Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Podsumowanie

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

Streszczenie

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 States^1-2. 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.

Wprowadzenie

Heart failure is common and deadly. It is a progressive disease that results in decreased contractility of the heart, which impairs blood flow to organs and leaves the metabolic demands of the body unmet. It is estimated that 5.7 million Americans have heart failure and it is the primary cause of hospitalization in the United States⁹. The collective cost of treating patients in heart failure in the United States exceeds $300 billion dollars per year ^9-10. 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 States^1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.². Clearly, this organ shortage needs to be addressed as other strategies are 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.

Mammalian adult cardiomyocytes demonstrate a limited regenerative capacity upon injury but recent evidence suggests that mammalian neonatal hearts maintain a remarkable regenerative capacity following injury^5-8. Specifically, following partial surgical resection, a regenerative window has been discovered between birth and postnatal day 7. This regenerative period is characterized by a lack of fibrotic scar, formation of neovascularization, release of angiogenic factors from the epicardium, and cardiomyocyte proliferation ^5-8,11. This regenerative window of time provides the potential for using the neonatal heart as a novel source of material for the development of a bioartificial heart.

The extracellular matrix is known to provide important cues to promote cardiomyocyte proliferation and growth. Distinct differences in the availability of molecules in the neonatal and adult matrices¹² and their ability to promote regeneration have been explored¹³. Decellularized adult matrices have been used in several studies to provide an ECM scaffold for cellular repopulation and the generation of a bioartificial heart. While these studies, and new discoveries in stem cell technologies, are advancing rapidly, several hurdles have yet to be met. For example, limitations in preserving native structure of the matrix, cellular integration into the matrix wall, and ability to support proliferation and growth all limit the success of this approach. While superior regenerative attributes have been ascribed to the neonatal heart, the practical aspects of using such a tissue have limited its exploration.

Based on the demonstrated regenerative capacity of the neonatal heart, we have developed novel matrices by developing a technique of decellularization for the P3 mouse heart. The P3 heart was chosen for these studies as it is within the window of cardiac regeneration as previously determined⁶ but the heart is large enough to harvest, decellularize and recellularize. The goal of this study is to demonstrate the feasibility of creating a matrix from a neonatal mouse heart. Our studies provide evidence for the feasibility of decellularizing a minute, neonatal heart while maintaining the structural and proteinaceous integrity of the ECM. We also demonstrate the ability to recellularize this cardiac ECM with mCherry expressing cardiomyocytes and we have examined these cardiomyocytes for expression of various cardiac markers following recellularization. This technology will allow for the testing of the superiority of a neonatal matrix for the development of a bioartificial heart.

Protokół

All mouse experiments were performed in accordance with US Animal Welfare Act and were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.

1. Method for Mouse Heart Isolation

1. Euthanize a neonatal mouse by decapitation with a single use blade.
2. Swab the thorax with 70% ethanol.
3. Dissect the skin from the chest by cutting it away from the chest wall with standard scissors while pulling the skin laterally with a pair of #5 forceps.
4. Perforate the abdomen just inferior to the sternum with the scissors by cutting through the abdominal wall. Grasping the xiphoid process with #5 forceps, retract the sternum rostrally from the body while cutting though the ribs on either side of the chest with the scissors. The rib cage is reflected superiorly with the forceps to reveal the heart.
5. Bluntly dissect the two main lobes of the thymus by pulling laterally with #5 forceps, exposing the arch of the aorta, as well as the caval and pulmonary veins.
6. Transect the major arteries from the aortic arch, and the aorta itself, with the 10 cm spring scissors. Retain the aorta between the base of the heart and the brachiocephalic artery. Grasp the ends of the severed vessels with the #5 forceps to reflect the heart forward, separating it from the trachea and esophagus.
7. Transect the pulmonary vasculature and other major veins, by cutting between the lungs and the heart on both sides of the heart with the 10 cm spring scissors. The veins remain open to provide drainage.
8. Remove the heart from the mediastinum with the #5 forceps, grasping the severed ends of the major vessels. Place the heart in a 60 mm culture dish containing sterile phosphate buffered saline (PBS) for catheterization.

