Abstract
While most of the in vitro cultures are carried out on bi-dimensional (2D)
substrates, most of the in vivo extracellular matrices are threedimensional
(3D). Consequently cells behave differently on 2D substrates
as a way to self-adaptation to a non-physiological environment. This fact
has encouraged the development of more relevant culture conditions
seeking to provide more representative models for biomedicine (e.g.
cancer, drug discovery and tissue engineering) and further insights into
any dimension-dependent biological mechanism. Different 3D culture
systems have been established though their variability and complexity
hinder their standardisation in common cell culture procedures. So, this
thesis deals with the dimensionality issue in cell/material interactions and
introduces sandwich-like microenvironments as a versatile tool to study
cell behaviour. Cells cultured within this system use both dorsal and
ventral receptors to adhere and spread, undergoing important changes
with respect to the 2D cultures and approaching to 3D conditions.
Stimulation of dorsal receptors has been previously addressed by
overlaying a protein gel on cells already attached on a 2D surface. Here we
propose a sandwich-like system that consists of two 2D surfaces so that
wider spectra of conditions can be investigated by changing the nature of
the substrate (material, topography…) and the protein coatings of both
ventral and dorsal sides.
Since sandwich culture provides an altered cellular adhesion
compared to the traditional 2D substrates by the excitation of the dorsal
receptors, changes in the intracellular signalling are expected, which
might alter important processes such as proliferation, morphology,
migration and differentiation. Hence this thesis evaluates the effect of
different sandwich culture parameters in cell behaviour.
First, cell fate upon adhesion was evaluated in terms of
morphology, proliferation and adhesion. Different conditions were studied
such as materials with different properties or protein coatings (dorsal and
ventral substrates), as well as the effect of sandwiching cells just after
seeding or after been allowed to adhere to the ventral substrate.
Interesting results were obtained such as the relationship between the
ability of cells to reorganise the ECM with cell morphology, proliferation
and adhesion, similarly as observed in 3D hydrogels (degradable vs nondegradable
systems).
Then, cell migration within sandwich culture was studied by live
imaging of a wound healing assay. Results revealed the key effect of both
ventral and dorsal substrates in determining the migration rate as well as
the migration mode used by cells. Moreover cells within the sandwich
culture migrating in the wound healing assay adopted an elongated cell
morphology that resembled cells migrating in other 3D systems. Beyond
differences in cell morphology and migration, dorsal stimulation
promoted cell remodelling of the extra-cellular matrix (ECM) over simple
ventral receptor activation in traditional 2D cultures.
Finally the effect of sandwich culture on cell differentiation was
evaluated. First we showed an increase in C2C12 myogenic differentiation
when cultured within the sandwich system. This enhancement was shown
to be dorsal stimulation dependent and related to an alteration of the
signalling pathway and the growth factor release. To determine if
sandwich culture leads only to myogenic differentiation or whether it
allows differentiation to other lineages, 4 different human mesenchymal
stem cells (hMSCs) lines were cultured under the same conditions. Results
showed the same sandwich environment triggered different cell
differentiation. This points out the importance of the microenvironment
cell niche in vivo, which highly influence cell fate, and thus the need of
mimicking it properly in vitro.
Overall, sandwich-like microenvironments switch cell behaviour
towards 3D-like patterns, demonstrating the importance of this versatile,
simple and robust approach to mimic cell microenvironments in vivo. / Ballester Beltrán, J. (2014). Sandwich-like systems to engineer the cellular microenvironment [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48166 / TESIS
| Identifer | oai:union.ndltd.org:upv.es/oai:riunet.upv.es:10251/48166 |
| Date | 20 March 2015 |
| Creators | Ballester Beltrán, José |
| Contributors | Rico Tortosa, Patricia María, Salmerón Sánchez, Manuel, Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia |
| Publisher | Universitat Politècnica de València |
| Source Sets | Universitat Politècnica de València |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/acceptedVersion |
| Rights | http://rightsstatements.org/vocab/InC/1.0/, info:eu-repo/semantics/openAccess |
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