W7 Prac PDF - Pluripotent Stem Cell Culture

🔬 W7 - Prac Part 1: Pluripotent stem cell culture – passaging techniques Confluence refers to the percentage of the surface area of a culture dish that is covered by cells. It’s used to describe the density of the cells in cultu...

🔬 W7 - Prac Part 1: Pluripotent stem cell culture – passaging techniques Confluence refers to the percentage of the surface area of a culture dish that is covered by cells. It’s used to describe the density of the cells in culture, typically expressed as a percentage. 0% confluence means no cells are covering the dish surface. 50% confluence means about half of the dish surface is covered by cells. 100% confluence means the dish is fully covered with a monolayer of cells, and the cells are densely packed. Experimental questions: At what confluence should your pluripotent stem cell colony be passaged? Pluripotent stem cell colonies should typically be passaged when they reach approximately 70-80% confluence. This ensures that the cells are W7 - Prac 1  actively growing but not overly crowded, which could lead to differentiation or stress. What observation may indicate a culture under stress? A culture under stress may show signs such as irregular morphology of the cells, slower growth rate, or increased spontaneous differentiation. These are indications that the cells may not be in optimal conditions for maintaining pluripotency. You’re going away to a conference for a week. How do you manage your cells? Before leaving, it's important to passage the cells to an appropriate confluence. Ensure that the cells are not too dense or too sparse. Also, leave detailed instructions for your colleagues on how to maintain the cultures, including the timing of the next passage if needed. Store the cells in an incubator with the appropriate conditions and ensure they have enough media to sustain them until your return. Why did you only count the white (not blue) cells? Trypan blue is used to stain non-viable cells. In cell counting using a hemocytometer, viable cells (which exclude trypan blue) are counted because they are the ones capable of proliferating and contributing to the ongoing culture. Counting only viable cells gives an accurate representation of the cell density and viability for subsequent passages or experiments. Part 2: Pluripotent stem cell culture – passaging techniques So for the protocol to create the culture - you need to determine the volume of the resuspended cells that will contain 10,000 cells. Add this volume into a new Eppendorf tube W7 - Prac 2  So for example, if you count 200 cells, how many microliters of your resuspended cell mixture contains exactly 10,000 cells, and then you use that specific volume for making the hanging drops. Average count: Say you counted 200 cells in the haemocytometer. Calculate total cells: This count of 200 cells is multiplied by the volume factor (10,000 cells/ml) to estimate how many cells you have in your suspension. This results in 2,000,000 cells per milliliter (2 million cells/ml). Determine volume for 10,000 cells: Since there are 2,000 cells in each microliter (µl) of the suspension (2,000,000 cells/ml ÷ 1,000 µl), you need 5µl (10,000 cells ÷ 2,000 cells/µl) to get 10,000 cells. Questions: How many cells are in the starting population of each EB you have made? From the protocol, you are using a 30µl drop of the suspension, which contains 10,000 cells/ml. Convert 30µl to milliliters: 30 μl=0.03 ml Calculate the number of cells in each drop: 10,000 cells/ml × 0.03 ml = 300 cells Thus, each embryoid body (EB) starts with approximately 300 cells. Why might an embryoid body provide a niche environment? An embryoid body (EB) provides a niche environment because it allows cells to grow in a 3D structure that mimics the conditions found during early embryonic development. Key reasons include: 1. Cell-Cell Interactions: In 3D, cells can interact with each other in ways that are more similar to how they would in an organism. This promotes the communication required for differentiation and development of different cell types from pluripotent stem cells. 2. Gradients of Growth Factors and Nutrients: The 3D structure allows for the establishment of gradients of signaling molecules, oxygen, and nutrients. These gradients can influence the W7 - Prac 3  differentiation of cells into specific lineages, similar to how tissues develop in an embryo. 3. Microenvironment: Embryoid bodies provide a controlled microenvironment where cells experience varying mechanical forces and biochemical signals that can influence their behavior and fate. 4. Mimicking Early Development: The spherical, multi-cellular structure resembles early-stage embryonic development, which can encourage pluripotent cells to differentiate into cells of all three germ layers (ectoderm, mesoderm, and endoderm). Thus, embryoid bodies create an environment that promotes differentiation and the formation of various tissue types, similar to how cells behave in vivo. W7 - Prac 4