# Whole Organ Engineering: Approaches, Challenges, and Future Directions
## 5 Recent Developments and Accomplishments  
### Heart  
The quest of engineering a viable and functional whole-heart replacement via whole organ engineering necessitates the development of various approaches for cellular removal due to the high density, large mass, and anatomic complexity of the native human heart. Preclinical studies that utilize perfusion decellularization of rodent hearts have presented encouraging results, but applying this technique to larger animal models such as porcine and bovine that would translate to clinical use of this technique are still a challenge [38]. In a 2010 study conducted by [PERSON] et al., a porcine heart was decellularized reproducibly and efficiently using retrograde aortic perfusion with a mixture of solvents, but reseeding this scaffold efficiently requires further research [97]. Decellularization and recellularization approaches are prevalent in whole heart engineering due to the heart's complex geometry and electromechanical functions making the native ECM architecture difficult to replicate in vitro. [PERSON] et al., expanded upon [PERSON] et al. 's original whole heart decellularization work in 2008 according to standardized protocols developed in the Ott lab to decellularize donated human hearts, and successfully removed over 99% of double-stranded DNA, as determined by endonuclease perfusion [98]. Micro-computed tomography (CT) scans found no statistically significant difference in vascular density between cadaveric and decellularized cardiac matrix, though the decellularized hearts did have lower vascular densities, especially in the endocardium, showing that the decellularization process preserved vasculature but that full-depth vascular recruitment is more difficult in a decellularized heart [98]. The decellularized constructs were repopulated with cardiomyocytes derived from human iPSCs in a custom bioreactor. Analysis of cell activity revealed glucose consumption and lactate production that suggested metabolic activity, electrical stimulation produced visible contractions, and histological analysis showed over 90% cell viability, which indicated that coronary perfusion was providing adequate nutrition and gas exchange [98]. [PERSON] et al., utilized a porcine model, using de-cell/re-cell procedures on porcine hearts and then heterotopically implanting them in living pigs to leverage the body's natural ability to repopulate the scaffold to a greater extent than if only ex vivo recellularization were used [99]. The heterotopic hearts were rapidly endothelialized by host cells, which could potentially reduce clot formation after transplantation, which is a concern with the heart transplant process [99]. Using a decellularization approach to generate whole heart cell scaffolding is promising, but does have its challenges, as assessed in a study by [PERSON] et al., which used the data from 39 heart decellularization procedures to identify critical troubleshooting points in the process [100]. Bottom-up heart engineering techniques, namely 3D bioprinting, are an alternative avenue of research for pursuing whole heart engineering without the challenges of de-cell/re-cell approaches [58; 92]. [PERSON] et al., developed a novel, personalized bioink fabrication method for extrusion bioprinting thick, vascularized cardiac tissue [92]. The personalized bioink was composed of thermoresponsive ECM hydrogel and cardiomyocytes derived from iPSCs, both of which were derived from omental tissue [92]. A sacrificial support material was used for printing, allowing complex geometries to be generated while the structure maintained its shape; thus, allowing cellularized cardiac patches with built-in vasculature and miniature hearts to be printed [92]. This technique's ability to generate vascularized constructs with complex geometry and its use of cellularized bioink derived from patient emmentum, a readily available cell source, make it a promising approach to whole organ engineering. Another bottom-up approach to cardiac tissue engineering was published by [PERSON] et al.