La función que cumple la generación de conocimiento científico desde el enfoque de sistemas

Corline heparin product’s interactions with Human Microvascular Endothelial Cells (HMECs) were determined. Specifically, we (1) determined the cytotoxicity, if any, of the Corline product on HMECs, (2) determined if the Corline product has an impact on endothelial cell attachment, and (3) determined if the Corline Heparin product can be applied on top of the HMECs after seeding.

Figure 32: 40,000 HMECs were seeded onto collagen coated plates with the addition of CHC or free Heparin for a length of 2 and 4 days. Results show that free heparin inhibits the proliferation of HMECs, but CHC, at low concentrations, does not. Furthermore, higher cell proliferation was seen when CHC was added before seeding compared to adding CHC after seeding.

To test the effect of CHC upon endothelial cell attachment, native porcine hepatic vessels were isolated from healthy market weight pigs. These vessels were decellularized using our established published protocol for the decellularization of whole livers. Next, the decelled vessels were either coated with the Corline heparin conjugate or not coated. HMECs were cultured on the vessels for 4 and 7 days and fixed in 10% neutral buffered formalin.

At both 4 and 7 days there does not appear to be a difference in HMEC cell attachment, viability, or confluence when comparing the uncoated decellularized liver vessels with Corline coated decellularized liver vessels.

Figure 33: HMECs Cultured on uncoated decellularized liver vessels (left) and Corline coated decellularized liver vessels (right) for four (top) and seven (bottom) days. At both 4 and 7 days there does not seem to be a difference in HMEC cell attachment, viability, or confluence when comparing the uncoated decellularized liver vessels with Corline coated decellularized liver vessels.

To experimentally test the effect of CHC applied after HMEC seeding, native porcine hepatic vessels were isolated from healthy market weight pigs. These vessels were decellularized using our established published protocol for the decellularization of whole livers. Next, we coated the decellularized liver vessel with CHC, and HMECs were culture on it (count as day0). On day2 media containing CHC was added. Samples were collected on day 4 and 7.

The samples were fixed in 10% neutral buffered formalin and were stained with H&E to assess their attachment and viability. At both 4 and 7 days there was nodifference in HMEC viability or confluence between the samples with no CHC added on day 2 vs. samples with CHC added on day 2.

Figure 34. HMECs Cultured on CHC coated decellularized liver vessels with no CHC added on day 2 (left) and Corline coated decellularized liver vessels with CHC added on day two (right) for four (top) and seven (bottom) days. At both 4 and 7 days there does not appear to be a difference in HMEC viability or confluence between the samples with no CHC added on day 2 vs. samples with CHC added on day 2.

7.4.8 Reconstruction of the Venous Network

Following the first set of experiments conducted, in which we investigated key endothelial cell seeding variables, we then developed a new method for delivering endothelial cells within a decellularized rat liver. For one decellularized rat liver, endothelial cell seeding was performed on 3 separate days (10 million per day) as opposed to the previous method of 30 million cells seeded at the same time. Histologic assessment of the graft showed endothelial coverage of several major vessels within the decellularized rat liver as well as sinusoidal space. This microvascular formation within the sinusoidal space was not seen with previous seedings.

Figure 35. (A) Representative H&E images from our newly developed method of seeding microvascular endothelial cells within a decellularized rat liver. Endothelial cell coverage of major vessels as well as the sinusoidal space can be seen throughout the seeded scaffold. (B) HMECs delivered via the portal vein (red) co-localized with HMECs delivered via the hepatic vein (green)

7.4.9 Reconstruction of the Arterial Network

Figure 36. Isolation and cannulation of the hepatic artery allowed for endothelial cell delivery throughout the hepatic arterial network. After 3 days of culture an arterial network was formed and could be visualized via H&E. TUNEL stained showed a majority of cells are viable.

7.5 DISCUSSION

One of the major challenges for whole organ engineering is to produce large volume tissues capable of being connected to the recipient’s blood supply for clinical applications. Pervious work in the field of bioengineer liver tissue has been met with many obstacles including cell sourcing, efficient cell seeding, vascularization of the engineered tissue, and provision of authentic cues for tissue development. The experiments conducted in this chapter were aimed at developing a technology that will provide meaningful progress toward engineering a fully functional hepatic vascular network within a decellularized liver scaffold. To do so, we presented here a method of decellularization that was used to fabricate a naturally derived whole-organ bioscaffold. We used the vascular channels as a conduit for reseeding human microvascular endothelial cells inside the bioscaffold. The three-dimensional liver bioscaffold provided spatial information for cell localization and engraftment, and supported cellular proliferation and phenotype maintenance. These developed methods offer a potential technique for fabrication of human liver tissue that can be readily transplanted into host animals or used for studies of liver cell biology, physiology, toxicology, and drug discovery with further development.

