Periodic Reporting for period 2 - CapBed (Engineered Capillary Beds for Successful Prevascularization of Tissue Engineering Constructs)
Reporting period: 2020-05-01 to 2021-10-31
The demand for donated organs vastly outnumbers the supply, leading every year to the death of thousands of people and the suffering of millions more. Engineered tissues and organs following Tissue Engineering approaches are a possible solution to this problem. However, a strategy to irrigate complex engineered tissues and assure their survival after transplantation is currently elusive. In the human body, organs and tissues irrigation is achieved by a network of blood vessels termed capillary bed which suggests such a structure is needed in engineered tissues. Previous approaches to engineer capillary beds reached different levels of success but none yielded a fully functional one due to the inability in simultaneously addressing key elements such as correct cell populations, a suitable matrix and dynamic conditions that mimic blood flow.
CapBed aims at establishing a new technology to fabricate in vitro capillary beds that include a vascular axis that can be anastomosed with a patient circulation. Such capillary beds could be used as prime tools to prevascularize in vitro engineered tissues and provide fast perfusion of those after transplantation to a patient. Cutting edge techniques will be for the first time integrated in a disruptive approach to address the requirements listed above. Innovative fabrication technologies such as 3D printing and laser photoablation will be used for the fabrication of the micropatterned matrix that will allow fluid flow through microfluidics. The resulting functional capillary beds can be used with virtually every tissue engineering strategy rendering the proposed strategy with massive economical, scientific and medical potential.
CapBed aims at establishing a new technology to fabricate in vitro capillary beds that include a vascular axis that can be anastomosed with a patient circulation. Such capillary beds could be used as prime tools to prevascularize in vitro engineered tissues and provide fast perfusion of those after transplantation to a patient. Cutting edge techniques will be for the first time integrated in a disruptive approach to address the requirements listed above. Innovative fabrication technologies such as 3D printing and laser photoablation will be used for the fabrication of the micropatterned matrix that will allow fluid flow through microfluidics. The resulting functional capillary beds can be used with virtually every tissue engineering strategy rendering the proposed strategy with massive economical, scientific and medical potential.
Stromal vascular fraction (SVF) cells were isolated from fat and part was used to obtain adipose stem/stromal cells (ASCs). These cells and SVF cells directly from isolation were used to produce cell sheets and test various media cocktails and culture conditions to maximize the production of extracellular matrix (ECM). One potential drawback of long culture times at confluent densities is spontaneous detachment from the culture surface which was mitigated by the use of cell contractility modulators. Protein was extracted from cell sheets and characterization and quantification were done by Western Blot analysis. Different profiles of expression of angiogenesis-relevant proteins were found. Proteomic analysis will be made in order to complete the characterization of the extracted ECM.
Several materials have been tested to fabricate hydrogels compatible with the laser ablation process. Some hydrogels, while apparently transparent, were found to scatter laser beams and were therefore ineligible to be used with laser ablation. Others have shown to be fully compatible with laser ablation within the tested concentrations but protocols to improve mechanical properties have to be applied. These materials were then used to ablate pre-designed microchannel networks in the interior of hydrogels. To achieve this, programming tools coupled with external triggering components were employed. More complex 3D geometries that better mimic native capillary beds are being now created and perfused with fluorescent beads, demonstrating the interconnectivity of the different channels.
Concurrently, several methodologies to produce model tissues that can be combined with the capillary bed are being developed. Biomaterial-based sponges are being used as 3D matrices for SVF culture. Prevascular networks were successfully produced in said matrices without the use of extrinsic growth factors.
One other potential application of the capillary bed is cancer research. Vascularized multicellular assembloid models of melanoma that can potentially be integrated with the capillary bed have been created. Some of these models are also being used to screen the effects of laser ablation, which is used as a tool to ablate tumors from patients.
Finally, novel protocols to extend the shelf life of engineered tissues, such as the capillary beds, are being developed by using hypothermic preservation. Several existing solutions and compounds are being tested and compared.
Several materials have been tested to fabricate hydrogels compatible with the laser ablation process. Some hydrogels, while apparently transparent, were found to scatter laser beams and were therefore ineligible to be used with laser ablation. Others have shown to be fully compatible with laser ablation within the tested concentrations but protocols to improve mechanical properties have to be applied. These materials were then used to ablate pre-designed microchannel networks in the interior of hydrogels. To achieve this, programming tools coupled with external triggering components were employed. More complex 3D geometries that better mimic native capillary beds are being now created and perfused with fluorescent beads, demonstrating the interconnectivity of the different channels.
Concurrently, several methodologies to produce model tissues that can be combined with the capillary bed are being developed. Biomaterial-based sponges are being used as 3D matrices for SVF culture. Prevascular networks were successfully produced in said matrices without the use of extrinsic growth factors.
One other potential application of the capillary bed is cancer research. Vascularized multicellular assembloid models of melanoma that can potentially be integrated with the capillary bed have been created. Some of these models are also being used to screen the effects of laser ablation, which is used as a tool to ablate tumors from patients.
Finally, novel protocols to extend the shelf life of engineered tissues, such as the capillary beds, are being developed by using hypothermic preservation. Several existing solutions and compounds are being tested and compared.
Several strategies have been proposed for the prevascularization of tissue engineered constructs. However, most of them fail due to an underestimation of the complexity of the angiogenic process that requires the action of several cell types (primarily ECs and stabilizing perivascular cells) and a precise spatiotemporal deployment of growth factor gradients to reach stable and functional blood vessels or capillaries. Furthermore, even if a completely functional and stable capillary network could be achieved, the time to spontaneously anastomose with the host’s circulation after transplantation would likely be too long, compromising the functionality of the engineered tissue. The strategy proposed in CapBed will address all the shortcomings identified above. Using cells from the SVF will allow a completely extrinsic growth factor-free strategy to endothelize pre-designed networks, unlike any strategy presented before. The standalone capillary bed with a vascular axis that will be created will be able to be anastomosed with the host’s circulation following standard surgical techniques, assuring immediate perfusion. Finally, since we are starting from the capillary bed, virtually any engineered tissue, scaffold-free or not, can be combined with it, endowing it with a great clinical potential. To achieve all of this, new methods for the laser-based micropatterning of hydrogels are being developed, which will have a wide impact in the bioengineering area. At the same time, methodologies for the gentle preservation of tissues are being created having not only the capillary beds in mind but also any type of engineered tissue, which may facilitate the clinical application of tissue engineering products in general. Ultimately, CapBed will provide critical tools that can be used not only to streamline the clinical application of already created engineered tissues but also to aid in the development of other more advanced solutions for the problem of organ and tissue shortage.