Preparation of the HIBCPP cell culture and conditions

The cell culture medium that was used for the HIBCPP incubation (37°C, 5% CO2 and increased humidity due to an installed water bowl) was used in two variants - one with antibiotics and one without antibiotics. For all HIBCPP incubations that took place before the S. suis infections, the cell culture medium was composed as follows:

15% FCS – cell culture medium:

500 ml Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 1:1 with phenol red (DMEM/F‐12; ThermoFisher, Waltham, USA) was used as basis, supplemented with 90 ml heat-inactivated fetal calf serum (FBS Superior S 0615; Biochrom GmbH, Berlin, Germany). The inactivation was performed by heating at 56°C for 30 minutes in a water bath. In addition, 12 ml of 200 mM L-glutamine (ThermoFisher, Waltham, USA) and 250 μl insulin (concentration 10 mg/ml) (Sigma-Aldrich Corp., St. Louis, USA) was added. This mixture was stored at +7°C in the refrigerator for maximal 1 month.

Weekly portions of 50 ml were aliquoted and were supplemented with 500 μl of penicillin/streptomycin (100x-concentrate; 10000 µg/ml; Biochrom GmbH, Berlin, Germany).

The shelf life was a maximum of 1 week.

The cell culture medium that was used one day before and during the infection experiments was prepared as follows:

52 1% FCS – cell culture medium (free of antibiotics):

500 ml Dulbecco's Modified Eagle Medium/Nutrient Mixture F‐12 1:1 without phenol red (DMEM/F‐12; ThermoFisher, Waltham, USA) was used as basis, supplemented with 5 ml heat-inactivated fetal calf serum (FBS Superior S 0615; Biochrom GmbH, Berlin, Germany) and 250 μl insulin (concentration 10 mg/ml) (Sigma-Aldrich Corp. St. Louis, USA).

The human choroid plexus papilloma cells (HIBCPP) (Ishiwata et al. 2005; Schwerk et al. 2012) were stored as a cryostock in -150°C. To use the HIBCPP for the cell culture model, the cryostocks were first defrosted in a water bath at 37 ° C until the ice started to melt. It was then immediately transferred to a tissue culture flask T-75 (SARSTEDT AG & Co. KG, Nümbrecht, Germany) filled with 15 ml of 37 ° C prewarmed cell culture medium (15% FCS-medium). The cells grew with regular change of the medium – three times per week - for about 3 weeks until reaching about 90% of confluency. During that time the cells were examined regularly macroscopically and microscopically examined for any hints of contamination, such as turbidity, color changes of the cell medium or odor. Mycoplasma contamination was also regularly excluded by PCR analysis during routine processes (Mycoplasma Detection Kit;

SouthernBiotech, Birmingham, USA). The cells were harvested at time of about 90% of confluency to maintain the cell line. Detachment was carried out according to the following principle:

The cell culture medium was carefully aspirated with a sterile Pasteur pipette. The cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS; ThermoFisher, Waltham, USA). Subsequently, 3 ml of 0.25% trypsin-EDTA (ThermoFisher, Waltham, USA) was added to the cells for 15-20 minutes. Separation of the cells was sometimes assisted by punching carefully the cell culture flask. After the cells became detached, 7 ml of cell culture medium was added and mixed thoroughly with the cells by pipetting up and down. The suspension was then centrifuged at 250g for 10 minutes at room temperature without acceleration or deceleration modus. The supernatant was removed with a sterile Pasteur pipette.

Afterwards, the pellet was taken up in a total volume of 1 ml of cell medium. Then, 10 μl of the suspension was added to 90 μl 0.4% trypan blue (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and mixed thoroughly. A volume of 10 μl of this mixture was transferred into a Neubauer chamber. With a 10x objective one of the main squares is focused and viable and

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dead (blue) cells are counted. Cells that touch the upper and left border are counted, cells that touch the right and lower border are excluded. The counting is performed in 4 main squares and the cell number of viable HIBCPP is averaged.

The actual amount of HIBCPP in the suspension was calculated for 1 ml according to formula 2.1 from chapter 4.1.3.

According to following procedures either a harvest to maintain the cell line until a maximum of 35 passages (application of 1-week culture or 2-week culture with 5x10⁶/cells or 1x10⁶/cells pro flask) was then carried out or a transfer to translucent ThinCert™Cell Culture Inserts for 24 well plates (membrane:, 3 µm pore diameter) (Greiner Bio One International GmbH, Kreismünster, Austria) - hereinafter referred to as "filter" - was performed. For the former, the volume (VCells) to be supplemented into the T-75 flask was calculated using the following formula:

Eq. 2.2 𝑉_{𝐶𝑒𝑙𝑙𝑠} [𝑚𝑙] = 𝑠ℎ𝑜𝑢𝑙𝑑 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 [𝑐𝑒𝑙𝑙𝑠]

ℎ𝑎𝑣𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 [^{𝑐𝑒𝑙𝑙𝑠}_𝑚𝑙]

For seeding on filter, the cell suspension was adjusted to 1x10⁶ cells/ml. The appropriate addition of medium (VMedium) was calculated using the formula 2.3:

Eq. 2.3 𝑉_{𝑀𝑒𝑑𝑖𝑢𝑚} [𝑚𝑙] = (ℎ𝑎𝑣𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 [^{𝑐𝑒𝑙𝑙𝑠}_𝑚𝑙 ]

𝑠ℎ𝑜𝑢𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 [^{𝑐𝑒𝑙𝑙𝑠}_𝑚𝑙 ]) − 1

Afterwards the cells were harvested and 100 µl were transferred on turned around filter into a 12-well plate (Greiner Bio One International GmbH, Kreismünster, Austria) with an amount of 1x10⁵ cells per filter. The filters were totally filled obliquely from the downside with cell culture medium until a touch at the membrane was occurring. Subsequently the filters were moistened with 100 µl medium from the upside and were reincubated for the next 24 hours.

To prepare the anatomical correct alignment, the filter were flipped and transferred to 24 well plates (Greiner Bio One International GmbH, Kreismünster, Austria) for further growing the next 4 days to form a tight cell barrier with an upper “blood-compartment” and a lower “CSF-compartment” (figure 2.1). Volumes of the medium was always 1 ml in the lower part and 500 µl in the upper part. After 3 days, the medium was refreshed. One day before the infection

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the filter were washed twice with DPBS and transferred into a new 24 well plate. The medium from this step on was free of antibiotics (1% FCS-medium).

Figure 16 The inverted transwell filter system for the HIBCPP

One day after seeding the HIBCPP on the membrane of a turned around filter, the filter is flipped. The cells are incubated in a hanging alignment for the next 4 days to form a tight barrier that separates the volumes of the well into a “blood compartment”

and a “CSF compartment”. By this setup the correct in vivo alignment of the cells for infection is created.