An in vitro model for studying mouse early embryo development

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Università di Pisa

Facoltà di Scienze Matematiche Fisiche e Naturali

Corso di Laurea Magistrale in Scienze Fisiche

Anno Accademico 2014/2015

Tesi di Laurea Magistrale

An in vitro model to study mouse early embryo

development

Candidato

Relatori

Sara Bonavia

Benoit Sorre

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1 Introduction

The aim of this project is to set up a method for growing in vitro mouse Epi-blast Stem Cells (mEpiSC). EpiEpi-blast 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 dieren-cies, both cells population are pluripotent (they can dierentiate to any tissue) and their pluripotency can be indenetely maintained in vitro.

There are also numerous protocols to force them to dierentiate to any fate and any tissue in culture. 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 dierentiation and to advance in the direction of tissue engeneering and in vitro organogenesis. Previous studies (van der Brink et al., 2014, Development, Warmash 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 connement to circular surfaces is sucient 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 connement 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 connement, but in a 3D and closed environment.

We set up a device, adapted from [1], for producing microtubes and micro-capsules of tunable dimensions, in which we encapsulate cells. We have chosen alginate as material for making cells connemnt 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 can freely diuse through it and waste produced by cells can diuse out.

The device used, to produced alginate tubes and hollow spheres, is composed of two coaxial glass capillaries, connected to two syringe pumps. The inner capillary is connected to a syringe lled by a cell suspension (cells suspended in their culture medium, eventually plus Matrigel). The outer capillary glass is connected to a syringe lled 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 ' 0.75P a · s, much higher than water viscosity at 20°C that is measured to be ' 1mP a · 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) ow of cells surrounded by alginate solution. We extrude the two phase ow directly in the reservoir where the crosslinking takes place.

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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 rst tried to chemically modify alginate, by binding to its lateral chain a peptide which is found in bronectin, 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 conned environment, but open to diusion of nutrients and growth factors. This system will allow investigation of physical contraints on stem cells dierentiation 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 rst 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 dierent cell lines are shown. The fth chapter is dedicated to conclusions and future perspectives of the project.

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2 Mouse early embryo development

Hereafter a method for studying mouse Epiblast Stem Cells (EpiSC) in a 3D conned environment is developped, adapted from [1]. Mouse Epiblast Stem Cells are extracted from the epiblast of a peri-implantation blastocyst. The life cycle of the mouse takes place over nine weeks from fertilization of the egg to the mature adult. This short time development plus the possibility to genetically modify its genome makes it a suitable model in order to study mammalian development. In Fig.1 all the stages from fertilization (zygote) until the primitive streak (PS) formation and beginning of gasrulation process are represented. For completeness of information in Fig.2 all the stages of mouse embryo development untill the adult formation are briey represented, even if stages subsequent to gastrulation are beyond the subject of this work.

Fertilization takes place in the oviduct and implantation in uterus only takes place at day 4.5 from fertilization. As the zygote is formed, it starts dividing and making the morula, a clump of identical and totipotent cells, that at this stage are called blastomeres. Totipotency means that from these cells all the embryonic and extra-embryonic tissues will be generated. From the 8-cells stage the morula undergoes a compaction that is the rst symmentry breaking process in the embryo. As a result of the compaction cells polarize and two dierent sub-population of cells appear: the inner cell mass (ICM) a clump of pluripotent cells from which all embryonic tissues will be generated, and the trohectoderm (TE) a supercial layer of cells, that has the morphology of an epithelium from which only extraembryonic tissues will be generated, such as the placenta, important for the nutrition of the future embryo. At this stage (3.5 days after fertilization) the blastocyst is formed and cells from the Inner Cell Mass (Embryonic Stem cells, ESC) can be extracted and cultivated in vitro.

Subsequently the Inner Cell Mass undergoes another two cell-fate decision: it dierentiates toward Epiblast (EPI) or Primitive Endoderm (PE), that will dierentiate further to extra-embryonic structures. The late blastocyst then is ready for implantation into the uterine walls.

At this stage, the Epiblast, which is going to develop the proper embryo, is cup-shaped, which is a typical feature of the mouse embryo, that we intend to mimic with the encapsulation procedure presented in the next sections. The Epiblast is a tissue that has the morphology of an epithelium, i.e. a tissue whose cells are tightly bounded between each other. At this stage the Epiblast contains around 1000 cells[18].

At 6.5 days after fertilization gatrulation takes place. Gastrulation is a complex process where collective cells migration shapes the embryo, that self-organizes in the way of setting the main axis (antero-posterior axis, dors-ventral axis) of the body. Gastrulation leads to the formation of the three germ layers, from which originate the dierent organs of the embryo: endoderm, mesoderm and ectoderm. The srt step in gastrulation is the formation of the primitive streak (PS) at the border of the cup-shaped epiblast, which is shown in Fig.3. In Fig.4 a slice of a real mouse embryo show the deformation of the epiblast at this stage. This is the srt event of spatial organization and symmetry breaking taking place during gastrulation and it sets the future posterior end of the embryo. The inner side of the cup-shaped Epiblast instead will become the dorsal side of the embryo. By cell proliferation and migration the Primitive

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spread along the primitive streak and form a intermediate layer of cells above the ectoderm, the mesoderm. Some epiblast cells will then form the nal endoderm. This three tisues will form dierent organs and apparatus in the embryo, as shown in Fig.5.

The mechanism underlying these precise commitment and driving spatial organization have not been completely understood yet nor it has been possible to observe these self-organization processes in vitro. Here we present a method to grow in vitro Epiblast Stem Cells, that we expect to capture some feature of the embryo in vivo, as its three-dimesional aspect, that are not represented by traditional culture protocol on 2D Petri dishes.

Figure 1: Mouse Early Embryo development: from fertilization (zygote) to peri-implantation and gastrulation stage. Here the rst two step of dier-entiation and fate decision are shown. After 8-cells stage where cells are all identical, two dinstinguishable populations appear: Trophectoderm cells (PE), that will generate only extra-embryonic tissue (placenta) and Inner Cell Mass (ICM), that will generate both Embryonic and Extra-embryonic tissue. ICM dierentiate to Primitive Endoderm (extra-embryonic) and Epiblast (embry-onic). After implantation Primitive Streak formation (PS), a thicknening zone, is the rst step of gastrulation stage, when the Epiblast origins the three germ layers from which all tissues in body will origin. Image is taken from http://www.devbio.biology.gatech.edu

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Figure 2: Mouse Embryo Development. From fertilization to organogenesis and adult formation. Image adapted from http://www.mun.ca/biology/

2.1 Embryonic Stem Cells and Epiblast Stem Cells

The fundamental properties of Stem Cells are Self-renewal and Pluripotency, i.e. Stem cells are capable of maintaining their pool by symmetric division or dierentiate to dierent tissue progenitors by asymmetric division, as shown in Fig.6. By the way, Pluripotency is only transient in vivo: during develop-ment stem cells undergo lineage commitdevelop-ment and their developdevelop-ment potential becomes less wide at each step in cell-fate decision.

Since the master genes and molecular pathways responsible for pluripotency are quite known it is possible to extract cells at their pluripotent stage and let them proliferate indenetely in vitro, keeping inalterate their developmental potential. It is also possible to force cells to dierentiate to almost any fate by exposing them to dierent mixture of morphogens.

