The authors are indebted to all members of Pinto-do-&#211; laboratory for relevant critical discussion. This work was supported by Programa MIT-Funda&#231;&#227;o para Ci&#234;ncia e Tecnologia (FCT) under the project &#34;CARDIOSTEM-Engineered cardiac tissues and stem cell-based therapies for cardiovascular applications&#34; (MITP-TB/ECE/0013/2013). A.C.S. is a recipient of a FCT fellowship [SFRH/BD/88780/2012] and M.J.O. is a FCT Fellow (FCT-Investigator 2012).

All the methodologies described were approved by the i3S Animal Ethics Committee and Dire&#231;&#227;o Geral de Veterin&#225;ria (DGAV) and are in accordance with the European Parliament Directive 2010/63/EU.
1. Preparation of the decellularization solutions
NOTE: All decellularization solutions should be filtered through a 0.22 &#956;m membrane filter and stored for a maximum of 3 months, except specified otherwise.
<ol>
	<li>For 1x PBS: Mix 8 g of NaCl, 1.01 g of Na2HPO4&#160;200&#160;mg of KCl, and 200&#160;mg of KH2PO4 with 900 mL of deionized water (DI water). Adjust solution pH to 7.5 and the final solution volume up to 1 L. Filter, autoclave, and store the solution at room temperature (RT).</li>
	<li>For Hypotonic Buffer (10 mM Tris, 0.1% EDTA): Dissolve 1.21 g of TrisBASE and 1 g of EDTA in 900 mL of deionized water. Adjust solution pH to 7.8 with NaOH/HCl and the final solution volume to 1 L. Filter, sterilize by autoclave, and store at RT.</li>
	<li>For 0.2% SDS solution (0.2% SDS, 10 mM Tris): Add 0.6 g of TrisBASE and 1 g of sodium dodecyl sulfate (SDS) in 400 mL of DI water and stir at 60 &#176;C until complete solute dissolution. Let solution to cool down to RT. Adjust solution pH to 7.8 and the final solution volume to 500 mL. Sterilize solution by filtration. 
	NOTE: The SDS solution should be prepared prior to use. Filter solution only after complete SDS dissolution, otherwise the SDS insoluble particles will saturate the filter, changing the final SDS concentration and consequently affecting tissue decellularization efficiency.</li>
	<li>For Hypotonic Wash Buffer (10 mM Tris): Mix 1.21 g of TrisBASE in 900 mL of DI water. Adjust solution pH to 7.8 and the final volume to 1 L. Filter, sterilize by autoclave and store at RT.</li>
	<li>For DNase treatment (20 mM Tris, 2 mM MgCl2, 50 U/mL DNase): Dissolve 2.42 g of TrisBASE and 2 mL of 1 M MgCl2 in 900 mL of DI water. Adjust pH to 7.8 and the final solution volume to 1 L. Filter and sterilize solution by autoclave. Store solution at RT. Add DNase I (50 U/mL) prior to use.</li>
	<li>For DNase I stock solution: Prepare a DNase buffer containing 50% glycerol, 20 mM Tris-HCl pH 7.5, 1 mM MgCl2, and adjust to a final solution volume of 10 mL with DI water. In a laminar flow hood, add the DNase I powder to the DNase buffer and mix gently until complete dissolution. Sterilize solution through 0.22 &#956;m filter. Prepare 1 mL aliquots and store at -20 &#176;C.</li>
</ol>
2. Tissue harvesting and cryopreservation
<ol>
	<li>Euthanize adult pregnant females at 18 days of gestation (E18 fetuses) via CO2 asphyxiation. Perform a cesarean section on the females with a surgical scissor and collect the uterine horns bearing the fetal mice to a Petri dish with ice-cold 1x PBS using two serrated tweezers with curved tips (one tweezer to each horn end).
	<ol>
		<li>Open the uterine horns and amniotic sac to isolate the fetuses. Transfer the fetuses to a new Petri dish with ice-cold 1x PBS to anesthetize them and decapitate with a scissor.</li>
		<li>Euthanize 7-8 weeks old male C57BL/6 mice using CO2 asphyxiation. Perform an incision on each mouse chest and collect the heart.</li>
		<li>Briefly rinse fetal and adult hearts with ice cold 1x PBS.</li>
	</ol>
	</li>
	<li>Remove the atria of adult heart with the help of a scalpel. Perform an incision on the right ventricle and remove it using straight small scissors. Expose the left ventricle inner wall by removing the septum. In a new Petri dish, divide the left ventricle free-wall in longitudinal strips with 2 mm thickness and remove the papillary muscle using a scalpel and serrated tweezers with curved tips.
