Strategies and Processes to Decellularize and Recellularize Hearts to Generate Functional Organs and Reduce the Risk of Thrombosis

Abstract

Heart failure is one of the leading causes of death in the United States. Current therapies, such as heart transplants and bioartificial hearts, are helpful, but not optimal. Decellularization of porcine whole hearts followed by recellularization with patient-specific human cells may provide the ultimate solution for patients with heart failure. Great progress has been made in the development of efficient processes for decellularization, and the design of automated bioreactors. Challenges remain in selecting and culturing cells, growing the cells on the decellularized scaffolds without contamination, characterizing the regenerated organs, and preventing thrombosis. Various strategies have been proposed to prevent thrombosis of blood-contacting devices, including reendothelization and the creation of nonfouling surfaces using surface modification technologies. This review discusses the progress and remaining challenges involved with recellularizing whole hearts, focusing on the prevention of thrombosis.

Introduction

The use of decellularized porcine whole-organ scaffolds combined with autologous human patient-specific cells would provide an essentially unlimited supply of organs for transplant. Great progress has been made in culturing patient-specific human cells (e.g., induced pluripotent stem cells [iPSCs]) and the promise of constructing a whole human organ from autologous human cells and decellularized porcine organ scaffolds is getting closer to reality.¹ The two steps in this process are decellularization of porcine organs while preserving the extracellular matrix (ECM) and recellularization with a human patient's cells. To fully develop this technology, there is a need to optimize decellularization processes and equipment to reliably and safely produce acellular hearts in large quantities for combination with human cells.² The processes for recellularization and preconditioning the organs in bioreactors prior to implantation also must be optimized. Finally, the organs must be characterized to make sure they are safe and meet all performance requirements.

One of the critical requirements for effective performance of recellularized blood-contacting scaffolds is the prevention of thrombosis. Thrombosis leads to the disruption or blockage of intravascular blood flow and is a consequence of uncontrolled clotting (the formation of fibrin networks) and activation of platelets due to flow disturbances and surface chemistry. The process of thrombus formation includes adhesion of platelets to surfaces, platelet aggregation, and the combination of platelets with fibrin and other cellular components. Thrombi that are released from a surface become thromboemboli, which may lodge in downstream blood vessels and interrupt blood flow to an area of tissue such as the brain, creating a stroke as cells in the brain begin to die. If anticoagulants, antithrombotics, fibrinolytics, and thrombolytics (e.g., coumarins and heparin), or antiplatelet agents (e.g., aspirin) are used during the implantation of recellularized organs to prevent clotting, then this may result in uncontrollable hemorrhage. Developing a reliable method for ameliorating the thrombogenicity of recellularized organs prior to implantation would be a major advance for this technology.

In reviewing the literature, numerous articles exist on the decellularization and recellularization of whole organs. These include 11 review articles on hearts, kidneys, pancreas, lungs, livers, and other organs^1–11 totaling 880 citations. Of these citations, 13 articles were identified as most relevant to the design of an automated system for decellularizing whole hearts.^12–24 The types of detergents, concentrations of solutions, flow rates, pressures, incubation temperatures, total number of steps, and organ exposure times were identified as major factors for consideration. These parameters are summarized in Table 1. Based on these recommendations, an optimized porcine heart decellularization protocol was conducted with excellent results.²⁵ Additional articles from the review of the literature identified the best techniques to introduce cells back into the decellularized hearts, growth and differentiation factors required to control cell phenotypes, bioreactor designs, and evaluation methods that have been developed.

Table 1.

Summary of Decellularization Protocols of Heart Tissues

Species	Predecellularization solutions	Decellularization solutions	Postdecellularization solutions	Decell time (days)	Total time (days)	Ref. No.
Rat	Heparinized PBS, 10 μM adenosine	1% SDS (w/v)	DI water, 1% TX-100, antibiotic solution	0.5	6	12
	PBS containing 1% pen/strep	8 mM CHAPS, 1.8 mM SDS (in 1 M NaCl, and 25 mM EDTA, PBS)	12% FBS endothelial growth media	2	4	13
	None	1% SDS (w/v)	DI water, 1% TX-100, PBS	0.8	1	14
	Heparinized PBS, 10 mM adenosine	1% SDS (w/v), 1% SDC (w/v), 0.05% NaN₃ in DI water; 1% saponin, 0.05% NaN₃ in DI water	20% glycerol, 0.05% NaN₃, 25 mM EDTA in 0.9% NaCl, DI water, 200 IU/mL DNase, MgCl in PBS	0.5	1.5	15
	Heparinized PBS, chilled PBS	1% SDS (w/v) in PBS	10% DMSO in PBS	1	1	16
	PBS	1% SDS (w/v)	DI water, 1% TX-100, antibiotic solution, MCDB-131 medium	0.5	1	17
Mouse	DI water	1% SDS (w/v)	DI water, 15 min, 1% TX-100, 5% pen/strep in PBS, DMEM	0.5	4	18
	DI water, PBS, −80°C	0.2% trypsin/EDTA; 1% SDS (w/v) in NaN₃	Peracetic acid, ethanol, PBS, DI water	0.03	0.1	19
Pig	Water, −80°C	DI water, 2× PBS, 0.02% trypsin in EDTA and NaN₃, 3% TX-100, 4% SDC, DI water, PBS	0.01% peracetic acid, 4% ethanol, PBS	0.1	0.33	20
	PBS, 2% pen/strep, amphotericin-B	1.1% NaCl, 0.7% NaCl, 0.02% EDTA, 0.05% trypsin, 1% TX-100, 0.1% NH₄OH	Sterile saline, 70% ethanol	3	4	21
	DI water, −80°C, 2× PBS	DI water, 2× PBS, 0.2% trypsin in EDTA and NaN₃, 3% TX-100, 4% SDC, DI water, PBS	0.01% peracetic acid, 4% ethanol, PBS	0.1	0.5	22
	PBS	0.1% SDS (w/v), 0.01% trypsin, 1 mM PMSF	70% ethanol	2	18	23
	PBS, −80°C	0.02% trypsin, 0.05% EDTA, 3% TX-100	None	7	8	24
	DI water, −20°C	DI water, 0.5% (w/v) SDS	None	1	1	25

CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DI, deionized; EDTA, ethylenediaminetetraacetic acid; PBS, phosphate-buffered saline; pen/strep, penicillin/streptomycin; PMSF, phenylmethylsulfonylfluoride; SDC, sodium deoxycholate; SDS, sodium dodecyl sulfate; TX-100, Triton X-100.

The overall purpose of this review article is to evaluate and highlight optimal strategies for decellularization, recellularization, growth factor delivery, bioreactor design, evaluation of whole organs, and prevention of thrombosis of blood-contacting porcine whole-organ scaffolds. Reviewed articles on decellularization (Table 1) were supplemented with articles on cells available for recellularization (Table 2), growth factor delivery (Table 3), bioreactor design (Table 4), evaluation of tissue-engineered organs (Table 5), and preventing thrombosis (Table 6).

Table 2.

Cell Sources for Regenerative Medicine

Species	Cell type	Comments	Ref. No.
Murine	Transdifferentiated fibroblasts into cardiomyocytes without inducing pluripotency	Direct reprogramming may be the most promising approach	36
	iPSCs	Immunosuppression enhances engraftment	37
	Transdifferentiated	Inefficient to reprogram	38
	iPSCs	High-quality iPSCs differentiate better	39
	STAP	Low pH induces pluripotency	40,41
	Cardiac telocytes	Injecting telocytes improved the function of hearts after infarction	42
	Neonatal cardiomyocytes, RAECs	Cardiomyocytes were injected into the decellularized scaffold, RAECs were infused through the brachiocephalic artery and vena cava of decellularized rat hearts	17
Murine/Human	iPSCs	Viral infection and correct stoichiometry of 4 transcription factors affect efficiency	43
	hESCs, mouse cardiac cells	Combined cells with urinary bladder matrix for cardiac repair	44
Porcine	Aortic valve interstitial cells	Primary cells were obtained from porcine leaflet explants and reseeded onto decellularized heart valves	45
Human	ESCs	ESCs were differentiated into hMECs and cardiac cells in decellularized mouse hearts	18
	iPSCs	Cells were differentiated into cardiac progenitor cells, ECs, SMCs, and cardiomyocytes in decellularized mouse hearts	19
	Saphenous vein endothelial cells and myofibroblasts	Decellularized porcine heart valves were reseeded with human cells	46
	Stem cells	Umbilical cord matrix was proposed as a source of stem cells	47
	iPSCs	Sendai RNA virus is efficient, and does not integrate into host	48
	iPSCs	Progress toward virus-free iPSCs	49
	iPSCs	Need to increase efficiency, reduce risk of teratoma, develop methods for cell delivery	50
	iPSCs	High-purity enrichment by FACS	51
	CPCs	Genetic modification	52
	iPSCs	Cardiomyocytes useful for drug screening, toxicity studies	53
	Macrophages	Macrophages are a primary responder after myocardial infarction	54
	Fibroblasts	Fibroblasts are the most abundant cell in the myocardium	55
	Endothelial cells	A functional nonthrombogenic layer of ECs on collagen was constructed	56
	ASCs, BMMNCs, MSCs, ADSCs, HFSCs, UCSCs, MDSCs, EPCs	Sources for stem cells	57
	ASCs, ESCs, iPSCs, BMMNCs, PBMNCs	Many cell types are available for potential use in tissue engineering cardiac tissue	58
	ESCs, BMAPCs, MSCs, HSCs	Differentiation agents for hESCs	59
	iPSCs	Useful for understanding disease mechanisms, screening new drugs, and cell replacement therapy	60
	ASCs, ESCs, iPSCs	Describes 4 ways to engineer organs	61

