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Cellular microenvironment

Extracellular matrix

The Extracellular Matrix (ECM) provides mechanical supports and biochemical signaling to cells which regulate many cell functions such as cell division, motility, embryonic development, and wound healing [12, 13]. In addition, the ECM plays an important role in forming the mass of skin, bones, and tendons. The ECM also forms extracellular structures such as the cornea of the eye and filtering networks in the kidney. A simplified diagram of the composition of the ECM is illustrated in Fig. 1.4.
The ECM is composed of ECM biopolymers such as collagen, elastin, laminin perlecan and other proteins proteoglycans. Collagen is made of long fibrous glycoproteins with a triple helix structure, which leads to a high tensile strength and great elasticity. Proteoglycans are consisted of small proteins attached to long polysaccharides, regulating movement of molecules through the matrix and also the binding of cations and water. Among others, fibronectin, a high-molecular weight (~440 kDa) ECM glycoprotein, can bind efficiently to the membrane-spanning receptor proteins called integrins and to the ECM, which decide the overall communication of the ECM [14–16]. Thus, fibronectin is of great importance in cell attachment and in the wound healing processes. It is also widely used for substrate treatment before cell culture. Elastin is the component that allows the ECM to be more “rubbery” and form elastic structures. While laminin is found in between the cells and it forms webs that help hold them together [17].

Cell-ECM interaction

The effects of the ECM to cells are primarily mediated through receptor-mediated signaling. Integrins (Fig. 1.4), a family of cell surface receptors, whose structure consists of the heterodimeric non-covalent association of α and β subunits. While β subunits seem to have a non-specific role in ligand binding activity, α subunits on the contrary confer high specificity of signal transduction. In their inactive state, integrins are freely diffusive within the cell membrane until they encounter an available binding domain in the ECM. Integrins change conformational once ligand binding, which leads to the recruitment cytoplasmic proteins [19, 20].
A premature adhesive junction between intercellular and extracellular molecules, formed by localized integrins cluster and structure proteins, activates the intracellular signaling events. As soon as ECM molecules bind to their specific integrin or non-integrin receptors, a change in cytoplasmic domain of the receptor occurs, which associates with the cytoskeleton at focal adhesion sites. Consequently, an assembly of the focal contact proteins occurs with other intercellular components, such as phosphorylated proteins. These changes can promote cytoskeleton rearrangement, which may determine differential interactions of chromatin and nuclear matrix at the nuclear level. The dynamic association of integrin receptors with the actin cytoskeleton may also induce changes in cell shape, which in turn alters the ability of cells to proliferate or differentiate [21].

Cell-cell interaction

Cell-cell interactions allow cells to communicate with each other in response to changes in their microenvironment, which is of great importance in the development and maintenance of multicellular organisms. Interactions between cells can be stable through cell junctions which have the ability of sending and receiving signals. Stable cell-cell interactions are essential for the communication and organization within tissues, otherwise the loss of communication between cells can result in uncontrollable cell growth and cancer spread.
Intercellular junctions normally have three main types, i.e., tight junctions, desmosomes and gap junctions (shown as Fig. 1.5), classified by the strength of the cell-cell interaction, which depends on the mixture, concentration, distribution and also cytoskeletal linkages of adhesion molecules on the cell surface. The main tight junction forms a seal between the two membranes. This seal consists of the transmembrane proteins and provides transcellular and paracellular transport of molecules, but prevents passive flow between cells. Desmosomes, adherent or anchoring junctions, build an encircling structure, with the main function of strengthening and stabilizing the circular occluding bands. Another important function is the linking cytoskeletons of adjacent cells. This is made possible by the transmembrane glycoprotein cadherin, which functions in presence of Ca2+. The major function of gap junctions, consisted of proteins named connexin, is the direct transfer and exchange of nutrients and signal molecules between the cells [23, 24]. The complex of connexin has a hydrophilic pore with the diameter of 1.5 nm, which permit the exchange of larger molecules.

Cell-substrate interaction from mechanotransduction aspect

Cells in the human body have different tissues in the body have specific cell types and characteristic microenvironments that work together to achieve their function. Since cells exploit mechanical forces to explore their environments and recognize material properties, the physical characteristics of their microenvironment profoundly impact cell behavior and fate [25]. Traditionally, researchers have tried to understand disease through altered cell phenotype, while paying comparatively little attention to the cell environment. Recently, considerable evidence has emerged that many diseases are associated with transformations of the tissue-specific cellular microenvironments. This is a classical “chicken and egg” problem: which comes first? Do unhealthy cells remodel their microenvironment, or do diseased microenvironments drive changes in cell behavior? It is never fully possible to disentangle this complex feedback. The popular approach is to manipulate key elements inside (i.e., cytoskeletal or nuclear elements) or outside the cell (i.e., ECM stiffness, strain, or phosphorylation state) and then analyze the resulting signaling in both directions. Insights into mechanobiology will help us to better understand both normal physiology and disease states [26–28].