	<ol>
		<li>Excise small tissue explants with the help of a scalpel using a 2 mm x 2 mm grid (Detailed description in supplementary Figure 15). Briefly rinse tissue explants with 1x PBS.</li>
	</ol>
	</li>
	<li>Add ~250 &#181;L of the optimal cutting temperature (OCT) compound to autoclaved 1.5 mL microcentrifuge tubes. Transfer whole fetal hearts and adult cardiac explants to the tubes with 250 &#181;L of OCT and freeze them with dry ice-cooled isopentane and store at -80 &#176;C (to a maximum of 6 months). 
	NOTE: The use of cardiac tissue explants cryopreserved for more than 6 months will significantly reduce the decellularization efficiency, especially in the adult heart tissue explants.</li>
</ol>
3. Tissue decellularization
NOTE: Cardiac tissue decellularization is performed in a 24-well tissue culture plate with one sample per well. 1 mL of each decellularization solution is added to each individual well. All decellularization steps should be performed with agitation at 165 rpm (incubator shaker with an orbital diameter of 20 mm) and at 25 &#176;C, unless specified otherwise. For more details, please consult the scheme on Figure 1A. Amphotericin B (e.g. fungizone) and gentamicin are freshly added to all decellularization solutions before use to a final concentration of 2.5 &#956;g/mL and 0.01 &#956;g/mL, respectively. To quantify the amount of DNA retained within decellularized tissues, the sample mass needs to be determined before starting the decellularization protocol. The DNA quantification protocol is further detailed in section 5.1.
<ol>
	<li>DAY 1
	<ol>
		<li>Remove tissue samples from the -80 &#176;C freezer. Leave the 1.5 mL microcentrifuge tubes at RT until OCT becomes partially melted. Transfer the cardiac tissue block of the still frozen OCT to a Petri dish with 1x PBS.</li>
		<li>After the OCT melts, move the cardiac tissue to a new Petri dish with 1x PBS. Wash samples in 1x PBS and at 60 rpm with a shaker for 10-15 min. Repeat at least twice.</li>
		<li>Add 1 mL of working Hypotonic Buffer per well of a sterile 24-well tissue culture plate.Transfer one sample per well with the help of forceps.</li>
		<li>Move the 24-well tissue culture plate with the samples to an incubator shaker at 25 &#176;C and start the 18 h incubation with Hypotonic Buffer.</li>
	</ol>
	</li>
	<li>DAY 2
	<ol>
		<li>Prepare the 0.2% SDS solution as described in section 1.3.</li>
		<li>Aspirate the Hypotonic Buffer and wash the samples with 1x PBS for 1 h. Repeat 3 times. 
		NOTE: PAUSE POINT: Tissue under decellularization can be kept in 1x PBS at 4 &#176;C for 18 h in static conditions.</li>
		<li>Remove the 1x PBS, add 1 mL of freshly made SDS solution (step 1.3) per well and incubate samples for 24 h. 
		NOTE: (CHECK POINT) Post SDS incubation, ensure that samples exhibit a white to translucent appearance. At this step, SDS treated samples are drenched in DNA, showing a gelatin-like consistency. Remove SDS solution slowly to avoid sample adhesion to the pipette tip.</li>
	</ol>
	</li>
	<li>DAY 3
	<ol>
		<li>Wash the samples with Hypotonic Wash Buffer (1 mL per well) for 20 min. Repeat 3 times. 
		NOTE: At this step, it is normal to observe a decrease in the sample size due to the removal of cell remnants. PAUSE POINT: Tissue under decellularization can be kept in Hypotonic Wash Buffer at 4 &#176;C for 18 h in static conditions.</li>
		<li>Add 1 mL of DNase Treatment solution per well and incubate the samples for 3 h at 37 &#176;C.</li>
		<li>Aspirate the DNase Treatment solution and wash the samples with 1x PBS (1 mL per well) for 20 min. Repeat 3 times. Perform a final wash with 1x PBS (1 mL per well) overnight at RT&#160;and shaking at 60 rpm.</li>
