The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki. Patients provided informed consent for the use of the tissue for research purposes, and the process of tissue collection was approved by the Institutional Review Board and ethics committee of Charit&#233; - Universit&#228;tsmedizin Berlin (EA4/028/12).

The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki and was approved by the institutional review board and ethics committee of Charit&#233; Medical University. All patients provided written, informed consent for use of heart tissue for experimental studies.
1. Preparation of Human Myocardium for Sectioning
<ol>
	<li>Obtain left ventricular myocardium (size varies depending on the type of surgery) directly from the operating room and transport in cold PBS in a sterile container.</li>
	<li>Remove all fat tissue with a sterile scalpel with a no. 10 blade under sterile conditions.</li>
	<li>Cut the myocardium in cubes of approximately 1 x 1 x 1 cm using a scalpel.</li>
	<li>Place the cubes in sterile 50 mL tubes. 
	NOTE: To pause, perform step 1.5, otherwise continue with step 2.2. Avoid repeated freeze-thaw cycles to prevent protein degradation and a decrease in tissue quality.</li>
	<li>Store the tubes containing the cubes at -80 &#176;C. The tissue can be stored for several months.</li>
</ol>
2. Sectioning of the Myocardium Cubes into Slices
<ol>
	<li>Take the tube(s) containing the prepared cubes out of the freezer.</li>
	<li>Transport on ice to the cryostat.</li>
	<li>Set the object and chamber temperature to -15 &#176;C. 
	NOTE: The right temperature is crucial to guarantee perfect sectioning.</li>
	<li>Chill empty 50 mL tubes and stamps in the chamber or on dry ice.</li>
	<li>Add a layer (approximately 0.5 - 1.0 mL) of cryosection medium onto the cold stamp and let it freeze until it is white and solid.</li>
	<li>Add a second layer of cryosection medium and place a myocardium cube onto this layer.</li>
	<li>Make sure the cryosection medium and myocardium are completely frozen before you continue. 
	NOTE: When the sample and cryosection medium are not frozen the sectioning will be impaired. Completely frozen cryosection medium turns from fluid and transparent to solid, white, and non-transparent. The myocardial cubes need at least 30 min up to 1 h to completely freeze if continuing immediately from step 1.4. When the tissue is frozen it should not deform when touched/handled with tweezers.</li>
	<li>Insert stamp with frozen myocardium into the holder and tighten all screws.</li>
	<li>Trim the surface of the tissue until an even sectioning surface is obtained.</li>
	<li>Set sectioning thickness to 300 &#181;m.</li>
	<li>Section the frozen myocardium with automatic sectioning. This guarantees uniform cut sections. Approximately 30 - 40 sections can be sliced from one cube.</li>
	<li>Place each slice after sectioning loosely in a precooled 50 mL tube. 
	NOTE: Do not press the slices or pack tightly. Approximately 40 slices can fit in one tube.</li>
	<li>Close the filled tubes and place on ice. 
	NOTE: To pause go on with step 2.14, otherwise continue with step 3.2.</li>
	<li>Store myocardium slices at -80 &#176;C. The slices can be stored for several months.</li>
</ol>
3. Decellularization of Human Myocardium Slices
<ol>
	<li>Take the necessary number of 50 mL tubes containing myocardium sections out of the -80 &#176;C freezer and transport on ice.</li>
	<li>Transfer the frozen sections of all 50 mL tubes from step 3.1 into one sterile beaker. Approximately 40 slices are added to the beaker per 50 mL tube. The beaker should be three times the volume of the 50 mL tubes; for example, for decellularization of one 50 mL tube use a 150 mL beaker.</li>
	<li>Add 50 mL lysis solution (10 mM Tris, 0.1% w/v EDTA, pH 7.4 in H2O) per 50 mL tube with myocardium slices.</li>
	<li>Shake the myocardium slices in a sterile beaker at 100 - 150 rpm continuously for 2 h at room temperature.</li>
	<li>Strain the liquid with the tissue slices through a coarse strainer. Transfer the tissue slices with a blunt tweezer to a new sterile beaker. Add 50 mL of 0.5% SDS in PBS per initial 50 mL tube of myocardium slices (e.g., use 100 mL SDS solution when two 50 mL tubes of myocardial sections are being decellularized).</li>
	<li>Shake continuously for 6 h at 100 - 150 rpm at room temperature.</li>
	<li>Strain the liquid with the tissue slices through a coarse strainer. Transfer the tissue slices with a blunt tweezer to a new sterile beaker and wash 3 times with 50 mL PBS for 10 min each while shaking at 150 rpm. Next, wash overnight in PBS with 1% Penicillin/Streptomycin and 1% Nystatin (PBS-P/S-N) at 100-150 rpm and 4 &#176;C while shaking. 
