Cell cultures are frequently preferred in the industrial production of high value-added biopharmaceuticals on a large scale. In the production of biopharmaceuticals, the selection of the appropriate cell line, the cell culture medium, and the culture conditions are very important. In medical products to be offered for human use, authorized institutions do not allow the use of serum which increases the culture yield, added to the culture. For this reason, the cell lines to be used must be adapted to the serum-free medium. In this study, first, the direct and gradual adaptation of the THP-1 cell line, which has a high potential for use in biopharmaceutical production, to a locally produced chemically defined serum-free medium was carried out. Then, a comparison of the production efficiency in the shake flasks with the commercially available two different serum-free media was performed. Finally, for the first time, THP-1 cells were produced in spinner flasks and stirring tank bioreactor to simulate the large scale within the scope of this work.

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer\'s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells.

Microinjection is a technique that allows for the delivery of a diverse array of compounds and biomolecules into live mammalian cells. As a result, the behavior of injected biomolecules and their resulting effects can be observed in the native environments of mammalian cells in real time. This capability allows for more accurate observations and conclusions about biological systems of interest. This chapter discusses the protocol and guidelines to successfully microinject live mammalian cells to maintain their viability. This chapter outlines considerations into the preparation of samples and the microscopy setup as well as a description of a stepwise presentation of the methods for proper execution of a microinjector. The presentation of the technique serves as a starting point to understanding how to apply microinjection to a cellular system compatible with a wide array of live-cell imaging techniques.

As a noninvasive molecular analysis technique, ultraviolet resonance Raman (UVRR) spectroscopy represents a label-free method suitable for characterizing biomolecules. Using UVRR spectroscopy, we collected spectral fingerprints of UV absorbing cellular components, including proteins, nucleic acids, and unsaturated lipids. This knowledge was used to guide the assignment of spectra derived from intact human cell lines (i. e., HSC-3 and HaCaT) and from the apoptotic events induced by cisplatin. Notably, a jet-flow system was employed to generate flowing cell suspensions during UVRR measurements, minimizing UV-induced damage. A spectral marker is established based on the ratio of Raman intensities at 1488 and 1655 cm-1 ; this ratio correlates to the level of cell death due to apoptosis. Collectively, this work demonstrates that UVRR spectroscopy is a sensitive and informative probe of cellular physiology and molecular composition. The molecular insight obtained from UVRR measurements can be used to improve understanding of therapeutic treatment and to guide drug development and the choice of therapeutic agents.

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5-15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48-72 h.