ADSCs, adipose-derived stem cells; ASCs, adult stem cells; BMAPCs, bone-marrow-residing adult progenitor cells; BMMNCs, bone marrow mononuclear cells; CPCs, cardiac progenitor cells; ECs, endothelial cells; EPCs, endothelial progenitor cells; ESCs, embryonic stem cells; FACS, fluorescence-activated cell sorting; hESCs, human embryonic stem cells; HFSCs, hair-follicle-derived stem cells; hMECs, human mesendodermal cells; HSCs, hematopoietic stem cells; iPSCs, induced pluripotent stem cells; MDSCs, muscle-derived stem cells; MSCs, mesenchymal stem cells; PBMNCs, peripheral blood mononuclear cells; RAECs, rat aortic endothelial cells; SMCs, smooth muscle cells; STAP, stimulus-triggered acquisition of pluripotency; UCSCs, umbilical-cord-derived stem cells.

Table 3.

Growth and Differentiation Factors

Factor	Purpose	Comments	Ref. No.
BMP-4, Activin A, bFGF, VEGF, DKK1	iPSC-derived cardiac progenitor stem cell, endothelial cell, smooth muscle cell, and cardiac myocyte differentiation	Cardiac progenitor stem cells were derived from iPSCs in culture flasks and then differentiation into various cardiac cells was performed in the acellular ECM of a mouse heart	19
bFGF	Induces the proliferation of endothelial cells, fibroblasts, smooth muscle cells, and hepatocytes	bFGF was released from alginate with initial 40% burst followed by sustained delivery, increasing vascularization of alginate scaffold	68
TGFβ1	Mediator of inflammatory and fibrotic response	Mediates the cardiomyocyte proliferation and production of ECM by fibroblast	69,70
VEGF	Regulates new vessel growth	PEG scaffolds releasing VEGF were completely remodeled into vascularized tissue	71
HGF	Mitogen of hepatocytes, increases proliferation of endothelial cells but not vascular smooth muscle cells	HGF bound by a collagen binding domain from fibronection was released from a collagen sponge, increased vascularization	72
PDGF	Multiple roles in growth of fibroblasts, SMCs, and glia cells; also implicated in various diseases	PDGF was shown to be effective in periodontal regeneration	73,74
BMP	Critical to development and differentiation of cells to cardiomyocytes	Fibroblasts were found to differentiate into cardiomyocytes by BMP	75
Angiopoietin-1	Regulates vascular integrity and quiescence	Provides a connection between quiescient and active endothelial cells	76

bFGF, basic fibroblast growth factor; BMP-4, bone morphogenetic protein-4; DKK1, Dickkopf-related protein 1; ECM, extracellular matrix; HGF, hepatocyte growth factor; iPSC, induced pluripotent stem cell; PEG, polyethylene glycol; SMCs, smooth muscle cells; PDGF, platelet-derived growth factor; TGFβ1, transforming growth factor beta 1; VEGF, vascular endothelial growth factor.

Table 4.

Bioreactors

Size	Media	Comments	Mechanical specification	Estimated cost	Ref. No.
250 mL (whole rat heart)	Decell: SDS, SDC, NaN₃, Saponin	O₂, CO₂, N₂ in a gas mixer with chamber-controlled pH. Mechanical stimulation improves cell alignment.	Left ventricular 3D stretching latex balloon, controlled with pressure and a membrane pump	$10,000	30
	Recell: FCS, DMEM
	Decell: SDS, TX-100	O₂, CO₂, electrodes for stimulation of cardiomyocytes, recording ECG and pressure	Pulsatile flow from peristaltic pump, controlled pressure and flow	$70,000	12
	Recell: IMDM, FBS, HS
100 mL (biopsy of porcine heart)	Recell: DMEM, 10% FBS, HS	CO₂, electrical stimulation, and ECG recording	Rotating bioreactors were used to culture cardiomyocytes in 3D	$10,000	86
40 mL (rat aorta)	2 reservoirs for external media and internal media	Small rotating bioreactor that can be put in CO₂ incubator	Pulsatile flow	$5000	84
1–2 mL (small vascular grafts and stents)	VascuLife EnGS EC culture media	Can be put in CO₂ incubator, not suitable for long cultures because of no media perfusion	Rotated culture at 5 rpm, no perfusion	$3 per device	85

3D, three-dimensional; DMEM, Dulbecco's modified Eagle's medium; EnGS, endothelial cell growth supplement; FBS, fetal bovine serum; FCS, fetal calf serum; HS, horse serum; IMDM, Iscove's modified Dulbecco's medium.

Table 5.

Evaluation Methods

Type of analysis	Method	Criteria for success	Ref. No.
Protein	Cytoplasmic protein (e.g., β-actin) removal as detected by immunoblotting	Residual cytoplasmic proteins should approach 0%	13
	Noncollagenous protein content, including laminin and elastin, detected by histology staining and western blotting	Less than 7% of native noncollagenous proteins retained	15
	Monitor total protein content in the effluent	Approach 0% after 6 h	16
GAG	Total GAG content	More than 200–250 μg/g preserved	15
DNA	Quant-iT PicoGreen dsDNA assay after using serum nucleases to remove DNA	Residual DNA was 0%	13
	DNA labeling with fluorescent markers	Less than 4% remains	12
	DNA quantification kit (Qiagen, Hilden, Germany)	Less than 3 μg/g (0.5%) of native DNA remained	15
	Quant-iT PicoGreen dsDNA assay	Less than 8% remains	20
Mechanical	Burst pressure of human umbilical arteries	No significant difference in burst pressure of blood vessels after decellularization	13
	Stress–strain test	Very similar to native	12
	Ball burst test	Maximum force endurance the same as native	20
Cell viability	Colorimetric method for testing tissue-engineered tracheas prior to implant	Average cell coverage of 40%	87
	Live/dead assay after 24 h culture with C2C12 myoblasts	>95% cell viability	15

GAG, glycosaminoglycan.

Table 6.

Antithrombotic Strategies

Tissue or substrate	Decellularization process	Antithrombotic strategy	Results	Key observations	Ref. No.
Rat hearts	SDS	ECs	Thrombomodulin activity was increased six- to eightfold	EC coverage reduced thromobogenicity	17
Porcine heart valves	TX-100, SDC, Igepal CA-630, ribonuclease digestion	ECs	Seeding with endothelial cells abolished platelet adhesion and activation	Decell porcine matrix activated platelets	90
	TX-100, SDC, Igepal CA-630, ribonuclease digestion	ECs	Decell process removed α-gal epitope	α-Gal epitope remained on Synergraft^® heart valves, possibly causing rejection	91
	0.5% TX-100, 0.5% SDC, 0.02% EDTA at 4°C for 48 h, then ribonuclease A, DNase at 37°C for 2 h	Polyelectrolyte layers+EPCs	Substantial reduction in platelet adhesion and activation	Heparin-chitosan multilayer film reduced platelet activity	93
Pulmonary valves	0.5% SDC, 0.5% SDS, PBS, DNase I	ECs	Surfaces were free of thrombi at 3 months after implantation in a large-animal model	First report of in vivo efficacy of reendothelialized decellularized heart valves	92

ECs, endothelial cells; EDTA, ethylene diamine tetracetic acid; EPCs, endothelial progenitor cells; PBS, phosphate-butter saline; SDC, sodium deoxycholate; SDS, sodium dodecyl sulfate; TX-100, triton X-100.

Decellularization

The process of decellularization begins with harvesting a heart from a heparinized animal under general anesthesia or from an animal at an abattoir immediately after exsanguination. For the latter, heparinized physiologic saline is used for antegrade coronary arterial perfusion and endothelial lavage to remove blood from the cardiac chambers. Heparinized salt solutions are typically used to remove circulating blood from the heart and to prevent coagulation in the smaller vessels. The heart may be transported at room temperature or on ice, and stored in the refrigerator or freezer for up to a year. Once initiated, the decellularization process is generally continuous, with the exchange of various fluids containing detergents or enzymes to disrupt the cell membranes and detach the cells from their underlying ECM. The detergents are then removed by washing steps. Hypotonic and hypertonic solutions may be used to further disrupt the cell membranes by osmotic shock. Sterilization may be performed prior to recellularization using antibiotics and acidic solutions. Various processes requiring different solutions, times, number of steps, or order of the reagents have been investigated as summarized in Table 1.

