Isolation, Characterization, and Vascular Potential of Porcine Cells in a Three-Dimensional Decellularized Liver Matrix Model

Sara Morini^1,2,3Sandra Melitón Barbancho³Álvaro Blanes Rodríguez³Iris Pla-Palacín³Pilar Sainz-Arnal^3,4Natalia Sanchez-Romero^3,4,5María Victoria Falceto⁶Olga Mitjana⁶Antonio Romero⁶Marcela Andrea Del Río Nechaevsky^7,8,9María Lourdes Begonchea Martínez¹⁰Emma Olmedo Arbizu¹⁰Sara Lorente¹⁰Angel Lanas^10,11,3Ana Fernandes-Platzgummer^1,2Pedro M Baptista^11,12,3,7*

• ¹Department of Bioengineering and iBB – Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
• ²Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
• ³Instituto de Investigación Sanitária de Aragón (IIS Aragón), Zaragoza, Spain
• ⁴Cytes Biotechnologies SL, Barcelona, Spain
• ⁵Facultad de Ciencias de la Salud, Universidad San Jorge, Villanueva de Gállego, Spain
• ⁶Departamento de Patología Animal, Instituto Agroalimentario de Aragón-IA2 (Universidad de Zaragoza-ITA),, Zaragoza, Spain
• ⁷Carlos III University of Madrid, Biomedical Engineering Department, Madrid, Spain
• ⁸CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Madrid, Spain
• ⁹Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain
• ¹⁰Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain
• ¹¹Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas (CIBERehd), Madrid, Spain
• ¹²Fundación ARAID, Zaragoza, Spain

The bioengineering of solid tissues and organs to mitigate the organ donor shortage has become a critical area of research in tissue engineering and regenerative medicine, where establishing a functional vascular network is crucial, particularly for complex organs such as the lung, kidney, and liver. This requires the isolation and characterization of various vascular cell types. In this quest, pigs have emerged as the preferred experimental animal model in this field, highlighting the importance of procuring and characterizing porcine vascular cells to create organs with functional vasculature for transplant. However, species-unique differences present challenges. Although some of the processes for isolating, expanding, and characterizing porcine vascular cells have been published, these are less established than those for human cells, requiring in our view and experience, additional research. Furthermore, no reliable and comprehensive models currently exist for testing vascular cell interactions in co-culture in vitro.In this study, we developed effective methods to isolate and further characterize distinct porcine vascular cell types from various sources. We also introduced a straightforward and practical three-dimensional model for testing vascular cell co-culture, organization and function in vitro. This proof-of-concept study demonstrates the potential of our co-culture strategy, employing a decellularized liver extracellular matrix disc scaffold microenvironment to assess cell interactions and vascular potential on a small scale in vitro.