<h5 id="91-stem-cell-culture">9.1 Stem Cell Culture</h5> <ul> <li> <p>Stem cells are undifferentiated cells with the ability for self-renewal and differentiation into specialized cell types.</p> </li> <li> <p>They can be classified into embryonic, fetal, and adult stem cells based on their source. Embryonic stem cells are pluripotent and have the potential to differentiate into any tissue of the body, while adult stem cells are multipotent and can differentiate into specific cell types.</p> </li> <li> <p>Embryonic stem cells have distinctive features such as the ability to self-renew, pluripotency, stable diploid chromosome, and the absence of X-chromosome inactivation in undifferentiated cells.</p> </li> <li> <p>Adult stem cells are present in most organs and tissues, such as bone marrow, liver, brain, and blood vessels. They are responsible for preserving and repairing the tissue in which they reside.</p> </li> <li> <p>Stem cells can be classified based on their differentiation potency into totipotent, pluripotent, multipotent, and unipotent stem cells. Totipotent stem cells have the highest potential for differentiation, while unipotent stem cells can only produce cells of their own type.</p> </li> </ul> <p>======</p> <h5 id="912-characteristics-of-stem-cells">9.1.2 Characteristics of stem cells</h5> <ul> <li>Adult stem cell plasticity is the ability of tissue-specific stem cells to become cell types different from their tissue of origin.</li> <li>Differentiation requires massive molecular rearrangement within the cells, influenced by the competence of the responding cells and micro-environmental cues.</li> <li>Alternative fate-changing pathways include trans-determination, transdifferentiation, dedifferentiation, heterogeneity, pleiotropy, and fusion.</li> <li>Trans-determination is the switch of a stem cell from producing certain lineages to another stem cell, giving rise to the cell types of that precursor.</li> <li>Transdifferentiation is the process where a differentiated cell achieves the phenotype of another differentiated cell. Dedifferentiation is rare in mammals but common in lower vertebrates. Heterogeneity is the most acceptable explanation for the plastic nature of adult stem cells. Fusion and pleiotropy are other proposed mechanisms for plasticity in adult stem cells.</li> </ul> <p>======</p> <h5 id="913-maintenance-of-stem-cell">9.1.3 Maintenance of Stem Cell</h5> <ul> <li>Stem cells should be maintained at controlled dissolved oxygen partial pressure lower than atmospheric pressure, and continuously monitored for parameters like pH and pO2.</li> <li>Fluorophores or intrinsic cellular fluorophores can be used for non-invasive sensing and monitoring of cell redox state and oxidative stress.</li> <li>The culture of human stem cells requires additional attention to retain their self-renewal and potency characteristics.</li> <li>Regular monitoring of stem cell cultures is recommended for cell line sterility, authenticity, and stability.</li> <li>Testing for microbial contaminations, cross-contamination, and genetic/phenotypic changes should be performed at regular intervals.</li> </ul> <p>======</p> <h5 id="914-applications-of-stem-cells">9.1.4 Applications of stem cells</h5> <ul> <li>Stem cell therapies aim to repair damaged tissues that cannot heal themselves.</li> <li>They involve more than just transplanting cells; persuading the body’s own stem cells to produce new tissue is also a focus.</li> <li>Many stem cell therapeutics are in experimental stages, but bone marrow transplantation is an exception.</li> <li>Stem cells show promise in treating neurological diseases, wound healing, cardiovascular diseases, and auto-immune disorders like Type 1 diabetes and multiple sclerosis.</li> <li>Challenges in stem cell research include immunological rejection and the behavior of stem cells, which can induce tumor growth. Safety is also a concern due to susceptibility to microbial infections.</li> </ul> <p>======</p> <h5 id="92-organ-culture">9.2 Organ Culture</h5> <ul> <li>Organ culture is the development of part or whole organ from tissue culture techniques, where parts of an organ are explanted and grown in vitro.</li> <li>The anatomical relationship and physiological functions of several tissue components are preserved in the culture on artificial medium, simulating its parent tissue.</li> <li>Careful handling of tissues is crucial for successful organ culture, minimizing deterioration during transport to the laboratory.</li> <li>Immunochemistry, autoradiography, and histology are some methods used for the analysis of organ cultures.</li> <li>Organ culture attempts to partially mimic the tissue microenvironment, overcoming limitations of conventional cell cultures such as lack of cell-to-cell interaction and in vivo structural complexity.</li> </ul> <p>======</p> <h5 id="921-characteristics-of-organ-culture">9.2.1 Characteristics of organ culture</h5> <ul> <li>The structural integrity of the native tissue is maintained in organ culture, allowing associated cells to interchange signals via cell interaction or adhesion.