Simple Liver Cell Culture Procedure PDF

Practical session for cell and tissue culture The principle of cell cultures Creating and maintaining an environment that mimics the physiological conditions necessary for cells to survive, grow, and function. Principle include: 1. Sterility Objective: Prevent contamination by microorgani...

Practical session for cell and tissue culture The principle of cell cultures Creating and maintaining an environment that mimics the physiological conditions necessary for cells to survive, grow, and function. Principle include: 1. Sterility Objective: Prevent contamination by microorganisms (bacteria, fungi, viruses, and mycoplasma). Practice: Use aseptic techniques, sterilized equipment, and antibiotics (if necessary). 2. Suitable Growth Environment Nutritional Support: Cells require specific media containing essential nutrients (e.g., amino acids, glucose, vitamins, and minerals). pH Maintenance: Buffered media maintain the pH (typically ~7.2–7.4). Gas Exchange: CO₂ and O₂ levels are regulated in the incubator (e.g., 5% CO₂ for most mammalian cells). 3. Temperature Control Optimal Temperature: Most mammalian cells thrive at 37°C, matching body temperature. 4. Adherence or Suspension Adherent Cells: Most cells require attachment to a surface (e.g., tissue culture-treated plastic, extracellular matrix). Suspension Cells: Some cells, like blood cells, grow freely in the medium. 5. Subculturing (Passaging) Purpose: Prevent overcrowding, maintain cell health, and sustain exponential growth. Process: Detach adherent cells using trypsin or mechanical methods, reseed at appropriate density. 6. Monitoring Cell Health Morphology: Healthy cells exhibit characteristic shapes (e.g., fibroblast-like, epithelial- like). Growth Rate: Evaluate doubling time and adherence. Contamination: Look for turbidity or abnormal growth under the microscope. 7. Cryopreservation Principle: Long-term storage in liquid nitrogen (-196°C) with cryoprotectants (e.g., DMSO) to prevent ice crystal damage. 8. Specificity of Culture Media Tailored Media: Different cell types require specific growth factors, hormones, and supplements. 9. Ethical Considerations Handle primary cells (e.g., from tissue biopsies) and stem cells responsibly, following ethical and regulatory guidelines. Description of the procedure of simple cell culture such as liver cell Culturing liver cells (hepatocytes or liver-derived cell lines like HepG2) requires careful attention to sterile techniques, appropriate media preparation, and optimal environmental conditions. Methods Materials 1. Liver Cell Source: o Primary hepatocytes (isolated from liver tissue).  2. Cell Culture Media: o For Primary Hepatocytes: Williams' E medium or DMEM supplemented with fetal bovine serum (FBS), insulin, glucagon, and dexamethasone. 3. Plasticware: o T25 or T75 culture flasks or 6-well/12-well plates (preferably collagen-coated for primary hepatocytes). 4. Reagents: o Phosphate-buffered saline (PBS). o Antibiotics/antimycotics (optional) to prevent contamination. 5. Equipment: o Biosafety cabinet (laminar flow hood). o CO₂ incubator (37°C, 5% CO₂). o Centrifuge. o Hemocytometer or automated cell counter. o Inverted phase-contrast microscope. Procedure 1. Preparation Sterilize all materials and reagents to maintain aseptic conditions. Pre-warm culture media, PBS, and trypsin (if applicable) to 37°C. Prepare the biosafety cabinet by wiping it down with 70% ethanol and turning on the airflow for 15 minutes before use. 3. Seeding Cells For Primary Hepatocytes: o Coat culture plates with collagen if not pre-coated. o Seed hepatocytes at an appropriate density (e.g., 1-2 × 10⁵ cells/cm²) in a minimal volume of medium to encourage adherence. 4. Cell Maintenance Incubate the culture vessel in a CO₂ incubator at 37°C with 5% CO₂ and 95% relative humidity. Monitor cells daily under an inverted microscope to check for: o Adherence and morphology. o Contamination (e.g., bacterial or fungal). Replace culture medium every 2-3 days, being careful not to disturb adherent cells. 5. Cryopreservation (Optional) For long-term storage:  1. Harvest cells during log-phase growth and centrifuge. 2. Resuspend cells in freezing medium (e.g., 90% FBS + 10% DMSO). 3. Aliquot into cryovials and freeze gradually (-1°C/min) using a controlled-rate freezer or isopropanol chamber. 4. Transfer cryovials to liquid nitrogen for storage. Key Notes Sterility: Maintain a sterile environment to prevent contamination. Specificity: Adjust media composition and supplements for the cell type being cultured. Observation: Regularly inspect cells for health and morphology, as stressed or dying cells will exhibit changes such as rounding or detachment. Adherence Support: Primary hepatocytes often require extracellular matrix coatings (e.g., collagen) for proper attachment and function. Applications 1. drug testing, 2. metabolism studies, 3. Liver disease modeling.