		<li>(Check point) After DNase treatment, ensure that the decellularized samples have lost the gelatin-like consistency. 
		NOTE: After DNase treatment, remove and add 1x PBS gently since samples can be easily entrapped and attached to the pipette tip.</li>
	</ol>
	</li>
</ol>
4. Assessment of decellularized tissue cell removal
<ol>
	<li>Fix the samples in a 24-well tissue culture plate, 1 sample per well, by adding 1 mL of freshly made 10% formalin neutral buffer with 0.03% aqueous eosin for 2.5-3 h at RT. 
	NOTE: Eosin is added to the fixative to stain the decellularized tissue and to ease sample visualization during sectioning and histological staining. The addition of the eosin neither interferes with the histological stains nor with immunofluorescence techniques. Alternatively, the fixation can be performed overnight at 4 &#176;C.</li>
	<li>Remove the fixative solution and add 1 mL of 1x PBS to wash the sample.</li>
	<li>Encapsulate the fixed bioscaffolds in histology processing gel using a disposable vinyl specimen mold according to the manufacturer&#39;s instructions.</li>
	<li>Remove the histology gel with the sample embedded from the mold and transfer to Biopsy Processing/Embedding Cassettes with Lid. 
	NOTE: An alternative to tissue embedding in histology and processing gel is to process each sample in two biopsy sponges with small pores. The samples should be localized to the center of the biopsy sponges to enable detection. Of note, some samples can be difficult to localize using this approach.</li>
	<li>Process the samples for paraffin embedding through successive incubations (30 min per solution) in series of alcohol concentrations (70%, 80%, 90%, 100%, 100%), isoparaffinic aliphatic hydrocarbon solution (2 incubation stations) and paraffin (2 incubation stations) at 56 &#176;C.</li>
	<li>Mount the samples in a paraffin block.</li>
	<li>Cut 3 &#956;m-thick paraffin sections on a microtome and collect the sections on glass microscope slides.</li>
	<li>Dry paraffin sections overnight at 37 &#176;C. 
	NOTE: (PAUSE POINT) Paraffin sections are stored at 4 &#176;C until further use.</li>
	<li>To verify protocol efficiency, perform Hematoxilin and Eosin (H&#38;E) and Masson&#39;s Trichrome staining (MT) of the decellularized sample sections and to sections of non-manipulated tissue according to manufacturer&#39;s instructions. 
	NOTE: H&#38;E staining stains cell nuclei blue/purple, cytoplasm dark pink/red and collagen pale pink and MT stains nuclei black, cytoplasm red, and collagen and mucin blue. Efficient decellularization is achieved when a porous light pink (H&#38;E, MT) and blue (MT) network is observed, in the absence of a nuclear stain.</li>
</ol>
5. Assessment of decellularized tissue nuclear material removal
NOTE: The quantification of the DNA content on decellularized tissue must be performed in comparison to the respective non-manipulated tissue.
<ol>
	<li>Before decellularization, weigh a 1.5 mL microcentrifuge tube on a high precision digital scale. Transfer the cardiac tissue to the tube, remove the extra amount of 1x PBS and weigh the tube with the samples. Calculate the wet weight of the samples: 
	Samplewet weight = weight tube with samples &#8211; weight empty tube</li>
	<li>Decellularize the samples as described in Step 3.</li>
	<li>After decellularization, collect the decellularized tissues into a microcentrifuge tube.
	<ol>
		<li>Due to the small dimensions and mass of the individual cardiac samples and kit specifications, pool decellularized tissues of each sample condition (fetal heart: 5 whole hearts; adult LV explants: 15 explants). Perform the same procedure with the non-manipulated tissue. 
		NOTE: (PAUSE POINT) The samples can be stored at -20 &#176;C until DNA extraction and quantification are performed.</li>
	</ol>
	</li>
	<li>Cut the non-manipulated and decellularized tissues in small explants with a help of a scalpel in a clean Petri dish. 
