:104px;">Stem cell technology</td>
			<td style="width:119px;">7920</td>
			<td></td>
		</tr>
	</tbody>
</table>

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;

All animals recovered within 1 hr&#160;after thoracotomies. The wound healing was rapid. No infection or edema was observed.
The double left thoracotomy allowed optimal access to the heart (Figure 1). Pain and post-surgery mortality were low. The animal recovered quickly from the surgery and gained weight (Figure 2). Kaplan Meir survival percent was 96% for the first thoracotomy. Four rats over 104 died within the 24 hr following treatment application via<...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

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

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 ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K Views.  University of Fribourg. 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.

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

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

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) ...