A Backup for Rainy Days

In the last blog, we talked about the process of picking your stem cell colonies. Now that we have cells that morphologically look like stem cells, we need to create a bank of these cells before we can characterize them to ensure that they are truly induced pluripotent stem cells (iPS cells) derived from your skin biopsy.

Alex: I am always looking forward to hearing about the progress my cells make with your expertise. Can you explain what a “bank” of cells is?

Lauren: A bank of stem cells consists of many vials of your cells frozen and stored in liquid nitrogen for future use. It is like a backup for rainy days. We need these cells for the characterization process and eventually for neuronal differentiation, but we also want to make sure there are enough cells cryopreserved for future experiments. Banking at this stage also allows us to preserve cells that are younger since long-term tissue culture can lead to abnormalities of growth and survival of cells. Lastly, it takes a lot of time and effort to make your iPS cells, a bank of cells will ensure that we do not have to go through this process again.

Alex: That makes a lot of sense. How do you create a bank of my stem cells?

Lauren: All of your stem cell lines that I picked over the last three to four weeks started from one single colony of cells and each stem cell line was ultimately derived from only one cell from your skin biopsy. Now we need to expand this one colony into many more colonies so that we can freeze them and still have some to further expand. Expanding your stem cells or –we like to be short and use jargon “iPS cells”- is similar to expanding skin cells as we talked about in the blog: Alex’s cells growing out of their “clothes”. But there are two main differences between expanding skin cells and expanding iPS cells. The first difference is that iPS cells do not grow as quickly. The second difference is that we will manually passage your iPS cells whereas we used the enzyme trypsin to break apart and dissociate your skin cells.

Expanding and banking pluripotent stem cells

Alex: What does “expanding” actually mean in this context? How do my cells expand?

Lauren: Cells are constantly dividing or multiplying. In the last blog you saw the development of a colony. It takes only one cell that has divided many times to create a colony of cells. In the picture below you can see how your cells divide. Each red circle shows a different time point in the division or mitosis of your cells. The blue in the image is the nucleus of the cell containing the genetic material in form of large DNA structures, called chromosomes, and the green is the rest of the cell. Circle 1 shows a cell that has started the division process and all blue chromosomes align in the middle of the cell, which is called metaphase. Circle 2 shows a cell in which the blue chromosomes starting to pull apart from each other, a stage that is known as telophase. Then in circle 3 we can see two cells that have just completely separated from each other. All of these cells divide many times which helps to expand or increase the number of cells we have so that we can create a cell bank. If you look closely at the image, you can see additional cells dividing.

Stem cell colony with dividing cells

Alex: Fascinating. I can identify more cells dividing. Another question: you also used the term “manually passaging” iPS cells? That sounds tedious.

Lauren:“Manual passaging” is similar to what I did when I was picking your stem cell colonies. As the cells grow, the colonies get larger, when the colonies are large enough I cut them with a needle into smaller pieces by cutting the colonies in squares like a grid, as you can see in the picture below, lift them off the plate with a pipette and transfer them into a new plate. The entire banking process will take me a few weeks, but in the meantime, I will start characterizing your stem cells.

Example of how to manually passage with a needle

Alex: Thank you, Lauren, this needs a lot of diligence and attention to detail. I am glad you are taking good care of my cells.

Lauren:You are very welcome. Next time, I will explain how to characterize your iPS cells.