Fig. 1.9 shows the schematic diagram of cell mechanical stimulation with substrate. Mechanical forces stimulate cells through the activation of mechanosensors, including the receptors that respond to ligand [29]. Cells are exposed to different types of forces: extracellular forces such as shear forces through fluid flow over the cell surface, tensile/traction forces through the ECM, intercellular forces through contact with neighboring cells, and intracellular cytoskeleton generated contractile forces (actomyosin contraction, microtubule polymerization and depolymerization, osmotic forces) [30]. In particular, the forces generated through cell–substrate interactions are important to initiate a cellular response to any adherent substrate. For cell–substrate interactions, integrin binding to an ECM protein, commonly fibronectin, laminin, or collagen or peptides derived from them, is a primary contributor to cell recognition. Binding of the cell to the substrate is the first stage to transmitting force across the cell membrane, and through this binding, integrins interact with the extracellular environment. Next, adaptor proteins bind to actin in the cytoskeleton, linking it to the cell membrane. Then, forces are transmitted from the actin filaments through the myosin head. Overall, integrins and adaptor proteins, such as those that form focal adhesions, initiate signaling pathways for various cell functions, such as survival, proliferation and differentiation.
In the study of these various cellular responses, the focal adhesions, an important component of substrate-mediated mechano-transduction, formed through integrin clustering is considered as regulator of the Rho signaling pathway (Fig. 1.10) [29], which controls myosin II phosphorylation and affects the cytoskeleton tension in adherent cells [31–35]. Other mechanotransduction pathways involve Src family kinases (SFKs), which respond to integrin clustering to activate the Rho GTPases family, Rac, Cdc42, and Rho, and also control cytoskeleton organization by regulating actin at focal adhesions and ERK1/2, which is activated by focal adhesion kinase (FAK) to participate in actin filament formation. FAK, activated by intrgrin binding, is one of the first mechanosensitive proteins and has recently been demonstrates as a link between mechanotransduction and differentiation, where increased FAK phosphorylation correlated with increased MAP2 expression, a mature neuronal marker [36, 37].
Figure 1.10 Rho family GTPase pathway. The Rho family GTPases, Cdc42, Rac, and Rho, control the cell shape, focal adhesions, and migration through signaling mechanisms to regulate actin filaments and the cytoskeleton [29].

Substrate stiffness

The ability of cells to sense the rigidity of the ECM affects the regulation of cellular activities in development, wound healing and even malignant transformation [31, 38, 39]. Integrins are transmembrane adhesion receptors that anchor cells to the ECM and transduce ‘outside-in’ or ‘inside-out’ biochemical signals [40]. For outside-in signaling, integrins transmit information about a cell’s adhesive state through a cascade of biochemical reactions that in turn regulates subsequent cellular responses such as motility and differentiation. In contrast, inside-out signaling involves intracellular reactions that result in changes in the integrin affinity for its extracellular ligand [41]. Interestingly, recent evidence suggests that integrins can undergo changes in affinity that depend on the stiffness of the culture substrate [42]. Because of their mechanotransductive properties, integrins have thus long been thought to be responsible for sensing (and responding to) the rigidity of the cell’s microenvironment.
Cells sense matrix rigidity through integrin binding dynamics. At low rigidities (soft substrates), the matrix deforms as the cell pulls on it, causing the rate of force loading of the integrin–fibronectin bond to be so low that the bonds detach stochastically because of their intrinsic unbinding rate (koff). In this case, the unbinding of the integrin-fibronectin bond is controlled by koff (schematic demonstrated in Fig. 1.11). On substrates with intermediate rigidities, the matrix effectively resists the cell-generated force, resulting in increased bond loading and force-enhanced dissociation. In this regime, koff > kon, and thus bonds break before new bonds are formed, which results in decreased adhesive forces. On high-rigidity substrates, the high force-loading rate exceeds the threshold value for integrin recruitment, where integrins cluster at the site of force application [43]. This reinforcement mechanism compensates for the increased unbinding, hence providing enhanced traction force. Therefore, cellular-traction forces reach a local minimum at intermediate substrate rigidities.


