Device fabrication and microfluidic techniques 

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Cells and stem cells

Cells

Cells are the smallest units of living organisms and they share some common features. For example, most cells have one nucleus which contains the chromosomes of the cell’s gene, and the nucleoli that produce ribosomes. The cell membrane is on the cell surface which controls the entry and exit of substances. The cytoplasm of the cell is composed of liquid substances and organelles. The ribosome produced in the nucleus can produce proteins, which can work inside the cell or leave the cell. The endoplasmic reticulum is for substance transport inside cells. Mitochondria is for energy production for cell activity. Lysosomes are for enzymatic breakdown of particles that enter cells. The centrioles play essential roles in cell division.
The human body is composed of many different types of cells, including neurons, cardiomyocytes, alveolar, airway, muscle, skin and cells, etc. As shown in Figure 1.1, they have different forms and different functions. Some cells such as blood cells can move in the blood vessels without sticking to each other and some others such as muscle cells are tightly connected to each other. Some cells such as skin cells can divide and proliferate quickly and some others such as neurons will not divide or proliferate. A variety of cells are dedicated to producing hormones, enzymes, milk, insulin, etc. Some alveolar cells secrete mucus, and some cells in the mouth produce saliva. Muscle cells do not produce substances but produce movement. Neurons produce both electrical impulses and neurotransmitters to ensure connections to the nervous system in the brain and the rest of the body.
In addition to the cells, a huge number of microorganisms live in different parts of the human body to help the food digestion or to prevent the growth of more dangerous bacteria, for example [1, 2]. Figure 1.1 Schematic of cellular structure, showing cell membrane, organelles, cytoplasm, nucleolus, and epithelial cells, nerve cells, muscle cells and connective tissue cells [3]

Stem cells

In the human body, most cells are “professional” to suit their specific functions, but the stem cells are not. They can be renewed and differentiated to a particular type of professional cells as shown in Fig 1.2 [4-7]. In particular, they can repair damaged tissue by replacing damaged cells or stimulate the regeneration function of native cells [8-14].
Figure 1.2 Stem cells and examples of stem cell-derived adult cells, including immune cells, epithelial cells, nerve cells, blood cells, etc.
Embryonic stem cells (ESC) can be differentiated to adult stem cells (ASC) and then specific adult cells [15-17]. ESCs can be self-renewed many times and differentiated into all tissue cells, including germ cells. ASCs such as umbilical cord stem cells and bone marrow stem cells are located in specific tissues of adults, which can be differentiated into cells with specific functions.
Depending on their differentiation ability, stem cells are called totipotent, pluripotent or unipotent. Totipotent stem cells are capable to generate fertilized eggs so a complete individual, pluripotent stem cells (PSC), including both embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC), can be used to produce various tissues and blood cells, and finally, unipotent stem cells such as neural stem cells and hematopoietic stem cells are restricted to differentiate to only limited cell types.

Induced pluripotent stem cell

Induced pluripotent stem cells (iPSCs) are obtained by reprogramming somatic cells and they are capable, quite like ESCs, of self-renewing for many generations and deafferenting into many cell types. The iPSCs were obtained from mouse fibroblast then form human fibroblast by the team of Professor Yamanaka of Kyoto University in Japan in 2006 [18], for which he was awarded the 2012 Nobel Prize [19, 20] The reprogramming factors used by his team (also called Yamanaka factors) are four transcription factors (Oct4, Sox2, cMyc, and Klf4). Many studies have shown that a set of small molecules can be used for reprogramming and that many types of adult stem cells and matured adult cells, including umbilical cord blood cells, bone marrow cells, peripheral blood cells, fibroblasts, keratinocytes, etc. can be reprogrammed and then turned into other types of adult stem cells and matured adult cells. Similar to ESCs, iPSCs can be used to generate all three primary germ layers (ectoderm, mesoderm, and endoderm) and all of their derivatives (Figure 1.3).
Compared to ESCs, the use of iPSCs does not pose any ethical problem and iPSCs can be derived from cells of either a healthy donor or a patient, thereby also reducing the risk of immune rejection [21-23]. The emergence of iPSCs has stimulated a strong response in research fields such as stem cells, epigenetics, and biomedicine, giving people a new understanding of the regulatory mechanism of pluripotency, and reducing the distance between stem cells and clinical diseases. iPSCs have great potential in cell replacement therapy and pathogenesis research, screening of new drugs, and treatment of clinical diseases such as neurological diseases and cardiovascular diseases [24-29].
Figure 1.3 Schematic of differentiation of human PSCs to all three embryonic germ layers and adult cells by using different sets of cell factors.