Suppressing the dierentiation program then is a key step in maintaining cells at the pluripotent stage. This can be achieved in culture providing mor-phogens and growth factors that activate the pluripotency program or inhibit the dierentiation signal.

For these features, Embryonic Stem Cells are suitable systems to study in vitro molecular processes that happen in vivo. Embryonic stem cells can be extracted from the mouse embryo at dierent stages, as shown in Fig.7.

Mouse Embryonic Stem Cells (mESC) are extracted from the Inner Cell Mass of an early pre-implantation mouse blastocyst at 3.5days after fertilization. Epiblast Stem Cells (mEpiSC) are extracted from a late blastocyst epiblast at the peri-implantation stage. The possibility to run in vitro investigations is helpful to overcome the accessibility problem in mammal embryogenesis, due to the fact that embryogenesis takes place iside the mother. Furthermore, being the in vitro system a simplication of what happens in vivo, it makes easier to

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Figure 3: Gastrulation begins with Primitive Streak (PS) formation. The loca-tion of the Primitive Streak will set the Antero-posterior axis. It is then the rst step in embryo morphogenesis. In the picture the sense of the arrows denotes the direction of the collective motion of Epiblast cells that will establish the three germ layers (endoderm, mesoderm and ectoderm) at the end of gastrulation. The image is adapted from Principles of Development, Lewis Wollpert.

to idetify in vivo, where a way more complex network of events takes place. For now, a big eort in research is focused on the possibility of observing in vitro part of the complexity of events that occur in vivo, since this would allow scientists to investigate on the molecular level the intercellular comunication and signalling pathways that are responsible for pattern formation in embryos. This would result also in progress in regenerative medicine and tissue engeneering.

Finally the possibility to genetically modify ES cells and to produce then transgenic lines makes them available for the study of dierent genes role in development and to use dierent markers/reporters to track dierent events.

Hereafter we develop a culture method to meet suitable conditions for mouse Embryonic Stem Cells (mEpiSc), but we do not exclude to extend this to inves-tigate also Embryonic Stem Cells. It is important to stress, that even if mouse Embryonic Stem Cells (mESC) and human Embryonic Stem Cells (hESC) both come from the same stage in development, they present quite dierent mor-phology in culture and they do not respond to the same treatment for self-renewal. While mESc are kept pluripotent and proliferate in culture by adding LIF (Leukemia Inhibitor Factor) and Serum or BMP (Bone morphogenetic pro-tein), mEpiSC are do not respond to LIF stimulation with self-renewal. Instead they have to be treated, as in the case of hES, with dierent growht factors as FGF (Fibroblast Growth Factor) and Activin for keeping Pluripotency and Self-renewal. For both these aspects hESC are considered to be closer to EpiSc, even if the latter come from a subsequent stage in development.

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Figure 4: Deformation of the Epiblast Associated to Primitve Streak Formation in the mouse embryo (A) transverse sec4on of a mouse embryo at the pre---streak stage, Staining for Hex expression (blue) marks the anterior side of the embryo (Anterior Visceral Endoderm, AVE). (B,C) transverse sec4ons of a mouse embryo at the early primi4ve streak stage showing buckling of the epiblast associated with the primi4ve streak forma4on (C) A black line underlines the contour of the epiblast. Scale bars 50µm. Adapted from Perea-Gomez et al. 2004

Figure 5: The three primary germ layers originate dierent tissues and organs in the mature embryo. The Endoderm will originate internal organs such as lungs and pancreas, the Mesoderm the gut, the skeleton and the muscles and the Ectoderm the skin and the neural system. The picture is adapted from http://www.sigmaaldrich.com/life-science/stem-cell-biology.html.

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Figure 6: Symmetric and Asymmetric divisions in Stem Cells. The picture shows the main characteristic of Stem Cells. They can divide simmetrically and originate two identical daughters in order to maintain their pool (self-renewal) or asymmetrically and originate two dierent daughters in order to give rise to progenitors of dierent tissues (dierentiation).

3 Why a 3D environment for cell culture

Since decades, cell culture protocols have been based cells growht on plastic or glass 2D sti surfaces. These cell culture techniques have lead and are leading also today to big achievements in cellular and molecular biology, but fail to capture an essential feature of the real environment experienced by cells: their tridimensionality. Today a big eort in research is put in the direction of setting up 3D environments suitable for cell culture , since they are recongnized to be more physiologically and biologically relevant than any 2D counterpart. Grow-ing cells on 2D surface may indeed alter their methabolic behaviour, as well as other functionalities, and may then provide responses that may signicantly dier from events occurring in vivo. Signicant changes have been reported [17] in comparing 2D and 3D culture systems in relevant biological processes in can-cer cells reasearch. Developping new 3D systems may then help in improving the predictiveness of results and having a more faithful representation of what happens in vivo.

Moreover the development of new 3D culture systems may allow researchers to run cost eective high-throughput drug tests, move step forward in the direc-tion of tissue engeneering and in vitro organogenesis, better understand

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mech-Figure 7: The origin of Embryonic Stem Cells (ESC) and Epiblast Stem Cells (EpiSC). Image adapted from http://dev.biologists.org/content/137/15/2455

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anism underlying development, overcoming the problem of in vivo accessibility and availability.

Many dierent materials have been tested for use in 3D cell culture. Partic-ularly hydrogels, hydrophilic networks of polymer chains, that polimerize and undergo liquid-gel transition under dierent conditions, have been favoured due to their characteristics that closely resemble those of the Extra-cellular Matrices (ECM). Extracellular matrix is a complex collection of molecules and proteins secreted by cells. Basically all cells forming tissues reside and attach on Ex-tracellular matrices. ECMs have structural functionalities, they support and segregate dierent tisssues, but play also a role in cell growth and cell migra-tion, gene expression and cell dierentiation.

Hydrogels are then use to partially mimic this complex environment. Hydrogels may be of syntethic, plant or animal origin. Synthetic hydrogels despite their reliability due to x structure and homogeneous nature have been avoided due to the harsh condition under which they polymerize and form gel structures (e.g. photopolymerization by UV irradiation). Hydrogels of animal origin have been avoided due not only to the variability depending on their composition and origin, but especially also for risks of pathogen transmission, that restrict and limit the possibility to use them in clinical research.

Hydrogels of non animal origin, such as Sodium Alginate, that is extracted from brown algae, have been selected as ideal substrate, despite the variability due to their natural origin, for their biocomatibility and their mild and phys-iological gelation condition. Moreover Alginate has properties that makes it a suitable material to be employed in cell culture. It is transparent to light, so that it is open to microscopy investigation, and it is extremely porous, with the dimension of the mesh depending on the concentration, but in the range 5-200nm (see Fig.8), so that oxygen, proteins and nutrients can freely diuse through it and waste produced by cells can easily diuse out.

3.1 Use of Alginate gels in 3D cell culture

In what follows, a method for growing cells in a 3D closed microenvironment made of Sodium Alginate is introduced, adapted from[1] and [5].

This section is about Alginate physical and chemical properties and how it has been used in cell culture up to now.