	NOTE: Use an individual scalpel and Petri dish for each sample. Mechanical disruption of the samples with a scalpel facilitates their posterior enzymatic digestion and subsequent DNA extraction.</li>
	<li>For DNA extraction, use a spin-column based DNA extraction method according to the manufacturer&#39;s instructions.
	<ol>
		<li>Collect the mechanically dissociated samples from the Petri dish with the master digestion buffer (digestion buffer and proteinase K) according to the manufacturer&#39;s instructions using a wide orifice pipette tip. 
		NOTE: Tissue lysis with Proteinase K digestion is faster in decellularized samples. To process distinct samples at the same time, keep lysed samples on ice, until all samples are lysed to minimize degradation. Complete samples lysis is achieved between 4-6 h.</li>
		<li>Proceed with the protocol according the manufacturer instructions.</li>
		<li>To increase the yields of extracted DNA of decellularized and non-decellularized tissue samples, elute the DNA on 25-50 &#956;L and 100 &#956;L of elution buffer, respectively. Collect the eluted DNA and load the spin-column. Incubate for 1 min at RT. Centrifuge the samples according to the manufacturer&#39;s instructions. 
		NOTE: (PAUSE POINT) The DNA extracted from the samples can be store at -20 &#176;C until further use.</li>
	</ol>
	</li>
	<li>Quantify the extracted DNA from the samples with a fluorescent dsDNA detection kit following the manufacturer&#39;s instructions.</li>
	<li>Normalize the DNA content as nanograms of DNA per milligram of initial sample wet weight (before decellularization).</li>
</ol>
6. Decellularized scaffolds cell seeding
NOTE: All solutions/reagents need to be sterile and the entire procedure performed at sterile conditions.
<ol>
	<li>Prepare 1x DPBS with 2.5 &#956;g/mL of amphotericin B and 1% P/S (penicillin, 100 I.U.; streptomycin 100 &#181;g/mL).</li>
	<li>Remove the 1x PBS solution from the last decellularization washing step (step 3.3.3) and add 500 &#956;L per well of the freshly prepared 1x DPBS/2.5 &#956;g/mL of amphotericin B/1% P/S solution. Store samples at 4 &#176;C from 1-7 days. 
	NOTE: Samples storage at 4 &#176;C is important to maintain sterility.</li>
	<li>Add P/S to cell basal media (without FBS and supplements) to a final concentration of 1%.</li>
	<li>Aspirate 1x DPBS/2.5 &#956;g/mL of amphotericin B/1% P/S solution from the decellularized samples. Add 500 &#956;L per well of cell basal media/1% P/S and let samples equilibrate for 1 h at 37 &#176;C, or at 4 &#176;C for more than 1 h.</li>
	<li>Split the cells of interest and prepare small aliquots of cells at the cell density desired. 
	NOTE: The decellularized tissue seeding must be performed under a stereoscopic microscope and in sterile conditions. Cell culture media should contain 1% P/S.</li>
	<li>Pipette 3-4 &#956;L of DPBS to the center of a well of a 96-well plate. Using thin and straight tweezers, transfer the decellularized scaffolds to the DPBS drop, remove the extra DPBS by aspiration, and make sure that the sample is not folded or wrinkled. Let it become dry at the edges to adhere to the well surface. 
	NOTE: Detached scaffolds have lower seeding efficiency.</li>
	<li>Add cells to the scaffold by pipetting the single-cell solution slowly against the edges of the well. 
	NOTE: For example, neonatal rat cardiomyocytes are seeded with at a density of 7,500 cells/mm2 and immortalized mouse Lin&#8722; Sca-1+ cardiac progenitor cells (iCPCSca-1) at a density of 60-1,500 cells/mm2. Adjust cell density according the experimental rationale.</li>
	<li>Add DPBS to the neighboring wells to avoid fast media evaporation.</li>
	<li>24 hours post cell seeding, if the cell type used adhered to the tissue culture polystyrene plates (TCPS), transfer bioscaffolds to a new well. Otherwise, replace the medium to remove non-adherent cells.</li>
	<li>Change carefully the media according to specific requirements of the cell type.</li>
</ol>