2. Method for Decellularization by Langendorff Perfusion

1. Prepare the catheter assembly in advance. Draw a 4 cm section of PE 50 tubing in a small alcohol flame to create a smaller, thinner catheter. Trim the ends with a blade to meet the dimensions (Approx. 300 µm outside diameter O.D. with a flange of approx. 500 µm O.D.) shown in Figure 1. This will provide two symmetrical catheters from each side of the pull.
2. Point the cut end of the tubing into the flame briefly, to melt a flange onto the opening at the thin end.
3. Fill the 12 ml syringe with PBS and assemble the catheter by placing a 3-way stopcock on the syringe. Add a 22 G x 1 needle to the stopcock and drive the needle through the septum. Slide the drawn PE tubing catheter onto the needle (Fig. 1).
4. Irrigate the assembled parts with PBS, assuring that all air bubbles have been removed.
5. Insert the catheter, prepared as outlined above, into the aorta of the isolated heart, not extending past the aortic valve and ligate with one tie of 7-0 suture. Rest the flange of the catheter proximally against the tie, making a tight seal and preventing the heart from coming off the catheter.
6. Observe the catheterization under magnification, while gently perfusing the heart using the syringe containing PBS. Ensure that there are no leaks in the system and the tissue homogeneously blanches as latent blood is removed.
7. Place the septum into the neck of the inlet adaptor and seal it by folding the sides over the glass.
8. Attach the reservoir filled with 60 ml of 1% sodium dodecyl sulfate (SDS) in distilled water (dH₂O) via a line of sufficient length to produce a column that generates 20 mm Hg pressure as shown in Fig. 1. Calculate the pressure from the liquid column height based on the relationship of 1 mm Hg is equal to 1.3595 cm H₂O.
   Caution: SDS is a highly flocculent powder with irritant properties. Contact should be avoided. Handle the powder using appropriate protective clothing.
9. Perfuse the heart with 1% SDS for 14 hr. The heart will be translucent in appearance with no observable remaining tissue.
10. Flush the system up to the stopcock of the remaining SDS solution and replace it with 10 ml dH₂O. Perfuse the heart with 10 ml 1% Triton X-100 (diluted in distilled water), followed by 10 ml dH₂O, followed by 60 ml PBS containing 1x penicillin streptomycin (Pen-Strep).
11. Store the heart in PBS with 1x Pen-Strep at 4 °C. If the intended post decellularization application requires perfusion then the catheter in the aorta should be maintained, otherwise cut the suture tie and remove the catheter.

3. DNA Determination

1. Prepare a digest of the decellularized ECM or control heart using 200 μg/ml proteinase K in 50 mM KCl, 2.5 mM MgCl₂, 0.45% Tween 20 in 10 mM Tris (pH 8.3).
2. Incubate the tissue at 55 °C with agitation until the tissue is dissolved, typically 4-5 hr.
3. Quantitate the DNA content of the homogenate with a DNA binding assay^4,11.

4. Fixation and Sectioning of Tissue

1. Fix decellularized, recellularized or control P3 hearts in 4% paraformaldehyde in PBS for 30 min at room temperature.
2. Wash the tissue in PBS 3x.
3. Place the tissue in a solution of 7.5% sucrose in 0.1 M phosphate buffer at 4 °C until it sinks.
4. Change the solution to 15% sucrose in 0.1 M phosphate buffer at 4 °C, again, until it sinks.
5. Warm the tissue to 37 °C, replace half of the volume with gelatin solution in 15% sucrose & 0.1 M phosphate buffer and equilibrate overnight. Concentrations of gelatin are increased stepwise through 1%, 2.5%, 5% and finally 7.5% twice.
6. Place the samples in cryomolds using fresh 7.5% gelatin. To restore the shape of the decellularized samples, liquid gelatin can be perfused into the chambers as the hearts are molded. Freeze by floating the cryomolds on liquid nitrogen. Store at -80 °C.
7. Cut (10 µm thick) sections with a cryostat. Bring the slide close to the section surface and observe the section move off from the knife onto the surface due to the charge on the electrostatically treated slides. Dry the slides overnight and store at -80 °C for future analysis.
8. Remove the slides from the freezer, equilibrate to room temperature and then place in a Coplin jar containing PBS for 20 min at 37 °C to dissolve the gelatin.
9. Stain the cut sections from Step 4.7 with Hematoxylin and Eosin. Place slides in a Coplin jar. Expose the slides hydrated in Step 4.8 to Hematoxylin for 45 sec, tap water for 1.5 min, buffer for 3 min, tap water for 1 min, bluing agent for 1.5 min, 80% ethanol for 1 min, Alcoholic Eosin Y for 8 sec, 80% ethanol for 1 min, 100% ethanol for 1 min 2x, clearing agent (xylene substitute) for 1 min 3x, coverslip with resin based mounting medium. Examine the slides microscopically.
10. To confirm the retention of ECM protein reactivity of the heart ECM, stain for ECM structural proteins such as collagen IV by placing the hydrated slides in a humidified chamber and treat with 10% normal donkey serum (NDS) in phosphate buffered saline with 0.1% Triton-X100 (PBST) for 1 hr at room temperature (RT). Use a sufficient volume to cover the tissue sections.
11. Replace the solution with the primary antibody of choice at an empirically determined dilution in 5% NDS / PBST. For example, the Collagen IV antibody was diluted at 1:150. Incubate overnight at 4 °C.
12. Wash the slides with PBST 3x and apply a secondary antibody of choice conjugated with a fluorescent dye. Dilute the antibody by an empirically determined amount. Incubate for 1 hr at RT. Wash with PBS 3x.
13. Stain the slides with a DNA binding dye such as DAPI to verify absence of cell nuclei by using a DAPI containing mounting medium when cover slipping the slides.
14. Examine the slides with a fluorescent microscope at 50 to 400X.