Alginates are usually extracted from brown seaweeds. They are polysaccha-rides formed by two dierent monomers, mannuronic acid (M) and guluronic acid (G), which are present in dierent percentage (see Fig.9 for chemical struc-ture of Alginate). The amount of G content varies between 30% and 70%. The blocks can be either of the kind (-MMM-) or (-GGG-), either alternating G and M monomers (-GMGM-). These characteristics are variable and depend on the origin of the alginate and aect the chemical properties of alginate. One remarkable property of Alginate is that it is soluble in water and forms ionic gels in presence of divalent cations. In solution Alginate polymer chains present a random coil conformation, i.e. the monomer subunits despite being boundend to one another present a random spatial orientation, which results in highly viscous solutions even at low concentration (1-2% (w/v)).

Pure Alginate crosslinks in presence of divalent ions and a hydrogel is formed. In this work Calcium is chosen, as usual for biological purposes, being it already present in most cell culture media. The gel is formed by chelation of ions between

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Figure 8: Porousity of Alginate hydrogels. (A) Alginate beads. (B) Alginate solution 2% (C) Alginate solution 1% (D) Alginate solution 0.5%. Adapted from Wang et al., 2009.

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two consecutive monomenrs and binding between monomers of dierent polymer chains as shown in Fig.10.

The physical properties of the gel depends on the amount of G-monomeres in Alginate, since G-blocks are the structural elements in alginate hydrogels. It is possible then to tune elasticity and stability of the gels, by choosing alginate with suitable G-content. Increasing the concentration of G-blocks, the length of G-chains and the molecular weight will increase the elasticity and the porousity of the resulting gel.

Once gel is formed, it is also possible to dissolve it, by mean of an ion chelator, such as ethylenediaminetetraacetic acid (EDTA).

Ionic crosslinking is quite fast: alginate gelation takes place in few seconds when alginate solutions are extruded or dripped into a gelation bath by diusion of Calcium ions into alginate. Here, Calcium chloride (CaCl2) is used as gelation

bath, as it is usually done in literature. The concentration of ions available for crosslinking is another parameter that aects stability and elasticity of the gel: here the request for gentle gelation condition impose an upper bound to Calcium Chloride concentration.

Alginate itself is very poorly adhesive to cells, which characteristic my come as an advantage for encapsulation, but represents a disadvantage in case of cells that need to attach to survive. It is possible by the way to chemically function-alize alginate to make its surface adhesive or to use alginate in combination to other substitutes of extracellular matrices. We will go through the necessary steps in detail in the following sections.

There are several approaches to the use of Alginate in 3D cell culture[2]. Most of them rely on immobilize monoclonal or policlonal spheroids of cells into beads of alginate. These methods consist in the formation of a unique solution composed by two dierent phases, the alginate solution and the cells or tissue suspension, which are later xed by gelation in a reservoir. Usually, beads dimensions varies in the range 100−1000µm depending on their purpose. Alginate is also used to build porous and elastic scaolds, in which cells can be seeded, attach (upon chemical modication of alginate) and grow. 3D printed molds can be use to model alginate to precise shapes as honeybox structure.

Moreover alginate can also be use as bioink in 3D printing, since it has been shown to be a good choice as bioink, due to its biocompatibility and the capacity of form hydrogels within few seconds.

3.2 Previous results in Embryonic Stem Cells research

The method here presented for epiblast stem cells culture in 3D environment has the characteristic to encapsulate cell in a closed environment of denite and tunable geometry. In this section some previous results are introduced, that suggest that physical constraints and mechanical stimulation play a role in the symmetry breaking events occurring during early stages in embryo development. Previous studies investigating the eects of physical constraints and me-chanical stimuli in pattern fomation focused on in vivo model, especially then on animal models whose embryos develop autonomously (such as Drosophila or Xenopus). These by the way restrict the eld of research to a few animal models and exclude mammals, since embryogenesis takes place inside the mother and it is hard to study in vivo.

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Figure 10: Alginate gel formation in presence by chelation of divalent ions (Ca2+_{). Adapted from [2].}

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Even if Embryonic Stem cells (ESC) can be indenitely mantained in a pluripotent state in culture and dierentiated to almost any tissue upon specic conditions, it was impossible untill recently to recapitulate in vitro some of the spatial organization events, that take place in vivo. It has been shown that dierentiation spontaneously occurs in ESC cultured in small aggregates ('Em-bryoid Bodies, EB') by mean of hanging drop culture technique , a technique used in embriology to generate 3D spheroids of cells. Dierentiated cells though appeared randomly in the clump, without formation of any organised pattern.

Recently some studies (van der Brink et al. 2014, Development, Warmash et al. 2014, Nature Methods) have shown that upon specic conditions it is possible to observe and reproduce in vitro some spatial organisation and sym-metry breaking in mouse Embryonic Stem Cells and human Embryonic Stem Cells colonies.

Van der Brink et al. have proved that mouse Embryoid bodies can set a co-ordinate reference and self-organise in a way reminescent of the spatial ordering that take place in early development. These events take place upon specica-tion of culture condispecica-tions that promote the dierentiaspecica-tion into mesoderm-like lineages and in way dependent on aggregate dimension. It has been shown that axis formation and gastrulation-like processes occur for aggregates that count roughly ' 300 cells, which is close to the number of cells contained in a mouse embryo at the peri-implantation stage, indicating that the eect of geometry on patterning formation is not scaling linearly with cells number.

Furthermore it has been shown [15] that connement into circular geometry and stimulation with BMP4 (Bone Morphogenetic Protein, it induces dierenti-ation) are sucient to trigger gastrulation-like spatial self-organization in human Embryonic Stem Cells. In response to stimulation with BMP4, colonies forced to grow on micropatterned circular surfaces are able to give rise to spatially or-dered germ layers (Fig.11). They were found to dierentiate reproducibly into an external tropectoderm-like ring and inner ring of cells expressing markers of ectodermal and mesodermal fate. It has to be noticed that the circular geome-try resembles the real environment of a human embryo since human embryonic cells organize into structures similar to disks at the gastrulation stage, which makes it dierent from the cup-shaped epiblast in mouse embryo at the same stage.

Both these results highlight that geometric cues do play a role in early pat-tern formation in embryo development. They show that stem cells have an intrinsic tendency in spatially organizing themselves to form ordered structures and that these features may be controlled by controlling the physical and geo-metrical environment that they experience in culture.

The method presented hereafter is intended to provide another instrument to investigate the role of such contraints in mouse Epiblast Stem Cells.

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Figure 11: Self-organization in human Embryonic Stem Cells on micropatterned surface. (Top) circular micropatterned surface vs traditional culture. (Bottom) dierent markers of dierent cell fate are expressed in concentric circular do-main. CDX2, SOX17, SOX2 and BRA are genes that are expressed respectively by Trophectoderm, Endoderm and Endoderm and Mesoderm. Bottom images show imunouorescence data for germ layers markers. Their expression allows to identify cell type. Image taken from Warmash et al., Nature Methods. Scale bars 100µm

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4 The experimental setup

The aim of the instrument here introduced is to produce Alginate microtubes (or capsules) inside which cells can be encapsulated.

As shown schematically in Fig.12, the device is composed of a system of two coaxial glass capillaries. The capillaries are connected by a system of Teon tubes to two syringe pumps. Teon tubes and glass syringes have been chosen whenever possible in order to better control uid ows and sharply interrupt them because of their stiness. Using plastic syringes and tubings leads to some leakage when the ows are interrupted due to the elasticity of the materials.

