RTP Time Variation for Cell Culture
In an attempt to improve membrane life in cell culture, different RTP times were tried on a single wafer. Samples from W514 were RTPed in Ar at 800C for 1, 5, and 10 minutes, respectively. Cells were then seeded at confluence on membranes, with membrane stability monitored for breakage. Discoloration was monitored on samples without cells and documented at membrane failure with cells.
Initial failure started at 21 hours and continued to 52 hours for complete failure of samples RTPed for 10 minutes. Samples treated for 1 and 5 minutes did not all fail as observed at 116 hours; however obvious failure ended at 52 hours. The remaining membranes shown as not failed in the above chart may have failed at some point with possible explanations including:
- the cells as a confluent monolayer are holding the membrane intact
- or the membrane is partially/completely gone and, because the cells are confluent, the bead assay can’t be used to “see” membrane failure
Below is an image containing the progression of three different samples. Cells were seeded at time 0 with initial images taken after 3 hours. Within 21 hours the cells on the membrane were confluent. In the first column is a membrane RTPed for 1 minute that seems to have survived 116 hours (~5 days) intact. This is a long time, longer than we have seen before. In the middle column is typical failure observed during these experiments. In the third column, partial membrane failure seems to have occurred, as shown by the lack of cells in certain areas and beads below these areas. However when I imaged this sample at 116 hours a confluent monolayer seemed to have formed.