5. P1 Neonatal Murine Ventricular Cardiomyocytes for Recellularization

1. Spray each pup with 70% ethanol and decapitate with a single use blade.
2. Hold each pup between the thumb and forefinger while the thorax is split at the midline with small sterile scissors to expose the thoracic cavity. Apply pressure to allow the heart to protrude freely from the chest while it is cut free of the major vessels and the atria leaving only the ventricular tissue.
3. Place each heart directly into a 50 ml tube containing 20 ml ice-cold Calcium, Bicarbonate free Hank's buffered saline solution with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (CBFHH) until all the hearts are collected.
4. Aspirate the CBFHH and add 10-15 ml of fresh CBFHH (ice-cold) to the tube. Wash the tissue by swirling the tissue in the tube several times.
5. Decant the solution containing the hearts into a 60 mm plastic Petri dish.
6. Dissect any latent atrial tissue from the hearts under magnification on ice by trimming it away from the hearts with the spring scissors. Mince up the ventricles into homogenously sized small pieces. Remove any separated non-ventricular tissue from the dish with #5 forceps.
7. Pipet as much of the CBFHH solution as is needed to transfer the tissue fragments into a small sterile vial that contains a sterile micro stir bar.
8. Allow the tissue to sediment to the bottom of the vial and remove the CBFHH solution.
9. Make up 50 ml of an enzyme solution 1.75 mg/ml trypsin, 20 µg/ml DNase II in CBFHH. Pipet 5 ml of this enzyme solution and add it to a tube containing minced ventricular tissue and place on a magnetic stirrer. Stir at a constant rate (340 rpm) at room temperature for 8 min.
   NOTE: The stirring action should be gentle and yet allow for the suspension of the slurry.
10. Remove the vial from the stir plate and allow the slurry to sediment for 3 min. Pipet and discard the initial digest supernatant, as it primarily contains blood cells and tissue debris.
11. Pipet a second 5 ml aliquot of the enzyme solution and add it to the digesting ventricle. Stir for 8 min and allow it to sediment for another 3 min. Prior to beginning the digests, prepare two 50 ml tubes (labeled 1 and 2) each containing 12 ml ice-cold fetal bovine serum (FBS). Place a 40 µm cell strainer atop each tube. Pipet this supernatant through the filter on tube 1.
12. Repeat the digestion process an additional 8 times, alternating the placement of the digest supernatant between the two tubes containing FBS. Split the last supernatant equally between tubes 1 and 2 to ensure equal volumes in each tube.
    NOTE: Each collection tube will now contain 32.5 ml total volume of cell suspension.
13. Centrifuge the tubes containing the supernatant 150 x g for 6 min at 4 °C.
14. While collecting the digestion aliquots, prepare five 100 mm tissue culture plates with 6 ml of media (Dulbecco's Modified Eagle's Media (DMEM) with 10% FBS, 1x Pen-Strep, 1x L-glutamine) per plate. Incubate these aliquots at 37 °C, 5% CO₂ for 30 min to equilibrate the media.
15. Aspirate the CBFHH enzyme mixture and resuspend the cells into the 30 ml of culture media prepared above, combining the cells from both collection tubes.
16. Return 6 ml of cell suspension to each of the 100 mm plates and incubate for 45 min at 37 °C.
17. At the end of the incubation, transfer the non-adherent cells to 5 new dishes and re-incubate for 45 min (the fibroblast fraction will have begun to adhere to the dishes and can be grown out separately if desired).
18. At the end of the second round of pre-plating, collect the media and non-adherent cells from the dishes and spin the media at 150 x g for 6 min.
19. Draw off excess media to bring the cells to the approximate desired concentration, (4.0 x 10⁵ cells per construct in 100 µl of perfusate). Remove an aliquot for counting and or viability testing¹⁴.