Substrate topography

The pervious researches show that mimicking in vivo microscale and nanoscale topography in implantable biomaterials is critical to guide cellular behaviors [44–46]. Recently, advances in micro- and nanofabrication methods are enabling researchers to develop sophisticated substrates with micro/nanotopography [47, 48]. Techniques such as photolithography [49], electron beam lithography [50], electrospinning [51] and 3D printing [52] have been utilized to recreate certain ECM topographical features at specific length scales or exactly replicated complex and hierarchical topography in vitro. The primary example of cells response to the substrate topography is the adhesion formation, which resides predominantly on the ridge structures [53]. The constraint of focal adhesions to the ridges of gratings could be due to the cell membrane stiffness which prevents a radical bending and hence favoring the plasma membrane to bridge the grooves rather than entering them. While larger grooves may allow enough bending of the membrane, lead to cell penetration into the grooves to form adhesions, which results in cell alignment as schematically shown in Fig. 1.13a. Moreover, recent studies reveal the intriguing observation that topographical cues may drive the differentiation of stem cells [54–56]. For instance, hMSCs cultured on electrospun aligned fibers show upregulation of myogenic markers Pax-3, Pax-7 and myogenin to indicate myogenic lineage commitment, whereas cells cultured on flat film composed of the same polymer do not [57].
Figure 1.13 Schematic depictions of representative topography geometries. Three basic nanotopography geometries include (a) grooves, (b) post array, and (c) pit array [58].

2D versus 3D culture

Currently, the understanding of majority of biological processes is based largely on studies of cells cultured on two-dimensional flat substrates, i.e., glass slide or plastic petri dish. Nevertheless, cells, in vivo, primarily exist within a complex and information-rich environment, interacting with multiple ECM components and medley factors. The striking disparity between traditional monolayer culture and the in vivo scenario lead to the different cell function, such as cell adhesion, migration and mechanical forces, etc [59–63].
Cells assume two-dimensional (2D) or three-dimensional (3D) geometries largely on the basis of whether integrin-mediated adhesions to the ECM form on one face of the cell or all around the cell surface. Different cellular responses in 2D versus 3D culture could arise from these variations in the spatial distribution of adhesions [64, 65]. One of the most striking differences observed when comparing cells in 2D and 3D is the dissimilarity in morphology [66–68] (Fig. 1.12). Cells grown in a monolayer are flat, and can adhere and spread freely in the horizontal plane but have no support for spreading in the vertical dimension. One consequence of this is that cells cultured on 2D surfaces have a forced apical-basal polarity [69]. This polarity is arguably relevant for some cell types, such as epithelial cells, but is unnatural for most mesenchymal cells, which-when embedded in a 3D ECM-assume a stellate morphology and only polarize from front to rear during migration [70]. These changes in cell geometry and organization can directly impact cell function.
Mechanical stresses generated or experienced by cells as they adhere to the ECM and to their neighbors represent a central component of how cells transduce adhesion-mediated signaling and process [71]. Traditional 2D culture on glass or plastic substrates places cells in a static mechanical environment that is supra-physiological in terms of stiffness. Recognizing the disparity between these artificial conditions and the markedly more-compliant microenvironment of most tissues, recent work using soft 2D gels has confirmed that ECM stiffness can influence adhesions, morphogenesis, and stem cell differentiation and maintenance [72–74]. Low stiffness is not necessarily an intrinsic property of 3D environments; however, it is a feature common to most 3D systems and a factor that should be taken into consideration when differences are noticed between cell behavior in 2D and 3D.

Technology of induced pluripotent stem cells

The induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are embryonic stem cells like obtained by reprogramming somatic cells. Typically, the reprogramming procedure involves ectopic expression of four transcription factors: Oct3/4, Sox2, c-Myc and Klf4, which are all crucial for the maintenance of pluripotency in embryonic stem cells [75]. The resulting iPS cells show close resemblance to the embryonic stem cells regarding to their morphology, i.e., high nucleus to cytoplasm ratio, generating flat compact colonies in culture with defined edges, their gene expression, i.e., SSEA3/4, Tra-1-60/80, Oct4, Sox2, Nanog, and their ability of differentiation into different types of somatic cells.
The iPSC reprogramming procedure was firstly introduced by Yamanaka and Takahashi in 2006, they managed to transform mouse embryonic and adult fibroblasts into a pluripotent state when introduced with viral vectors. A year after the generation of murine iPS cells, they showed that human somatic cells could be converted with the same set of factors, generating the first human iPS (hiPSCs) cells. At the same time, a different set of reprogramming factors, such as Oct4, Sox2, Nanog and Lin28, also revealed by the Thomson group. At present, the general knowledge is that Oct4 is an essential factor, while Sox2 and Klf4 can be omitted only if the genes endogenous expression is high in the somatic cells. Nanog, c-Myc and Lin28 are dispensable, but they do act to enhance conversion rate. Gurdon and Yamanaka received the Nobel Prize in medicine in 2012 for their historic contribution in iPS-technology, which inspired an astonishing flurry of follow-up studies, including successfully reprogramming of a wide variety of other cell types to pluripotency, differentiation to specific linages and also disease modeling [76– 78].
Human iPS cells are strikingly similar to human embryonic stem cells (hESCs). They replicate via mitotic division whilst maintaining an undifferentiated state, and they have the potential to differentiate into all somatic cell types when provided with the necessary stimuli. Furthermore, the arrival of iPS cell technology marked the start of an exciting new dimension of stem cell research, avoiding the ethical and immunological issues that typically hamper ES cell research, which expand the potential of regenerative cell medicine [79]. iPS-technology has produced great hope for an unlimited source of patient specific stem cells for tissue regenerative applications, the possibility to study disease specific cells and developmental processes without ethical concerns compared with the use of hESCs (Fig. 1.6).

