W7 Prac: Pluripotent Stem Cell Culture

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Questions and Answers

If you count 200 cells in your resuspended mixture, how many cells would be estimated in one milliliter of that suspension?

200,000,000 cells/ml

200,000 cells/ml

20,000 cells/ml

2,000,000 cells/ml (correct)

What volume of the resuspended cell mixture is needed to obtain exactly 10,000 cells if the concentration is 2,000 cells/µl?

5 µl (correct)

10 µl

2.5 µl

15 µl

How many cells are present in a 30 µl drop of suspension at a concentration of 10,000 cells/ml?

30 cells

100 cells

300 cells (correct)

1,000 cells

What is a key reason that embryoid bodies (EBs) provide a niche environment for cell growth?

They create a 3D structure mimicking early embryonic development. Signup and view all the answers

What is the primary benefit of allowing cells to grow in a 3D structure as seen in embryoid bodies?

More accurate modeling of in vivo cellular interactions. Signup and view all the answers

When preparing the culture with a haemocytometer, what must be done before determining the volume containing 10,000 cells?

Calculate the cells per microliter of suspension. Signup and view all the answers

Which calculation step follows after counting 200 cells to formulate the culture?

Multiply by the volume factor to estimate total cells. Signup and view all the answers

Which factor is critical in ensuring proper cell communication and differentiation in embryoid bodies?

Cell-Cell interactions in a 3D environment. Signup and view all the answers

At what confluence should pluripotent stem cell colonies typically be passaged?

70-80% Signup and view all the answers

What might indicate that a cell culture is under stress?

Slower growth rate Signup and view all the answers

What is the purpose of using Trypan blue in cell counting?

To differentiate between live cells and dead cells Signup and view all the answers

What should be done with pluripotent stem cells before leaving for an extended time?

Passage the cells to appropriate confluence Signup and view all the answers

What does 100% confluence indicate in a culture dish?

A fully covered monolayer of cells Signup and view all the answers

Which of the following best describes a culture that is too crowded?

Cells may start to differentiate Signup and view all the answers

Why is it important to leave detailed instructions for colleagues when managing cell cultures?

To maintain optimal culture conditions Signup and view all the answers

What does a culture shown at 0% confluence indicate?

Cells have just been plated Signup and view all the answers

Study Notes

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).

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Description

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.