6. Bioreactor Recellularization of P3 Heart Matrix

1. Pretreat the isolated heart matrix with culture media (see Step 5.15) overnight before adding the cells.
2. Assemble the elements of a Langendorff system as shown in Fig. 2. Autoclave the glass parts and ethylene oxide sterilize the plastic parts to assure sterility. Various sub units of the system can be assembled in a laminar flow hood before being mounted on the support stand. Circulating water bath and heated water flow path has been omitted for clarity.
3. Fill the assembled system with 120 ml of tissue culture media (see Step 5.15).
4. Place the catheterized heart matrix from Step 2.14 in a 60 mm culture dish under magnification and connect a 1 ml syringe with 22 G x 1 needle loaded with cell suspension from Step 5.20 to the catheter. Ensure that this assembly is free of air bubbles to avoid embolizing the heart.
5. Gently perfuse the 100 µl of cell suspension into the heart matrix via the coronary arteries. Once complete, detach the syringe/needle combination.
   NOTE: A slow perfusion of approx. 20 µl per min is required to prevent the cells from flowing out of the veins, transiting the heart.
6. With a 22g blunt stub secured to the Luer fitting on the underside of the bioreactor heart jar lid, push the catheter onto the stub.
7. Start the peristaltic pump and observe that the flow proceeds as outlined in Figure 2. Flow proceeds from the reservoir via the pump through the bubble trap to the catheter placed in the aorta in Step 2.5 (red path in Fig. 2). Observe the flow of the cardiac circulation out of the veins and drip from the apex, recirculating to the media reservoir.
   NOTE: The perfusion flow rate is dependent on individual factors such as the vascular resistance of the construct, and the size of the heart, but a rate of 50 to 100 µl/min is a good starting point. At intermediate time points, functional assessments can be executed. The size scale of the neonatal recellularized heart limits the assessments, that can be performed but we have determined that optically based systems can be used to quantify beating behavior just as they have been used in adult rat hearts.⁴ The construct can be perfused for extended periods of time (up to 23 hr).

Wyniki

Decellularization
On average, the time to decellularization of a P3 heart using this protocol is approximately 14 hr. given an average heart weight of 23 mg for the P3 neonate.

Acellularity
Figure 3a demonstrates a fully intact P3 neonatal heart (whole mount). Figure 3b shows the same heart following decellularization. Figures 4a and 4b show the hema...

Dyskusje

The dependence of this technique on repeated perfusions of the heart makes the avoidance of an embolism a critical component of a successful outcome. From the initial catheterization of the heart in Steps 2.2-2.6, to the changes of solution between Steps 2.8-2.14, there are manipulations that can allow introduction of air bubbles which compromise the flow of perfusate into the myocardium. Due to the diminutive size of the neonatal heart, even minute bubbles in the vasculature can cause a technical infarct, thus rendering...

