• 3D cell culture
• stem cell

The aim of this project is to set up a method for growing in vitro mouse Epiblast Stem Cells (mEpiSC). Epiblast Stem Cells are cells that are extracted from a mouse embryo at the peri-implantation stage (5.5days
after fertilization), while Embryonic Stem Cells (ESC) are extracted from a earlier pre-implantation stage (3.5days
after fertilization). Despite showing many biological differencies, both cells population are pluripotent (they can differentiate to any tissue) and their pluripotency can be indefinetely maintained in vitro.

There are also numerous protocols to force them to differentiate to any fate and any tissue in colture. It is still unclear though to what extent it is possible to recapitulate in vitro the symmetry breaking and pattern formation that occur in embryo development. Being able to reproduce these events in vitro would allow researchers to investingate molecular processes underlying differentiation and to advance in the direction of tissue engeneering and in vitro organogenesis.

Previous studies (van der Brink et al., 2014, Development, Warmflash et al., 2014, Nature Methods) have shown that it is possible to observe in vitro some spatial organization in small aggregates of mouse Embryonic Stem Cells (mESC) and that geometric confinement to circular surfaces is sufficient to trigger in human Embryonic Stem Cells (hESC) ordered germ layers formation.

Traditional 2D culture systems though fail to capture the 3Dness of a real embryo and the confinement that it experiences by other extraembryonic tissues.

With this preliminary work we want to provide another instrument that allows researchers to investigate the role of geometric confinement, but in a 3D and closed environment.

We set up a device, adapted from , for producing microtubes and microcapsules of tunable dimensions, in which we encapsulate cells. We have chosen alginate as material for making cells confinemnt due to its largely acknowlegdged biocompatility.

Alginate is a polysaccharide soluble in water that is extracted from seaweeds and whose physicochemical properties depend on its origin. It crosslinks and form a soft elastic gel when exposed to the presence of divalent ions. Here we use as crosslinking bath a calcium chloride solution 0.1M
as calcium is already present in many culture media. Alginate hydrogels are extremely porous, with a mesh size that depends on alginate kind and concentration, but anyway in the range 5-200nm
so that nutrients, oxygen and proteins canf freely diffuse through it and waste produced by cells can diffuse out.

The device is composed of two coaxial glass capillaries, connected to two syringe pumps. The inner capillary is connected to a syringe filled by a cell suspension (cells suspended in their culture medium, eventually plus Matrigel). The outer capillary glass is connected to a syringe filled by Alginate solution. Alginate solution viscosity depends on a number of parameters among whom the molar concentration. The viscosity of 2% (w/v) solution has been measured to be 0.75Pa*s, much higher than water viscosity at 20°C that is measured to be 1mPa*s. I have chosen to work when possible with 1% (w/v) Sodium Alginate since increasing the concentration increases dramatically the viscosity. We have then a coaxial (instable) flow of cells surrounded by alginate solution. We extrude the two phase flow directly in the reservoir where the crosslinking takes place. Since crosslinking is an instantenous process we are able to encapsulate cells in alginate gel tubes. Cells in tubes may grow in standard culture conditions for mammals cells (37°C, 5% CO2) for days.

Once established a working device, we have adjusted parameters as to meet Epiblast Stem Cells culture conditions. Epiblast Stem Cells form epithelial tissue, then they need to attach to a surface to spread and grow. Cells do not attach on pure alginate, as they have no receptors to recognize it as extracellular matrix. We first tried to chemically modify alginate, by binding to its lateral chain a peptide which is found in fibronectin, a protein that is found in the extracellular matrix and that drive cells adhesion among other functions. While this worked well for some test cell lines, it failed to produce results with mEpiSc that could be comparable to usual culture dishes.

By adding Matrigel, a gelatinous protein mixture that is commonly used to mimic extracellular matrix, to cell suspension we obtained a way better cell adhesion. Cells can be seed in Alginate tubes coated with Matrigel. They attach and form colonies that spread and grow in a way that resemble their behaviour on traditional plastic surfaces.

We set up then a method for growing Epiblast Stem Cells in a 3D confined environment, but open to diffusion of nutrients and growth factors. This system allows investigation of physical contraints on stem cells differentiation and mechanical stimuli (it is possible to evaluate the role of mechanical stress such as stretching and compression due to elasticity of alginate).

The work is organized as follows: in a first chapter the aim of the project is presented and basic concepts in mouse developmental biology are introduced. In the second chapter the state of the art in 3D cell culture and stem cells research is presented. A third chapter is dedicated to description and characterization of the device. In the fourth chapter results of experiments with different cell lines are shown. The fifth chapter is dedicated to conclusions and future perspectives of the project.