Cellular microenvironment

Cell behaviors are largely influenced by the cellular microenvironment, includes the extracellular matrix (ECM), soluble factors and cell-cell contacts.

ECM

The extracellular matrix (ECM) is a complex network out of the cell and composed of a variety of specific molecules synthesized and secreted by cells as shown in Fig 1.4 [30-33]. These molecules such as collagen, fibronectin, and laminin are mostly glycoproteins and account for 1.0% to 1.5% of proteins in mammalian proteomes.
Figure 1.4 Interaction of extracellular matrix (growth factors, collagen fibers, proteoglycan, fibronectin) and cell membrane proteins (receptors, plasma membrane).
The animal extracellular matrices are divided into two categories, i.e., the interstitial matrix and the basement membrane. The interstitial matrix is located in the intercellular spaces and largely composed of polysaccharides and fibrous proteins, acting as a compression buffer against the stress. Basement membranes (BMs) are sheet-like ECM composed of specific ECM proteins, supporting as substrate the formation of epithelial or endothelial cell layers. Each type of connective tissue in animals has a type of ECM: collagen fibers and bone mineral comprise the ECM of bone tissue; reticular fibers and ground substances comprise the ECM of loose connective tissue; and blood plasma is the ECM of blood.
ECM provides not only a physical support for cells but also important signaling through interactions between αβ integrin heterodimer and ECM peptide motifs. Therefore, cell migration, proliferation, differentiation and survival are all ECM dependent [34-37] (Figure 1.5A).
BMs are also important and found underneath all epithelial and endothelial cell layers [38-41]. They support and help the organization of cell layers and act as a barrier, together with the attached cell layer, to protect the organ, limit the loss of large molecules but allow selectively small molecules. BMs are made principally by type IV collagen [42, 43], laminin [43, 44], nidogen (previously called entactin) [45, 46] and perlecan [45, 47, 48]. Each of them plays a role in the structural assembly, which is characterized by two distinct but tightly linked polymer networks, one made of type IV collagen and another of laminin to ensure the BM’s stability. Different from other types of collagen which are all fibrous, type IV collagen cannot form fibrous with its triple helix composed of three α-chains. Laminin is characterized by a heterotrimeric three-pronged fork made of α-, β-, and γ-chains. More than two laminin isoforms were found with different combinations of five α-chains, three β-chains, and three γ-chains. Laminin isoforms also provide different structural variations, which contribute to a functional diversity of BMs. Type IV collagen and laminin networks are connected by nidogen and perlecan, which are secreted by cells and cannot self-assemble into a network (Figure 1.5B). Finally, epithelial and endothelial cells adhere to a BM surface by forming integrin-containing focal adhesion and hemidesmosomes.
Figure 1.5 Illustration of basement membrane (laminin, type IV collagen, perlecan, nidogen) and cell-basement membrane interaction via integrin-laminin coupling.

Soluble factors

In the cell and extracellular matrix, there are many different types of soluble factors [49, 50] including growth factors for the development, cytokines for immune-modulating, hormones for transferring signals.
Growth factors refer to the natural protein which can stimulate cell proliferation and cell differentiation [51-57]. They regulate various activities and functions of cells. As signaling molecules, growth factors can activate specific receptors on the surface of target cells. These factors usually promote cell differentiation and maturation. Different growth factors have diverse functions, for example, bone morphogenetic protein stimulates the differentiation of bone cells; while vascular endothelial growth factor stimulates the proliferation of vascular endothelial cells. Cytokines comprise interleukins, interferons, tumor necrosis factors, chemokines, and lymphokines [51, 58-62]. They are produced by macrophages, mast cells and B and T lymphocytes, endothelial cells, fibroblasts and a large variety of stromal cells.
Some growth factors may cause apoptosis of target cells and some cytokines may have inhibitory effects on cell growth and proliferation.
Hormones are chemical messengers that pass from one cell to another and change the cell’s metabolism with a small dose. They are produced by cells, glands, or organs that can affect the activities of other cells in the body [63-69]. They are usually transported to designated locations in the body through the blood, and cells respond to hormones through their special receptors.
After the hormone molecule binds to a receptor protein, it opens a signaling pathway for signal transduction and eventually causes the cell to respond specifically. Hormone molecules secreted by the endocrine system are usually released directly into the blood, mainly into blood capillaries. Although the amount of hormones in the human body is quite small, it has a huge impact on health [70-73]. Lack of or excessive secretion causes various diseases, for example too much growth hormone secretion will cause gigantism, too little secretion will cause dwarfism; and excessive secretion of thyroid hormone will cause palpitations, hand sweats and fast metabolism, too little secretion will easily lead to obesity, drowsiness, etc.; insufficient insulin secretion will lead to diabetes.