During the decellularization process, the cells are disrupted and detached from the basement membrane protein structure, or ECM. Detergents are perfused through the vascular network of the organ to solubilize cell contents and membrane components. Lipids, sugars, soluble proteins, and DNA are removed. Insoluble proteins, such as collagen, laminin, fibronectin, and elastin, that form the structural features remain, as well as some signaling molecules that are important for guiding tissue regeneration after recellularization. All of these changes result in a tissue construct that is uniquely modified by the process. Standardization of the process, and the use of common assays to confirm the results, is required to create a fully functional human heart replacement. The reduction of time is a critical factor for optimization in the decellularization process, in order to limit damage to the ECM by exposure to the detergents, reduce the risk of contamination, and minimize material and labor costs.

The first report of perfusion decellularization of whole hearts was by Ott et al. in 2008.¹² In their study, they used sodium dodecyl sulfate (SDS) to decellularize rat hearts for 12 h, followed by 15 min of deionized (DI) water, and then Triton X-100 (TX-100) for 30 min to remove SDS and renature the ECM. Phosphate-buffered saline (PBS) with antibiotics was then perfused through the heart for 124 h. This group demonstrated that a rat heart could be decellularized to form an ECM scaffold that could be reseeded with a recipient's cells and grown into a functional heart. They also compared different methods for decellularizing rat hearts using SDS, TX-100, and polyethylene glycol. They concluded that SDS was most effective in removing cells from the heart, but challenges remained, including removal of excess SDS from the matrix. After decellularization, they recellularized the rat heart with freshly isolated neonatal cardiac cells through intramural injection. Perfused organ culture was maintained for 8–28 days. By day 8 after cell seeding, they reported that the heart constructs showed electrical and contractile responses to stimulations and heart beats were observed.

Since the pioneering work of Ott et al.¹² to decellularize and recellularize rat hearts, multiple groups have applied decellularization technology to rat,^13–17 mouse,^18,19 pig,^20–25 and even human hearts.^2,26–28 For example, Sanchez et al. reported the decellularization of 23 whole human hearts with SDS for 4–5 days.²⁷ Guyette et al. also reported the decellularization of porcine and human hearts.^26,28 The methods used for decellularizing human hearts were developed based on published protocols by the same authors for decellularizing hearts from rats¹² and pigs.²⁰ Retrograde perfusion of 1% (w/v) SDS through the aorta was performed after perfusing the organs with heparin solution at constant pressure of 60 mm Hg. TX-100 and PBS were then perfused through the hearts to complete the decellularization and remove detergents.²⁹ All steps were performed aseptically to prevent contamination of the organs. The upcoming sections review the key articles that have reported results for decellularizing whole mammalian hearts.

Development of Decellularization Protocols in Rodent Models

Heart models were first developed using rodent models since large numbers are available commercially, and the small size gives better control of the decellularization environment with fewer cells required for recellularization. Fischer F344,^12,13 LEW/Crl,¹⁵ Wistar,^30,31 and Sprague Dawley^14,16 rats, average body weight of 300 g, have been used in these studies as well as mice with average body weight of 30 g.^18,19

One of the key concerns in the use of decellularized organs has been the recipient immune response to residual proteins, DNA, and cell debris remaining in the ECM. Although ECM proteins are considered nonimmunogenic for transplantation, DNA residues more than 50 ng per mg of lyophilized tissue and residual cell proteins in tissues can induce negative immune responses in the host body.^22,32 Gui et al.¹³ used serum nucleases to remove residual DNA from decellularized tissues. Harvested hearts from Fischer F344 rats were cut along the longitudinal and circumferential axes and then incubated with CHAPS, SDS buffers, and endothelial growth media containing 2.5% to 12% fetal bovine serum (FBS) for 22, 22, and 48 h, respectively. A significant decrease in the amount of residual DNA was observed in samples treated with FBS. Quantification of β-actin, an indicator of residual cytoplasmic proteins, was used to evaluate treatment efficacy. From these studies, the use of detergents combined with rinses containing serum nucleases was superior in removing cell debris as indicated by the significantly lower amounts of β-actin remaining in the samples. Additional research will be required to confirm that these steps remove all immunogenic components.

In 2012, Witzenburg et al.¹⁴ examined the effect of decellularization on mechanical characteristics of the right ventricles of rat hearts. Hearts were harvested from adult female Sprague Dawley rats (9–13 weeks of age) with fresh hearts serving as controls for comparison to decellularized hearts treated with 1% SDS over 20 h. Strain tracking was performed within 48 h of dissection of the heart samples on a biaxial system and Verhoeff's stain was applied to examine the epicardial surface. From these tests, it was concluded that although decellularization produces quantitative differences in modulus, decellularized tissue still provides a useful model of the native tissue ECM. Further studies will be needed to confirm that decellularization processes will not detrimentally impact the mechanical structure of the heart.

Akhyari et al.¹⁵ compared three published protocols^12,20,33 with a novel protocol developed for whole-heart decellularization of LEW/Crl rats. Searching for an ideal protocol for myocardial decellularization, they introduced a protocol using SDS, sodium deoxycholate (SDC), glycerol, and saponin as detergents. Examining the decellularized hearts for remaining noncollagenous proteins and residual DNA content, as well as preservation of glycosaminoglycans (GAGs) and viability of C2C12 myoblasts after reseeding in the ECM, they concluded that each protocol had its advantages and disadvantages. Comparing all four protocols it was shown that none of the analyzed protocols produced a biological matrix entirely free of donor cell material and a scaffold with preserved ECM components. This study demonstrates the need for careful removal of cells to avoid damaging the ECM.

Crawford et al.¹⁶ reported successful recellularization of decellularized Sprague Dawley rat hearts after long-term cryopreservation, to determine whether long intervals between decellularization and reseeding with cells might be a viable option for commercial scales. In their study, hearts were harvested from Sprague Dawley rats, flushed with 2500 U of heparin in PBS, and then decellularized with 1% SDS in PBS for 24 h, and cryopreserved at −80°C with 10% DMSO in PBS. After up to 1 year, the hearts were thawed, treated with 1% Triton-X 100 in DI water, washed with DI water, and incubated in 6.7 U/mL nuclease at 25°C for 1 h. For decontamination, the samples were placed in 0.1% peracetic acid and 4% ethanol for 20 min and soaked in medium with FBS for 1 h to prepare for recellularization. Cell seeding was then performed by injecting 2×10⁷ canine endothelial cells with an 18-gauge needle directly through the myocardium at 5-mm spacing. The hearts were incubated under static conditions for 45 min and then moved to the recellularization bioreactor at 37°C and 5% CO₂; then, growth medium was pumped through the heart for 9 days. No DNA testing was reported for these decellularized samples to ensure complete removal of residual DNA; however, to ensure nontoxicity of the samples, canine peripheral blood progenitor cells were grown in contact with decellularized fragments in culture wells, and when compared with wells without tissue fragments, no differences in cell proliferation were observed, signifying no gross, growth-impeding toxicity. This study is an example of the successful use of cross-species cells and scaffolds. It provides encouragement for the use of porcine hearts with human cells.

Ng et al. used the same techniques and decellularized the hearts of 9–12-week-old male FVB/N mice.¹⁸ The decellularization was initiated with overnight perfusion of 1% SDS in distilled water followed by washing the decellularized organ with TX-100 in distilled water for 1.5 h to remove the SDS residue. Disinfection was accomplished by perfusing the decellularized hearts with a solution of 5% penicillin/streptomycin in PBS for 72 h at 4°C. Then, the hearts were reseeded by retrograde aortic injection with 3×10⁶ human embryonic stem cells (hESCs) and human mesendodermal cells derived from hESCs. After 14 weeks of static culture in H9c2-conditioned cell medium (Dulbecco's modified Eagle's medium with 10% FBS, 2 mM glutamine, and 1% penicillin/streptomycin), the hearts were implanted subcutaneously into severe combined immunodeficiency mice and then the hearts were dissected and characterized after 2, 4, and 6 weeks. They reported that the stem cells expressed cardiac-specific markers, such as cTnT, Nkx-2.5, Myl2, Myl7, Myh6, and CD31; however, the beating function was lacking.

In another use of cross-species ECM and cells, Lu et al. reported the use of human iPSC-derived cardiac progenitor cells in mouse hearts.¹⁹ The optimal decellularization procedure was completed in <2 h and, after 20 days of culture with cardiac multipotent stem cells derived from human iPSCs (Y1-iPSCs), the hearts began to beat. Differentiation into cardiac myocytes, smooth muscle cells, and endothelial cells on the ECM was reported and it was shown that ECM stimulates the proliferation of cardiac myocytes for a longer period of time than in three-dimensional (3D) environments without ECM. Also, the effect of isoproterenol on the beating, recellularized hearts was tested and demonstrated an increase in beats per minute by a factor of 2.