Materiały

Name	Company	Catalog Number	Comments
1. Materials For Mouse Heart Isolation
P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J)	Jackson Laboratories	21577	or equivalent
60 mm Culture dish	BD Falcon	353004	or equivalent
Phosphate buffered saline pH 7.4 (sterile)	Hyclone	SH30256.01	or equivalent
Single Use Blade	Stanley	28-510	or equivalent
Standard Scissors	Moria Bonn (Fine Science Tools)	14381-43	or equivalent
Spring Scissors 10 cm	Fine Science Tools	15024-10	or equivalent
Vannas Spring Scissors - 3mm Cutting Edge	Fine Science Tools	15000-00	or equivalent
#5 Forceps	Dumnot (Fine Science Tools)	11295-00	or equivalent
2. Materials For Decellularization
Inlet adaptor	Chemglass	CG-1013	autoclavable
Septum	Chemglass	CG-3022-99	autoclavable
1/8 in. ID x 3/8 OD C-Flex tubing	Cole-Parmer	EW-06422-10	autoclavable
Male luer to 1/8" hose barb adaptor	McMaster-Carr	51525K33	autoclavable
Female luer to 1/8" hose barb adaptor	McMaster-Carr	51525K26	autoclavable
Prolene 7-0 surgical suture	Ethicon	8648G	or equivalent
Ring stand	Fisher Scientific	S47807	or equivalent
Clamp	Fisher Scientific	05-769-6Q	or equivalent
Clamp regular holder	Fisher Scientific	05-754Q	or equivalent
60 cc syringe barrel	Coviden	1186000777T	or equivalent
Beaker	Kimble	14000250	or equivalent
22g x 1 Syringe Needle	BD	305155	or equivalent
12 cc syringe	Coviden	8881512878	or equivalent
3-way stop cock	Smith Medical	MX5311L	or equivalent
22 x 1 g needle	BD	305155	or equivalent
PE50 tubing	BD Clay Adams Intramedic	427411	Must be formable by heat. Polyethylene recommended
1% SDS	Invitrogen	15525-017	Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.
1% Triton X-100	Sigma-Aldrich	T8787	Make up fresh solution from a 10% stock and filter sterilize before use.
Sterile dH2O	Hyclone	SH30538.02	Or MilliQ system purified water.
1X Pen/Strep	Corning CellGro	30-001-Cl	or equivalent
3. Materials For DNA Quantitation
Proteinase K	Fisher	BP1700	>30U/mg activity
KCl	Sigma-Aldrich	P9333	or equivalent
MgCl*6H2O	Mallinckrodt	5958-04	or equivalent
Tween 20	Sigma-Aldrich	P1379	or equivalent
Tris base/hydrochloride	Sigma-Aldrich	T1503/T5941	or equivalent
Pico-Green dsDNA assay kit	Life Technologies	P7589	requires fluorimeter to read
4. Method for fixation and sectioning of tissue.
Paraformaldehyde	Sigma-Aldrich	P6148	or equivalent
Gelatin Type A from porcine skin	Sigma-Aldrich	G2500	must be 300 bloom or greater
5. Method for tissue histology
Cryomolds 10 x 10 x 5mm	Tissue-Tek	4565	or equivalent
Cryostat	Hacker/Bright	Model OTF	or equivalent
Microscope Slides  25 x 75 x 1 mm	Fisher Scientific	12-550-19	or equivalent
Hematoxylin 560	Surgipath/Leica Selectech	3801570	or equivalent
Ethanol	Decon Laboratories	2701	or equivalent
Define	Surgipath/Leica Selectech	3803590	or equivalent
Blue buffer	Surgipath/Leica Selectech	3802915	or equivalent
Alcoholic Eosin Y 515	Surgipath/Leica Selectech	3801615	or equivalent
Formula 83 Xylene substitute	CBG Biotech	CH0104B	or equivalent
Permount Mounting Medium	Fisher Chemical	SP15-500	or equivalent
Collagen IV Antibody	Rockland	600-401-106.1	or equivalent
α-Actinin Antibody	Abcam	AB9465	or equivalent
mCherry Antibody	Abcam	AB205402	or equivalent
NKX2.5 Antibody	Santa Cruz Biotechnology	SC-8697	or equivalent
Donkey anti-mouse AF488 Antibody	Life Technology	A21202	or equivalent
Donkey anti-chicken AF594 Antibody	Jackson Immunoresearch	703-585-155	or equivalent
Donkey anti-goat CY5 Antibody	Jackson Immunoresearch	705-175-147	or equivalent
Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594	Jackson Immunoresearch	111-587-003	or equivalent
Prolong Gold Antifade Mountant with DAPI	ThermoFisher	P36930	or equivalent
6. Isolation of neonatal ventricular cardiomyocytes using pre-plating.
HBSS (Ca, Mg Free)	Hyclone	SH30031.02	or equivalent
HEPES (1M)	Corning CellGro	25-060-Cl	or equivalent
Cell Strainer	BD Falcon	352340	or equivalent
50 mL tube	BD Falcon	352070	or equivalent
Primeria 100 mm plates	Corning	353803	Primeria surface enhances fibroblast attachment promoting a higher myocyte purity
Trypsin	Difco	215240	or equivalent
DNase II	Sigma-Aldrich	D8764	or equivalent
DMEM (Delbecco's Minimal Essential Media)	Hyclone	SH30022.01	or equivalent
Vitamin B12	Sigma-Aldrich	V6629	or equivalent
Fibronectin coated plates	BD Bioscience	354501	or equivalent
Fetal bovine serum	Hyclone	SH30910.03	or equivalent
Heart bioreactor glassware	Radnoti Glass Technology	120101BEZ	Must be sterilizable by autoclaving or gas.