The two pipettes are assembled on a system of sleeves and T-junctions that connect the tubes to Teon tubings and the syringes. 3D printed supports x the system of glass pipettes to a metallic bar that supports the whole structure. Previous design was based on glass pipettes glued on a glass slide. The new design allows the experimenter to change and replace rapidely one pipette inde-pendently of the other. This is particularly convenient since the outer pipette inner diameter sets at rst order approximation the outer diameter of the tube or capsule produced. The possibility then to easily change the dimensions of one pipette allows for testing dierent congurations and get dierent results coherently to the purpose.

The outer capillary is connected to the alginate solution, whether the inner one is fed by a test solution or a suspension composed by cells and culture medium or cells, culture medium and matrigel, as explained in full detail in the next sections. As test solution distilled water plus ink or DMEM (Dulbecco's modied Eagle medium) culture medium have been used to rapidely check the eective formation of tubes or capsules.

The crosslinking takes place in calcium reservoir, made of 0.1M Calcium chloride (CaCl2) solution. Here alginate solution is used mostly at 1% (w/v)

concentration in order to avoid dealing with highly viscous solutions, as it re-quires to have more powerful syringe pumps and can easily lead to clogging in the device. To increase the density of the solution and make easier and more stable the extrusion process Glycerol has been added to the alginate solution.

In order to ensure that the two pipettes are roughly coaxial (otherwise tubes are not formed, but only irregular alginate bers that do not have a lumen), four small cylinders are glued at the cardinal points of the inner glass pipette (see Fig.13). In order to have hollow tube is sucient that the inner pipette does not touch the outer one. If this happens but the pipettes are misaligned, the tubes produced will have a misaligned lumen. This anyway does not represent a problem in cell culture due to the porousity of alginate.

I have tested two dierent congurations of the device: in a rst congura-tion the outer pipette was not pulled, in a second one the outer pipette tip has been pulled with a micro-pipette puller (schematically shown in Fig.13). It is possible to reduce the diameter of the pipette by mean of a micropipette puller (P-1000 micropipette puller, Sutter Instrument Company) that warms up the glass pipette and pulls it in order to get two pipettes with a tip diameter that can be tuned by tuning some parameters such as heat and the strenght that pulls the pipette. This was done in order to get tubes with a smaller diameter, without having to reset the dimensions of both capillary pipettes.

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Figure 12: Scheme of the device. Two coaxial glass capillaries assembles through connectors and T-devices are connected to two syringe pumps. The outer pipette is fed with alginate solution, the inner pipette with cells suspension. The two phase ow is extruded inside a gelation bath, made of Calcium chloride solution.

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Figure 13: (a) conguration without pulling outer capillary glass. (b) outer pipette is pulled in order to obtain smaller diameter aginate tubes. It is possible to vary capillary sizes in the range 200 − 1000µm. The tip has been pulled to get a diameter in the range 150 − 250µm.

4.1 Visualizing results: chemical modication of Alginate

In order to easily and precisely measure the dimensions of the tubes, alginate has been chemically modied and labelled with a uorescent dye (protocol adapted from already published work). This has been done in order to use confocal uorescence microscopy to measure tubes dimensions, being it hard to measure tube diameter and particularly the walls thickness in bright eld images.

Fluoresceinamine (14), a synthetic uorophore commonly used in microscopy that absorbs blue frequencies and emit green frequencies, was covalentely bound to alginate. An amin bound was established between the amin group on the uorescent dye and the carboxilic acid on the alginate chain. EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), a water-soluble carbodiimide, has been used to activate carboxilic groups on alginate and makes possible the formation of the amide bound. Playing with EDC quantity in reaction allows for choosing how many sites to activate and then to labelled, making it possible to decide the amount of units to decorate among all the units. In Fig.15 a scheme of the reaction is shown.

Fluorescent images of Alginate tubes have been analyzed and measured with ImageJ software. In Fig.17 a tube section has been reconstructed from a z-stack of images taken with confocal uorescence microscopy. In Fig.16 a section of the tube in the middle plane is shown.

4.2 Characterization of the device

It is possible to tune the geometry of the tube (the outer diameter, the inner diameter and the wall thickness) by changing some parameters among which the diameter of the outer pipette, the concentration and viscosity of alginate solution, and particularly the ow rates of the two solutions. In Fig.18it is shown

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Figure 14: Chemical structure of Fluoresceinamine. The urophore (Fluores-cein) is functionalised with an amin group so that it can form a peptide bond with a carboxyl group.

how the diamensions of the tube can be change, by varying the ow rates. From mass conservation, an empirical relation between the geometry of the tubes and the ow rates can be established.

h Rout = 1 − r _q in qin+ qout (1)

Here h is the thickness of tube wall, Routis the outer diameter, while qinand

qoutare the ow rate of the inner solution and the alginate solution respectively.

In order to characterize the behaviour of the device, many samples have been produced, varying the parameters that was possible to control. In Fig.19-20 the outer diameter, the inner diameter and the thickness of the tubes walls are plot-ted versus the inner solution ow rate and the alginate ow rate respectively. Data represented in both graphs are taken in the same experimental congura-tion: alginate solution 1% (w/v) and tip diameter ' 225µm. Data have been acquired by mean of confocal uorescence microscopy and ImageJ software for image analysis.

It has been observed that alginate ow rates aects less the nal output than the inner ow rate (compare Fig.19and Fig.20). Anyway, increasing the ow rate slightly increases the outer diameter, while the inner diameter decreases accordingly to mass conservation.

Tuning the inner ow rates changes in a sensible way both outer and inner diameter of alginate tube.

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Figure 15: Labelling of alginate with uorescent dye. The role of EDC in forming an active intermediate is shown.

are greater than the pipette tip diameter. This can be explained as an example of the die swelling (or Barus eect) [14], an eect that occurs when polymers are extruded through a die, which is the case here since alginate solution is a polymer solution and the glass pipette works as a die. Polymer chains that are entangled at rest, when forced through a die organize into less entangled solu-tions (polymers chains disentangle and align). Once out of the die the polymer material immediately relaxes to a more chaotic conguration, that implies an outer diameter for the alginate tube greater than the pipette diameter. It has to be noticed in this regard, that increasing the inner ow rate increases the outer diameter until a certain value, above which value the outer diameter slightly decreases even if the inner ow rate is further increased (Fig.19). This can be possibly explained by taking into account the shear thinning behaviour of Al-ginate. Alginate solution is a non-Newtonian uid, whose viscosity decreases when the shear stress applied increases. Thus increasing the inner ow rate can be thought of as increasing the shear stress to alginate ow. This is found to counteract the extrudate swell eect that produces the enlargement of the outer diameter.

The die swell eect can possibly be limited, if necessary, by mean of longer glass pipettes. This will slow down the relaxation time. Since gelication is almost instantaneous, polymer chains have not suciet time to relax to a more cahotic conguration.

In Fig.21 observed data are compared and found in good agreement with theoretical expectations based on mass conservation, expressed by (1).

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Figure 16: A section of a tube image with confocal uorescence microscopy. Alginate is labelled with Fuorescein. External diameter is ' 800µm

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Figure 17: A picture of an alginate-uorescein tube imaged with confocal uo-rescent microscopy. The diameter of the tube is ' 800µm. The image has been reconstructed from a superposition of images from a z-stack of 87 images with ImageJ software.

