Permer 2017 Bachelor Thesis
|Permer M (2017) Respirometrische Charakterisierung kryopräservierter HEK 239T Zellen. Bachelor Thesis 1-56.|
Abstract: High Resolution Respirometry is the gold standard of methods for the investigation of mitochondrial respiration. Although the research on mitochondrial diseases has made considerable progess in the past years, it is still rather difficult to diagnose the right type of mitochondrial malfunction. In this regard High Resolution Respirometry could help in the process of diagnosing mitochondrial diseases in addition to other methods such as basial chemical assays, quantitative determination of plasma amino acids or a classical echocardiogram. To accomplish this goal there has to be a reference sample for the measurement of mitochondria, since results have to be comparable independent of the fact in which laboratory the measurement has been performed. Such as sample has to be stable for a certain amount of time and has to deliver the same results over and over again when measured in different laboratories. The culture of the examined cells and the respi rometric measurement have been done for the company Oroboros Instruments at the Daniel Swarowski Research Laboratory (Innrain 66 6020 Innsbruck Austria). HEK 293T cells have been cultured and were immediately frozen at -80°C. One bulk culture of those cells had been permeabilized with digitonine before freezing, the other bulk was frozen as intact cells. Later in the process also HUH7 cells have been cultured and were frozen at -80°C in intact form as well. For the measurement of mitochondrial activity for each titration protocol 2 Oxygraphs with 2 chambers have been used. To elaborate the impact of freezing and storage of the cells in the -80°C freezer measurements have been done in certain intervals. The process of shipping the cells has also been mimicked by storing cells in a -20°C freezer for the period of one week. Results showed that intact cells withstand the process of freezing and defrosting much better, with the number of cells and the viability showing little change over 2 months of cryopreservation. In line, the measurement in the Oxygraphs also showed comparable results over this period. In contrast, permeabilized cells seemed to suffer more from the process of freezing and thawing, with a consideravble loss of respiratory activity becoming evident within only 2 weeks of cryopreservation. Overall, with some more testing and a development of an adjusted titration protocol, the use of intact HEK293T cells for a stable reference sample appears to be a promising possibility.
• Keywords: RP1, RP2, CCP, Huh7 • Bioblast editor: Krumschnabel G • O2k-Network Lab: AT Innsbruck Oroboros
Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Other cell lines, HEK Preparation: Intact cells, Permeabilized cells
Coupling state: LEAK, ROUTINE, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, ROX HRR: Oxygraph-2k