	NOTE: Thorough washing is crucial to completely remove any residual SDS. Remaining traces of SDS are toxic when the ECM is used in combination with cells.</li>
	<li>Remove the washing solution and add 25 mL preheated FBS (37 &#176;C) with 1% Penicillin/Streptomycin and 1% Nystatin to the decellularized slices per initial 50 mL tube of myocardium slices.</li>
	<li>Incubate for 3 h at 37 &#176;C. In this step, any remaining DNA is removed from the matrix due to the intrinsic DNase activity of the FBS.</li>
	<li>Transfer the slices to a new sterile beaker and wash 3 times with 50 mL PBS for 10 min each while shaking at 150 rpm. 
	NOTE: The ECM slices can be stored in PBS-P/S-N at 4 &#176;C for several days. However, it is recommended to proceed immediately.</li>
</ol>
4. Processing the Decellularized cECM to a Powder
<ol>
	<li>Place the ECM slices loosely in a new sterile 6-well plate. Freeze the ECM at -80 &#176;C and lyophilize for 2 days in a lyophilizer. 
	NOTE: More than one slice can be placed per well. Arrange the slices to completely cover the surface of the well. Overlapping the slices does not affect the subsequent pulverization success.</li>
	<li>For the pulverization of the ECM slice, use specific milling tubes (see Table of Materials) containing 1.4 mm ceramic beads.Fill the tubes loosely with lyophilized ECM using sterile blunt tweezers. For optimal results, a total weight of ECM between 10 - 20 mg is recommended. 
	NOTE: If the ECM is packed too tightly in the milling tubes the pulverization process will be negatively affected.</li>
	<li>Snap-freeze the tubes in liquid nitrogen. 
	CAUTION: Liquid nitrogen is extremely low temperature, wear appropriate personal protection.</li>
	<li>Insert the frozen tubes into the milling machine and pulverize the ECM.
	<ol>
		<li>Set a duration of 30 s and a maximum speed and start the device.</li>
		<li>After finishing, take out the tubes and snap-freeze again in liquid nitrogen.</li>
		<li>Repeat five times.</li>
	</ol>
	</li>
	<li>Wash the microparticles from the tubes by adding 1 mL ddH2O per tube and shake or vortex for complete solubilizing. Filter the heterogeneous solution through a 200 &#181;m mesh into a 50 mL tube.</li>
	<li>Freeze the tube in -80 &#176;C or liquid nitrogen.</li>
	<li>Replace the standard lid of the tube with a 0.22 &#181;m filter. This filter lid prevents the powder from flowing out of the tube but allows air circulation for water evaporation.</li>
	<li>Insert the tubes in the lyophilizer and lyophilize again to remove the water from the step (step 4.5) 
	NOTE: Lyophilization at this step can take up to 3 days.</li>
	<li>Store powder at -80 &#176;C until further processing or continue with step 5.1.</li>
</ol>
5. Enzyme Based Homogenizing of cECM Micro-Particles
<ol>
	<li>Dissolve porcine pepsin in 0.01M HCl (pH 2.0) to a concentration of 1 mg/mL. Place on a rotary shaker at room temperature until it is completely dissolved.</li>
	<li>Dissolve the lyophilized human ECM powder (Step 4.8) in the pepsin solution to a final concentration of 10 mg/mL. Mix thoroughly through pipetting. The total volume depends on the amount of ECM solution needed. For example, dissolve 10 mg of ECM powder in 1 mL pepsin solution. 
	NOTE: Dissolving particles can be supported through gentle vortexing (1,000 - 1,500 RPM). Remaining lumps of microparticles will negatively affect the digestion.</li>
	<li>Transfer the solution to 2 mL tubes with a volume of 1 mL per tube.</li>
	<li>Place the tubes in a tube shaker for 48 h at 27 &#176;C and 1,200 rpm.</li>
	<li>Take the tubes out of the shaker and place on ice or a pre-cooled tube holder.</li>
	<li>Mix 1/9 of the volume of ECM solution of cold 10x PBS (pH 7.4) with 1/10 of the volume of ECM solution of cold 0.1M NaOH in a precooled tube and place on ice. This mixture will neutralize the digested ECM solution and irreversibly inactivate the pepsin.</li>
	<li>Add the ECM solution to the neutralization mixture and mix thoroughly through pipetting or vortexing.</li>
	<li>Set the ECM to the desired concentration with 1x PBS (pH 7.4). The pH of the solution can be checked with pH paper. 
	NOTE: Example calculation for 10 mL digested ECM solution: 
	Add 10 mg porcine pepsin to 10 mL HCl (pH 2.0). 
	Add 100 mg ECM powder to pepsin solution. 
	NOTE: Example calculation for neutralizing 10 mL digested ECM solution and setting the concentration to 8 mg/mL: 
	VolFinal = cstart x Volstart / cFinal = 10 mg/mL x 10 mL / 8 mg/mL = 12.5 mL 
	Vol10xPBS = Volstart / 9 = 1/9 from 10 mL = 1.11 mL 10x PBS 
	VolNaOH = Volstart / 10 = 1/10 from 10 mL = 1 mL 
	Vol1xPBS = VolFinal - Volstart - Vol10xPBS - VolNaOH = 12.5 mL - 10 mL - .11 mL - 1 mL = 0.39 mL</li>
</ol>

