Bioengineering strategies of the uterus towards improving current investigative models and female reproductive health
- Autores: Hannes Campo
- Directores de la Tesis: Antonio Pellicer Martínez (dir. tes.), Irene Cervelló Alcaraz (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: Pedro Batista Corbal (presid.), Amparo Ruiz Jorro (secret.), Mats Hellstöm (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
In this thesis, we intended to develop tissue-engineering approaches based on decellularized (DC) uterine tissues obtained from whole organs to improve several aspects within reproductive medicine. We hypothesized that that the decellularization of whole uteri from different species has not only the potential to, one day, create tissue-engineered, transplantable organs but that the DC endometrial fraction can also be processed further into thin sections, ECM hydrogels and coatings that can be used as a biocompatible tissue-specific substrate for cell and embryo culture. Moreover, we intended to corroborate if the differences in the cyclically and drastically changing endometrium are translated to these hydrogels and coatings, possibly affecting the development of embryo culture.
In the first part of this thesis, we developed a perfusion-based protocol usable for large pig uteri. The effect of a prior freeze/thaw step was also assessed. For this, three frozen-thawed and three fresh uteri were subjected to a DC protocol we optimized for the pig uterus consisting of SDS and Triton X-100 cycles. Furthermore, the F/T step did not noticeably affect the ECM architecture in our experience. DC efficiency was tested by histology techniques and by DNA and protein quantification. Vascular corrosion cast, immunofluorescence (Collagen I & IV, elastin, laminin and fibronectin), scanning and transmission electron microscopy were performed to assess the effect on organ vasculature, ECM composition and its ultrastructure respectively. Finally, in vitro biocompatibility was tested by recellularizing endometrial sections using human endometrial Side Population stem cell lines.
In the second part of the thesis, we created coatings and hydrogels from different DC endometrial tissues and compare in vitro embryo development between these substrata, with commercial coatings and with standard culture conditions. For this, DC uteri without ovarian stimulation and synchronous uteri with day 3 embryos were obtained. In total eight different embryo culture conditions were tested: on top of biological surface coatings and hydrogels made of non-synchronous, synchronous acellular endometrium and Matrigel and compared to standard culture. After that, hatching rates, morphometry and expression of three core pluripotency markers were analyzed and compared.
Our results seem to demonstrate that synchronous endometrium coating would retain and release compounds that permit, in part, to mimic the endometrial environment under in vitro conditions.While DC organs could be used to create transplantable organs, endometrial ECM hydrogels and coatings have the potential to become a platform used in the culture of stem/progenitor cells and primary culture cells to better maintain their tissue-specific phenotype, improving in vitro models. They can also have in vivo applications, such as the treatment of ashermans syndrome and endometrial atrophy.