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The authors have nothing to disclose.

The extracellular matrix (ECM) is a highly dynamic and complex meshwork of fibrous and adhesive glycoproteins, consisting of a reservoir of numerous bioactive peptides and entrapped growth factors. As the major modulator of cell adhesion, cytoskeleton dynamics, motility/migration, proliferation, differentiation and apoptosis, ECM actively regulates cellular function and behavior. Knowing that cellular behavior differs in 2D and 3D cultures, there have been efforts to develop novel organotypic models that can accurately replicate natural tissue environments. In the last years, tissue decellularization has emerged as an alternative technique for tissue engineering and regenerative medicine. Thus, tissue and organ decellularization is currently the tool of choice to better dissect tissue-specific microenvironmental parameters (biochemical, structural and mechanical) and biological activity in vitro and in vivo.
We developed a protocol that combines the use of a hypotonic buffer with a detergent of anionic surfactant properties followed by a DNase treatment5,12,13. The present protocol constitutes a simple and reproducible method to perform comparative analysis between decellularized fetal and adult mouse cardiac tissue. Our experience with other tissues, namely with tumor samples derived from cancer patients&#39; surgical resections and mouse lung tissue, shows that this protocol is easily adaptable and successful in other conditions and models12,13. The application of the same decellularization procedure on different samples allows comparative studies of the ECM composition, biomechanical properties, architecture and cellular modulatory properties in a 3D context.
One of the most difficult challenges of tissue decellularization is the balance between outright cell removal, ECM meshwork preservation and tissue biocompatibility. Hence, several critical steps need to be carefully considered, such as the time of tissue cryopreservation before decellularization, the correct explant size, the use of fresh solutions, and the manipulation of the final decellularized tissue. During our studies, we observed a direct correlation between long storage time of cryopreserved tissue and the use of longstanding SDS solutions with decellularization inefficiency. Long term tissue storage leads to inefficient cellular content removal rendering remnants of thick cellular areas (without nuclei) among the complex ECM meshwork. A similar undesirable effect is attained when long stored SDS solutions are used for tissue decellularization, likely because SDS solutions have a short stability due to reduced solubility, hydrolysis, and pH alterations over time15,16. The use of explants of the correct size (~1.5 mm x 1.5 mm x 1.5 mm) is also essential for successful adult tissue decellularization, since larger tissue resections are more difficult to decellularize, displaying cell debris entrapped at the surface and arrested between the dense ECM network. Although this protocol provides an efficient method of cardiac tissue decellularization, a minor fraction of adult explants may present cell remnants, in particular those of a larger size. Histological analysis of these samples eases their identification and subsequent exclusion from the study. Ultimately, the protocol herein is versatile and readily applicable to distinct specimens with slight adjustments to the tissue explant size, SDS concentration (0.1-0.2% SDS) or solution incubation time5,12,13.
The major limitation of the present method is that the manipulation of small size samples, i.e. murine fetal heart and adult heart explants, requires some handling skills. In fact, both fetal and adult decellularized tissues are delicate structures that can be permanently deformed when dried during the procedure or entrapped by the thin pipette tip or during handling with forceps5.
The major novelty of this protocol, besides the parallel decellularization of fetal mouse heart and adult left ventricle explants for comparative assessment of the ECM composition (biomechanical analyses) and associated biological function, is its simple translation to distinct tissues and models5,12,13. The decellularization of tissues of distinct ages by applying standardized methodology was described only on the rhesus monkey kidney, and the transverse sections of fetal, neonatal and adult tissues, which were subjected to a 1% SDS treatment for 10 days6. In a cardiac setting, although fetal, neonatal and adult tissues have been decellularized, the methods differed on the degree of mechanical dislodging, SDS concentrations and time of application7,8. As each decellularization procedure affects the ECM in a unique manner, the comparison of decellularized samples obtained from different protocols may lead to misleading conclusions. Hence, applying the same decellularization procedure across different samples enables reliable comparative analysis.
The large majority of the commercially available decellularized tissues derive from adult specimens17. Despite the growing recognition for an increased ability of fetal microenvironments in providing pro-regenerative signals in comparison to their adult counterparts, decellularization of fetal tissues was only reported in few studies5,7,18,19,20,21. Understanding ECM dynamics during the aging process will be crucial to identify unique features of pro-regenerative microenvironments which, in turn, will impact the development of higher efficiency biomimetic-materials.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56924/comparable-decellularization-fetal-adult-cardiac-tissue-explants-as">Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies</a>.&#160; The&#160;affiliations were&#160;updated.
The fourth&#160;affiliation&#160;was corrected from:
Gladstone Institutes, University of California San Francisco
to:
Gladstone Institute&#160;of Cardiovascular Disease
An additional affiliation was added for Todd C. McDevitt:
University of California San Francisco