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The authors have nothing to disclose.

When preparing human myocardial ECM, the goal is to achieve the following: removal of relevant immunogenic cellular material, preservation of ECM integrity and bioactivity, sterility, non-toxicity of the end-product, GMP-process compatibility, and suitability of the product for a given application in terms of handling. By combining our 3-step decellularization protocol with further processing into microparticles or self-assembling hydrogel, human cardiac ECM material is obtained that possesses specific biological activity, is easy to handle, and has the potential for multiple applications in vitro and in vivo, similar to what has been shown for ECM products from several animals species11,13,20,21.
During the initial processing of the myocardial tissue, it is important to section myocardial slices of identical thickness, because compound concentration and treatment duration have been titrated for 300 &#181;m slices. Thicker slices will be incompletely decellularized and thinner slices will suffer unwanted breakdown of ECM components with loss of bioactivity. This is particularly important with regards to the SDS treatment, where toxicity can negatively affect ECM properties and cellular analyses16. Furthermore, prolonged SDS treatment can lead to complete absence of fibronectin as seen in the study by Godier-Fournement et al.22, while it is preserved with our method17. The cECM sections are suitable for use in experiments without further processing (i.e., in 3D culture models analyzing cell differentiation or cell-ECM interactions, or for &#34;tissue engineering&#34; cellular repopulation approaches). The use of FBS for DNA removal is highly efficient, but could potentially lead to some residual FBS proteins in the ECM sections. This could influence any FBS sensitive assays. The translation to a clinical grade GMP protocol should be possible with the use of certified sera, however an adaptation might be necessary depending on regulations. This could either be confirming the absence of FBS residues in the ECM or an alteration to the protocol will be necessary to replace the FBS with DNase treatment. Lastly, repeated freeze-thaw cycles of the tissue and ECM sections are to be avoided to prevent degradation of the matrix.
When producing the cECM hydrogel, pepsin digestion is the most critical step in the process. In our initial experiments, pepsin digestion at pH 1 for 48 h at 37 &#176;C led to a loss of bioactivity, which could be prevented by modifying the incubation to pH 2 for 48 h at 27 oC. Also, bear in mind that the hydrogel is produced from lyophilized cECM microparticle suspension, not from freshly decellularized wet cECM slices. In addition to the improved general handling of the hydrogel as compared to cECM slices, the hydrogel has the advantage that it can be diluted for use in different concentrations, for instance at a higher concentration in combination with other materials or for 3D culture, or at a lower concentration for coating of cell culture dishes or as an additive to culture medium.
This protocol has been established as a laboratory-grade SOP for human cECM preparation to help fill the gaps in translational regenerative medicine that were recently described by Saldin et al.10 While decellularized cECM slices may be applied as sheets on the epicardial surface of the diseased heart, as recently described by Sarig et al., with porcine cECM in a rat model of myocardial infarction23, processing into a hydrogel allows for more diverse in vivo applications, such as the injection of cECM into myocardium as shown by Singelyn et al.13,24 In order to proceed toward clinical translational activities, the current protocol will have to be converted to GMP-grade procedures. In principle, all materials used are also part of approved medical device/ATMP production processes (i.e., decellularized heart valves or blood vessels) and no major obstacles are to be expected. However, information on the safe storage duration and absence of pathogens will certainly be required. Ultimately, a reliable source of larger quantities of fresh and sterile heart tissue from donors without heart disease is needed. Here, hearts from deceased organ donors that are not suitable for transplantation are the most likely scenario.