Figure 18: Section of two uorescein-alginate tubes. The geometry is aected by the dierent alginate ow rate in the two samples. The other parameters (Alginate solution concentration 2% (w/v), outer pipette diameter 700µm, inner ow rate 62.9ml/h) were kept constant, meaning that tuning the ow rates makes it possible to partially control the geometry. Outer diameter ' 1mm, lumen diameter ' 500µm for the tube on the left. Outer diameter ' 1.2mm, lumen diameter ' 250µm for the tube on the right.

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Figure 19: Variation of tubes characteristics (inner diameter, outer diameter, wall thickness) as inner ow rates change. Error bars represent the standard deviation from the mean of n=3 measuares on dierent parts of the tube.

The length of the diameter of the tubes has been measured in dierent parts of the tube. The data shown result from the average of n=3 measures and the error bars represent the standard deviation from the mean.

4.3 Other attempts in device microfabrication

Dierent alternatives to the device presented in the previous section were also considered and partially implemented, adapted from examples found in litera-ture for production of tubes and bers by mean of hydrogels. Microuidic chips for producing coaxial ows were designed and tested.

For completeness of information, here are reported some essays. Two dif-ferent PDMS (Polydimethylsiloxane) chips have been designed. Fig.22 shows the masks for a multilayer PDMS chip that I have designed adapting it from a not published work. The masks have been designed with AutoCAD software. I produced the masks used as molds for PDMS chips in clean room with pho-tolythography technique. The chip is made of three dierent layers, that share a common hole in the middle. At the extrusion hole three uids meet: the inner uid (cells suspension or test uid), an intermediate uid (alginate solution) and the outer uid (crosslinking bath). At the hole, tube is directly formed, focused by the ow of the calcium chloride. Being the ow unstable though, it was hard to control the proper conditions for tube making.

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Figure 20: Alginate tubes dimensions (inner diameter, outer diameter, wall thickness) versus outer ow rate. Error bars represent the standard deviation from the mean of n=3 measuares on dierent parts of the tube.

channels (alginate and cell suspension) toward a common exit where the tube is formed, as shown schematically in Fig.23. This second chip was more reliable than the previous one and has been partially used during this work. The main device was in the end preferred because of not requiring 3D printing or the use of the clean room, but it is possible to further improve some of these solutions in the future.

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Figure 21: Ratio of tubes wall thickness versus outer tube diameter varying with ratio of outer ow rate versus inner ow rate according to (1). Error bars represent the standard deviation from the mean of n=3 measuares on dierent parts of the tube.

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Figure 22: Chip design. On the left, the design for one of the three layer is shown. On the right, the superposition of three layers, centred on a common hole. The dimension of the chip is around3cm×4cm. The widht of the channels is in the range 750 − 250µm. AutoCAD software has been used for the disign.

Figure 23: Scheme of a two ows PDMS chip. A section of glass pipette is inserted in the chip to help the focussing of the inner solution by alginate solution and make the ow coaxial. The width of the channel is 250µm for alginate solution and 150µm for the inner channel. The molds have been 3D printed and the PDMS layers have been Plasma bonded.

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5 Cell culture experiments

Once the device was proven to correctly work and continuously produce tubes with a clear lumen of tunable diameter, I started working on cells encapsulation and on adjusting culture conditions for this particular conguration. In this section, results in dierent cell lines cultures are presented. In order to gradually add complexity to the system I started with cells that do not require adhesion for growing, then I moved to cells that grow in adhesion and I started working on modications of alginate to meet this request. For testing this I started with HeLa cells, being them less expensive and easier to grow than Epiblast Stem Cells.

5.1 Dictyostelium cells essay

A rst essay was made to test whether it was possible to encapsulate cells in alginate tubes with the implemented device. Since cells do not recognize alginate as extracellular matrix material, they do not attach on it. As a preliminar test I have chosen to work with a cell line that does not require adhesion. Being them already present in the lab, Dictyostelium cells were used.

Dictyostelium belongs to the order of Dictyosteliida, also called Social Ameba. Their characteristic is to be at the border between unicellular and multicelluar organisms, since they start their life as unicelular organisms but can assemble in multicellular ones when starved to move in search for food. They are present in nature in many terrestrial ecosystems, being them part of soil microora. They are bacteriovores and perform the function of regulating bacterial presence in soil. In Fig.24 Dictyostelium are encapsulated in Alginate tube whose diameter is ' 800µm. After encapsulation, Alginate tubes are placed into 35mm Petri dishes, lled with culture medium and put in the incubator. Data and pictures have been acquired each day to check cells viability in this environment. In Fig.26 it is shown the growth of Dictyostelium in tubes up to ve days after en-capsulation. Bright eld images have been taken daily to monitor the behaviour of Dictyostelium. A sequence of images of the rst days after encapsulation is shown in Fig.25.

In order to precisely measure tubes diamensions pictures have been taken also with confocal uorescence microscopy, using the uorescein labelled Algi-nate. Dictyostelium were already labelled with RFP (Rhodamine Fluorescent Protein). An example is shown in Fig.27.

Timelapse microscopy has been used to ensure that cells were alive and that they were dividing and growing. Even if the aim of the present work does not concern Dictyostelium research, it is not to be excluded that the positive results obtained in growing Dictyostelium in this system might be of some interest in this eld of research.

5.2 HeLa cells essay

As a subsequent step in this project, after Dictyostelium tests, I focused my attention on a system more similar to Epiblast Stem Cells. HeLa cells have been chosen for being readily available and cost eective. HeLa cells were considered a good system in order to run preparatory tests and adjust the protocol for

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Figure 24: Dictyostelium encapsulated in Alginate tubes. Image in bright eld. Tube diameter ∼ 800µm. The picture is taken immediately after encapsula-tion.The irregular surface of the inner wall of the tube might be due to oscilla-tions in pressure and has been already observed in polymers extruded through a die.

Figure 25: A sequence of images taken in Bright Field microscopy that shows the growht of Dictyostelium in Alginate tubes during the days following the encapsulation. Culture medium has been changed daily.

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Figure 26: Dictyostelium encapsulation in Alginate tubes. Dierent pictures have been taken immediately after encapsulation (top, day 1) and on the fth day after encapsultion (bottom, day 5). Dictyostelium are found to correctly divide and grow in this new environment. The tube outer diameter is ' 800µm and the lumen diameter is ' 400µm .

the same culture conditions (37°C, 5% CO2) and they need an adhesive surface

they can attach on to survive in culture. Furthermore the procols for growing Stem Cells have many features that are common to standard mammalian cells protocols.

HeLa cell line is an immortal cell line. Immortal means that cells that commonly stop dividing and die, due to senescence, do not and keep dividing due to some genetic mutation. HeLa cells represent the rst human immortal cell line. They were rst extracted from a cervical cancer in 1951. It is the rst human cell line that could be kept alive in vitro. The name comes from the name of the donor, Henrietta Lacks.

In Fig.28 it is shown how HeLa cells look like in culture on traditional Petri dishes (plastic at surfaces). As shown in the picture, they have the morphology of an epithelial tissue.

Since sterility is a crucial factor in cell culture, all the operations had to be implemented under a laminar ow hood. All the materials have been previously autoclaved at 121°C or microltered with 0.22µm lters. When possible, plastic materials has been preferred to glass ones for matters of sterility. The device has been cleaned by owing inside abundant ethanol 70% in water before and after any use. Once anything had been set up under the hood, 30 minutes sterilization with UV rays has been run. In Fig.29 HeLa cells encapsulated in tubes are shown just after encapsulation.