Cell-cell contact

Cell-cell contact is critical to multicellular organisms. Some cell-cell interactions are stable such as cells in the epithelium and some others are transient such as the interactions between immune cells and that between immune cells and infected cells in a host tissue. Three types of cell-cell junctions are critical to the maintenance and function of an epithelium:
Tight junction is a protein complex that seals neighboring cells together in an epithelial sheet to prevent leakage of water and water-soluble molecules between them [74-76].
Desmosomes (Anchoring junction) is a cluster of proteins associating the intermediate filaments in one cell to those in a neighbor [77-79].
Gap junction (Communicating junction) is composed of transmembrane proteins called connexins and allows the passage of small water-soluble ions and molecules [80-83].

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Physiological systems

General notions

The human body is composed of trillions of physiological systems, from intracellular systems that operate at the molecular level to the highly developed central nervous system (CNS). In terms of complexity and scale, these systems range from systems contained within cells to systems responsible for coordinating the activities of millions of cells. However, every physiological system has a common purpose, which is to regulate all aspects of body functions to maintain homeostasis. From the perspective of macroscopic human organs, the human body is made of ten physiological systems [84, 85], i.e., the cardiovascular, digestive, endocrine, immune, muscular system, nervous, renal, reproductive, respiratory, and skeletal systems. Their functions and related organs and cells are listed in Table 1.

Lung and alveoli

The lungs contain approximately 300 to 500 million alveoli, connected to the alveolar ducts and respiratory airway bronchioles [111, 112]. The alveoli are the elementary units of the lungs that ensure respiration, i.e., the gas exchange or extracting oxygen from the atmosphere and transferring it into the bloodstream, and reversely, releasing carbon dioxide from the bloodstream into the atmosphere. Each alveolus is surrounded by a blood capillary network to ensure receiving deoxygenated blood from the heart for a maximum release of carbon dioxide and sending oxygenated blood with a maximum absorbed oxygen to the body [113, 114].
Figure 1.6 Illustration of alveolus made of an alveolar sac, a blood capillary, alveolar macrophages, fibroblasts, and associated basement membranes, and gas exchange function.
Microscopically, the lung alveoli are constructed with two types of alveolar epithelial cells, capillary endothelial cells, macrophages, and fibroblasts (Fig 1.6).
Alveolar epithelial type I cells (AT1) are thin squamous cells for oxygen diffusion from air sac to capillaries across the basement membranes which cover ~96% of the total lung surface area [115]. Alveolar epithelial type II cells (AT2) are cuboidal cells for producing surfactant proteins and lipids, which are important for keeping a low surface tension of the alveoli and preventing the collapse of the alveoli upon breathing [116, 117].
AT2 cells are also capable of responding to innate immune stimuli, thus having the functions in the immune response. AT1 and AT2 cells are both derived from alveolar progenitor cells [118]but AT2 cells themselves can also be self-renewed and become AT1 cells [119, 120].
Alveolar macrophages (AMs) are tissue-specific immune cells that are found on the luminal epithelial surface of the alveoli [121-123]. They are the first protective line against invading pathogens. AMs are heterogeneous in disease environments where a subset of monocyte-derived AMs express profibrotic genes [124]. It is known that many acute and chronic lung diseases are associated with inflammation of alveoli but many questions remain to be answered.