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56924/comparable-decellularization-fetal-adult-cardiac-tissue-explants-as">Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies</a>.&#160; An affiliation&#160;was updated.

Erratum: Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

The extracellular matrix (ECM) is a dynamic network of molecules that regulate important cellular processes, namely fate-decision, proliferation and differentiation1,2. The investigation of cell-ECM interactions has been performed mainly in two-dimensional (2D) in vitro cultures coated with ECM components, which constitute a simplification of native ECM properties found in vivo. Decellularization generates acellular 3D-like ECM bioscaffolds that largely preserve the extracellular architecture and composition of native tissues and organs3,4. In addition to serving as bioactive scaffolds for tissue engineering, decellularized 3D ECM biomaterials are emerging as novel platforms to assess cell-ECM biology that parallel the in vivo environment.
Assessment of the differential role of the ECM components of distinct tissues, organs and age will benefit with the use of similar protocols of generating native bioscaffolds. In the heart, we have developed a versatile protocol for decellularization of fetal and adult-derived samples, as an alternative approach to perform comparative studies of the organ microenvironment. Using this methodology, we captured the native cardiac microenvironment and showed that fetal ECM promotes higher repopulation yields of cardiac cells5. Decellularization further provided identification of resident structural differences between fetal and adult ECM at the level of basement lamina and pericellular matrix mesh arrangement and fiber composition5. Prior to this work, head-to-head comparison of tissues at different ontogenic stages using the same decellularization approach has only been reported for rhesus monkey kidneys and rodent hearts. In addition, a limited number of studies report fetal tissue/organ decellularization per se5,6,7. This has been achieved using SDS as a unique decellularization agent; however, distinct SDS concentrations were used for the decellularization of fetal and adult cardiac tissue7,8. SDS is one of the most effective ionic detergents for clearance of cytoplasmic and nuclear material, and widely used in the decellularization of different tissues and specimens9,10. Solutions containing high SDS concentrations and extended periods of exposure have been correlated with protein denaturation, glycosaminoglycan (GAGs) loss and disruption of collagen fibrils10,11, and therefore a balance between ECM preservation and cell removal is necessary. To apply the same procedure to fetal and adult heart tissue, the protocol described herein is divided in three sequential steps: cell lysis by osmotic shock (hypotonic buffer); solubilization of lipid-protein, DNA-protein and protein-protein interactions (0.2% SDS); and nuclear material removal (DNase treatment).
Our protocol shows several advantages: i) the possibility of equivalent decellularization of age-specific cardiac tissues by the application of the same decellularization strategy; ii) no requirements for specialized methods or equipment; iii) ready adaptation to other tissues and species as it has been successfully applied with minor alterations in human intestine biopsies12 and mouse lung13; and, importantly, iv) can address ECM biomechanical properties while enabling the assembly of 3D-like organotypic cultures that more closely mimic the molecular features of the native tissue microenvironment.