HeLa cells attach well (Fig.28) on tissue cuture treated plastic wells (poly-stirene chemically modied to ruduce its hydrophobicity and makes it easier for adherent type cells to attach) in presence of DMEM (Dulbecco's Modied

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Figure 27: An image of Dictyostelium encapsulated in uorescein-Alginate tube taken with confocal uorescence microscopy. Tube diameter ' 850µm. Lumen diameter ' 400µm.

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Figure 28: HeLa cells on plastic at surfaces. HeLa have been extracted from a human cervical cancer in 1951. The name comes from the donor's name Henrietta Lacks. The two pictures show cells in culture that have a dierent degree of conuence, i.e. a dierent amount of surface covered with cells. Image taken from https://www.lgcstandards-atcc.org

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Figure 29: HeLa cells encapsulated in Alginate tubes in DMEM culture medium after encapsulation. Tube diameter' 800µm. Images taken in bright eld microscopy.

they do attach on pure Alginate, I ran an experiment, whose result is shown in Fig.31 . Here it s possible to observe that while on Petri dishes cells are attached and spread with their peculiar stellar morphology, on a pure alginate substrate clumps of detached cells are visible. Those cells are probably dead cells that were not able to attach. Some adhesion though is still visible. To improve ad-hesion as a rst attempt the alginate structure was chemically modied. The chemistry in this process is the same as in the uorescent labelling protocol. I adapted a protocol already present in literature (Rowley et al., 1999).

5.2.1 Functionalized alginate with RGD peptide

Alginate does not interact with mammalian cells. It is not sucient to add Serum, since the proteins in it cannot mediate the adhesion, due to the fact that alginate does not promote protein absorption. Being the aim of the project to use Alginate as a substrate for cell culture and to conne cells inside alginate structure, it was necessary to modify it.

Cell adhesion is mediated by receptors, found on cellular membranes, that recognize adhesion molecules and interact with them to anchor cells to the substrate. The idea here is to covalently bind to alginate a cell adhesion ligand. Here is used a peptide that containes the RGD sequence (a sequence of three aminoacids), which is found in bronectin. Fibronectin is a protein that triggers cell adhesion by interacting with integrins, a class of transmembrane receptors. By mean of carbodiimide chemistry the uronic acids on alginate backbones are activated and covalently bound to the terminal amine of the peptide. A scheme of the reaction can be found in Fig. 30, which shows the role of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) in forming an intermediate state, before establishing an amide bound between alginate and the peptide.

In Fig. 31 the result on HeLa cells culture is shown. HeLa cells are found to attach on a substrate coated with functionalized alginate better than on a pure alginate substrate. The number of dead cells is found to decrease and clumps of

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Figure 30: Scheme of the rection that binds RGD peptide to alginate. Adapted from Rowley, 1999.

detached cells are less dense. Their morphology appear close to HeLa on plastic substrates.

5.3 mEpiSC essay

After having established that HeLa cells could attach and grow on functionalised alginate, I focused on mouse Epiblast Stem cells. These cell line requires a more complicated culture protocol than HeLa Cells. Growth Factors have to be added to culture media in order to maintain cells undierentiated, otherwise dierentiation spontaneously starts. Some dierentiation anyway is dicult to avoid and if present is generally localised at the borders of the colonies. In Fig.32 two pictures of mEpiSC culture dishes are shoOn the left image own. mEpiSCs are an epithelial tissue, that form in culture tightly packed colonies and grow in monolayers. On the border of the colonies, it is possible to observe some cells that have a dierent morphology: they are less packed with the neighbours and much more spread than the others. Those cells have dierentiated toward a neural-like fate.

First attempts in mEpiSCs encapsulation have been run using 1%(w/v) RGD-labelled Alginate. Resuslts are shown in Fig.33, where mEpiSCs growth in RGD-Alginate is followed up to the fourth day after encapsulation. Epiblast stem cells are seeded as single cells or small colonies. This is done by breaking the bounds between cells in the same colony, before starting the encapsulation procedure. To this aim collagenase is used to break the peptide bonds between cells. This is a necesessary step, because otherwise big colonies would clog the device. The images show that after encapsulation stage mEpiSCs are able to form small colonies inside the tube, which indicates that they attach and divide. After experiments using RGD-functionalised alginate, dierent alternatives to chemically modied alginate were considered, in order to make alginate sub-strate as similar as possible to the niche that cells experience in vivo. The possibility to add dierent ligands in two dierent ways directly to alginate was tested. As ligands, bronectin and Matrigel have been considered. Fibronectin is a protein of the Extracellular Matrix (ECM). It binds to membranes receptors of cells such as integrins. Cell adhesion is one among its functions.

Matrigel is a commercially available mixture of proteins of the extracellular matrix. It is secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and it contains most of the Basement Membrane proteins.

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Figure 31: HeLa Cells behaviour on dierent substrates. On pure Alginate they do not attach, as it can be seen from their round morphology (top). They attch on RGD functionalized alginate (middle). As a negative control HeLa on plastic Petri dish (bottom). Images are taken in bright eld microscopy magnication 10x.

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alginate was crosslinked by adding and then removing Calcium chloride and the on the top of alginate gel layer some bronectin was poured. In a third well, crosslinking of alginate was made by mean of a solution of Calcium chloride and Fibronectin, in order to chek whether or not bronectin was trapped by aginate chains. In a fourth well Fibronectin was directly mixed with alginate, that was then crosslinked by adding and then removing Calcium chloride. In a fth well Matrigel was poured on the top of a alginate gel layer. In a sixth well Matrigel was mixed to Calcium chloride an poured on the alginate solution layer. In a seventh well Alginate was directly mixed with Matrigel and then crosslinked by pouring Calcium chloride.

Then a suspension made of mEpiSC and their culture medium was poured in each well. The better results in cells adhesion and viability were reached in those wells containing Matrigel and among them in the one where Matrigel was mixed with Calcium chloride soution.

This was not surprisingly since Matrigel is commonly used in stem cells culture to coat the substrate and recreate the niche environment. It is a solu-tion that undergoes gelasolu-tion at 24-37°C, but becomes very viscous already at room temperature. Therefore working with it requires some further attention. Cooling down the system is recommended in order to avoid clogging the device. This particular condition was meant to simulate the conguration in which Matrigel is mixed to cells suspension during extrusion process. Therefore en-capsulation experiments have been run using cell suspension with 20% Matrigel, as shown schematically in Fig.34.

The results are shown in Fig.36. It is possible to observe than cells attach and spread better than on functionalised Alginate. Also, that rate at which cells divide is observed to increase in this conguration. A direct comparison between the two systems one day after encapsulation is shown in Fig.37. Therefore it is possible to conclude that providing Matrigel in suspension together with cells it is sucient to make the interior walls of alginate tubes a suitable substrate where Epiblast Stem Cells can grow. This situation is represented schemtically in Fig.35. It was already reported in literature (Alessandri K., 2013. PhD thesis) that some kind of anity between Matrigel and Alginate exists, such that when matrigel is injected inside alginate tubes it will automatically coat alginate tubes, even if provided mixed with cells suspension. The morphology of the colony on this substrate is closer to what is observed on traditional plastic substrates (Fig.38). Therefore this suggests that encapsulation in 3D alginate structures coated with Matrigel is a suitable system in order to study Epiblast Stem Cells.

