Controlled cell assembly is essential for fabricating in vitro models that mimic native tissue architecture. Conventional tissue engineering techniques are time-consuming and offer limited control over the spatial organization of cells within the assembled cell aggregates. We describe a label-free, scaffold-free, rapid cell manipulation technique to assemble cells into layered aggregates. Suspensions of cells in a culture medium with higher magnetic susceptibility are seeded into wells of a 96-well plate placed on a quartet magnet array. An FDA-approved paramagnetic agent is added to the regular cell culture medium to enhance the magnetic susceptibility. The inhomogeneous magnetic field and the susceptibility difference drive cells toward the lowest magnetic field region on the well surface. Two cell types are sequentially added to the wells to form layer-on-layer aggregates within 6 h. Next, the label-free technique is extended to develop a cell migration assay. Besides being time-consuming, the traditional scratch-based cell migration assay is not reproducible, whereas the alternate physical barrier-based method is expensive. Annular aggregates of human bronchial epithelial cells (HBEC3 KT) are formed within 3 h using a coaxially arranged ring-cylinder magnet array. The effects of the paramagnetic agent on cell viability, metabolism, and transcriptional profiles are investigated. The closures of the circular cell-free areas enclosed by HBEC3 KT are analyzed at different times in response to various signaling molecules and surface conditions. Further, we demonstrate the formation of the annular aggregates on human lung fibroblast-laden collagen hydrogel surfaces. The cell-free area closures on hydrogel surfaces in response to signaling molecules are analyzed. The high reproducibility and scalability of the label-free method make it amenable for preclinical research. / Thesis / Doctor of Philosophy (PhD) / Cell cultures are essential tools for studying cell functions under controlled conditions. A better understanding of cell behavior in tissues is required to develop effective treatments for diseases. The organized arrangement of cells in tissues controls tissue functions. The existing culture techniques are time-consuming and have limited control over the cellular arrangement. We describe a simple, rapid, and inexpensive bioprinting technique to arrange cells in layers, which resembles the cellular organization in tissues, such as the skin. The layered structures are formed in standard well plates within 6 h. Cell movement is an essential cell function in various biological processes, such as wound healing. Using the bioprinting method, we form ring-shaped cellular structures within 3 h to study cell movements in response to various signals. The ring structures enclose cell-free areas, which are populated over time as the cells move from the ring into the cell-free regions. The bioprinting method is easy to use and can rapidly form organized cellular structures for drug testing.

Tato diplomová práce se zabývá popisem principů kultivace embryonálních fibroblastových buněk myší (3T3), lidských endoteliálních buněk odebraných z pupečníkové žily (HUVEC) a epiteliálních buněk vaječníku čínského křečka (CHO) v mikrofluidních systémech simulujících kapiláry. Byly provedeny literární rešerše v oblasti realizací experimentu “Scratch Wound Healing Assay” v mikrofluidních systémech s použitím fibroblastů a endotheliálních buněk. V práci jsou dále popsány principy konfokální a fluorescenční mikroskopie a metody zpracování obrazů pro sledování buněčné migrace. Experimentální nastavení pro mikrofluidní realizaci “Scratch Wound Healing Assay” s použitím trypsinu – EDTA pro vytvoření rýhy, a konfokálního mikroskopu Leica TCS SP8 X pro následující snímání pořízených dat bylo navrženo a otestováno s dostatečným počtem opakování. Vhodný algoritmus pro analýzu buněčné migrace byl napsán v programovacím prostředí Matlab. Závěrem této práce je diskuze získaných výsledků.

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