In summary, in studies performed in rodent models to date, whole hearts have been successfully decellularized and recellularized with endothelial cells to prevent thrombosis and heart muscle cells to produce contractility. When these hearts are transplanted, however, the pumping force of the implanted heart is not sufficient to sustain life, and the endothelialization is incomplete, leading to potential thrombosis in the absence of anticoagulants, and the possibility of hemorrhage when anticoagulants are used.

Development of Decellularization Protocols in Porcine Models

Although Ott et al. mentioned in their original report¹² the attempt to decellularize a porcine heart, they did not provide details. Several other groups have subsequently attempted the decellularization of porcine hearts.^20–25 In general, the same concentrations of detergents and solutions used for murine hearts have been used for porcine hearts. Solution volumes have been increased, but exposure times have been optimized to <1 day. Summaries of five different protocols for porcine hearts are given in the following paragraphs.

In the first report of porcine heart decellularization, Wainwright et al.²⁰ attempted decellularizing a porcine heart with a complex protocol in order to achieve an intact ECM scaffold. In this procedure, the hearts were frozen at −80°C for at least 16 h for convenience of storage and to aid in cell lysis. The hearts were then thawed in type 1-reagent-grade water at room temperature. The aorta was cannulated with a 1/2′′ to 1/4′′ straight-barbed reducer and connected to 1/4′′ internal-diameter silicone tubing. Each heart was placed in a 4-L beaker containing 3 L of hypotonic water that was recirculated using a peristaltic pump for 15 min at 1 L/min. The water was replaced with 2× PBS at 1 L/min for 15 min. Three liters of 0.02% trypsin, 0.05% ethylenediaminetetraacetic acid (EDTA), and 0.05% NaN₃ solution was warmed to 37°C using a digital hotplate and then perfused through the myocardial vasculature at 1 L/min for 2 h. A 3% TX-100, 0.05% EDTA, and 0.05% NaN₃ solution was then used for perfusion followed by a 4% SDC solution at 1.3 L/min each for 2 h at room temperature. After each chemical solution was used as a perfusate, type 1-reagent-grade water was perfused through the heart for ∼5 min with no recirculation followed by recirculating 2× PBS for 15 min to aid in cell lysis and removal of cellular debris and chemical residues.

Eitan et al.²¹ decellularized 3-mm-thick ring samples dissected from the left ventricle of hearts harvested from slaughter-weight female pigs. The samples were incubated for 48 h in 1% TX-100+0.1% NH₄OH in PBS solution followed by washes in sterile saline solution, followed by immersion in 70% ethanol overnight for decontamination. The effects of decellularizing a porcine heart and reseeding with adult sheep cardiac fibroblasts, newborn Sprague Dawley rat cardiac myocytes, and mesenchymal stem cells on mechanical properties were examined, and these studies showed the viability and proliferation of cells from various species on decellularized samples. DNA or cell residue assays were not performed on these decellularized tissues to ensure complete removal of DNA and cell debris; however, in order to indicate the biocompatibility of these samples, they measured the stimulation of macrophages in vitro using basal nitrite concentration to demonstrate one aspect of their sample's biocompatibility.

Remlinger et al.²² reported a whole porcine heart decellularization based on the protocol of Wainwright et al.²⁰ Their modification was adding cycles of hypotonic/hypertonic solutions with type 1 distilled water and 2×PBS each for 10 min. They also increased the total enzyme and detergent exposure time from 6 to 8.5 h, and increased total decellularization time after thawing the hearts from <8 h to more than 12 h. Using a Pico green assay, they reported 350 ng DNA per mg of native ventricles and <50 ng DNA residues per mg of lyophilized decellularized heart, representing a significant decrease in residual DNA. Although DNA lengths of <200 bp and total content of <50 ng/mg have been proposed as acceptable threshold levels for DNA residues after decellularization,^5,34 it has not been shown that reductions in DNA levels lead to improvements in recellularization or acceptance by the host. Further work is required to demonstrate the effect of residual DNA on the performance of recellularized hearts.

Wang et al.²³ reported the decellularization of sections from porcine hearts in a rotating bioreactor. The bioreactor stimulated decellularized myocardium samples in square shapes both mechanically and electrically, and was used to improve differentiation of stem cells into myocardiac cells and recellularization of samples. They decellularized 20×20×3-mm³ myocardium sections with 0.1% SDS, 0.01% trypsin, 1 mM phenylmethylsulfonylfluoride (PMSF) as a protease inhibitor, 0.2 μg/mL RNase, and 0.2 mg/mL DNase in the presence of 100 U/mL penicillin/streptomycin for 2.5 weeks, renewing the solution every 2 days. Cell viability, histology, and immunofluorescence staining were performed to confirm the presence, number, and phenotype of cells following reseeding. The differentiated cells expressed myosin heavy chain, cardiac troponin T, and sarcomeric α-actinin, indicative of a cardiomyocyte phenotype. The rotating bioreactor maintained long-term viability of cells in a sterile environment with the ability to provide mechanical and electrical stimulus.

Merna et al.²⁴ reported the use of optical imaging to predict the change in mechanical properties during decellularization of porcine hearts. Each heart was frozen at −80°C and then thawed and decellularized with 0.02% trypsin and/or 3% TX-100 solutions containing 0.05% EDTA and 0.05% NaN₃ for 1 to 7 days. Multiphoton microscopy combined with image correlation spectroscopy was used to noninvasively characterize the mechanical properties of the hearts. It was found that TX-100 preserved the collagen matrix, whereas trypsin weakened the heart structure.

Using the previously reported conditions for decellularization, Momtahan et al.²⁵ used SDS and TX-100 to decellularize porcine hearts in <1 day. These hearts were subsequently characterized by SEM and histology and used to develop a thrombogenicity assay for whole organs.

Some important lessons have been learned from the prior work on optimization of decellularization. For example, trypsin or other enzymes are typically used to detach cells from surfaces, including tissue culture polystyrene. In employing the use of enzymes to remove cells from the whole-organ ECM, there is a concern that damage may occur to the ECM or insoluble signaling proteins. The article by Merna et al.²⁴ showed that trypsin weakened the heart tissue more than TX-100. Also, DNAse was used to break up and remove the DNA that could be potentially immunogenic. Several articles have indicated, however, that the removal of DNA by detergents, such as SDS and TX-100, is sufficient, and the presence of low amounts of DNA is not considered an impediment to recellularization or of concern for immunogenicity.⁶ This conclusion was based on the studies by Keane et al.³⁵ that described similar cell behavior on fully decellularized scaffolds compared with those after incomplete decellularization. Additional work is required to verify this conclusion. Overall, while there are various methods that have been successful for decellularization, the combination of SDS and TX-100 appears to be the best method reported so far. SDS is a strong ionic detergent that is very effective at removal of cells, but persists in the ECM. TX-100 is very effective at removing SDS and remaining cell debris. In our experience, retrograde perfusion with three batches of SDS for 2 h each, alternating every 2 h with DI water, and then perfusing with TX-100 for 4 h, followed by extensive rinsing in DI water for at least 4 h, produces a completely decellularized porcine heart.

Strategies for Recellularization

The process of recellularization involves the selection and proliferation of cells, as well as the provision of nutrients, growth factors, gases, and waste exchange in a bioreactor. Many cell types^{17–19,36–61} from autologous and allogeneic sources have been proposed for use in recellularization (Table 2).

It may be necessary to provide cocultures of multiple cell types, or if a pluripotent cell type is selected, then it may be possible for that single cell type to differentiate into all the cell types of the heart. Early attempts to engineer heart valves identified the aortic valve interstitial cells,⁴⁵ saphenous vein cells, and myofibroblasts⁴⁶ as potential cell sources. Cells that have been specifically considered for whole-heart engineering include embryonic stem cells,⁵⁷ skeletal myoblasts, resident adult cardiac stem cells, adipose-derived stem cells, peripheral blood mononuclear cells, bone-marrow-derived hematopoietic, and mesenchymal stem cells⁵⁸ to provide contractility and to produce ECM proteins during tissue repair and remodeling. Cardiomyocytes are required to populate the thick portions of the heart muscle; fibroblasts are required for generation, maintenance, and repair of the ECM; and endothelial cells are required to line the flow surfaces. In addition, smooth muscle cells that form the walls of blood vessels and pacemaker cells that control the heart rhythm must also be reintroduced. Care must be taken to prevent contamination from bacteria and fungi during recellularization, storage, and handling prior to implant. Ideally, the patient's own (autologous) cells would be used; however, this would most likely require the isolation and culture expansion of cells in a facility that is capable of simultaneously handling human cells from many patients under strict controls. Alternatively, allogeneic cells could be used, but they would need to be carefully selected or may require surface modification to prevent the cells from initiating an immune response.

