W7 Prac: Pluripotent Stem Cell Culture

Pluripotent Stem Cell Culture - Passaging Techniques

• Confluence: Percentage of culture dish surface covered by cells. Expressed as a percentage.

  • 0% confluence: No cells covering the dish
  • 50% confluence: Half the dish surface covered by cells
  • 100% confluence: Dish fully covered with a monolayer of densely packed cells.

• Passaging Pluripotent Stem Cells: Cells should be passaged when they reach approximately 70-80% confluence. This ensures active growth without overcrowding, which can lead to differentiation or stress.

Maintaining Cells During Absence

• Cell Management: Before leaving for a conference, passage cells to an appropriate confluence (not too dense or sparse).
• Instructions for Colleagues: Provide detailed instructions to colleagues on maintaining culture conditions, including when to perform the next passage.
• Storage: Store cells in an incubator with the correct conditions and ensure adequate media.

Cell Viability and Counting

• Trypan Blue: Used to stain non-viable cells in a hemocytometer for cell counting. Only viable (not stained) cells are counted, as these cells can proliferate.
• Cell Viability Assessment: Counting only viable cells accurately represents cell density and viability. This data is used for subsequent experiments.

Creating Stem Cell Cultures

• Determining Cell Volume: For a desired number of cells (e.g., 10,000), calculate the required volume of resuspended cells needed. This depends on the cell count per unit volume.
• Example Calculation (using hemocytometer): If the average cell count is 200, and you need 10,000 cells, one would need 5 µl.

Embryoid Body (EB) Formation

• EB Formation: Each EB starts with approximately 300 cells, derived from a 30µl drop of a suspension containing 10,000 cells/ml.
• Niche Environment: EB provides a niche that simulates early embryonic development, promoting differentiation.
  • Cell-Cell Interactions: Encourages differentiation of pluripotent stem cells within a 3D environment to produce different cell types.
  • Growth Factor Gradients: Allows for cell differentiation based on spatial gradients of signaling molecules, oxygen and nutrients.

EB Characteristics

• 3D Structure: Mimics the conditions in early embryonic development, similar to in vivo.
• Controlled Microenvironment: Mechanical forces and biochemical signals influence cell behavior and fate in a controlled manner.
• Early Development Mimicry: Multi-cellular structure resembles early-stage embryo development, encouraging pluripotent cells into specific cell lineages (all three germ layers).

This quiz covers the essential techniques for culturing pluripotent stem cells, including the management of cell confluence and passaging. Learn how to maintain optimal conditions for cell growth and provide instructions for colleagues handling cell cultures. Test your knowledge on best practices in stem cell culture.