Cell Culture
37°C, 5% CO2 incubator for 48 hours. For tri-cul- ture conditions, PBMC (AllCells Cat. No. PB003F)
were thawed into IMDM (Corning Cat. No. 10- 016-CM) with 10% FBS, centrifuged at 200 x g for 15 minutes, and resuspended in FB media. PBMC were added to the 384-well spheroid microplates, containing 48 hour mono- and co-culture A549 and FB spheroids, with 1800 cells in 10µL of FB media per well. As a control for mono- and co-cul- ture conditions without PBMC, 10µL of FB media was added per well. Spheroids were incubated for an additional 48 hours prior to fixing or staining.
Chemotherapeutic screening To determine the optimal seeding conditions, chemotherapeutic compounds doxorubicin and paclitaxel were used. The compounds included for chemotherapeutic testing are listed in Table 1. Immediately following media or PBMC addition, 10µL per well of compounds in FB media with 10% DMSO were added and mixed twice using a CyBi®-Well Pipettor. Vehicle control consisted of 10µL of FB media with 10% DMSO. Spheroids were cultured for an additional 48 hours prior to cell viability assays. For each compound, this was performed in triplicate two independent times. For cell viability assays, spheroids were assayed in the spheroid microplate using CellTiter-Glo® 3D (Promega Cat. No. G9683). 40µL per well of CellTiter-Glo 3D reagent was added directly to the spheroid microplates 48 hours after compound addition. Microplates were shaken for five minutes and then incubated for 25 minutes at room temper- ature. Luminescence was measured using a Tecan Infinite® M1000 plate reader. The concentration at which half of the cells were no longer viable, as
Drug Discovery World Winter 2017/18
measured by comparing luminescence to vehicle control wells, was calculated as toxicity potency (TC50) for each compound.
A549 cells and FB can be incorporated into co- culture spheroids using different seeding ratios. To determine the optimal cell seeding ratio for a chemotherapeutic screen, co-culture spheroids were formed using FB to A549 cell ratios of 9:1, 8:2, and 1:1. After 48 hours in culture, a dose series of the chemotherapeutic agents doxorubicin and paclitaxel were added, and the spheroids were cultured for an additional 48 hours. Each mono- and co-culture condition resulted in a single spheroid per well (Figure 2). After a total of 96 hours in culture, cell viability was assessed using CellTiter-Glo 3D cell viability assay. As shown in Figure 1, FB mono-culture demonstrated higher cell viability after 48-hour exposure to low dose doxorubicin than A549 mono-culture spheroids. A 9:1 ratio of FB to A549 cells also displayed this protective effect of FB from the low-dose doxoru- bicin exposure. FB mono-culture also displayed resistance to paclitaxel treatment at both high and low doses compared to the effects seen on A549 mono-culture spheroids, however the presence of FB did not demonstrate protective effects on A549 cells. The protective effects of FB on A549 cells in the presence of a low dose of doxorubicin can also be visualised using viability and cytotoxicity stains (Figure 2).
Chemotherapeutic screening of mono-, co- and tri-culture spheroids Dose series of several chemotherapeutic com- pounds were applied to mono-, co- and tri-culture spheroids after 48 hours of culture in the spheroid
43
Figure 1
A549 vulnerability to chemotherapeutics is affected by the presence of lung fibroblast cells. 48-hour mono- and co-culture A549 and lung fibroblast (FB) spheroids were exposed to doxorubicin (high dose = 862µM, low dose = 1.3µM) and paclitaxel (high dose = 1.2µM, low dose = 9nM) for 48 hours. FB monoculture demonstrated higher cell viability after 48- hour exposure to low dose doxorubicin, as measured using CellTiter-Glo® 3D cell viability assay, than A549 mono-culture spheroids. A 9:1 ratio of FB to A549 cells also displayed this protective effect from the low dose doxorubicin exposure. FB monoculture displayed resistance to paclitaxel exposure at both high and low doses compared to the effects seen on A549 mono-culture spheroids
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72