The extracellular matrix (ECM) provides not only structural support but is also important for biologic cell and tissue function1. In the heart, the ECM participates in the regulation of pathophysiologic responses such as fibrosis, inflammation, angiogenesis, cardiomyocyte contractile function and viability, and resident progenitor cell fate. In addition to its primary components - fibrous glycoproteins, glycosaminoglycans, and proteoglycans - it contains a host of secreted growth factors, cytokines, and membranous vesicles containing nucleic acids and proteins2,3.
It has recently become clear that acellular ECM preparations are not only invaluable for studying cell-matrix interactions, but also for potential therapeutic cell-based applications. The importance of providing an adequate environment to therapeutic cell products or engineered tissues is now widely acknowledged. Attempts have been made to combine cell suspensions or active compounds with defined biopolymeric hydrogels4,5,6 or with protein cocktails secreted by murine sarcoma cells (i.e., Matrigel, Geltrex)7. However, the former have limited bioactivity, the latter are problematic in GMP-grade processes, and both lack the tissue-specific bioactivity of cardiac ECM (cECM)8,9,10,11,12,13.
Decellularization of the myocardium has previously been performed by perfusion of the whole heart via the coronary vasculature14,15. While this is possible in animal hearts, intact human hearts are rarely available. Therefore, an immersion process that allows for handling tissue samples obtained in the operating room was favored. Our &#34;3-step&#34; protocol contains 3 separate incubation steps namely lysis, solubilization, and DNA removal. It yields human myocardial ECM with largely preserved protein and glycosaminoglycan composition16,17. These cECM slices allow for in vitro studies of cell-matrix interactions but are poorly suited for potential human-scale therapeutic applications. The manufacturing process was then extended to produce either lyophilized cECM microparticles or a cECM hydrogel18.
This protocol allows for the decellularization of human myocardium obtained from surgical samples, preserving the major components of myocardial extracellular matrix (ECM) and their biologic activity. This protocol is recommended when human cardiac ECM with preserved tissue-specific bioactivity is required for experimental studies of cell-matrix interactions, or when a suitable environment is needed for cell-based myocardial regeneration approaches. In principle, it is also possible to adapt this protocol to GMP-grade conditions, so that the use of processed cECM should be feasible in future therapeutic applications.