Previously, decellularization of tissue was performed by submersion of the tissue within a detergent solution under agitation to allow cell removal in bulk from the surface of the tissue moving inward. These approaches were successful for decellularization of smaller samples (up to 5 mm in thickness), whereas in thicker specimens the core of the tissue remained cellular. To circumvent this limitation, we took advantage of the native liver vascular network by perfusing the detergent through this network and distributing it throughout the entire liver. This gentle

procedure preserves the architecture of the liver matrix and vascular system. The choice of detergent for the production of whole organ bioscaffolds using perfusion may also impact the preservation of important biochemical cues. In chapters 5 and 6 we showed that harsh ionic detergents such as SDS facilitate rapid removal of cells from dense tissues and can yield a functional bioscaffold, but they may damage some ECM components. Therefore, we opted to use a mild nonionic detergent, Triton X-100. We found that this detergent could successfully decellularize the whole liver by the removal of a majority of cellular DNA. These studies highlight the utility of perfusion decellularization to generate whole organ bioscaffolds with significant potential for organ bioengineering.

Typically, neovascularization of bioengineered tissues was addressed by supplementing cells with angiogenic growth factors or fabricating scaffolds from synthetic material that allowed micro-patterning of vascular tree-like structures. When growth factors are used alone, they tend to create only a microvasculature consisting of small and fragile capillaries, and therefore this technique is only applicable for the engineering of smaller size tissues. An alternative fabrication method is using a micropatterning technique that can be scaled up to larger sizes by modular construction. However, currently this method cannot replicate the progressive complexity and ECM composition of the native liver vascular tree. The bioscaffolds generated from whole livers produced via our decellularization method retain the complexity of multiple size vessels that can deliver fluids from the larger vena cava or the portal vein and reach each individual liver lobule.

An additional advantage of this method is the retention of important ECM molecules required for site-specific engraftment and/or differentiation of different cell types that are present in the liver. Prior research showed that liver-specific stem cells can be isolated and differentiated to hepatic fate. We used human microvascular endothelial cells to recellularize the bioscaffolds.

The advantage of seeding human microvascular endothelial cells is that they are consistent in their proliferation rate, they are easy to maintain in culture, and they are associated with a low experimental cost. This allowed us to perform many recellularization experiments before moving to more clinically relevant iPSC derived human endothelial cells.

A major obstacle in producing large-volume tissues is the delivery of adequate numbers of cells to the entire thickness of the tissue. Commonly used methods for cell seeding into scaffolds employ static, dynamic, or perfusion bioreactor seeding, resulting in seeding of cells that can only penetrate several millimeters below the surface, with further engraftment dependent on active migration of the cells. Alternatively, previous methods to recellularize organ scaffolds have relied heavily on direct cell injection that damaged the scaffold microarchitecture and produced heterogeneous scaffold seeding. The perfusion method introduced here supports cell infusion through both the venous and arterial vascular networks and deposition throughout the thickness of the bioscaffold, achieving greater seeding efficiency without compromising the integrity of the bioscaffold. Furthermore, by accessing different vessels that feed into the liver, we were able to deliver cells selectively to different compartments of the liver tissue. Endothelial cells delivered through the vena cava selectively seeded larger and smaller blood vessels up to the pericentral area of the liver lobule, without reaching the periportal space of the lobule where the final branching vessels of portal vein are located. On the other hand, cells seeded through the portal vein, which delivers blood from the intestine and other organs to the liver, reached predominantly the periportal area of the liver lobule without extensive penetration to its pericentral space. These seeded endothelial cells cover the entire circumference of a vascular channel and maintained cell-cell junctions. Thus, simultaneous utilization of both vascular routes for cell seeding enables complete access to the entire length of the vascular network, which has

an essential importance for prevention of blood clotting and ultimately failure to transplant the bioengineered liver.

This study demonstrates that three-dimensional biologic scaffolds composed of liver extracellular matrix have the capability to become a completely re-endothelializes scaffold closely mimicking the native three-dimensional structure of liver tissue. The bioscaffold may also prove as a good tool to study normal tissue and organ development as well as liver pathology. Ultimately, this technology may bring us closer to the ultimate goal of providing bioengineered livers for transplantation.