Several articles have highlighted the progress and challenges associated with culturing cells for regenerative medicine.^{17–19,36–53,57–61} Five of these articles specifically reviewed the use of stem cells for cardiovascular engineering.^57–61 Since these reviews were published, multiple articles have addressed the unique issues pertaining to the use of iPSCs, which may ultimately be the best solution to provide patient-specific cells, but will require improvements in efficiency of dedifferentiation, the elimination of the risk of teratoma formation, improved culture techniques, and novel methods for delivering the cells to the decellularized matrix.

An alternative to the use of iPSCs may be the transdifferentiation of fibroblasts directly to cardiomyocytes without inducing pluripotency. This has been demonstrated by Ieda et al.,³⁶ in which three developmental transcription factors (Gata4, Mef2c, and Tbx5) were found to efficiently (20%) reprogram cardiac fibroblasts into cardiomyocytes in 3 days without inducing a pluripotent state. However, Chen et al. attempted to replicate this method with contrary results.³⁸

Bajpai et al.⁵⁷ and Jing et al.⁵⁹ also have reviewed the potential and challenges of stem cells for repairing damaged heart tissue. Stem cells are unique in that a certain fraction proliferate and retain their “stemness” while others differentiate. Types of stem cells include embryonic, bone-marrow-residing adult progenitor cells, side population, cardiac stem cells, skeletal muscle progenitors (satellite cells), and adipose-derived stem cells. Liu et al.⁶¹ described four ways to engineer organs: from a single stem cell, injecting stem cells into blastocysts, using a decellularized tissue scaffold plus stem cells, and combining stem cells with synthetic scaffolds.

There are many advantages to the use of iPSCs, including the avoidance of political and ethical issues related to embryonic stem cells. The discovery of the Yamanaka transcription factors (Klf4, Sox2, Oct4, and c-Myc) has allowed many labs worldwide to study iPSCs. Cell lines are now available for many diseases and cell types.⁴⁹ Some of the challenges of using iPSCs are the low efficiency, need for viral infection, and the possibility of teratoma formation.⁴³ iPSCs from various sources demonstrate variable efficiencies. Recent reports have claimed efficiencies in the creation of iPSCs of >90% after enrichment through cell sorting.⁵¹ There are also recent reports that a low pH environment may be sufficient to produce pluripotency.^40,41

Many other investigators have examined various ways to improve the performance of cells on engineered substrates. For example, the culture environment for endothelial cells has been shown to have a major role in whether antithrombogenic or procoagulation factors are produced.⁵⁶ In addition, Le et al.⁶² described the surface roughness needed to control protein and cell attachment. Higuchi et al.⁶³ demonstrated that heart matrix supports cardiomyocyte attachment. Tosun and McFetridge⁶⁴ proposed the use of gradients to improve cell seeding, and Daly et al.⁶⁵ discussed the initial binding of cells during recellularization.

Recently, telocytes, a type of interstitial cells that are found in myocardium, epicardium, endocardium, and in cardiac stem cell niches, were discovered. Though the functional role of these cells in myocardial tissue is not completely understood, it has been shown that they play an essential role as niche-supporting cells that nurse the cardiac stem cells and angiogenic cells in the myocardium.^66,67 Zhao et al.⁴² showed that cardiac telocytes can be identified using CD34 and c-kit markers as well as morphological techniques. They reported that 4 days after myocardial infarction, the number of telocytes in the heart are highly diminished, and injecting them back into the damaged hearts improved the function and reduced the size of scar tissue in the rat hearts.

To reduce the thrombogenicity and improve the function of the recellularized hearts, Robertson et al.¹⁷ recellularized rat hearts that were decellularized based on the previous protocol described by the same group¹² with rat aortic endothelial cells (RAECs) and neonatal cardiomyocytes. They infused either 2×10⁷ or 4×10⁷ cells to the decellularized hearts from the brachiocephalic artery (BA), aorta, or inferior vena cava (IVC). During the infusion from the aorta, retrograde perfusion of media was stopped for 1 h. Alternatively, endothelial cells were infused to the heart from the BA without any breakage in the MCDB-131 media perfusion. They reported that cells delivered only via the aorta were not uniformly distributed throughout the heart and the best result was delivering the cells through the IVC and BA simultaneously. Using both BA and IVC they observed that the endocardial surface of the left ventricle was predominantly recellularized with RAECs delivered via the BA, whereas the endocardial surface of the right ventricle was populated with RAECs delivered via the IVC. After 7 days of RAEC culture, they injected 1.3×10⁸ rat neonatal cardiac cells into the left ventricular wall in three to four parallel injections. One day afterward, electrodes were sutured to the apex and base of the hearts in order to stimulate the cardiac function. After 10 days of culture, a microtip pressure catheter was inserted into the left ventricle and the pressure generation by the cells was monitored. They observed that the pressure generated by the heart, at 4 Hz of stimulation, was six times higher in hearts that were cultured with RAECs prior to recellularization with cardiomyocytes.

Challenges remain in selecting human cell types for use in cardiac tissue engineering. These include combining cells with ECM proteins, promoting cell migration and homing to the proper location in the pre-existing ECM, ensuring electromechanical cell coupling for propagation of depolarization potentials, producing a robust and stable contractile function, and proper vascularization of the tissue.⁵⁸ Challenges also remain with the use of stem cells due to the possibility of teratoma formation, the need to culture and maintain large quantities of viable cells, and the necessity to deliver the cells in a manner that is conducive to their survival and integration into the heart. If stem cells are combined with a decellularized matrix, then there is also a need for the addition of growth and differentiation factors (Table 3), bioreactors designed to properly condition the heart so it is ready for transplant (Table 4), evaluation of the heart prior to implant (Table 5), and methods to integrate the heart into the body so it can perform its function. Another major challenge is the prevention of thrombus formation (Table 6).

Growth and Differentiation Factors

Soluble trophic factors may be delivered during the growth of the tissues to promote differentiation or proliferation. Generally FBS is added in cell culture, although the elimination of as many unknowns as possible has led multiple researchers to develop xeno-free formulas of cell media. In the case of adding growth factors to the porcine hearts, testing of the combination of factors and possibly their sequence of administration will be necessary to identify potential regeneration factors and eliminate immunogenic factors. Some of the growth and differentiation factors used in tissue engineering^19,68–76 are listed in Table 3.

Critical factors⁷⁷ that have been identified for evaluation include basic fibroblast growth factor (bFGF),^19,68 transforming growth factor beta 1 (TGFβ1),^69–70 vascular endothelial growth factor (VEGF),^19,71 hepatocyte growth factor (HGF),⁷² platelet-derived growth factor (PDGF),^73,74 bone morphogenetic protein (BMP),⁷⁵ and angiopoietin-1.⁷⁶

The temporal and spatial distribution of the factors is important, and encapsulation or controlled release strategies of the factors may be required. Lee et al.⁷⁸ found that synthetic particles that released VEGF in response to mechanical stress could control blood vessel density in newly forming tissue. Freeman et al.⁷⁹ demonstrated that the release of bFGF from alginate hydrogels doubled the formation of blood vessels, and Harel-Adar et al.⁸⁰ showed that modulation of cardiac macrophages by phosphatidylserine-presenting liposomes improved infarct repair.

Bioreactor Designs

Preconditioning of the organ with a physiologically relevant set of conditions must be performed prior to implant. Bioreactors that can operate in a range of temperatures, from room temperature to 37°C, allow for gas exchange, and provide nutrients during the growth phase have been designed.⁸¹ For hearts, the muscle must be allowed to grow and organize, and then be subjected to flow conditions that mimic normal blood flow dynamics in order to strengthen the muscle until it can perform its function of pumping with the appropriate force. One particular challenge with hearts is incomplete perfusion due to trabeculated atrial appendages that are potentially difficult to perfuse. For example, the left atrial appendage (LAA) has a narrow, sharply pointed shape. It is believed to function as a decompression chamber during left ventricular systole and other periods when left atrial pressure is elevated, and it has endocrine function as the primary source of atrial natriuretic factor (ANF). In contrast, the right atrial appendage is broad based and less distinct in appearance, with trabeculations (pectinate muscles) that extend toward the tricuspid valve and are not confined to the appendage.⁸² Al-Saady et al.⁸³ posited that obliteration or amputation of the LAA may help reduce the risk of thromboembolism in patients with primary cardiac disease, but this may result in undesirable physiological sequelae, such as reduced atrial compliance, reduced secretion of ANF in response to pressure and volume overload, and decreased stroke volume and cardiac output that may promote heart failure. Bioreactor designs must be robust, and potentially may need to include sensors to detect flow rates and pressures to provide a means to compensate for changes related to the presence of atrial appendages, while optimizing flow to help minimize thrombogenesis.