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Figure 32: mouse Epiblast Stem Cells in culture on traditional plastic sur-face.mEpiSC form tightly packed colony. On the border of the colony some cells can be observed that are less bound to the others. This is a signal that some dierentiation is taking place.

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Figure 33: mouse Epiblast Stem Cells encapsulated in alginate tubes. Cells do attach on RGD-functionalised alginate and grow. From the rst day, when they are seeded as single cells, they form spherical colonies during the following days. They divide at a slower rate with respect to plastic (∼16hrs).

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Figure 34: Encapsulation in Alginate tube with matrigel as Extracellular Ma-trix.

Figure 35: Scheme of mEpiSc encapsulation in Alginate. EC= Embryonic Cap-sule.

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Figure 36: mEpiSC encapsulation in Alginate tube. Matrigel is used for coating the interior of Alginate tubes. Picture are taken in bright eld microscopy (10x) during the three days following encapsulation. In the middle and bottom pic-tures, colonies are forming inside the tubes.Tube diameter is ' 400µm. Colonies appear tightly packed as expected to be from traditional culture experiments.

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Figure 37: A direct comparison between results of mEpiS cells culture in RGD-functionalised alginate (right) and alginate plus Matrigel (left). Cells are ob-served to attach and spread better on Matrigel substrate than on functionalised alginate. Bigger colonies and no dierentiation are found in Alginate tubes coated with Matrigel.

6 Conclusions and perspectives

A method for successfully growing mouse Epiblast Stem Cells in a 3D environ-ment of dened and tunable cylindrical geometry has been developped.

This work reperesents the rst step of a wider project aiming studying cel-lular processes in stem cells dierentiation that happen in vivo during the rst stages of embryo development, with particular regard to gastrulation and for-mation of the three germ layers, ectoderm, mesoderm and endoderm.

Here some possible follow up of the project are briey introduced.

6.1 Capsule formation

The previous method could be used also to produce hollow capsules in which encapsulate cells. In Fig.39 some preliminar results are presented, showing that it is already possible to get hollow capsules, using 1%(w/v) uorescein-alginate. Before starting to run encapsulation expertiments anyway, some parameters need to be adjusted.

Capsules are produced exploiting the Plateau-Rayleigh instability (capillary instability) which is responsible for jet breakups and droplet formation, as shown in Fig.40. The driving force of the process is the surface tension of the uid, which acts to minimize the surface of the uid. The breakup takes place outside the calcium chloride bath, so droplets are formed outside the gelation bath. Gelation only takes palce once capsules enter the reservoir. Stiness of Alginate and reservoir can be tuned by mean of surfactants, as SDS (Sodium dodecyl sulfate) or Tween20.

In order to produce capsules with a nicely spherical geometry, it is important that the gelation is very fast, in order to avoid the mixing of the alginate solution

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Figure 38: Comparison between traditional plastic surface (bottom) and algi-nate plus Matrigel substrate (top). Colonies are found to have a similar mor-phology on the two substrates. On Matrigel substrate dierentiation on the border of the colony is not observed.

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Figure 39: Two examples of Alginate capsules. Pictures taken with confcal uorescence microscopy. Capsules radius rout ' 600µm. Mixing of the two

phases of the ow produces the irregular interior capsule walls. On the right, a not perfectly spherical capsule is shown as a rersult of coalescence of two consecutive droplets.

with cell suspension. Mixing of the two phases is responsible for the irregular inner walls of the capsule, shown in Fig.39 on the left.

Improving this method and adjusting it for Epiblast Stem cells culture will provide another system to study Stem Cell dierentiation and early embryo biology.

6.2 Which role Do Mechanical and physical contraints

play in development?

Based on previous results showing that connment to at disks was sucient to trigger self-organization events in human Embryonic Stem cells, remines-cent of pattern formation occurring in early embryo, this project is meant to be preparatory to the investigation of the role of such a connement in three dimesions.

Here the protocol has been adapted to mouse Epiblast Stem cells, since cylin-drical geometry is reminescent of the egg-cylinder shape of the mouse embryo at the stage of implantation.

Previous results have shown that mechanical orces generated by the cells or by the ECM have an inuence on morphogenesis and dierentiation taking place during embryogenesis [10, 8, 9].

The presented system is suitable to study the role of mechanical external constraints in tissue patterning: this is intended to be done by mean of

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dier-Figure 40: : The capillary-driven instability of a water thread falling under the inuence of gravity. Adapted from http://web.mit.edu/.

ent mechanical stress that can be applied to alginate capsules and tubes with dierent techniques (osmotic shock, micropipette aspiration, compression under micro-plates.). This is intended to study whether and how mechanical inputs can be translated into signalling pathways that trigger dierentiation toward some particular fate.

Even if for the time being only mEpiSC have been used, it is reasonable to assume that the study can be extended also to Embryonic Stem Cells.

Moreover varying the percentage of Matrigel in the inner solution is another way to produce dierent systems with dierent characteristics: using 100% Ma-trigel plus cells as inner solution will produce again another conguration in which cells are trapped in Matrigel gel, but still free to move and self-organize, being Matrigel a softer gel than alginate.

In conclusion, the system presented is suitable to study many dierent as-pects in stem cells dierentiation and developmental biology cellular processes. The possibility to easily produce a large number of tubes (or capsules) makes it possible to run quantitative studies of the cellular processes underlying dier-entiation and to address many open questions in early embryo development.

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7 Appendix

How to bind RGD peptide to Alginate

Here the protocol to covalentrly bind RGD containing peptide to alginate polysac-charide is reported.

Prepare a 1% (w/v) Alginate solution in 0.5 M MES buer at pH 6.5 (add alginate powder slowly and while stirring with a mechanic stirrer) For 5 ml of solution add 2.7 mg of Sulfo-NHS (MW 217.14 g/mol) Add 2.4 mg of EDC (MW 191.7 g/mol)

After 5 minutes add 50 µL of peptide stock solution at concentration 5mg/ml (peptide full name Acetyl-Gly-Asp-Arg-Gly-Asp-Ile-Pro-Ala-Ser- Ser-Lys-Gly-Gly-Gly-Gly-Ser-Asp-Arg-Leu-Leu-Leu-Leu-Leu-Leu-Asp-Arg-Amide MW 2680.02 g/mol)

Let the reaction run for 20hrs

This protocol is meant to activate 5% of uronic acids in alginate. The concentration of EDC is then 5% of the concentration of Alginate. NHS and EDC are in ratio 1:1.