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubated Benchtop Shaker</td><td>Orbital Shakers</td><td>IKA:3510001</td><td>Recommended</td></tr><tr><td>Fluorimeter</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>Digital weight scale</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>Inverted Microscope</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>Cell culture incubator</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>Fridge (4&amp;ordm;C)</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>Deep freezer (-80&amp;ordm;C)</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>Microtome</td><td>-</td><td>-</td><td>Equipment available</td></tr><tr><td>&lt;strong&gt;Cirurgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Vannas Spring Scissors - 2.5mm Cutting Edge</td><td>Fine Science Tools</td><td>5000-08</td><td>Recommended</td></tr><tr><td>Dumont 5 Fine Forceps - Biologie/Inox</td><td>Fine Science Tools</td><td>11254-20</td><td>Recommended</td></tr><tr><td>Dumont 7 forceps</td><td>Fine Science Tools</td><td>11272-30</td><td>Recommended</td></tr><tr><td>Dissecting Scissors, straight</td><td>-</td><td>-</td><td>Tool available</td></tr><tr><td>Forceps, serrated, curved</td><td>-</td><td>-</td><td>Tool available</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24 well plates, individually wrapped</td><td>VWR</td><td>29442-044</td><td>-</td></tr><tr><td>96 well plates, individually wrapped</td><td>VWR</td><td>71000-078</td><td>-</td></tr><tr><td>Steriflip-GV, 0.22&amp;micro;m, PVDF, Radio-Sterilized</td><td>Millipore</td><td>SE1M179M6</td><td>-</td></tr><tr><td>Eppendorff</td><td>-</td><td>-</td><td>Material available</td></tr><tr><td>15 mL Falcon tubes</td><td>Fisher Scientific</td><td>430791</td><td>-</td></tr><tr><td>50 mL Falcon tubes</td><td>Fisher Scientific</td><td>430829</td><td>-</td></tr><tr><td>Four-Compartment Biopsy Processing/Embedding Cassettes with Lid</td><td>Electron Microscopy Science</td><td>70075-B</td><td>-</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Thermo Fisher Scientific</td><td>22-037-246</td><td>-</td></tr><tr><td>&lt;strong&gt;Tissue cryopreservation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shandon Cryomatrix embedding resin</td><td>Thermo Scientific</td><td>6769006</td><td>-</td></tr><tr><td>2-METHYLBUTANE ANHYDROUS 99+% (isopentane)</td><td>Sigma-Aldrich</td><td>277258-1L</td><td>-</td></tr><tr><td>Dry ice</td><td>-</td><td>-</td><td>-</td></tr><tr><td>&lt;strong&gt;Decellularization&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>BDH Prolabo</td><td>27810.364</td><td>-</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S-31264</td><td>-</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P5379-100g</td><td>-</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P8041-1KG</td><td>-</td></tr><tr><td>TrisBASE</td><td>Sigma-Aldrich</td><td>T6066-500G</td><td>-</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L-4390</td><td>-</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>MERCK</td><td>1.05833.1000</td><td>-</td></tr><tr><td>DNAse I</td><td>AplliChem</td><td>A3778,0050</td><td>-</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-049</td><td>-</td></tr><tr><td>Fungizone</td><td>Gibco BRL</td><td>15290-026</td><td>-</td></tr><tr><td>Deionized water (DI water)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 % formalin neutral buffer</td><td>Prolabo</td><td>361387P</td><td>-</td></tr><tr><td>Eosin Y AQUEOUS</td><td>Surgipath</td><td>01592E</td><td>Can be replaced by alcoholic eosin</td></tr><tr><td>Richard-Allan Scientific HistoGel Specimen Processing Gel</td><td>Thermo Fisher Scientific</td><td>HG-4000-012</td><td>-</td></tr><tr><td>Ethanol ethilic alcohol 99,5% anydrous</td><td>Aga</td><td>4,006,02,02,00</td><td>-</td></tr><tr><td>Deionized water (DI water)</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Clear Rite 3</td><td>Richard-Allan Scientific</td><td>6915</td><td>-</td></tr><tr><td>Shandon Histoplast</td><td>Thermo Fisher Scientific</td><td>RAS.6774006</td><td>-</td></tr><tr><td>&lt;strong&gt;Kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PureLink Genomic DNA Mini Kit</td><td>Thermo Fisher Scientific</td><td>K182001</td><td>-</td></tr><tr><td>Quant-iT PicoGreen&amp;nbsp;dsDNA kit</td><td>Invitrogen</td><td>P11496</td><td>-</td></tr><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DPBS</td><td>VWR</td><td>45000-434</td><td>-</td></tr><tr><td>Penicillin-Streptomycin Solution 100X</td><td>Labclinics</td><td>L0022-100</td><td>-</td></tr><tr><td>Fungizone</td><td>Gibco BRL</td><td>15290-026</td><td>-</td></tr><tr><td>Cell culture media of the cell of interest</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

