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Maya Sim - Week 7


This week, I designed an in vitro conditioned media experiment to test the effects of parathyroid gland (PTG)-derived CD34+ cells on stressed human islets. To stress these cells, I cultured them for 24 hours in either replete media (control), 1% diluted media, 1% oxygen (to induce hypoxia), or both stress factors. I added SCIPCs, PTG cells, CD34- cells, or CD34+ cells to one of each category, resulting in total of 16 wells.

This is the image of the well plates in the incubator for overnight culture.

The next day, I looked at the conditions of the cells using a microscope and I could visibly see that islets in wells containing CD34+ cells were doing much better than all the other wells. Then, I stained the cells using propidium iodide (PI), which binds to the DNA. However, it cannot cross the membrane of live cells and thus only stains dead cells. With PI staining, I could quantify cell viability of each well using flow cytometry. As I have seen with the microscope, the stressed islets’ survival percentage was much higher when cultured with CD34+ cells. For islets cultured in both nutrient deprived media and 1% oxygen, around 90% did not survive, but in the case of CD34+ cells, the number was around 60%. This experiment confirmed in vitro that PTG-derived CD34+ cells do release factors that improves islet viability. Although Nino had already completed a similar conditioned media experiment and got similar results, I was happy to design my own experiment and know that I did not make any major mistakes.

Continuing our work with mitochondria, we imaged the stressed human islets to see if they took in the free mitochondria mixed with the media. Before they were imaged with the electron microscope, we stained the mitochondria and islets. Surprisingly, the image showed healthy islets with mitochondria inside them, everywhere. Compared to islets that were not stressed, there was much more intake of mitochondria. Although we do not know the exact mechanism of how mitochondria enter the cells, we have now confirmed that when cells are stressed, they are able to survive better with the uptake of mitochondria.

Having received ten more PCL encapsulation devices with a rigid ring, we implanted five of them to NSG mice, which is one of the most immunodeficient strains. I forgot to attach a photo of these devices on my last post, so I'm attaching it here. In two weeks, either human islets or SCIPCs will be infused through the long, narrow part of the device, then heat-sealed so that only the circular part will remain containing cells. Unfortunately, the SCIPCs were contaminated in the lab that produces them, so we are still waiting to transplant them into mice that underwent pre-implantation of the empty device last week.

I cannot believe that I only have a week left here! I hoped to see the outcome of these new rigid devices but it is highly unlikely that I will still be at the lab. When I voiced my disappointment to Nino, he promised that he would send me the images as soon as he took them :)
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