<table><tbody><tr><td>Balance</td><td>DR Precisa, Dietikon, Switzerland</td><td>Precisa XR 205SM</td><td></td></tr><tr><td>Blades Nr.10 Skalpell Nr.3</td><td>InstrumenteNRW, Erftstadt, Germany</td><td>SK-10-004</td><td></td></tr><tr><td>Cell culture plates (6-well)</td><td>Greiner, Frickenhausen, Germany</td><td>657160</td><td></td></tr><tr><td>Cryostat CM</td><td>Leica, Wetzlar, Germany</td><td>3050S</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth, Karlsruhe, Germany</td><td>8043.3</td><td></td></tr><tr><td>Eppendorf reaction tubes (1.5 or 2 ml)</td><td>Greiner, Frickenhausen, Germany</td><td>616201, 623201</td><td></td></tr><tr><td>Falcon 15ml, 50ml</td><td>Greiner, Frickenhausen, Germany</td><td>188271, 227270</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biochrome, Berlin, Germany</td><td>S 0115</td><td></td></tr><tr><td>Freeze Dry System</td><td>Labconco, Kansas City, USA</td><td>7670520</td><td></td></tr><tr><td>Freezer (-80&amp;deg;C)</td><td>Thermo Scientific, Waltham, MA, USA</td><td>Forma 900 Series</td><td></td></tr><tr><td>HCl</td><td>Carl Roth, Karlsruhe, Germany</td><td>281.1</td><td></td></tr><tr><td>Microtome Blades Type 819</td><td>Leica, Wetzlar, Germany</td><td>14035838925</td><td></td></tr><tr><td>Minilys Homogeniser</td><td>PEQLAB Biotechnologie GmbH, Erlangen, Germany</td><td>91-PCSM</td><td></td></tr><tr><td>NaOH</td><td>Carl Roth, Karlsruhe, Germany</td><td>K021.1</td><td></td></tr><tr><td>Nystatin</td><td>PAN Biotech, Aidenbach, Germany</td><td>P06-07800</td><td></td></tr><tr><td>PBS</td><td>Thermo Scientific, Waltham, MA, USA</td><td>14190-094</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies, Darmstadt, Germany</td><td>15140122</td><td></td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich, Taufkirchen, Germany</td><td>P6887-1G</td><td></td></tr><tr><td>Precellys Keramik-Kit 1.4 mm</td><td>Peqlab Biotechnolgie, Erlangen, Germany</td><td>91-PCS-CK14</td><td></td></tr><tr><td>Rotamax 120 Plate shaker</td><td>Heidolph, Schwabach, Germany</td><td>544-41200-00</td><td></td></tr><tr><td>SDS</td><td>Carl Roth, Karlsruhe, Germany</td><td>CN30.3</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica, Wetzlar, Germany</td><td>M125</td><td></td></tr><tr><td>Steriflip-GP, 0,22 &amp;micro;m</td><td>Merck Millipore, Darmstadt, Germany</td><td>SCGP00525</td><td></td></tr><tr><td>Stuart analogue rocker &amp;amp; roller mixers</td><td>Sigma-Aldrich, Taufkirchen, Germany</td><td>Z675113-1EA</td><td></td></tr><tr><td>Tissue Tek O.C.T compound</td><td>Hartenstein, Wurzburg, Germany</td><td>TTEK</td><td></td></tr><tr><td>Transfusion set 200&amp;micro;m</td><td>Sarstedt, N&amp;uuml;mbrecht, Germany</td><td>798.200.500</td><td></td></tr><tr><td>TRIS</td><td>Carl Roth, Karlsruhe, Germany</td><td>5429.3</td><td></td></tr><tr><td>vedena Skalpellgriff Fig. 3, Standard, 125 mm</td><td>Medical Highlights, Rohrdorf, Germany</td><td>CV102-003</td><td></td></tr><tr><td>Vortex-Genie2</td><td>Scientific Industry, New York, USA</td><td>SI-0256</td><td></td></tr></tbody></table>

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.