comparable decellularization of fetal and adult cardiac tissue explants as 3d-like platforms for in vitro studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The decellularization efficiency should be assessed through three main techniques: macroscopic observation, histology and DNA quantification. The macroscopic appearance of samples post-SDS treatment indirectly affects the efficacy of cell removal. After SDS incubation, samples should appear as translucent to whitish (Figure 1C). Fetal (E18) decellularized tissues are characterized by a highly translucent structure while adult explants have a translucent to white appearance. A whiter appearance is generally correlated to an ECM network exhibiting higher fiber and collagen content, e.g. the adult ventricular and vascular vessels ECM mesh (Figure 1C). Hematoxylin &#38; Eosin (H&#38;E) and/or Masson&#39;s Trichrome (MT) stains are performed to confirm efficient cell removal by the observation of a porous mesh (light pink, H&#38;E; light pink and blue, MT) (Figure 1C). In addition, the MT stain highlights the collagen meshwork in blue5. Clearance of nuclear material after decellularization is accessed by DNA quantification and a reduction of approximately 99.8% is generally obtained, when compared to non-manipulated tissues (Figure 1C). The presence of nuclear material on decellularized scaffolds has been described as a trigger of undesired inflammatory response upon implantation14. For this reason, confirmation of efficient decellularization is essential prior to native 3D scaffold repopulation experiments. Decellularized scaffolds may be stored in sterile conditions up to the seeding with the cells of interest. Cell viability is monitored throughout in vitro culture by calcein staining (Figure 2A). Nevertheless, calcein stain can be cytotoxic to sensitive cell types. Terminal analysis of cell repopulation and distribution across scaffolds is performed post-paraffin processing; a snapshot of the bioscaffold repopulation assessed by H&#38;E staining at a central section of bioscaffolds is shown (Figure 2B, 2C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56924/56924fig1.jpg" /> 
Figure 1. Fetal (E18) and adult cardiac tissue decellularization procedure and confirmation of decellularization efficiency. (A) Protocol overview. (B) Decellularization protocol detailed. (C) Macroscopic analysis of cardiac fetal and adult tissue before and after decellularization. Scale bar: 2 mm. (D) Quantification of nuclear material in decellularized versus non-manipulated tissue. Data expressed as mean &#177; SEM. Student&#39;s t-test, two-tailed *p&#60;0.05. (E) H&#38;E and Masson&#39;s Trichrome histological analysis of cardiac fetal and adult tissue before and after decellularization. Scale bar: 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56924/56924fig1large.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/56924/56924fig2.jpg" /> 
Figure 2. Repopulation analysis of decellularized scaffolds seeded with Lin- Sca-1+ cardiac progenitor cell line (iCPCSca-1). (A) High cell viability observed at scaffolds surface during in vitro culture by calcein stain (green). Scale bar: 100 &#956;m. (B) Cell number and distribution across scaffolds assessed via H&#38;E staining. Scale bar: 100 &#956;m. (C) Orthogonal view of cells embedded in the decellularized ECM. Confocal image of 50&#956;m-thick paraffin section. Scale bar: 10 &#956;m (orthogonal view XZ, YZ) and 40 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56924/56924fig2large.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>Step</td>
			<td>Observation</td>
			<td>Possible reason</td>
			<td>Problem</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>3.1.2.</td>
			<td>Tissue with a brownish color</td>
			<td>Sample cryofixation; unstable freezing temperature</td>
			<td>Sample cryofixation will lead to inefficient removal of cytoplasmic proteins</td>
			<td>Store frozen tissue during shorter periods. Check freezer temperature and stability</td>
		</tr>
		<tr>
			<td>3.2.4.</td>
			<td>Pink-to-white opaque samples</td>
			<td>SDS solution was not well prepared; adult LV explants too big&#160;</td>
			<td>Inefficient removal of cytoplasmic proteins&#160;</td>
			<td>Ensure complete SDS dissolution. Decellularize samples of smaller size.</td>
		</tr>
		<tr>
			<td>3.3.4.</td>
			<td>Samples with a gelatin-like appearance</td>
			<td>Inefficient DNA removal</td>
			<td>Inefficient nuclear material clearance</td>
			<td>Increase DNase concentration and/or incubation time.</td>
		</tr>
		<tr>
			<td>4.6.</td>
			<td>Histology gel compaction</td>
			<td>Long waiting time in PBS1X/ethanol before paraffin processing</td>
			<td>Alteration of decellularized tissue structure</td>
			<td>Start histological processing as soon as possible</td>
		</tr>
		<tr>
			<td>6.6.</td>
			<td>Dried decellularized samples</td>
			<td>Samples were left to dry for too long</td>
			<td>Permanent collapse of decellularized tissue. Inefficient cell seeding</td>
			<td>Allow samples to slightly dry only in the periphery in order to become attached to the bottom of the well during seeding</td>
		</tr>
		<tr>
			<td>6.7.</td>
			<td>Decellularized sample detachment during seeding</td>
			<td>Samples were not sufficient dried prior to seeding</td>
			<td>Inefficient cell seeding</td>
			<td>Increase ECM drying time to allow better adherence prior to cell seeding</td>
		</tr>
	</tbody>
</table>
Table 1. Troubleshooting table for parallel decellularization of fetal (E18) and adult mouse cardiac tissue.

Watch this Scientific Journal Video about Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies at JoVE.com

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

comparable-decellularization-fetal-adult-cardiac-tissue-explants-as

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.9K Views.  Universidade do Porto. Deze methode kan helpen bij het aanpakken van belangrijke vragen in het cardiovasculaire veld over de impact van de extracellulaire matrix heeft tijdens verschillende fysiologische en ontwikkelingsstadia van het hart. Het belangrijkste voordeel van deze techniek is dat het een vergelijkende analyse tussen foetale en volwassen extracellulaire matrices mogelijk maakt door een vergelijkbare decellularisatiemethode toe te passen op beide soorten weefsel. Dus deze methode is succesvol toegepast op...

Video: Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

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

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

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 ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

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 ...

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 ...

Generation of Myocardium-Mimicking Extra Cellular Matrix for Cell Culture

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Author Spotlight: Studying Cardiac Cell-Matrix Interactions In Vitro

Fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. After decellularization, this substrate can be used for the culture and study of other cardiac cells and cell-matrix interactions.

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

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