</li> <li>A major limitation in organ culture is the lack of a vascular system, restricting the size and possibly the polarity of cells within the organ culture due to limited nutrient supply and gas exchange.</li> <li>To facilitate gas exchange and maintain nutrient access, tissues are maintained at a gas-liquid phase, with anchorage to a solid substrate to minimize alterations in geometry.</li> <li>Maintaining organ cultures at the interface of gaseous and liquid phase is crucial, as optimal liquid level ensures spherical geometry, while too superficial or deep levels can result in outgrowth or compromised gas exchange.</li> <li>Enhancing oxygen permeation in organ cultures can be achieved using hyperbaric oxygen or enhanced pure oxygen content.</li> </ul> <p>======</p> <h5 id="922-growth-and-differentiation">9.2.2 Growth and differentiation</h5> <ul> <li>Growth and differentiation are related processes in cell cultures, with differentiated cells often losing the ability to proliferate.</li> <li>The termination of growth can induce differentiation, and most organ cultures only proliferate on outer cell layers due to physical limitations.</li> <li>Organ cultures maintain suitable cellular communications, differentiations, and environment, with soluble growth factors added for differentiation.</li> <li>Whole embryo culture involves preparing a medium for the embryo, observing development under a microscope, and incubating the embryo for further development.</li> <li>Example: Spratt (1950s) studied the effect of metabolic inhibitors on developing chick embryos in vitro using whole embryo culture.</li> </ul> <p>======</p> <h5 id="923-types-of-organ-culture">9.2.3 Types of organ culture</h5> <ul> <li>Histotypic culture is a type of cell culture where characterized cell lines are proliferated at high density in the presence of soluble factors and extracellular matrix.</li> <li>Examples of histotypic culture include vascular endothelial cells forming capillary tubules in a collagen matrix and cells infiltrating a cellulose sponge coated with extracellular matrix components to form glandular structures.</li> <li>Organotypic culture is a type of cell culture where cells of different lineages are co-cultured to create a tissue-like structure, allowing for the evaluation of heterologous cell interactions.</li> <li>Organotypic culture is an easy way to maintain a co-culture of two cell types, such as fibroblast and epithelial cell clones.</li> <li>Organoids are self-organized 3D tissue cultures derived from stem cells, and have the potential to replace diseased or non-functional organs or deliver genetically altered cells into a patient’s body.</li> </ul> <p>======</p> <h5 id="924-applications-of-organ-culture">9.2.4 Applications of organ culture</h5> <ul> <li>Cultured organs can act as substitutes for transplantable organs, which is particularly relevant as the availability of transplantable organs is decreasing in developed countries.</li> <li>Organs produced through organ culture use a patient’s own stem cells, allowing for transplantation without the need for immunosuppressive drugs.</li> <li>Organ culture allows for the study of tissue behavior in an in vitro system and provides easier understanding of the biochemical and functional characteristics of an organ or tissue.</li> <li>This method is useful for studying hormones and their effects, both individually and in combination with other hormones.</li> <li>An example of a commonly cultured organ is the mammary gland of a mouse.</li> </ul> <p>======</p> <h5 id="925-limitations-of-organ-culture">9.2.5 Limitations of organ culture</h5> <ul> <li>Organ culture has limitations as it relies on histological techniques, not biochemical and molecular analyses.</li> <li>Biochemical analyses are challenging due to the lack of reproducibility, which is not achievable in organ culture.</li> <li>Organ culture exhibits higher variability and lower reproducibility compared to cell culture, requiring fresh organs from donors for each experiment.</li> <li>The primary reasons for high variability in organ culture include slight variations in geometry, handling, sampling, and cell proportions.</li> <li>Organ culture is expensive and challenging to prepare, and it only allows examination of integrated tissue behavior, not isolated cells.</li> </ul> <p>======</p> <h5 id="926-future-prospects">9.2.6 Future Prospects</h5> <ul> <li>The future challenge is to adapt cell-based assay protocols for 3D cell cultures.</li> <li>3D cell cultures include multicellular spheroids, scaffold hydrogels, organoids, organs-on-chips, hanging drop, microfluidics, magnetic levitation, microtissues, and 3D bioprinting.</li> <li>These 3D cell structures differ from traditional 2D cell cultures.</li> <li>Adapting protocols will involve accounting for the altered conditions of 3D cell growth.</li> <li>This adaptation is necessary for accurate analysis of cells in 3D structures.</li> </ul> <p>======</p>