The 3-step protocol for decellularization of human myocardium presented here results in near-complete removal of cellular material, while preserving the key ECM components and the fibrillar structure of ECM. After decellularization, the gross removal of cells from the tissue is evident by the change in color (Figure 1A). Histological analysis with H&#38;E and Masson Trichrome stains revealed the complete absence of residual intact cells (Figure 1B). Quantitative assays for individual ECM components revealed a more complete DNA removal and better preservation of total collagen, elastin, and glycosaminoglycans as compared with decellularization by SDS alone (Figure 2A). Functional evidence of the biologic activity of cECM is shown in Figure 2B. Here, RT-PCR was used to quantify the expression of structural cardiomyocyte proteins (Myh6, Tnnt2), early (Mef2c, Nkx2.5) and late (Gata4) cardiomyogenic transcription factors, and endothelial cell surface marker genes (von Willibrand Factor (vWF), VE-cadherin) in murine ESC undergoing spontaneous differentiation. Compared to both uncoated culture dish surface and Matrigel-ECM coating, it is evident that contact with cECM drives pluripotent stem cells preferably towards a cardiomyocyte-like phenotype.
Lyophilized ECM slices can be further processed into microparticles without using enzymatic or chemical reagents. Mechanical grinding or pulverizing using suitable kits allowed for production of microparticles with a uniform size (Figure 3A). Mass spectrometry provides an overview of all recognizable proteins present in cECM. Gene ontology analysis concentrating on cellular components revealed that the majority of ECM proteins are indeed derived from extracellular space, with few hemoglobin residues and a number of predominantly intracellular proteins (Figure 4). This pattern may determine the tissue-specific biologic function of cECM, but this hypothesis needs further research. A summary of the cECM protein composition is shown in Table 1. Processing the ECM into microparticles preserves its biological activity. HL-1 cells exposed to simulated &#34;ischemic&#34; conditions (hypoxia, glucose and serum deprivation) displayed an increase in cell metabolism (Figure 3B) and a decrease in cell death when cultured on cECM (Figure 3C).
Limited pepsin-based digestion of the cECM powder, based on a modified protocol developed for collagen purification19, results in a homogenized ECM solution. Phase contrast light microscopy pictures (Figure 5A) demonstrate the digestion process after 0 h and 48 h. This homogenizing step facilitates applications in regenerative medicine10. The resulting processed cECM is shown in Figure 5B. Below room temperature it remains liquid (Figure 5B, left) but at 37 &#176;C it forms a self-assembling hydrogel with stability suitable for an overlay with cell suspensions allowing 3D culture (representative picture, Figure 5B right, hydrogel layered with culture medium). Live/dead staining of human cardiac fibroblasts also shows the beneficial effects of culturing on cECM (Figure 5C). Both cECM microparticles and cECM hydrogel enhanced the metabolic activity of contractile HL1 cells in ischemic conditions as compared to cells in standard culture (Figure 5C, lower right panel).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56419/56419fig1.jpg" /> 
Figure 1: Decellularization of human cardiac myocardium. (A) Macroscopic analysis of cECM by stereo microscopy reveals the removal of cellular material with the 3-step decellularization protocol. This is evident by the increase in transparency. (B) Histology staining of myocardial slices before and after decellularization with HE- (left; Scale bar = 50 &#181;m) and Masson Trichrome staining (right; Scale bar = 200 &#181;m) shows successful removal of cells from the tissue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56419/56419fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56419/56419fig2.jpg" /> 
Figure 2: Content and effect of cECM slices. (A) Quantitative biochemical analysis of selected ECM components and DNA, comparing the 3-step decellularization protocol to 0.5% SDS-alone decellularization. Data are expressed as percentage of the content in native myocardium. With the 3-step protocol, the residual DNA content was nearly zero and significantly lower than that after a 9 h incubation with a standard combined 0.5% SDS/lysis buffer combination and FBS (SDS 9 h + FBS). Respective biochemical assays demonstrated a near-complete preservation of collagen, approximately 20% preservation of glycosaminoglycan content compared to native tissue, and a nearly 30% preservation of elastin content, all significantly higher than with the standard SDS reference protocol (Bars represent mean &#177; SEM). (B) mRNA expression of selected genes in induced pluripotent stem cells differentiating on cECM, compared to uncoated culture dishes and to Matrigel-coated surface. cECM data are normalized to those obtained on the respective control materials (control = 1, dashed line). The majority of cardiac genes are significantly more highly expressed when cultured on cECM (Bars represent mean &#177; SEM). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56419/56419fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56419/56419fig3.jpg" /> 
Figure 3: Appearance and biological activity of cECM microparticles. (A) cECM microparticle powder after lyophilization. (B) Metabolism (MTS) and (C) Cell death (LDH release) analysis of HL-1 cells cultured with cECM or gelatin. cECM significantly reduces cell death and increases metabolism. Bars represent mean &#177; SEM, statistical significance tested by one-way ANOVA and Bonferroni. Figure 3B-C have been modified from Kappler et al.18 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56419/56419fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56419/56419fig4.jpg" /> 
