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Maya Sim, Weeks 4-5


Unfortunately, I had to go back to Korea during my fourth week as there was some problem with my visa. After an interview at the US embassy and a few days of waiting, the problem was solved and I returned to the US. While I was missing my time in the lab, Nino finished up the experiment with encapsulation device-transplanted mice and started on an entirely new project. Recently, there has been news on a newborn baby who suffered from cardiac arrest and had little to no chance of survival. After an experimental procedure of transplanting a billion mitochondria into her damaged heart, she showed signs of improvement. We decided to take this approach and apply it to diabetes – if mitochondria had special healing capabilities, then can we transplant them in the pancreas to restore the beta cells?

Since both Nino and I were completely new to the field of mitochondria, we started very basic and decided to test if it was actually mitochondria responsible for cell survival after stress. To begin, I first thawed the cells from the parathyroid and stressed them with various kinds of chemicals. The surviving cells would produce or have something that other cells did not have, and my goal was to narrow down what this “something” was. To study the biogenesis, I transferred the surviving cells to two separate plates after an hour of exposure. In one plate, I washed the cells with some lysis buffer so that the cell membrane would burst. In the other plate, I left the cells untouched. For both plates, I filtered the solution with a 0.2 micrometer filter to narrow down the size. Then, I stressed the cells again and saw that all of the filtered cells died. This shows that whatever protected the cells before is bigger than 0.2 micrometer. When I repeated this process using a 0.45 micrometer filter, the results were the same, so I am guessing that it is an organelle.

The next day, we asked the assistance of another lab that was working closely with mitochondria and obtained a protocol to isolate the mitochondria. First we had to prepare a solution with 259mM sucrose, 20mM HEPES, 1mM EGTA at a pH of 7.2. Nino asked me if I knew molarity and pH, and I confidently replied, “Yes, of course!” With a calculator and a pen, I quickly figured out the mass needed for each chemical to make 50mL of the solution. Then, I set the pH of the solution to 7.2 using a pH probe and some NaOH.

Finally ready to start the isolation of mitochondria, we thawed parathyroid gland CD34- cells(PTG CD34-) and peripheral blood mononuclear cells(PBMC). We added the prepared solution and pipetted it up and down a few times to make sure the cells are now lysed. Then, we split each sample into two 1.5mL tubes and saved one as the control. Following the protocol, we centrifuged one tube of each sample at 3000rpm for 10 minutes at 4 degrees Celsius. At this step, the pellet will contain nuclei and unbroken cells, while the supernatant will contain mitochondria. After transferring the supernatant to a new tube, we resuspended the pellet and repeated the steps to achieve a higher mitochondrial yield. The supernatant we collected on two steps were mixed together and centrifuged at 10000rpm to create a pellet of the mitochondria. After resuspension, the mitochondria from each cell type as well as the controls were split into two new tubes. One tube was filtered using a 0.2 micrometer filter and the other remained unfiltered.

PBMC control
PBMC filtered
PBMC mitochondria
PBMC mito- filtered
CD34- control
CD34- filtered
CD34- mitochondria
CD34- mito- filtered




Now, with a total of 8 tubes, we stained them using MitoTracker which is a special dye that accumulates in live mitochondria. Using FACS, we analyzed our tubes, expecting very low number of events for the filtered tubes. The results matched our expectations as the controls showed significantly bigger numbers than the ones that were filtered. Our isolation of mitochondria was also successful as the results showed slightly lower values than the controls, which could be from losing a few mitochondria from the transfer processes. Although I had to finish my lunch within the ten minutes that the cells were being centrifuged, I was glad that the new protocol did not fail us!
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