Table of contents :

1.1 Cell and cellular microenvironment
1.1.1 Cell and cell cytoskeleton
1.1.2 Cellular microenvironment Extracellular matrix Cell-ECM interaction Cell-cell interaction
1.2 Cell-substrate interaction from mechanotransduction aspect
1.2.1 Mechanotransduction
1.2.2 Substrate stiffness
1.2.3 Substrate topography
1.2.4 2D versus 3D culture
1.3 Technology of induced pluripotent stem cells
1.3.1 The induced pluripotent stem cells
1.3.2 Differentiation of induced pluripotent stem cells Differentiation via EB formation Substrate for stem cell differentiation
1.4 Scaffold engineering
1.4.1 Biomaterials
1.4.2 Fabrication techniques
1.4.3 State of the art and future directions
1.5 Research objectives of this work
2.1 Introduction
2.2 UV photolithography
2.2.1 General principles
2.2.2 Mask design and exposure
2.2.3 Photoresist
2.2.4 UV expose and development
2.3 Soft photolithography
2.3.1 Mechanism and fabrication process
2.3.2 Micro-contact printing (μCP)
2.3.3 Replica molding
2.4 Electrospinning
2.4.1 Principles
2.4.2 Effects of various parameters on electrospinning Applied voltage Flow rate and tip to collector distance Alignment, collector-pattern and 3D structure
2.5 Conclusion
3.1 Introduction and motivation
3.2 Materials and methods
3.2.1 Materials
3.2.2 Fabrication of the substrate
3.2.3 Microfluidic device integration
3.2.4 Cell culture
3.2.5 Immunocytochemistry
3.2.6 Imaging and statistical analysis
3.3 Results and discussion
3.3.1 Fabrication of the micropillar substrate
3.3.2 Rigidity of the micropillar substrates
3.3.3 Cell motility and cell migration
3.3.4 Cell morphology and cell proliferation
3.3.5 Complex stiffness map
3.4 Conclusion
4.1 Introduction and motivation
4.2 Materials and methods
4.2.1 Materials
4.2.2 Fabrication of tri-layer scaffold PDMS mold fabrication Aspiration-assisted PEGDA frame production and ring mounting Electrospinning nanofibers
4.2.3 Cell culture
4.2.4 Immunocytochemistry
4.2.5 SEM observation
4.2.6 Live/dead assay
4.2.7 Cell proliferation
4.3 Results
4.3.1 Tri-layer scaffold formation Frame pattern and size Frame thickness Materials for electrospinning Nanofiber density
4.3.2 Cell attachment and spreading
4.3.3 Cell viability
4.3.4 Cell morphology and 3D cell culture
4.3.5 Cell migration Top to bottom migration Bottom to top migration Migration on thick scaffold
4.3.6 Cell proliferation
4.4 Conclusion
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials
5.2.2 Fabrication of elastomer micropillars
5.2.3 Fabrication of the PDMS stencil
5.2.4 hiPSCs culture
5.2.5 Scanning electron microscopy imaging
5.2.6 Immunocytochemistry
5.2.7 MTT assay
5.2.8 Intracellular Ca2+ transient assays
5.2.9 External electrical stimulation
5.3 Results
5.3.1 Sample characterization SEM imaging Effective stiffness of the elastomer pillars
5.3.3 Stencil assistant EBs formation The effect of the substrate stiffness The effect of cell seeding density Pluripotency of hemisphere EBs 3D view of the EBs
5.3.4 Cardiac differentiation on micropillar substrates Differentiation protocol Immunostaining of differentiated cardiomyocytes
5.3.5 Beating behavior of differentiated cardiomyocytes
5.3.6 Impact of external electrical stimulation
5.4 Conclusion
1 Introduction
2 Experiments
3 Results and discussion
3.1 Microwave and MLAs patterns
3.2 Rice leaf-like structure and the hydrophobic property
4 Conclusion


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