Figure 4: Protein composition of cECM microparticles. Following mass spectrometry, String DB analysis depicts the key components of cECM, which are mainly associated with the extracellular space (GO:0005615; red spheres p &#62;0.05). Lines indicate protein interactions based on experimental-biochemical data (purple), on co-expression data (black), on database interactions (blue), and on co-expression (green). String DB required confidence: score &#62;0.4. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56419/56419fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56419/56419fig5.jpg" /> 
Figure 5: Processing of cECM to a hydrogel preserve the biological effectivity. Phase contrast light microscopy pictures demonstrate that enzymatic digestion of the cECM for 48 h results in a more homogenized ECM solution; Scale bar = 500 &#181;m. (A) The ECM solution remains liquid up to room temperature (B, left) and contains the potential to self-assemble towards a hydrogel when incubated at 37 &#176;C (B, right). Live/dead stain of cardiac human fibroblasts cultured with and without cECM reveals a higher calcein staining intensity of living cells; Scale bar = 50 &#181;m (C). The cECM hydrogel increases the metabolic activity of HL1 cells in ischemic conditions in the same manner as the micro particles (C, lower right panel, modified from Kappler et al.18) Bars represent mean &#177; SEM, statistical significance tested by one-way ANOVA and Bonferroni. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56419/56419fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein Description</td>
		</tr>
		<tr>
			<td>5-hydroxytryptamine receptor 7 OS=Homo sapiens GN=HTR7 PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Actin-like protein 6B OS=Homo sapiens GN=ACTL6B PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Actin, aortic smooth muscle OS=Homo sapiens GN=ACTA2 PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Antithrombin-III OS=Homo sapiens GN=SERPINC1 PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Apolipoprotein C-III OS=Homo sapiens GN=APOC3 PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Apolipoprotein E OS=Homo sapiens GN=APOE PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Coiled-coil domain-containing protein 47 OS=Homo sapiens GN=CCDC47 PE=1 SV=1</td>
		</tr>
		<tr>
			<td>C-type lectin domain family 11 member A OS=Homo sapiens GN=CLEC11A PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Chloride intracellular channel protein 1 OS=Homo sapiens GN=CLIC1 PE=1 SV=4</td>
		</tr>
		<tr>
			<td>Collagen alpha-2(I) chain OS=Homo sapiens GN=COL1A2 PE=1 SV=7</td>
		</tr>
		<tr>
			<td>Complement C3 OS=Homo sapiens GN=C3 PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Collagen alpha-2(IV) chain OS=Homo sapiens GN=COL4A2 PE=1 SV=4</td>
		</tr>
		<tr>
			<td>Cyclic AMP-responsive element-binding protein 3-like protein 4 OS=Homo sapiens GN=CREB3L4 PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Dermatopontin OS=Homo sapiens GN=DPT PE=2 SV=2</td>
		</tr>
		<tr>
			<td>Fibronectin OS=Homo sapiens GN=FN1 PE=1 SV=4</td>
		</tr>
		<tr>
			<td>Glutathione peroxidase 3 OS=Homo sapiens GN=GPX3 PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Hemoglobin subunit alpha OS=Homo sapiens GN=HBA1 PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Hemoglobin subunit beta OS=Homo sapiens GN=HBB PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Ig kappa chain C region OS=Homo sapiens GN=IGKC PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Lumican OS=Homo sapiens GN=LUM PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Mimecan OS=Homo sapiens GN=OGN PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Nidogen-1 OS=Homo sapiens GN=NID1 PE=1 SV=3</td>
		</tr>
		<tr>
			<td>Pseudopodium-enriched atypical kinase 1 OS=Homo sapiens GN=PEAK1 PE=1 SV=4</td>
		</tr>
		<tr>
			<td>Pigment epithelium-derived factor OS=Homo sapiens GN=SERPINF1 PE=1 SV=4</td>
		</tr>
		<tr>
			<td>Mitochondrial coenzyme A transporter SLC25A42 OS=Homo sapiens GN=SLC25A42 PE=2 SV=2</td>
		</tr>
		<tr>
			<td>Serum amyloid P-component OS=Homo sapiens GN=APCS PE=1 SV=2</td>
		</tr>
		<tr>
			<td>Transcription initiation factor TFIID subunit 5 OS=Homo sapiens GN=TAF5 PE=1 SV=3</td>
		</tr>
		<tr>
			<td>Tudor domain-containing protein 3 OS=Homo sapiens GN=TDRD3 PE=1 SV=1</td>
		</tr>
		<tr>
			<td>Zinc finger matrin-type protein 4 OS=Homo sapiens GN=ZMAT4 PE=2 SV=1</td>
		</tr>
		<tr>
			<td>Zinc finger protein 101 OS=Homo sapiens GN=ZNF101 PE=2 SV=1</td>
		</tr>
	</tbody>
</table>
Table 1: Protein composition of cECM powder as determined by mass spectrometry.

Watch this Scientific Journal Video about Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications at JoVE.com

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.

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. ...

processing-human-cardiac-tissue-toward-extracellular-matrix-self

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.0K Views.  corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health. Dit protocol beschrijft de complete decellularization van menselijke myocard met behoud van de componenten van de extracellulaire matrix. Verdere verwerking van de resultaten van de extracellulaire matrix in de productie van deeltjes en een cytoprotective zelfassemblerende hydrogel.

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

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 ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Cancer Research

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

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

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

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

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

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.

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

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

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