Several investigators have proposed various designs^{12,30,84–86} for bioreactors (Table 4). Decellularization is often performed in a modified Langendorff apparatus¹² with retrograde perfusion preferentially used over antegrade flow. Geeslin et al.⁸⁵ discussed the bioreactor designs for vascular conduit regeneration, including duel reservoirs for endothelial cells and smooth muscle cells. These were connected in parallel with pumps and oxygenators. The smooth muscle cells were cultured on the exterior of a decellularized matrix, while the endothelial cells were cultured on the lumen. Avci-Adali et al.⁸⁶ discussed a rotating bioreactor based on ready-to-use medical disposables to reendothelialize a vascular conduit. Their results demonstrated that expanded polytetrafluoroethylene (ePTFE) grafts with albumin and heparin coatings produced the most extensive endothelial cell adhesion and spreading. Bursac et al.⁸⁴ discussed rotating bioreactors for culturing cardiomyocytes in 3D cultures. They found that cells cultured in 3D created better tissue than in two-dimensional monolayers. Huelsmann et al.³⁰ applied the concept of 3D stretching to provide mechanical stimulation to a whole decellularized heart matrix from Wistar rats. Their device included an inflatable latex balloon inserted into the left ventricle and inflated by a syringe pump, and was controlled with a pressure sensor and LabView software. Hearts seeded with C2C12 murine myoblasts were cultured for 24 h in static culture, and with biomechanical stimulation for another 72 h. Although the results demonstrated lower cell viability, there was an improvement in orientation of cells in the exercised hearts. Further work will be required to optimize the development of bioreactors for whole porcine hearts.

Evaluation of Organs

Each type of recellularized organ (kidney, lung, heart, etc.) will require a different set of tests to evaluate its functional performance prior to implant. For example, the proper performance of kidneys will require that the cells filter blood and create urine. Lungs will require that the cells exchange oxygen and carbon dioxide with circulating blood. Hearts must be preconditioned to pump blood at physiologically relevant pressures and flow rates. Although bioengineered, recellularized organs may eventually be able to perform desired endocrine functions, this physiologic property must be secondary, and is expected to be sacrificed during the development of transplantable organs with adequate biomechanical function to sustain life. Nevertheless, across all of these organs, the flow surfaces must be prepared for contact with blood. Endothelial cells naturally perform the function of preventing thrombosis. The logical goal of whole-organ recellularization, therefore, is to fully cover every blood-contacting flow surface with endothelial cells. Assuring that the blood-contacting flow surface has been thoroughly reendothelialized will require the development of strategies to measure endothelial cell viability, coverage, and functional performance in prevention of thrombosis, in addition to other analytical tests.

Analytical methods have been identified to detect proteins,^13,15,16 cellular debris, and DNA content after decellularization.^{12,13,15,17,20,22} Research has also been done on the evaluation of mechanical characteristics after decellularization and recellularization,^{12–14,20,21} demonstrating the potential for strengthening the muscles prior to implant. In addition, Jungebluth et al.⁸⁷ have demonstrated a viability assay for cells prior to transplantation. These strategies are summarized in Table 5.

Although hundreds of proteins comprise the ECM, the major proteins, including collagen, laminin, vitronectin, and fibronectin, have been known and studied extensively. The less-prominent proteins may be critical to successfully engineer an organ. Byron et al.⁸⁸ described the identification of ECM proteins using proteomics, and estimated that there are over 100 proteins comprising the ECM of most organs. Barallobre-Barreiro et al.⁸⁹ described the changes in porcine cardiac ECM after ischemia. They identified collagen α-1 (XIV), cartilage intermediate layer protein 1, matrilin-4, extracellular adipocyte enhancer binding protein 1, asporin, and polargin as contributers to cardiac remodeling. These proteins were identified by liquid chromatography tandem mass spectrometry. Although this type of analysis is useful, there is still a need for assays that verify the presence of these proteins in vivo. It is also imperative to demonstrate that the adhesive properties of the ECM have not been overly compromised after decellularization due to protein denaturation.

Strategies for Preventing Thrombosis

During the development of vascular grafts, intracoronary stents, the total artificial heart, and other blood-contacting medical devices, many strategies have been proposed for preventing thrombosis, including delivery of anticoagulants, the attachment of endothelial cells,^17,90–99 attachment of heparin,¹⁰⁰ the creation of nonfouling surfaces,^101–104 and other approaches. The use of anticoagulants (e.g., heparin) in the systemic circulation can be used to prevent acute thrombosis, but may lead to hemorrhage over the long term, and are not included in this review. The other strategies are summarized in Table 6 and are described in detail in the following paragraphs.

The process of preventing thrombosis is closely related to strategies for recellularization. If endothelial cell coverage could be perfectly achieved, and inflammation during healing at the anastomoses could be minimized, then theoretically thrombosis could be avoided. Kasimir et al.⁹⁰ described the decellularization of porcine heart valve matrix with TX-100 and SDC. The scaffolds were then recellularized with human umbilical vein endothelial cells (HUVECs) and tested with human platelets from volunteers. Platelet adhesion and activation were measured and antibodies to collagen and elastin were used to characterize the material. It was found that the decellularized scaffold was a platelet-activating surface. However, seeding the surface with endothelial cells abolished platelet adhesion and activation. They concluded that endothelial cells are essential to prevent thrombosis. Kasimir et al.⁹¹ then tested Synergraft valves (porcine) versus TX-100-decellularized grafts. Important factors that were identified included the α-gal epitope, monocyte migration, calcification of glutaraldehyde crosslinked valves, and the need for complete coverage by endothelial cells to prevent platelet activation, since platelets recruit leukocytes and express chemokines and cytokines that activate monocytes, leading to inflammation. The removal of the α-gal epitope, present on the surface of porcine vascular endothelium, is important since α-gal activates the classical complement pathway, resulting in hyperacute rejection and graft failure. In addition, other GAGs can interact with cytokines and chemokines and may also need to be eliminated to avoid an inflammatory or immune response.

Alternatively, GAGs may need to be preserved to properly attach the endothelial cells. The GAGs are part of the local environment surrounding each cell, called the pericellular matrix (PCM). The PCM may need to be reconstituted with the appropriate GAGs and proteins to retain adhesion molecules and insoluble signaling molecules while also removing the immune-activating molecules. McLane et al.⁹⁵ discussed the role of the PCM on chondrocytes and methods to noninvasively measure the ultrastructure of the hydrated zone that extends 100 to 500 nm from the cell surface. It was concluded that the PCM mediates cell interactions with surrounding tissue, and may influence important processes, such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. In reestablishing the PCM for cardiac cells, care must be taken to first provide a nominally nonthrombogenic or nonfouling surface. The cardiomyocytes and endothelial cells can then be added. If there is a break in coverage of the endothelial cells, then the platelet adhesion and activation may be mitigated by the nonthrombogenic constituents despite the presence of the underlying thrombogenic ECM. Lichtenberg et al.⁹² decellularized pulmonary valves using detergents, and DNAse I to remove >95% of the DNA. In the decellularized structures, collagen, elastic fibers, GAGs, and basement membrane were all preserved. It was hypothesized that the absence of endothelium may predispose the matrix surface to thrombosis. Adhesion ligands (collagen IV, laminin, and perlecan) in the basement membrane were thought to attract circulating endothelial cells or progenitor cells that covered the pulmonary valves. They provided evidence that preservation of GAGs followed by reendothelialization can prevent thrombosis and hyperplasia of tissue-engineered, blood-contacting structures.

Another approach has been to reattach specific GAGs to the blood-contacting surface. For example, heparin attached to ePTFE vascular grafts has been successful in preventing thrombosis.¹⁰⁰ The trade-off between controlling thrombosis with systemic heparin, and preventing the natural healing response at the anastomoses and pseudointimal hyperplasia in the lumen of the implant has always been a challenge for vascular grafts. Propaten^® vascular grafts successfully prevent early thrombosis due to the covalent end-point heparin attachment method developed by Carmeda^® and the high concentration of heparin attached to the surface. Ye et al.⁹³ proposed the use of a polyelectrolyte multilayer film on decellularized porcine aortic valves to prevent thrombosis. The alternating layers of heparin and chitosan provide another method of attaching heparin to the surface to prevent the initiation of thrombosis. A nonthrombogenic underlayer could provide a safety factor against thrombosis if the endothelial cell layer is incomplete or disrupted during initial or long-term use.

