TÍTULO DE LA PROPUESTA

2.3.1 Preparation of exosome-depleted media

Secreted exosomes were harvested from a variety of cell types including BT-20, HCC-1954 and MSCs. A schematic representation of the exosome isolation process is shown in Figure 2.3. The first step in this process was to ensure that cells were cultured in the presence of exosome-depleted media in order to determine the effect that cell-secreted exosomes would have on recipient cells. It was therefore necessary to deplete the foetal bovine serum (FBS), which is used as a source of growth factors in culture media, of exosomes prior to the culturing process. In order to do this, 20mL aliquots of FBS were placed in 25PC thick walled tubes (Hitachi Koki Centrifuge Ware) and ultracentrifuged for 16 hours overnight at 110,000 x g and 4 °C in order to separate out the exosome fraction. The exosome-depleted FBS was then removed and stored in 50mL aliquots at -20 °C until required. When preparing to carry out an exosome harvest, the exosome-depleted FBS was thawed and added to a fresh media bottle which was also supplemented with Pen/Strep.

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2.3.2 Isolation of exosomes from cell-conditioned media

In order to isolate exosomes from a chosen cell line, a suitable number of flasks (based on visual inspection and estimation of cell density) were trypsinised in the usual manner before re-suspending in exosome-depleted media and counting using the Nucleocounter. A standardised seeding density of 2x106 cells was used in all exosome setups. 10mL of exosome- depleted media was pipetted into each of 10 x T175cm2 flasks and rolled around the floor of the flask to ensure that all areas were adequately covered in media. Equal amounts of cells were added to each flask and the flask was again rolled to evenly coat the floor of the flask with cells. The cells were incubated overnight at 37 °C and 5% CO2, allowing them to adhere to the

flask. The exosome-depleted media was replaced with 10mL of fresh exosome-depleted media the following morning and placed in the incubator for 48 hours.

Following a 48 hour incubation period the conditioned media was decanted into 50mL falcons and centrifuged at 300 x g for 10 minutes (Figure 2.3).

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Figure 2.3 Schematic of exosome isolation protocol for cell conditioned media.

The supernatant was pipetted into new 50mL falcons and centrifuged at 2,000 x g for 10 minutes. In each of these steps, approximately 2.5ml of liquid was left behind above the pellet. These differential centrifugation steps remove large cells and debris. Next, the supernatant was passed through a 0.2µm sterile filter (Millipore, Billerica, MA, USA) with the purpose of removing any remaining cellular material and debris with a diameter in excess of 200nm. The filter allowed the integrity of the small

Cells cultured in exosome-depleted

media x48 hours

Cell-Conditioned Media Harvested

centrifuged at 300 x g, 10 mins

Supernatant centrifuged at 2,000 x g, 10 mins

Supernatant passed through a 0.2µm filter

Supernatant ultracentrifuged at 110,000 x g,

70 minutes

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exosomal membranes to be maintained throughout this process. The collected filtrate was placed into 25PC thick walled ultracentrifugation tubes in 20mL aliquots and ultracentrifuged at 110,000 x g for 70 minutes at 4 °C in a Hitachi Micro Ultracentrifuge CS 150FNX. If the exosomes were being isolated for the purpose of carrying out morphological analysis using Transmission Electron Microscopy, a primary fixative was added to the supernatant prior to ultracentrifugation (described in detail in Section 2.5.1). Following ultracentrifugation, the supernatant, now referred to as exosome- free conditioned media, was removed and approximately 2mL was retained for further analysis. The exosomes were scraped down from the side and bottom of the ultracentrifuge tube using 55µL of sterile PBS and a 100 µL pipette tip. The exosome-rich PBS was transferred to a taper-bottomed tube. Next, 5 µL of exosome suspension was placed in complete lysis buffer, which will be described in Section 2.2.2, for protein estimation in order to indirectly quantify the amount of exosomes that had been isolated. Both samples were then labelled and stored at -80 °C until required. If exosomes were required for Western Blot Analysis (Section 2.5.2), the entire sample was placed in complete lysis buffer (components listed in Table 2.2, Section 2.4) and stored at -80 °C.

The T175cm2 flasks were trypsinised, a cell count was performed and the cells were pelleted for later analysis. The cell pellet was labelled and stored at -80 °C.

2.3.3 Isolation of exosomes from serum

Various serum exosome isolation techniques were trialled in this study. Initial attempts involved the direct ultracentrifugation of serum in 1.5mL microtubes without prior dilution. The technique which was validated involved the dilution of serum in PBS prior to the ultracentrifugation process. In brief 300-500µl of serum was thawed on ice, diluted with 12mL of PBS and inverted to mix (Figure 2.4). Next the samples were centrifuged at 800 x g for 10 minutes and then 2000 x g for 10 minutes. The supernatant

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was drawn off after each differential centrifugation step leaving no more than 0.5mL of precipitant containing cellular debris behind. It was then filtered using a 0.2 µm filter into a 25PC thick walled ultracentrifugation tube. The exosome pellet was then recovered at 110,000 x g for 2 hours at 4 °C in a Hitachi Micro Ultracentrifuge CS 150FNX. The pellet was re- suspended in 55µl of PBS as before, and 5µl of this was placed into 20µl of complete lysis buffer for protein estimation. In cases where the sample was required for Western Blot, the entire re-suspended pellet was placed in complete lysis buffer (Table 2.2, Section 2.4) and stored at -80 °C. As with cell-secreted exosomes, serum-derived exosomes that were destined for visualisation using Transmission Electron Microscopy were ultracentrifuged in the presence of a primary fixative and then re-suspended in PBS.

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Figure 2.4 Schematic of exosome isolation protocol for human or animal serum samples.