Animal BT Studying Notes (7) PDF

What are the merits and advantages of 2D (monolayer & suspension) cell culture? - Absolute control on cell environment - Cell observation, measurement, and manipulation are easier What are the limitations of 2D cell culture? - Cells loses their histological organization, polarity, and differe...

What are the merits and advantages of 2D (monolayer & suspension) cell culture? - Absolute control on cell environment - Cell observation, measurement, and manipulation are easier What are the limitations of 2D cell culture? - Cells loses their histological organization, polarity, and differentiation - Lack of host-tissue microenvironment - Altered gene expression & growth characteristics due to a deficiency in cell-cell and cell-matrix interactions - Increased drug sensitivity - Use of suspension culture is also limited by the sensitivity of some cell lines to shear stress What are the merits and advantages of 3D (Organ & Spheroid) culture? - Sufficiently well characterized to simulate the pathophysiological cellular microenvironment - Show enhanced reliability & predictability of clinical efficacy & minimize studies w/ animal models - Reconstitute a tissue-like cyto-architecture with cell-cell and cell-matrix interactions, growth, differentiation & therapeutic responses similar to tissue in vivo - Gene expression profiles of 2D model reflect clinical expression profiles of tumors What are the limitations and disadvantages of 3D culture? - Lack of vasculature, host-immune interactions - Diffusional transport limitations: O2 and other essential nutrients may not reach to all of the cells; accumulation of toxic waste products within the scaffold space What is a multicellular tumor spheroid and what does it do? - Sphere-shaped cell colonies that permit growth & functional studies of diverse normal & malignant tissues - Mimics the growth of naturally occurring human tumors - Extracellular matrix & network of cell-cell and cell-matrix interactions are similar to in vivo conditions What are the layers of multicellular tumor spheroids? 1) Proliferative 2) Quiescent 3) Necrotic What are the different methods for generating multicellular tumor spheroids? 1. Liquid overlay method 2. Hanging drop method 3. Agitation method 4. Microfluidic technology 5. Matrices & scaffolds Discuss on the process of liquid overlay method on generating tumor spheroids. - Cells are seeded on non-adhesive surfaces to avoid cell attachment - Super-hydrophobic agar//agarose are frequently applied to make non-adherent surfaces Elaborate on the process of the hanging drop method of creating tumor spheroids. - Following cell seeding, the tray is inverted and aliquots of cell suspension turn into hanging drop that are kept in place due to the surface tension. Cell accumulate at the tip of the drop, at the liquid-air interface, and are allowed to proliferate Discuss the process of the agitation-based approaches on creating tumor spheroids. - Cell suspension is placed into a container and the suspension is kept in motion, that is, either it is gently stirred, or the container is rotated - Continuous motion of the suspended cells allows cell-cell interactions Spinner flask bioreactors Rotating cell culture bioreactors A container to hold the cell suspension & a The culture container itself is rotated. The stirring element to ensure that the cell constant motion prevents cells from adhering suspension is continuously mixed to the chamber walls Elaborate microfluidic technology on generating tumor spheroids. - Miniaturized devices that include chambers & channels with fluid flow - Precise//accurate manipulation & handling of microenvironments in terms of pressure & shear stress on the cells - Offers the scale-up of spheroids formation -suitable for high-throughput screening Elaborate on the matrices and scaffolds methods of generating tumor spheroids. - Highly porous (sponge-like) scaffolds are useful in generating immobilized 3D arrangement of spheroids - Cnbe produced with natural polymers and synthetic polymers What are the challenges in the applications of multicellular spheroids? 1. Uniformity & Reproducibility a. How to consistently produce MCTs of homogeneous shape & size? 2. Assessment a. How to establish a valid eval method for MCTs growth & drug efficacy? 3. High-throughput a. How to develop high-throughput MCTs culture & drug screening platform? Discuss the challenges in terms of uniformity and reproducibility. - The cells in MCTs formed strong interaction between cell-cell and cell-environments - Factors affecting spheroids variability: cell type, culture techniques, medium composition & volume, & cell density Elaborate on the challenges in the assessment of MCT. 1. Growth Characteristics assessments a. Size - diameter; volume b. Circularity c. Tools - Optical microscopy, SEM & TEM 2. Drug//Therapeutics Efficacy a. Size - Diameter, volume b. Viability - 2D & 3D assay, Spheroids, Cell disruption c. Apoptosis d. ATP assay, Metabolic activity What is the problem in the high-throughput platforms in creating MCTs? - Extensive use for drug screening is still limited because the traditional spheroids forming system takes a long time to culture & produces spheroids of various sizes - Solution: spheroids generation in microfluidic device What are other 3D cultures? 1. Microcarriers 2. Filter well inserts 3. Hollow Fibers Bioreactors What are microcarriers? - Hydrogels can also form the base for cell-compatible microcarriers (beads) - High surface area to volume ratio - Enables most anchorage dependent animal cells to grow in suspension cultures - Can be used to increase the surface area of traditional monolayer cultures - Applicable for 3D cell culture in a stirred tank bioreactor What are filter well inserts? - A commercialization product of filter-based culture system - Filters can pre-coated with collagen, laminin, fibronectin, or Matrigel - The filter substrate provides an environment for studying cell interaction, stratification, polarization, & tissue modeling. What are the application of filter well insert and its limitations? - Enable the study of 3D chemotactic responses of the cells cultured on filters or in gels placed on the filters - Lack the ability to image at high resolution in real time or to control many aspects of the local cellular microenvironments What are hollow fibers bioreactors? - Development of a perfusion chambers with a bed of plastic capillary fibers - Gas & nutrient permeable, supporting cell growth on their outer surface What is the application of hollow fibers systems? - Ideal system for studying the synthesis & release of biopharmaceuticals and are now being employed on a semi-industrial scale What are the concepts of tissue engineering? - Construction of tissue equivalent cultures allow tissue replacement therapy possible: skin equivalent cultures have been used in burn repair What are the applications of 3D cultures? 1. Basic cellular processes a. Modeling of cellular microenvironment allows study of cellular organization of tissue-like structures (tubules, aggregates, and cysts), and disease states such as cancer propagation & mestasis, inflammatory toxicity-driven pathways, and angiogenesis 2. Drug discovery a. To study the penetration of cytotoxic drugs, antibodies, or other molecules used in targeted therapy b. To study the effect of drug dosages: in 3D model, cells from antural barriers to drugs