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Cytogenetics

Culture Maintenance

Once a culture is initiated, it is not a “set it and forget it” process. Human cells in vitro are entirely dependent on the laboratory scientist for their metabolic needs. Culture maintenance involves a scheduled regimen of feeding, sub-culturing, and environmental monitoring to ensure the cells remain in the “Log Phase” of growth (exponential division) until the day of harvest. Neglecting maintenance leads to nutrient depletion, pH toxicity, and cell death

Feeding Strategies (Media Renewal)

Cells consume glucose and amino acids (like glutamine) and excrete toxic metabolic byproducts (lactate/ammonia). “Feeding” is the process of replenishing the nutrients and diluting the toxins

  • Suspension Cultures (Blood/Marrow)
    • Short-term (24–72 hours): Generally do not require feeding. The initial volume of media is sufficient for the short duration
    • Long-term (Lymphoblastoid lines): Requires “splitting.” The cells settle to the bottom. The laboratory scientist removes half the supernatant and replaces it with fresh media, or splits the volume into two new flasks to reduce cell density
  • Monolayer Cultures (Amnio/Tissue)
    • Feeding Schedule: Typically every 2 to 3 days
    • Partial Feed: Removing 50% of the old media and adding fresh media. This retains some of the “conditioned factors” (growth factors secreted by the cells) which promote division
    • Complete Change: Removing 100% of the media. This is done if the media has turned yellow (acidic) or to remove debris/RBCs (e.g., cleaning up a bloody amniotic fluid sample)

Sub-Culturing (Passaging)

For anchorage-dependent cells (fibroblasts/amniocytes), growth is limited by surface area. When cells cover the entire surface (Confluence), they stop dividing due to Contact Inhibition. To keep them dividing, they must be moved to a larger vessel or split into multiple vessels

  • Trypsinization
    • The Enzyme: Trypsin: is a proteolytic enzyme that digests the adhesion proteins attaching the cells to the plastic
    • The Process: Remove media \(\rightarrow\) Rinse with saline (to remove serum, which inhibits Trypsin) \(\rightarrow\) Add Trypsin \(\rightarrow\) Incubate briefly (\(37^\circ\text{C}\)) \(\rightarrow\) Tap flask to dislodge cells \(\rightarrow\) Add fresh media (with serum) to neutralize the Trypsin
  • Splitting Ratio
    • Typically 1:2 or 1:3. One confluent flask is split into two or three new flasks. This provides empty space for the cells to resume division (re-entering the cell cycle)
    • Note: Cytogenetics labs rarely passage cells more than 2–3 times. Excessive passaging can induce in vitro chromosomal mutations (cultural artifacts) or senescence

Environmental Monitoring

The incubator is the life-support system. Its parameters must be checked and documented daily (even on weekends/holidays)

  • Temperature (\(37^\circ\text{C}\)): Checked against a certified NIST-traceable thermometer inside the unit. The digital display on the door is not sufficient proof; it must be verified physically
  • CO2 Levels (5%): Verified using a “Fyrite” gas analyzer or digital probe. Fluctuations in CO2 cause pH shifts in the bicarbonate-buffered media
  • Humidity / Water Pan: The water pan must be kept full to maintain \(>90\%\) humidity. If the pan runs dry, the media evaporates. This increases the osmolarity (salt concentration) of the remaining media, causing cells to shrink and die from hypertonic stress
  • Gas Supply: Ensuring the CO2 tanks are not empty. Many labs use automatic tank switchers, but manual verification of tank pressure is a daily task

Growth Assessment (Microscopy)

The laboratory scientist must visually assess the “health” of the culture using an inverted phase-contrast microscope

  • Confluence: Estimating the percentage of the surface covered by cells (e.g., “50% confluent”). Harvest is typically targeted when cells are 70–80% confluent (still dividing actively) rather than 100% (contact inhibited)
  • Morphology: Healthy fibroblasts are elongated and spindle-shaped. Healthy amniocytes form discrete colonies with clear borders. Unhealthy cells appear round, granular, or detached (floating)
  • Mitotic Index: In some cultures (like CVS direct), the laboratory scientist looks for “rounded up” refractile cells, which indicates cells in metaphase. A high number of rounded cells suggests the culture is ready for harvest