The use of in vitro endothelialization⁹⁷ is another proposed way to prevent thrombosis in tissue-engineered and synthetic vascular grafts. However, attempts to endothelialize vascular grafts have generally been unsuccessful due to the cost and complexity of maintaining living grafts during shipping and storage. Several strategies have been employed to increase endothelial attachment. For example, fibronectin was added to decellularized aortic grafts to accelerate reendothelialization and prevent thrombosis.⁹⁸ Wissink et al. developed a new method for collagen crosslinking using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) in order to improve endothelial cell seeding on human umbilical veins.⁹⁹ Lord et al.¹⁰¹ proposed the use of perlecan to reduce thrombogenicity and demonstrated that perlecan did not permit aggregation of platelets when the heparin sulfate chains attached to perlecan were intact. Elastin has also been proposed as a nonthrombogenic material.¹⁰² Other nonfouling synthetic materials, including polyethylene oxide (PEO), have been proposed.¹⁰³ Similarly, Smith et al.¹⁰⁴ at Semprus^® Biosciences (a division of Teleflex) developed a technology to attach sulfobetaine molecules to the surface of blood-contacting devices that minimizes thrombus formation. The Semprus technology employed zwitterionic sulfobetaine molecules that coordinate water near the surface of blood-contacting catheters to create a nonfouling surface. Both bacteria and platelets were prevented from attaching. All of these approaches to improve reendothelialization could be considered for adaptation to decellularized organs. Each approach would require further optimization, and may require new chemistry and process conditions to provide benefit without damaging the ECM.

It may also be possible to genetically modify the cells that are used for recellularization to be more thromboresistant. For example, Kader et al. demonstrated that overexpression of endothelial nitric oxide synthase (eNOS) by endothelial cells produced antithrombotic conditions and decreased platelet aggregation by 46%.⁹⁶ Alternatively, some have proposed that it may be possible to implant decellularized tissues into the recipient's body without the need for reendothelization. Assmann et al.³¹ dissected thoracic aortas from Wistar rats and decellularized them with four 12-h cycles of 0.5% SDS+0.5% SDC and then rinsed the aortas with distilled water for 24 h to wash off the detergents. The aortas were then rinsed with three 24-h cycles of 1% penicillin/streptomycin in PBS. These engineered grafts were implanted in recipient rats and connected to their circulatory system. After 8 weeks they extracted and examined the samples and reported a confluent luminal cell layer, purportedly from circulating cells in the peripheral blood. Although this approach is promising, it has not been shown that these results can be extrapolated to smaller blood vessels (<4 mm) where artificial grafts have consistently failed due to thrombosis and hyperplasia.

In order for reendothelialization to be effective, the specific attachment of endothelial cells to the surface may be required, while avoiding the attachment of platelets. Insoluble signaling proteins, such as fibronectin, are believed to be required in order to reestablish correct spatial orientation of cells during recellularization. Specific sequences of amino acids, such as arginine-glycine-aspartic acid (RGD) peptides from fibronectin, are involved in cell attachment, and, if disrupted, then they may not permit proper positioning of the new cells on the matrix. Bellis⁹⁴ recently reviewed the use of RGD peptides for directing cell attachment. In the late 1980s on through the 1990s, a number of labs were successful in attaching RGD peptides to a variety of surfaces to promote endothelialization.^{103,105–110} Although the first approach for specificity in the attachment of endothelial cells to synthetic matrices was based on the use of RGD peptides, these peptides were found to be nonspecific and also bound platelets in addition to endothelial cells. In addition, although RGD peptides have been shown to promote the attachment of cells in vitro, once a surface is exposed to blood the surface is rapidly remodeled and covered by circulating proteins (Vroman effect). Therefore, combining peptides that promote the adhesion of endothelial cells with nonfouling surfaces or nonthrombogenic molecules may be a more likely way to succeed. The creation of a nonfouling, nonthrombogenic surface followed by RGD, YIGSR, GFOGER, cyclic RGD, REDV, or other specific peptides to attach endothelial cells has been proposed.⁹⁴ This approach will require further investigation. To be successful, cells should attach, integrate, promote regeneration, and be interactive and biomimetic. Multiple modification steps with various molecules may be needed to achieve all of these objectives.

It is clear that in order to advance the field of whole-organ decellularization and recellularization, there are many possible ways to accelerate the processes, and there are many variables that can be optimized. In the pioneering work of Ott et al.,¹² an in vivo animal model assay was used to test the performance of bioengineered hearts. It would be cost prohibitive to test all the possible combinations of seeding strategies and to compare all of the other variables in animal models. Therefore, rapid, reliable, and relevant in vitro assays that are amenable to whole-organ testing will be essential tools to investigate the various strategies for recellularization and thrombosis prevention. The multiple modalities, blood components, and conditions that affect thrombus formation on a surface necessitate concomitant sophistication in the test methods to assess this phenomenon. For example, coagulation may be more pronounced in areas of low flow and stagnation (e.g., atrial appendages) while platelet activation, adhesion, and aggregation may be elevated in regions of high shear (e.g., coronary arteries). Thus, assessment of whole-organ performance may require a diverse panel of tests to identify specific problems that will help expedite development of a nonthrombogenic organ. The ISO 10993-4 standard prescribes such a panel of in vitro tests for the assessment of blood-contacting device hemocompatibility¹¹¹ that may serve as a useful guide when adapted suitably for whole-organ thrombosis assessment. Assessments of fluid-phase markers of coagulation cascade activation (e.g., thrombin-antithrombin ELISA) and platelet activation (e.g., β-thromboglobulin ELISA and p-selectin expression by flow cytometry) are some of the recommended tests.¹¹² Evaluation of surface-bound platelets and thrombus using radiolabeled platelets and scanning electron microscopy are highlighted as important tests.¹¹³ The roles of the complement pathways and leukocytes in thrombosis have also been recognized and associated assays are considered valuable.¹¹⁴

In addition to the tests outlined in the ISO 10993-4 standard, several other test models have been reported in the literature. Hanson and Sakariassen¹¹⁵ reviewed the experimental models of thrombosis used to measure antithrombotic drug efficacy. They demonstrated that flow of the blood in a circuit can also activate platelets. Shankarraman et al.¹¹⁶ have utilized thromboelastography to quantify thrombogenicity of blood-contacting surfaces. In vitro blood flow models have been used to assess thrombosis and thromboembolism associated with artificial hearts and vascular implants such as stents.^{112,113,117,118} Ensley and Nerem¹¹⁹ studied the effects of shear stress on performance of endothelial outgrowth cells. McGuigan and Sefton⁵⁶ seeded HUVECs on collagen and developed thrombogenicity assays using whole fresh blood from consenting donors. They perfused constructs with blood, demonstrating that HUVECs prevented the attachment of platelets.

In the first report of measuring thrombogenicity in decellularized whole organs, Taylor's research group has shown that recellularizing rat hearts with RAECs prior to adding neonatal cardiomyocytes decreases the thrombogenicity and improves the beating function of the heart.¹⁷ To measure the thrombogenicity of the recellularized organs, they examined protein C activation as an indicator for activation of the anticoagulation pathway. After 7 days of RAEC culture, they observed a six- to eightfold increase in thrombomodulin and protein C activity in recellularized hearts compared with acellular hearts. They also performed a heterotopic transplantation of the reendothelialized hearts into recipient rats that were on anticoagulation therapy with sodium heparin, 100 IU/kg twice on day of transplant and 200 IU/kg subcutaneous for the next 2 days and daily coumadin (0.25 mg/kg) in the drinking water. After 7 days the hearts were dissected and they reported the observation of fewer blood clots in the aortas and ventricles of the reendothelialized hearts compared with acellular hearts. They also observed the presence of endothelial cells in acellular hearts that were not recellularized with RAECs before transplantation.

In our experiments, we have found that the thrombogenicity of decellularized and recellularized whole hearts can be assessed using bovine blood with an assay¹²⁰ that has become a standard performance measurement for the medical device industry. In this in vitro flow model of thrombogenicity, suitably anticoagulated bovine blood is pumped through the heart at a controlled flow rate. The thrombus that forms in the heart is characterized after a period of blood flow by high-resolution digital photography and measured by radiolabeled platelets that accumulate in the thrombus. Scanning electron microscopy of select regions is also used to investigate the morphology of thrombi and to understand their origin and progression on the test surfaces. One of the unique features of studies conducted with advanced in vitro flow models is that relevant conditions can be controlled more precisely than is possible in animal and clinical studies.¹²¹ Parameters that significantly influence thrombosis, such as hemostatic conditions (i.e., anticoagulation), can be controlled precisely in the in vitro setting. Yet another benefit of an in vitro setting is the ability to directly quantify thrombosis (e.g., with¹¹¹ Indium-labeled platelets). In future studies, the in vitro blood flow model can be used to assess thrombogenicity of tissue-engineered hearts and test the effect of various decellularization process conditions and cell sources.

