Hepatocyte proliferation and thermally induced cell detachment on non-woven PP, PET and nylon three-dimensional scaffolds

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THREE DIMENSIONAL CELL CULTURE SYSTEMS 

THEIR APPLICATIONS: A REVIEW Mammals are comprised of a large number of cell types with a variety of specialised functions including tissue scaffolding and structure; signalling and sensing; digestion and absorption; transfer of molecular oxygen; immunity; mobility and reproduction to name a few. One of the major developments in science and particularly cell biology has been the development of systems allowing the growth of these various cell types in vitro, literally meaning ”in glass” or outside the body.
In vitro, biologists have been able to study amongst others cell differentiation, function, development, growth, regeneration and death. Tissue culture was first developed in the early 1900’s as a method for studying the behaviour of tissue fragments and gradually biologists developed techniques to study the behaviour of single cells and changed the name to cell culture. In its simplest form, cell culture involves the dispersal of cells in an artificial environment composed of nutrient solutions, a suitable surface to support the growth of cells, and ideal conditions of temperature, humidity, and gaseous atmosphere. In such a system, a researcher can measure the cells’ response to culture and genetic alterations, physiological signalling molecules, prospective drugs, interaction with other kinds of cells, carcinogenic agents and pathogens.
As our ability to grow, manipulate and analyse cells in this way has developed, so has our knowledge of cell function and physiology. Increasingly the concept of three-dimension(al) (3D) is being applied in relation to in vitro cell culturing. In vivo all tissues reside in an extracellular matrix (ECM), which comprises of a complex 3D fibrous meshwork. Additionally, depending on the cell type the 3D microenvironments differ significantly (1). For example, osteoblasts are found on the surface of bone organised in sheet-like structures as cuboidal cells; hepatocytes are packed closely together in hexagonal shaped lobules and lymphocytes are suspended freely in blood or lymph vessels (1).
Today, there is increasing awareness of the drawbacks of 2D in vitro cell culturing and the related effect on the value of the research being performed as the dynamic range of structural organisation of various cell types in vivo cannot be accurately emulated using 2D cell culture technology. As a result, altered metabolism and reduced functionality are observed in 2D in vitro cell culture (2, 3) thus, 3D cell culture matrices also known as scaffolds were introduced to overcome these limitations (1).
An analysis of the number of publications in the field of 3D cell culture indicates an increasing interest in this field. Not surprisingly, more and more scientists are shifting their focus to cells cultured in 3D as intuitively one can appreciate that with cells, form affects function (4). Figure 1.1 provides a graphical representation of the increase in the number of publications arising from studies performed by using cells cultured in 3D. This review looks at 3D cell culture and its growing importance in in vitro biology as well as the various parameters used to design and construct 3D scaffolds. Various applications of 3D cell culture in the fields of in vitro toxicology, anti-cancer drug screening and host-pathogen interactions are reviewed and the future of 3D cell culture is discussed. essential requirement for implantable 3D scaffolds as the scaffolds should degrade at the rate that in-growing tissue replaces them (1).
Synthetic materials generally degrade by hydrolysis (21) and natural materials generally undergo enzymatic degradation (22). Mechanical properties of the bulk materials are an important set of characteristics to consider in 3D matrix design, especially in tissue engineering for structural tissues i.e. bone. The scaffold must be able to endure sufficient loads so as not to fracture but, should not be too strong thus damaging adjacent tissue. Mechanical properties also directly shape surface mechanical properties such as surface stiffness or elasticity, which elicit clear cellular responses (1). Natural ECM is a fully hydrated gel, thus wettability is another bulk material consideration; a hydrophilic biomaterial is better at mimicking the in vivo aqueous environment. Transparency is another important parameter for 3D in vitro modelling applications where cellular behaviour within the scaffolds is to be visualised using optical, fluorescence or confocal microscopy (1). Many types of biodegradable polymeric materials have been used for scaffold fabrication in tissue engineering applications. The gradual degradation of a biodegradable polymer is an important feature of a scaffold to aid the integration of the cells they carry with host tissues. Natural-based materials include polysaccharides (starch, alginate, chitin/chitosan, hyaluronic acid derivatives) or proteins (soy, collagen, fibrin gels, silk) and, as reinforcement, a variety of bio-fibres such as lignocellulosic natural fibres (23).
Synthetic polymers include poly(lactic acid), poly(glycolic acid), poly(ε-caprolactone) (PCL) and poly(hydroxyl butyrate) (24). Many advantages and disadvantages characterise these two different classes of biodegradable polymers. The synthetic polymers have relatively good mechanical strength and their shape and degradation rate can be easily modified, but their surfaces are typically hydrophobic and lack cell-recognition signals. Naturally derived polymers have the potential advantage of biological recognition that may positively support cell adhesion and function, but they generally have poor mechanical properties (24). Synthetic polymers can be produced under controlled conditions and therefore exhibit, in general, predictable and reproducible mechanical and physical properties such as tensile strength and elastic modulus. A further advantage of synthetic polymers is the control of material impurities. Possible risks such as toxicity, immunogenicity and favouring of infections are lower for pure synthetic polymers with constituent monomeric units having a well-known and simple structure (23).