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List of Figures

1 Mouse Early Embryo development: from fertilization (zygote) to peri-implantation and gastrulation stage. Here the rst two step of dierentiation and fate decision are shown. After 8-cells stage where cells are all identical, two dinstinguishable popula-tions appear: Trophectoderm cells (PE), that will generate only extra-embryonic tissue (placenta) and Inner Cell Mass (ICM), that will generate both Embryonic and Extra-embryonic tissue. ICM dierentiate to Primitive Endoderm (extra-embryonic) and Epiblast (embryonic). After implantation Primitive Streak for-mation (PS), a thicknening zone, is the rst step of gastrulation stage, when the Epiblast origins the three germ layers from which

all tissues in body will origin. Image is taken from http://www.devbio.biology.gatech.edu 6 2 Mouse Embryo Development. From fertilization to organogenesis

and adult formation. Image adapted from http://www.mun.ca/biology/ 7 3 Gastrulation begins with Primitive Streak (PS) formation. The

location of the Primitive Streak will set the Antero-posterior axis. It is then the rst step in embryo morphogenesis. In the picture the sense of the arrows denotes the direction of the collective motion of Epiblast cells that will establish the three germ lay-ers (endoderm, mesoderm and ectoderm) at the end of gastru-lation. The image is adapted from Principles of Development, Lewis Wollpert. . . 8 4 Deformation of the Epiblast Associated to Primitve Streak

For-mation in the mouse embryo (A) transverse sec4on of a mouse em-bryo at the pre--streak stage, Staining for Hex expression (blue) marks the anterior side of the embryo (Anterior Visceral Endo-derm, AVE). (B,C) transverse sec4ons of a mouse embryo at the early primi4ve streak stage showing buckling of the epiblast as-sociated with the primi4ve streak forma4on (C) A black line un-derlines the contour of the epiblast. Scale bars 50µm. Adapted from Perea-Gomez et al. 2004 . . . 9 5 The three primary germ layers originate dierent tissues and

or-gans in the mature embryo. The Endoderm will originate internal organs such as lungs and pancreas, the Mesoderm the gut, the skeleton and the muscles and the Ectoderm the skin and the

neu-ral system. The picture is adapted from http://www.sigmaaldrich.com/life-science/stem-cell-biology.html. . . 9 6 Symmetric and Asymmetric divisions in Stem Cells. The picture

shows the main characteristic of Stem Cells. They can divide simmetrically and originate two identical daughters in order to maintain their pool (self-renewal) or asymmetrically and origi-nate two dierent daughters in order to give rise to progenitors of dierent tissues (dierentiation). . . 10 7 The origin of Embryonic Stem Cells (ESC) and Epiblast Stem

Cells (EpiSC). Image adapted from http://dev.biologists.org/content/137/15/2455 11 8 Porousity of Alginate hydrogels. (A) Alginate beads. (B)

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9 Alginate chemical structure. Image taken from [2] . . . 13 10 Alginate gel formation in presence by chelation of divalent ions

(Ca2+_{). Adapted from [2]. . . 15}

11 Self-organization in human Embryonic Stem Cells on micropat-terned surface. (Top) circular micropatmicropat-terned surface vs tradi-tional culture. (Bottom) dierent markers of dierent cell fate are expressed in concentric circular domain. CDX2, SOX17, SOX2 and BRA are genes that are expressed respectively by Trophecto-derm, Endoderm and Endoderm and Mesoderm. Bottom images show imunouorescence data for germ layers markers. Their ex-pression allows to identify cell type. Image taken from Warmash et al., Nature Methods. Scale bars 100µm . . . 17 12 Scheme of the device. Two coaxial glass capillaries assembles

through connectors and T-devices are connected to two syringe pumps. The outer pipette is fed with alginate solution, the inner pipette with cells suspension. The two phase ow is extruded inside a gelation bath, made of Calcium chloride solution. . . 19 13 (a) conguration without pulling outer capillary glass. (b) outer

pipette is pulled in order to obtain smaller diameter aginate tubes. It is possible to vary capillary sizes in the range 200 − 1000µm. The tip has been pulled to get a diameter in the range 150 − 250µm. . . 20 14 Chemical structure of Fluoresceinamine. The urophore

(Fluo-rescein) is functionalised with an amin group so that it can form a peptide bond with a carboxyl group. . . 21 15 Labelling of alginate with uorescent dye. The role of EDC in

forming an active intermediate is shown. . . 22 16 A section of a tube image with confocal uorescence microscopy.

Alginate is labelled with Fuorescein. External diameter is ' 800µm 23 17 A picture of an alginate-uorescein tube imaged with confocal

uorescent microscopy. The diameter of the tube is ' 800µm. The image has been reconstructed from a superposition of images from a z-stack of 87 images with ImageJ software. . . 24 18 Section of two uorescein-alginate tubes. The geometry is

af-fected by the dierent alginate ow rate in the two samples. The other parameters (Alginate solution concentration 2% (w/v), outer pipette diameter 700µm, inner ow rate 62.9ml/h) were kept constant, meaning that tuning the ow rates makes it pos-sible to partially control the geometry. Outer diameter ' 1mm, lumen diameter ' 500µm for the tube on the left. Outer diam-eter ' 1.2mm, lumen diamdiam-eter ' 250µm for the tube on the right. . . 24 19 Variation of tubes characteristics (inner diameter, outer

diame-ter, wall thickness) as inner ow rates change. Error bars repre-sent the standard deviation from the mean of n=3 measuares on dierent parts of the tube. . . 25 20 Alginate tubes dimensions (inner diameter, outer diameter, wall

thickness) versus outer ow rate. Error bars represent the stan-dard deviation from the mean of n=3 measuares on dierent parts of the tube. . . 26

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21 Ratio of tubes wall thickness versus outer tube diameter varying with ratio of outer ow rate versus inner ow rate according to (1). Error bars represent the standard deviation from the mean of n=3 measuares on dierent parts of the tube. . . 27 22 Chip design. On the left, the design for one of the three layer is

shown. On the right, the superposition of three layers, centred on a common hole. The dimension of the chip is around3cm × 4cm. The widht of the channels is in the range 750−250µm. AutoCAD software has been used for the disign. . . 28 23 Scheme of a two ows PDMS chip. A section of glass pipette is

inserted in the chip to help the focussing of the inner solution by alginate solution and make the ow coaxial. The width of the channel is 250µm for alginate solution and 150µm for the inner channel. The molds have been 3D printed and the PDMS layers have been Plasma bonded. . . 28 24 Dictyostelium encapsulated in Alginate tubes. Image in bright

eld. Tube diameter ∼ 800µm. The picture is taken immediately after encapsulation.The irregular surface of the inner wall of the tube might be due to oscillations in pressure and has been already observed in polymers extruded through a die. . . 30 25 A sequence of images taken in Bright Field microscopy that shows

the growht of Dictyostelium in Alginate tubes during the days following the encapsulation. Culture medium has been changed daily. . . 30 26 Dictyostelium encapsulation in Alginate tubes. Dierent pictures

have been taken immediately after encapsulation (top, day 1) and on the fth day after encapsultion (bottom, day 5). Dictyostelium are found to correctly divide and grow in this new environment. The tube outer diameter is ' 800µm and the lumen diameter is ' 400µm. . . 31 27 An image of Dictyostelium encapsulated in uorescein-Alginate

tube taken with confocal uorescence microscopy. Tube diameter ' 850µm. Lumen diameter ' 400µm. . . 32 28 HeLa cells on plastic at surfaces. HeLa have been extracted

from a human cervical cancer in 1951. The name comes from the donor's name Henrietta Lacks. The two pictures show cells in culture that have a dierent degree of conuence, i.e. a dif-ferent amount of surface covered with cells. Image taken from https://www.lgcstandards-atcc.org . . . 33 29 HeLa cells encapsulated in Alginate tubes in DMEM culture

medium after encapsulation. Tube diameter' 800µm. Images taken in bright eld microscopy. . . 34 30 Scheme of the rection that binds RGD peptide to alginate. Adapted

from Rowley, 1999. . . 35 31 HeLa Cells behaviour on dierent substrates. On pure Alginate

they do not attach, as it can be seen from their round morphology (top). They attch on RGD functionalized alginate (middle). As a negative control HeLa on plastic Petri dish (bottom). Images are taken in bright eld microscopy magnication 10x. . . 36