Development of novel apoptosis-assisted lung tissue decellularization methods

Abstract

Decellularized tissues hold great potential for both regenerative medicine and disease modeling applications.The acellular extracellular matrix (ECM)-enriched scaffolds can be recellularized with patient-derived cells prior to transplantation, or digested to create thermally-gelling hydrogels for 3D cell culture. Current methods of decellularization clear cellular components using detergents, which can result in loss of ECM proteins and tissue architectural integrity. Recently, an alternative approach utilizing apoptosis to decellularize excised murine sciatic nerves resulted in superior ECM preservation, cell removal, and immune tolerance in vivo. However, this apoptosis-assisted decellularization approach has not been optimized for other tissues with a more complex geometry, such as lungs. To this end, we developed an apoptosis-assisted lung tissue decellularization method using a combination of camptothecin and sulfobetaine-10 (SB-10) to induce apoptosis and facilitate gentle and effective removal of cell debris, respectively. Importantly, combination of the two agents resulted in superior cell removal and ECM preservation compared to either of the treatments alone, presumably because of pulmonary surfactants. In addition, our method was superior in cell removal compared to a previously established detergentbased decellularization protocol. Furthermore, thermally-gelling lung ECM proteins supported high viability of rat lung epithelial cells for up to 2 weeks in culture. This work demonstrates that apoptosis-based lung tissue decellularization is a superior technique that warrants further utilization for both regenerative medicine and disease modeling applications. IntroductionAdvances in tissue engineering have led to broad use of naturally-derived biomaterials such as collagen, fibrin, hyaluronan, and decellularized tissue extracellular matrix (ECM) scaffolds, for regenerative medicine as well as for ex vivo disease modeling 1-4 . Naturally-derived biomaterials offer unique advantages in both applications because they provide natural cell attachment sites and are biocompatible, biodegradable, and cause little to no immune response post-implantation 2 . Additionally, natural biomaterial scaffolds allow assessment of cell behavior in a physiologically relevant manner as two-dimensional (2D) substrate coatings and as threedimensional (3D) cell culture scaffolds 1 . Studies have collectively demonstrated the importance of ECM-derived cues in cell response in healthy and diseased states, further highlighting the importance of environmental relevance when understanding cell behavior and translating the results to clinical applications.Among different naturally-derived biomaterials, decellularized tissues and organs offer unique advantages in generating acellular scaffolds with high retention of the majority of tissue-specific ECM components while removing immunogenic cellular debris 5 . As such, decellularized tissues have been widely used to repair damaged tissues and replace lost or non-functional tissues/organs...

Decellularization for the retention of tissue niches

“…70 Other zwitterionic detergents Sulfobetaine 10 and Sulfobetaine 16 (SB10 and SB16, respectively) induce cell apoptosis, resulting in improved cell removal, which eliminates the need for vigorous washing steps. 71 Physical agitation is used to gently assist in coaxing apoptotic cells from the matrix. 71,72 SB-10 and SB-16 have been shown to result in better retention of ECM basement membrane integrity and higher rates of cell removal when compared to ionic detergents.…”

“…71 Physical agitation is used to gently assist in coaxing apoptotic cells from the matrix. 71,72 SB-10 and SB-16 have been shown to result in better retention of ECM basement membrane integrity and higher rates of cell removal when compared to ionic detergents. 73 Both detergents are typically followed by enzymatic treatments, such as DNase, to promote DNA fragmentation and reduce immunogenicity of the ECM construct.…”

“…73 Both detergents are typically followed by enzymatic treatments, such as DNase, to promote DNA fragmentation and reduce immunogenicity of the ECM construct. 39,71 In the case of CHAPS treatment, immunogenicity can also be reduced by induction of a physiologically accurate pH during decellularization to reduce inflammation upon later implantation. 74,75 Song et al 71 describes that cellular apoptosis is more quickly induced when SB-10 and SB-16 are combined with another apoptosis-inducing agent, camptothecin, while McCrary et al 57 found that SB-10 and SB-16 treatment could improve SDC decellularizations.…”

“…It has been utilized in several studies and has shown great promise for decellularization of sciatic nerve, as it preserves anatomical architecture while retaining small bioactive molecules. 71,151 Cornelison et al 151 reported that camptothecin was necessary for cellular removal through hypertonic/hypotonic buffers. Further treatment with DNase over hypotonic buffers led to effective DNA removal for nerve tissue.…”

“…Interestingly, the use of potassium laurate for decellularization resulted in an increased recellularization potential and a significantly reduced immune response upon implantation compared to lung decellularized by SDS. 63 Song et al 71 found that the zwitterionic detergent, SB10 following treatment with the cytotoxic drug, camptothecin, was able to successfully decellularize rat lung while significantly reducing the damage to the ECM collagen structure when compared to a commonly utilized Triton X-100 and SDS treatment. 71 The potential of using potassium laurate and SB10 for detergent-based decellularization of thin tissues is worth further investigation (Figure 2).…”