Lecture on Disaggregation of Tissues and Creating Primary Culture

Instructor: Drew Collup

Introduction

• Dissection of chicken embryo for educational purposes.
• Purpose: Instruct biotechnology students on creating primary cultures.
• Viewer discretion is advised due to dissection content.

Disaggregation Process

Purpose

• Break apart organs into individual cells for primary culture.

Observations

• Heart:
  • Contains sinoatrial node, can beat outside the body.
• Kidney, Liver, Gallbladder, Stomach, Intestine, Brain, Eyes:
  • Various organs displayed and briefly described.

Techniques for Disaggregation

1. Explant Technique

• Process:
  • Tissue chunks placed in a dry dish.
  • Tissue adheres to the dish for cell migration.
• Incubation:
  • 37 degrees for 30 minutes.
• Outcome:
  • Cells migrate away from the tissue chunk.

2. Cold Trypsin Technique

• Process:
  • Tissue chunks placed in cold trypsin at 4 degrees.
  • Trypsin infuses tissue overnight.
• Activation:
  • Trypsin activated at 37 degrees.

3. Mechanical Disaggregation

• Best for: Soft tissues (e.g., liver, brain).
• Process:
  • Tissue cut into small pieces.
  • Pressed through a cell strainer using a syringe plunger.
  • Media washes cells through the strainer.
• Outcome:
  • Cells collected at the bottom of a conical tube.

Cell Culture and Observation

Preparation of Culture

• Media: Minimal Essential Media (MEM).
• Seeding:
  • Different concentrations tested for optimal growth.

Observation

• Microscopy:
  • Inverted microscope with camera for viewing.
  • Explant should look darker; cells should be clear and elongated.

Challenges in Primary Culture

• Cells:
  • Heart cells observed to migrate from explant.
  • Risk of cells being senescent and not surviving.

Conclusion

• Disaggregation methods are crucial for primary culture.
• Success depends on proper technique and conditions.
• Importance of experimentation for establishing continuous cell lines.

Additional Resources

• Encourage viewing related experiments (e.g., ostrich vs chicken egg experiment).

Note: For practical lab applications, ensure sterile conditions to prevent cross-contamination.