Cell culture substrate

Conventionally, culture dishes, flasks, multi-well plates, etc., are used for cell culture and cell-based assays. To get a better performance, a variety of cell adhesion molecules are used to coat the cell culture surface. However, these coatings may not be sufficient to achieve particular functionalities because of 2D characteristics of the culture substrate.
The problem emerged for culture and differentiation of pluripotent stem cells, co-culture of more than two types of cells, tumor spheroids, organoids, etc. In such cases, cells may not be always organized in 2D and more sophisticated ECM proteins organizations have to be introduced to achieve tissue or organ-like higher functions. In the following, we discuss cell culture-related material properties.

Hydrogels

A hydrogel is a network of cross-linked polymer chains which is intrinsically hydrophilic and water permeable. Due to their flexibility in synthesis, high biocompatibility, large range of constituents, and unique physical characteristics, hydrogels became one of the best choices for biomaterial engineering and tissue engineering [125-127]. Hydrogels can also be used for drug and protein delivery as well as adhesives or barriers between tissue and material surfaces [128, 129].
Poly (ethylene glycol) (PEG) and derivatives: PEG is a synthetic polymer largely used in biomedical and biotechnological fields [130]. The molecular weight of PEG covers a broad range between 102 and 107 g/mol. The chemical structures of PEG such as linear, branched, or combined chains are available. The synthetic procedure determines the polymer’s end groups which allow the introduction of different specific or functional groups as well as linkage to other polymers. Nevertheless, PEG-based biomaterials are resistant to hydrolytic or enzymatic degradation which causes concern about accumulation effects. Cell-binding sites like integrin need to be incorporated into the polymeric matrix to enable cell adhesion. The formation of PEG-based hydrogels is usually based on various mechanisms including ionic, physical, or covalent crosslinking. PEG-based hydrogels, however, hold the highest mechanical stability by covalent crosslinking, for example, by introducing diacrylate units into PEG chains which results in poly (ethylene glycol) diacrylate (PEGDA). In contrast to classic polyesters, these materials do not release acidic by-products during their degradation and also are reported capable of cell adhesion [131].
Agarose: Agarose is a linear polysaccharide extracted from seaweed. Most commonly known as a component of agar, it is formed by a disaccharide of β-D-galactose and 3,6-anhydro-α-L-galactopyranose [132]. Agarose can be dissolved in hot water using a water bath or simply a microwave oven. Agarose gel can be formed upon cooling. The gelation process is thermoreversible at gelation temperatures, which are specific depending on different types of agarose or the methoxyl content. Agarose can form strong gels at concentrations even below 1%. As for suspension culture, agarose can form a thin gel layer on the surface coating. Agarose gel also offers a high diffusion rate and is commonly used as filters, or purification, due to its porous structure and adjustable pore sizes (100-500 nm) which are determined by agarose concentration.
Gelatin: Gelatin is produced by acidic, alkaline, or hydrolysis of collagen from bovine or porcine skin, thus it is a biodegradable protein [133]. By hydrolysis, random gelatin chains can be produced by breaking up the collagen’s triple helix structure. Gelatin can be also classified into two types depending on the origin and extraction reaction.
Gelatin Type A is processed by acid, generally with a low viscosity but high bloom.
Gelatin Type B is processed by alkali, generally with a high viscosity but low bloom.
Upon cooling, a three-dimensional hydrogel network can be formed by reassembling random gelatin chains into a triple helix structure. Both in vivo and in vitro, gelatin is enzymatically degraded without producing harmful species. Moreover, gelatin still contains integrin-binding sites which promote cell adhesion [134].
Gelatin methacrylate (GelMA): Gelatin’s gelation is a temperature-dependent, reversible process. However, the use of gelatin hydrogels as scaffold materials is limited due to their rapid degradation property [135-137]. Thus, the vinyl methacrylate groups in the gelatin polymeric backbone can result in stable and covalent crosslinking networks, since they can react between themselves or with vinyl groups of other small molecules, oligomers, or polymers through photo- or temperature-initiated radical polymerization.
Matrigel: a commercial hydrogel containing protein mixture isolated from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, an ECM-protein rich tumor [138]. Matrigel is thereby composed of several ECM proteins like laminin and collagen, making it an excellent substrate or hydrogel for both two-dimensional cell adhesion and three-dimensional cell culture or organoid culture. Some growth factors and proteins are also included in Matrigel. It polymerizes rapidly into a 3D network at temperatures between 22 and 35 ℃. At 4 ℃, Matrigel starts to thaw. Consequently, the preparation and processing of Matrigel-based samples need to be performed under cold conditions.

