Cell culture techniques

2.2.10.1 Preparation of hamster glial cell cultures

Isolation and culture of glial cells is based on a procedure from Lima and colleagues [Lima et al., 2007] and was carried out with modifications as described by Pritzkow et al. (2011) and Boerner et al. (2013). All equipment was sterile and aseptic techniques were used where possible. For the preparation of glial cell cultures, normal neonatal Syrian hamsters were used as donors of brain tissue. Animals were sacrificed 2-3 days after birth by decapitation. Brains were removed and kept in PBS/glucose on ice until further processing. The cerebellum and the meninges were removed using a disposable

scalpel and a stereo microscope, and the remaining brain tissue was cut into pieces and kept in PBS/glucose afterwards. The tissue was centrifuged for 3 min at 1000 rpm (Varifuge 3.0 R Heraeus) and ambient temperature. Brain tissue was washed twice with 9 ml of glial cell isolation medium (GIM) and was subsequently resuspended in 5 ml fresh GIM. Using serological pipettes (5 ml and 2 ml pipettes) the tissue pellet was sheared carefully to separate cells until a homogeneous single cell suspension was obtained. The solution was drained trough a cell strainer (pore size 40 µm) to remove cell debris and was subjected to centrifugation. The pellet was resuspended in 10 ml growth medium (GM) and density of cells was determined by vital staining with trypan blue. Cell count was determined using a Neubauer hemocytometer. 3x10⁶ cells per one plastic cell culture flask (175 cm², NUNC) were grown in 20 ml GM. All culture incubations were performed in a humidified 37 °C, 5 % CO2 atmosphere incubator.

Cells were allowed to adhere to the bottom of the culture flask over night. The next day, cells were washed with 10 ml PBS to remove unattached cells. Incubation was continued for further seven days in fresh GM. Subsequently, cells were washed again with PBS and enzymatically detached from the bottom of the culture flask. Cells were incubated with 2.5 ml trypsin for approx. 5 min until they were floating. 20 ml GM was added and the cell suspension was subjected to a subsequent centrifugation to clear from trypsin. The cell pellet was resuspended in 10 ml GM and homogenized to obtain a single cell suspension using serological pipettes and a cell strainer. For cryopreservation, 3x10⁶ cells were diluted in 1 ml freezing medium per cryovial. Vials were cooled down over night to -70 °C using a freezing container with a cooling rate of -1 °C/min. The next day, cryovials were transferred to liquid nitrogen for long-term storage.

2.2.10.2 Plate cultivation

Cryovials were thawed quickly in order to avoid toxic effects of DMSO on cells and were diluted with 9 ml GM and subjected to centrifugation for 3 min at 1000 rpm (Varifuge 3.0 R Heraeus) and ambient temperature. Cells were resuspended in GM, separated and the number of vital cells was determined by staining with trypan blue using a hemocytometer as described above. For cell infection assays, cells were cultivated in 6-well plates at a density of 1.5x10⁴ cells per well and 3 ml GM for 2-3 days prior to infection.

2.2.10.3 Infection of glial cell cultures

Cell cultures were exposed to the indicated amounts of infectious or control hamster brain tissues or PMCA products. Cultures for negative controls were exposed to normal hamster brain tissue. Cells were inoculated with hamster brain tissue that was homogenized in PBS. PMCA products contained Triton X-100 and thus were toxic to cells. They were subjected to ultracentrifugation at 45000 rpm (Optima Max Ultracentrifuge, Beckmann Coulter) and 4 °C for 2.5 h. The pellet was resuspended in 100 µl PBS in a ultrasound waterbath. Cells were inoculated with 10 µl suspension per culture well that was added to the culture medium. After three days post initial exposure (DPE), the inoculum was removed and the cells were washed once with PBS.

Subsequently, cells were cultured further and harvested at the indicated time points.

During cultivation the medium was renewed once a week. Cells were examined regularly for vitality and microbial contaminants with an inverted microscope.

Metal ion solutions for cell culture applications were prepared with sterilized ddH₂O.

When cells were cultured in the presence of metal ion solutions this was done at final concentrations of metal ions of 20 µM or 50 µM.

2.2.10.4 Harvesting

Cell cultures were harvested at the indicated DPE as previously described [Boerner et al., 2013]. Cells were washed with PBS and detached with a cell scraper and collected in 1 ml PBS. Cells were pelleted by quick spin. The supernatant was discarded and the pellets were stored at -70 °C until further processing.

2.2.10.5 Limited proteolysis with PK

Pellets from cells were resuspended in 50 µl PBS and were subjected to enzymatic digestion with proteinase K. However, in the course of infection experiments it was found that the processing of cell products by PK can be optimized by resuspending pellets in NBH instead of PBS. In this way, the sensitivity of the detection of PrPres from cell cultures infected with BSE-H associated PrPres could be increased. Pellets were resuspended using a vortex mixer and additionally by two sonication steps at approx. 250 W for 40 sec in an ultrasound waterbath. Caps of reaction tubes were sealed tight with parafilm tape to prevent contamination of samples with water from the waterbath. 15 µl of the cell suspension were mixed with 3 µl PK and 1.5 µl sarkosyl for a final concentration of 150 µg/ml PK and 1 % (v/v) sarkosyl, respectively. Samples were incubated for 1 h at 37 °C and 450-500 rpm and were subsequently mixed with an equal volume of sample loading buffer (Table 2-2) and incubated for 10 min at 100 °C.

1 µl PMSF was added to a final concentration of 2.5 mM. In this way samples could be

stored at -20 °C prior to further processing. Samples were subjected to SDS-PAGE (chapter 2.2.7) and Western blotting (chapter 2.2.8) as described above.

2.2.10.6 Deglycosylation with PNGase F

Cell pellets were subjected to limited proteolysis with PK and subsequently deglycosylated with PNGase F as described in chapter 2.2.6.

2.2.11 Extraction of PrPres from PMCA products for Fourier transform-infrared