Preparation of Single Cell Suspension from Chicken Liver (Primary Cell Culture)

 

EXPERIMENT NO: 6

TITLE: Preparation of Single Cell Suspension from Chicken Liver (Primary Cell Culture)

Objective:

To prepare a single cell suspension from chicken liver tissue for primary cell culture, which involves the isolation, disaggregation, and suspension of cells from the liver tissue.

Materials Required:

• Chicken liver tissue (freshly obtained)
• Phosphate-Buffered Saline (PBS)
• Enzymes: Collagenase, Trypsin-EDTA
• Sterile Petri dishes
• Sterile scissors and forceps
• Scalpel
• Sterile gauze
• Tissue culture media (e.g., DMEM, RPMI-1640)
• Fetal Bovine Serum (FBS)
• Centrifuge
• Sterile conical centrifuge tubes (15 mL and 50 mL)
• Pipettes and sterile pipette tips
• Cell strainer (70 µm or 100 µm mesh)
• Hemocytometer
• Incubator (37°C, 5% CO2)
• Personal Protective Equipment (PPE): Lab coat, gloves, safety goggles

Procedure:

1. Collection of Chicken Liver Tissue:

• Tissue Collection:
• Obtain a fresh chicken liver and immediately place it in a sterile container with cold PBS to transport it to the laboratory.
• Disinfection:
• Rinse the liver tissue in sterile PBS containing antibiotics to reduce the risk of contamination.

2. Preparation of Tissue:

• Dissection:
• Place the liver tissue in a sterile Petri dish. Using sterile scissors and forceps, remove any connective tissue, blood vessels, or fat.
• Mincing:
• Mince the liver tissue into small pieces (1-2 mm³) using a sterile scalpel. The smaller the pieces, the more efficient the enzyme digestion will be.

3. Enzymatic Digestion:

• Collagenase Treatment:
• Transfer the minced tissue into a sterile centrifuge tube and add collagenase solution (typically 0.1% to 0.2% in PBS or media).
• Incubate the tissue with collagenase at 37°C for 30-60 minutes with gentle agitation to facilitate the breakdown of the extracellular matrix.
• Trypsin-EDTA Treatment (Optional):
• After collagenase digestion, you can treat the tissue with trypsin-EDTA (0.05% trypsin, 0.02% EDTA) for 5-10 minutes to further disaggregate the cells.
• Monitor the digestion carefully to avoid over-digestion, which can damage the cells.

4. Mechanical Disaggregation:

• Gentle Pipetting:
• After enzymatic digestion, gently pipette the tissue suspension up and down with a wide-bore pipette to mechanically break down the tissue into a single cell suspension.
• Filtration:
• Pass the cell suspension through a sterile cell strainer (70 µm or 100 µm mesh) into a clean centrifuge tube to remove undigested tissue and large debris.
• Rinse the strainer with sterile PBS or media to collect as many cells as possible.

5. Washing and Centrifugation:

• Centrifugation:
• Centrifuge the cell suspension at 300-400 g for 5-10 minutes at room temperature to pellet the cells.
• Supernatant Removal:
• Carefully aspirate the supernatant without disturbing the cell pellet.
• Resuspension:
• Resuspend the cell pellet in fresh tissue culture media (e.g., DMEM supplemented with 10% FBS) to prepare the final cell suspension.

6. Counting and Viability:

• Cell Counting:
• Take a small aliquot of the cell suspension and count the cells using a hemocytometer under a microscope.
• Viability Assessment:
• Assess cell viability using trypan blue exclusion or a similar method. Mix the cell suspension with trypan blue and count the viable (unstained) and non-viable (stained) cells.

7. Plating the Cells:

• Seeding:
• Plate the cell suspension in tissue culture flasks or Petri dishes at the desired density.
• Incubate the culture at 37°C in a humidified incubator with 5% CO2.
• Medium Replacement:
• After 24 hours, carefully replace the medium to remove non-adherent cells and debris. Add fresh medium and continue to culture the cells.

Observations:

• Observe the appearance of the cell suspension after each step, noting any clumps or debris.
• Record the cell count and viability percentage.
• Monitor cell attachment and proliferation over the next few days.

Results:

A single cell suspension should be obtained with a high percentage of viable cells. The cells should attach to the culture dish or flask and begin to proliferate if the conditions are optimal.

Precautions:

• Handle all tissues and reagents under aseptic conditions to prevent contamination.
• Avoid over-digestion during enzymatic treatment, as this can damage the cells.
• Use gentle mechanical techniques to prevent cell damage during disaggregation.

Conclusion:

A successful single cell suspension from chicken liver tissue has been prepared, suitable for establishing a primary cell culture. This method allows for the isolation and cultivation of liver cells for various experimental applications.