Table of contents :

Chapter 1 Introduction 
1.1 Cells and stem cells
1.1.1 Cells
1.1.2 Stem cells
1.1.3 Induced pluripotent stem cell
1.2 Cellular microenvironment
1.2.1 ECM
1.2.2 Soluble factors
1.2.3 Cell-cell contact
1.3 Physiological systems
1.3.1 General notions
1.3.2 Lung and alveoli
1.4 Cell culture substrate
1.4.1 Hydrogels
1.4.2 Nanofibers
1.4.3 Surface treatment
1.4.3.1 Cell-attachment treatment
1.4.3.2 Ultra-Low attachment treatment
1.4.4 From 2D to 3D culture substrate
1.4.5 Air-liquid interface
1.5 Microfluidic technologies
1.5.1 Introduction of microfluidic device
1.5.2 Fabrication methods of microfluidic device
1.5.2.1 Lamination
1.5.2.2 Molding
1.5.2.3 3D printing
1.6 Cell-based assays
1.6.1 Organ-on-a-chip
1.6.1.1 Lung-on-a-chip
1.6.1.2 Liver-on-a-chip
1.6.1.3 Kidney-on-a-chip
1.6.1.4 Gut-on-a-chip
1.6.1.5 Heart-on-a-chip
1.6.1.6 Multi-organ-on-a-chip
References
Chapter 2 Device fabrication and microfluidic techniques 
2.1 Photolithography
2.2 Vacuum-assisted molding
2.2.1 PDMS mold fabrication
2.2.2 PEGDA molding
2.3 Electrospinning
2.3.1 Electrospinning
2.3.2 Chemical crosslinking
2.3.3 Thermal crosslinking
2.4 Micro-milling
2.5 Cutting plotter
2.6 Parylene deposition
2.7 Culture patch, basement membrane mimics and accessories
2.7.1 Culture patch
2.7.2 Ultrathin artificial basement membrane
2.7.3 Chamber for improved cell seeding on patch
2.7.4 Patch handler for Air-liquid interface (ALI) culture
2.8 Microfluidic devices
2.8.1 Device configuration
2.8.2 Mechanical clamping
2.8.3 Concluding remarks
References
Chapter 3 Automatic stem cell differentiation 
3.1 Introduction
3.2 Development of the system
3.3 Dynamic cell culture
3.4 Cardiac differentiation
3.4.1 Fabrication of the culture patch
3.4.2 Preparation of the culture media with different factors
3.4.3 Protocol implementation
3.4.4 Results
3.5 Neuron network maturation
3.5.1 Protocol implementation
3.5.2 Operation details
3.5.3 Results
3.6 Conclusion and discussions
References
Chapter 4 Fabrication of alveolar tissue constructs 
4.1 Introduction
4.2 Fabrication of alveolar organoids
4.2.1 Preparation of medium and soluble factors
4.2.2 HiPSCs maintenance
4.2.3 Definitive endoderm
4.2.4 Anterior foregut endoderm
4.2.5 Bud tip progenitor organoids
4.2.6 Maturation of alveolar organoids
4.3 Manipulation of alveolar organoid
4.3.1 Passage of alveolar organoids
4.3.2 Cryopreservation of alveolar organoids
4.3.3 Thawing of alveolar organoids
4.3.4 Dissociation and replating
4.3.5 Air-liquid interface (ALI) culture
4.4 Characterization of alveolar organoids and derived epithelium
4.4.1 Immunocytochemistry
4.4.2 TEER monitoring
4.5 Conclusion
References
Chapter 5 Response of hiPSC derived cells to S-proteins of SARS-CoV-2 
5.1 SARS-CoV-2: Physiological aspects
5.2 SARS-CoV-2: Virus-host interaction
5.3 ACE2 and renin-angiotensin system
5.4 Effect of S-protein
5.5 ROS monitoring
5.6 Response of iPSC derived cardiomyocytes
5.7 Discussion
5.8 Conclusion
References
Chapter 6 Conclusion and perspectives 
Appendix
Appendix A: Fabrication of micro-cage devices for spheroid handling
Appendix B: Fabrication of ultrathin artificial basement membrane
Appendix C: Effect of periodic deformation on alveolar cell layer on nanofibers
Appendix D: Realization of a bioreactor for patch-based hiPSC differentiation
Résumé en français
Abbreviation list
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