Kultur Sel Hewan - Primary Cell Culture and Establishing Cell Line

Introduction

This tutorial will guide you through the process of primary cell culture and the establishment of a cell line, as discussed in the video "Kultur Sel Hewan" by Sri Wijayanti Wulandari. Understanding these techniques is crucial for biological research and pharmaceuticals, as they allow scientists to study cellular behavior and test drugs in a controlled environment.

Step 1: Preparing the Workspace

• Ensure a clean and sterile environment to prevent contamination.
• Gather all necessary materials:
  • Cell culture dishes or flasks
  • Growth media suitable for the specific cell type
  • Sterile pipettes and tools
  • Incubator set at the appropriate temperature for cell growth

Step 2: Obtaining Cell Samples

• Acquire tissue samples from the desired organism.
• Use aseptic techniques to minimize contamination:
  • Wear gloves and a lab coat.
  • Work near a laminar flow hood if possible.

Step 3: TIssue Dissection

• Carefully dissect the tissue to isolate the cells:
  • Use sterile scissors and forceps.
  • Minimize damage to the cells during dissection.

Step 4: Cell Digestion

• Prepare a digestion solution (e.g., trypsin or collagenase) to break down the extracellular matrix.
• Add the digestion solution to the tissue samples and incubate:
  • Follow the recommended incubation time specific to the tissue type.

Step 5: Cell Suspension

• Gently triturate (mix) the digested tissue to release individual cells.
• Filter the cell suspension through a sterile mesh or filter to remove debris.

Step 6: Cell Counting and Viability Assessment

• Use a hemocytometer or cell counter to determine the cell concentration.
• Assess cell viability with a dye exclusion method (e.g., trypan blue):
  • Live cells will exclude the dye, while dead cells will take it up.

Step 7: Plating Cells

• Dilute cells to the desired concentration using growth media.
• Transfer the cell suspension to culture dishes or flasks:
  • Ensure even distribution across the surface.

Step 8: Incubation and Monitoring

• Place the cells in an incubator set to the optimal temperature and CO2 concentration (typically 37°C and 5% CO2).
• Regularly check for contamination and cell growth:
  • Observe the morphology under a microscope.

Step 9: Subculturing (Passaging)

• Once cells reach 70-80% confluence, they need to be subcultured:
  • Use trypsin to detach cells from the surface.
  • Dilute and transfer to new culture vessels.

Conclusion

In this tutorial, you learned the essential steps for primary cell culture and establishing a cell line. From preparing your workspace to subculturing, each step is critical for successful cell growth. Ensure to maintain sterile conditions throughout the process to avoid contamination. For further applications, consider exploring different cell types or enhancing your techniques with advanced cell culture methods.