For these studies, fresh blood is obtained by inserting a cannula directly into the heart of cows during exsanguination at a local abattoir and collected into a collapsible reservoir containing an anticoagulant (heparin or sodium citrate). The negative controls in these studies are fully decellularized hearts that would be expected to have a very high amount of thrombus formation. The positive controls are freshly harvested hearts prior to decellularization that would be expected to have a low amount of thrombus formation. The test hearts are decellularized under various conditions, modified with nonthrombogenic molecules, and optimally recellularized with endothelial cells. Test hearts are attached to appropriate polymer tubing whose tips are inserted into the blood reservoirs (maintained at 37°C) to complete the flow circuit. Roller pumps control blood flow at a desired flow rate, and this is monitored noninvasively using an ultrasonic flow probe. The blood collected from a single animal is divided into multiple blood reservoirs (as many as there are hearts to compare simultaneously). Incomplete endothelialization may be detected via this in vitro assay, as long as contact of blood from one animal with endothelial cells from another animal does not lead to immune-response-mediated thrombosis or other confounding adverse responses. Preliminary studies have shown that this method can distinguish differences between decellularized and freshly harvested porcine hearts.²⁵ Additional experiments will be needed to test the thrombogenicity of hearts after recellularization.

Studies can also be conducted with recalcified citrated blood flowing through the heart at a lower flow rate (e.g., 1 L/min) for a shorter duration (e.g., <30 min) to focus the assessment on coagulation. Other studies can be conducted with heparinized blood flowing through the heart at a higher flow rate (e.g., 3 L/min) for a longer duration (e.g., 2–3 h) to focus the assessment more on platelet-mediated thrombosis. Assessment of plasma markers of complement activation, platelet activation, and inflammation can also be conducted with human hearts and human blood. While in vitro tests of thrombosis and associated markers are valuable tools, direct extrapolation of in vitro results to the in vivo setting may not be possible, and the relevance of certain in vitro tests is still being debated.^121,122

Discussion

One of the goals of tissue engineering research with decellularized whole organs is to adapt the published protocols for decellularization of blood-contacting scaffolds for automation and scaling to whole porcine hearts. Other goals include the development of strategies for recellularization, growth factor delivery, bioreactor design, quality analysis, and measurement of thrombogenicity in vitro prior to implantation. The possibility of using decellularized whole porcine hearts combined with human cells to create viable hearts for transplant is getting closer to reality.¹ Porcine hearts are similar to human hearts in size and anatomy, and the use of porcine heart valves in humans has a long history of success.¹²³ The development of the decellularization process in rats¹² has been successfully scaled to porcine hearts.²⁰ Optimizing the decellularization process further will potentially limit damage to the ECM. Since the ECM signals modulate inflammatory and reparative pathways, these signals affect cell survival phenotype and also gene expression.¹²⁴ Expediting the decellularization process will also reduce the risk of contamination and reduce the cost of reagents and labor. In reviewing the literature, we found many articles that described decellularization methods, but very few described the optimization and automation of recellularization strategies to prevent thrombosis in whole organs, which motivated us to initiate the development of a novel thrombosis assay.²⁵

This scope of this review covered the progress that has been made to decellularize whole hearts, including rodent, porcine, and human hearts, since the first attempt was reported in 2008,¹² as well as the methods for preventing thrombosis in medical devices. Many variables have been studied for the process of decellularizing whole hearts, including various detergents, concentrations, rinses or washes, flow rates, pressures, and times. This review highlights the most important parameters for optimization in future efforts to automate decellularization and recellularization processes, and prevent thrombosis of blood-contacting organs. We also described a method for the assessment of these strategies through the use of an in vitro blood flow thrombogenicity assay.¹²⁰

In addition to the previous articles that have been reviewed, much can be learned from the history of decellularization of porcine heart valves and small intestine submucosa (SIS), which have been approved as commercial products. Several reviews have been written on these products. For example, Badylak et al. have reviewed the development of SIS^32,125–127 and also recently reviewed whole-organ decellularization¹²⁵ and the use of porcine bladder¹²⁸ for use as a matrix. Gilbert et al.³ reviewed the decellularization process for tissues and organs, focusing primarily on the development of SIS. The use of mechanical agitation, sonication, and osmotic shock with hypertonic/hypotonic solutions was discussed. SDS and TX-100 were proposed as detergents that could be used. A call was made for the development of assays to determine the extent of decellularization.

Stem cells are a possible strategy for recellularization, since they are pluripotent, and may be guided to differentiate into a variety of cell types. Use of iPSCs has been proposed since they could be patient specific, but still have pluripotency. The efficiency of creating iPSCs is improving, and the use of environmental cues may eliminate the need for the viral introduction of transcription factors.^40,41 Transdifferentiation may be a better strategy than using iPSCs, since transdifferentiation may decrease the risk of teratoma formation associated with inducing pluripotency. At the very least, the iPSCs must be shown to be terminally differentiated prior to implantation, as has been done by Advanced Cell Technology, Inc. (ACT), in a human clinical trial using terminally differentiated hESCs for treating dry age-related macular degeneration.¹²⁹

Although it was not the focus of this review, other strategies have been proposed for culturing cells into 3D structures without the use of decellularized scaffolds, mimicking embryological development. Although the initial development of organs during embryonic formation does not require the introduction of a separate scaffold, the size of these organs is very small relative to an adult organ. The embryological organs create their own ECM as they develop. As a child grows and matures into an adult, all of the organs constantly remodel and replace the smaller versions with larger organs. This provides a rationale for growing new hearts using neonatal or adolescent cells.

The source of cells may be autologous or allogeneic. Ideally, the cells will be autologous, but there may be situations in which autologous cells are not preferred. For example, one may not want to use an elderly patient's cells for recellularization, especially if they are diseased. Even patient-specific stem cells may not be good candidates to recellularize a bioengineered heart for transplantation in individuals with systemic disease. As cells age, they lose their youthfulness, purportedly due to telomere shortening.¹³⁰ Juvenile cells that have many years of life ahead of them could be a great source for recellularization. It may be preferable to grow a heart with a younger patient's cells, if they are properly immunologically matched. Cells could be isolated from neonatal or young patients whose families agree to donate their organs. In the best case, juvenile, autologous, patient-specific stem cells can potentially be used from tissue banks, if the patient or the patient's parents had the foresight to bank cells at a young age, or retained the umbilical-cord-derived stem cells at birth.

During recellularization, the coordination of multiple cell types in the matrix may require extensive engineering of the matrix proteins. Suggestions of adding RGD peptides (a three-amino-acid sequence from fibronection responsible for cell adhesion) to the matrix have been made.⁹⁴ However, RGD peptides are not specific; therefore, other peptides such as YIGSR (a sequence from laminin) may be better, and not attract platelets, just the desired endothelial cells.

There are numerous alternative strategies to the use of decellularized scaffolds and patient-specific cells for creating new hearts. For example, 3D printing using cells and ECM proteins has been proposed as a possible method for engineering a new tissue. These technologies hold the promise of exciting possibilities and are also worthy of additional research. In addition, cell sheet technology has been developed by Professor Okano's lab¹³¹ and L'Heureux et al.¹³² have described the use of cell layers rolled on a mandrel to create a blood vessel.

Other organs have been decellularized and recellularized, including the trachea,¹³³ lungs,^65,134–140 liver,^141,142 pancreas,¹⁴³ blood vessels,¹⁴⁴ and kidneys.^{141,145–148} Many of these articles have outlined the design criteria for bioreactors for decellularization and recellularization. The general principles addressed in this review pertaining to the heart also apply to these other organs. Also, much can be learned from these articles on the processes used for decellularization and recellularization of other organs that can be applied to the heart.

With all of these tissues, there is a need to prevent transmission of disease. Allografts have been used for many years, and procedures are in place to decontaminate donated tissues, including using acids and alcohols. Hodde et al. have described a method to evaluate tissues for viral infection,¹⁴⁹ the effects of sterilization on ECM composition and architecture,¹⁵⁰ and the effects of sterilization on ECM bioactivity and matrix interaction.¹⁵¹ All of these sterilization techniques will need to be adapted to decellularized whole organs.

The use of endothelial cells for recellularization is the ideal solution for preventing thrombosis. In the case that there is not complete covering of the flow surface with endothelial cells, a modified subendothelial surface would require the use of nonfouling molecules or heparin molecules attached to the surface. However, this approach may prevent reendothelization or alter the Vroman effect of protein adsorption to the surface, and great care must be taken if these strategies are employed.

The current status of this work is that hearts can be decellularized, and initial success has been obtained with attachment of cells to the scaffold, but full recellularization to provide function while preventing thrombosis has not been achieved. Additional work is required to optimize both the decellularization and recellularization processes. Once the processes for decellularization and recellularization are optimized, other remaining issues with the use of porcine hearts will need to be addressed, including the need for culturing large quantities of immunologically matched, patient-specific cells; the development of aseptic protocols to maintain the organs free from contamination; reinnervation of the heart muscle; shipping and storage protocols; and the development of surgical techniques to implant porcine hearts into humans. It is our hope that these challenges may soon be overcome, and the promise of regenerative medicine may be realized for heart failure patients.

Footnotes

Disclosure Statement

Nima Momtahan, Beverly L. Roeder, and Alonzo D. Cook: No competing financial interests exist. Sivaprasad Sukavaneshvar: Vice President of Thrombodyne, Inc.

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