TABLE OF CONTENTS :

• Submission Declaration
• Plagiarism Statement
• Acknowledgements
• Summary
• Table of Contents
• List of Figures
• List of Tables
• Abbreviations
• Appendix A
• CHAPTER 1 Three dimensional cell culture systems and their applications: A review
  • 1.1. 2D vs 3D: Why do we need 3D?
  • 1.2. 3D Scaffolds
    • 1.2.1. Materials of choice
    • 1.2.2. Bulk modifications
    • 1.2.2. Surface modifications
  • 1.3. Applications of in vitro 3D cell culture
    • 1.3.1. In vitro anti-cancer drug screens: Tumour tissue models
    • 1.3.2. In vitro toxicology models
    • 1.3.3. In vitro host-pathogen interactions
  • 1.4. 3D cell culture: Future directions
  • 1.5. Objectives
• CHAPTER 2 Hepatocyte proliferation and thermally induced cell detachment on non-woven PP, PET and nylon three-dimensional scaffolds
  • 2.1. Introduction
    • 2.1.1. Scaffold selection
    • 2.1.2. Cell culture automation
    • 2.1.3. Objectives
  • 2.2. Materials and Methods
    • 2.2.1. Scaffold fabrication
    • 2.2.2. Grafting methods
    • 2.2.3. Cell-scaffold interaction
    • 2.2.4. Cell viability and proliferation
      • 2.2.4.1. AlamarBlue® assay
      • 2.2.4.2. DNA quantification using Hoechst
    • 2.2.5. Imaging cell-scaffold-interaction
    • 2.2.6. Hepatocyte metabolic activity measurement
      • 2.2.6.1. Bradford standard curve
      • 2.2.6.2. Albumin assay
    • 2.2.7. Cytochrome P450 mRNA expression
      • 2.2.7.1. RNA extractions
      • 2.2.7.2. cDNA synthesis and qRT-PCR
    • 2.2.8. Thermal release of cells from the various scaffolds
    • 2.2.9. Automated cell culture and thermal cell release
  • 2.3. Results
    • 2.3.1. Grafting methods
    • 2.3.2. Cell viability and proliferation
    • 2.3.3. Fluorescence microscopy and albumin quantification
    • 2.3.4. Cytochrome P450 mRNA expression
    • 2.3.5. Thermal release
    • 2.3.6. Automated cell culture device
      • 2.3.6.1. Prototype
      • 2.3.6.2. Final system design and testing
• 2.4. Discussion

• CHAPTER 3 Application of A 3D scaffold: A sporozoite-hepatocyte model
  • 3.1. Introduction
    • 3.1.1. Malaria
    • 3.1.2. Sporozoite hepatocyte invasion
    • 3.1.3. In vitro culturing of hepatocytes for malaria sporozoite invasion
  • 3.2. Materials and Methods
    • 3.2.1. In vitro cultivation of asexual P. falciparum cultures
    • 3.2.2. In vitro cultivation of sexual stage P. falciparum parasites (gametocyte cultivation)
      • 3.2.2.1. Candle jar method
      • 3.2.2.2. Flask method
      • 3.2.2.3. Monitoring of exflagellation
    • 3.2.3. Mosquito rearing
    • 3.2.4. Mosquito feeding and dissections
      • 3.2.4.1. Mosquito preparation
      • 3.2.4.2. Mosquito mid-gut dissections and sporozoite isolation from salivary glands
    • 3.2.5. Populating 3D scaffolds and 2D wells with hepatocytes
    • 3.2.6. Seeding sporozoites into 2D hepatocytes and 3D scaffolds
    • 3.2.7. gDNA isolation
    • 3.2.8. TaqMan® assay
  • 3.3. Results
    • 3.3.1. Gametocyte production and exflagellation
    • 3.3.2. Sporozoite invasion in 2D and 3D
      • 3.3.2.1. Mosquito feeding and dissections
      • 3.3.2.2. Sporozoite invasion of HC04 cells
      • 3.3.2.3. Quantification of sporozoite invasion in HC04 cells
• 3.4. Discussion
• CHAPTER
  • Concluding Discussion
  • References
  • Appendix A

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