Evaluate/Subculture Monolayer Cells

The management of monolayer cultures - specifically amniocytes, chorionic villus fibroblasts, and solid tissue fibroblasts - requires a distinct skillset compared to suspension cultures. These cells are anchorage-dependent, meaning they must attach to a substrate to survive and proliferate. The laboratory scientist must expertly evaluate their growth kinetics to determine the precise moment to intervene: either to subculture (expand) them to prevent overcrowding or to harvest them to capture metaphases. Misjudging this timing results in culture failure due to contact inhibition (stalled growth) or senescence

Evaluation: The Inverted Microscope

Because monolayer cells grow on the bottom surface of the flask or coverslip, they are evaluated using an Inverted Phase-Contrast Microscope. Light is projected from the top, passing through the culture media and cell layer to the objective lens below. This visualization allows the laboratory scientist to assess three critical parameters:

• Confluence (Density): The percentage of the growth surface covered by cells
  • Lag Phase (<20%): Cells are sparse and recovering from inoculation. They appear flattened and spread out
  • Log Phase (50–80%): The “Sweet Spot.” Cells are dividing rapidly. Mitotic figures (round, refractile cells) are visible. There is still open plastic space for expansion
  • Stationary Phase (100%): The surface is a solid sheet of cells. Contact Inhibition: sets in; cells sense their neighbors and stop dividing. A 100% confluent culture will yield zero metaphases during harvest because the cells are locked in G0/G1
• Morphology (Health)
  • Healthy: Fibroblasts are spindle-shaped (fusiform) and transparent. Amniocytes form discrete “fried egg” colonies or cobblestone sheets. Membranes are smooth
  • Unhealthy (Senescent/Toxic): Cells appear granular (dark spots in cytoplasm), vacuolated (bubbles), or ragged. If cells are rounding up and floating without being in mitosis, they are dying/detaching
• Colony Quality (For In Situ Amnio)
  • The laboratory scientist counts the number of independent colonies. For diagnosis, typically 15+ independent colonies: are required. If only 2 large colonies are present, the culture must be subcultured to generate more biomass, even if the colonies look healthy

The Subculture Decision (To Split or Not to Split)

Subculturing (Passaging) is the process of moving cells from one vessel to multiple vessels to provide more surface area

• When to Subculture
  • Overcrowding: When a flask approaches 80–90% confluence but is not yet ready for harvest (e.g., waiting for backup flasks). Splitting it 1:2 keeps the cells in the log phase
  • Rescue: If an in situ coverslip culture has too few colonies (e.g., only 3 colonies), they cannot be analyzed directly. They are trypsinized and moved to a flask to expand the population for a suspension harvest
• When NOT to Subculture
  • Harvest Ready: If the flask is 70% confluent and actively dividing, it should be harvested immediately. Subculturing stresses the cells and adds a 24–48 hour lag time for re-attachment
  • Senescence: If cells are old, large, and granular, trypsinizing them might kill them. They may not have the energy to re-attach

The Subculture Protocol (Trypsinization)

The chemical detachment of cells must be precise. Prolonged exposure to trypsin destroys the cell membrane proteins, killing the cells

1. Rinse (The Saline Wash): The old media is aspirated. The monolayer is rinsed with sterile Calcium/Magnesium-free HBSS or PBS
   • Why? Culture media contains Serum (FBS). Serum contains Alpha-1 Antitrypsin, a natural inhibitor of trypsin. If you don’t wash the serum away, the trypsin won’t work
2. Digestion (Trypsin-EDTA): A minimal volume of Trypsin-EDTA is added to cover the monolayer. The flask is incubated at \(37^\circ\text{C}\) for 1–5 minutes
   • Observation: The laboratory scientist watches the flask. The cell sheet will start to “slough” off or cloud the fluid. Tapping the side of the flask helps dislodge stubborn cells
3. Neutralization: Immediately upon detachment, fresh “Complete Media” (containing Serum) is added
   • Why? The serum instantly inactivates the trypsin, stopping the digestion before it damages the cell membranes
4. Reseeding: The cell suspension is divided into new flasks (e.g., a 1:2 split) and topped up with fresh media. The cells will settle and re-attach within 4–24 hours

Special Considerations for Amniocytes

• The “Clean Up” Subculture: Sometimes an amniotic fluid specimen is extremely bloody. The RBCs coat the bottom of the dish, preventing amniocytes from attaching
  • Technique: At 24–48 hours, the media (containing the suspended RBCs) is gently removed and replaced. The heavy/sticky amniocytes remain on the glass, while the RBCs are washed away. This is not a “true” subculture (no trypsin), but a critical maintenance step
• Amniocyte Types: Amniotic fluid contains varying cell types:
  • E-Type (Epithelioid): Grow slowly, often from the fetal urinary tract
  • F-Type (Fibroblastic): Grow rapidly, from fetal skin/connective tissue. These are the desired cells for cytogenetics
  • AF-Type: Derived from amniotic membranes
  • Laboratory scientists prioritize the harvest of F-Type colonies as they yield the best chromosome spreading