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Cytogenetics

Prepare Media

The culture medium is the lifeblood of the cytogenetic analysis. It must act as a surrogate for the human body, providing all the necessary nutrients, growth factors, and environmental stability required for cells to survive and divide in vitro. Unlike routine bacterial cultures that grow on simple agar, human cells are fastidious; they require complex, buffered, and supplemented media. The preparation of this media is a precise quantitative process, as imbalances in pH, osmolarity, or mitogen concentration can arrest the cell cycle

Base Media Selection

The foundation is a commercially balanced salt solution containing amino acids, vitamins, glucose, and inorganic salts. The choice of base media depends on the cell type

  • RPMI-1640: The standard “workhorse” for blood and bone marrow (suspension cultures). It was originally developed at Roswell Park Memorial Institute for lymphocytes. It is rich in phosphate and is robust for suspension cells
  • MEM (Minimal Essential Medium) / Alpha-MEM: Often used for amniocytes and solid tissues (monolayer cultures). Alpha-MEM contains additional nucleosides and vitamins that support the rapid growth of adherent cells
  • Chang Medium / AmnioMAX: Specialized, commercially optimized “complete” media specifically designed for prenatal samples (Amnio/CVS). These contain proprietary blends of hormones and growth factors to maximize colony growth in short turnaround times

Critical Supplements

Base media alone is insufficient. It must be “completed” with biological additives before use

  • Serum (Fetal Bovine Serum - FBS)
    • Purpose: Provides essential growth factors, hormones, carrier proteins (albumin), and trace elements that are not defined in the chemical recipe
    • Concentration: Typically 10% to 20%: of the final volume
    • Quality Control: Serum varies by lot number. It must be “heat-inactivated” (56°C for 30 min) prior to use to destroy complement proteins that could lyse the human cells
  • L-Glutamine
    • Purpose: An essential amino acid used as a major energy source and carbon donor
    • Stability: It is unstable at \(37^\circ\text{C}\) and degrades into toxic ammonia over time. Therefore, it is usually added fresh just before use or purchased as a stable dipeptide (L-Alanyl-L-Glutamine)
  • Antibiotics
    • Purpose: To prevent bacterial contamination
    • Common Cocktail: Penicillin/Streptomycin: (Pen/Strep)
    • Special Cases: Gentamicin (broader spectrum) or Kanamycin may be used for “dirty” samples like skin or POCs. Mycoplasma: is not killed by Pen/Strep
  • Buffering System (Sodium Bicarbonate)
    • Purpose: Maintains physiological pH (7.2–7.4). The bicarbonate buffer works in equilibrium with the CO2 in the incubator (\(CO_2 + H_2O \leftrightarrow H_2CO_3 \leftrightarrow H^+ + HCO_3^-\)). If the CO2 level drops, the pH rises (alkaline); if CO2 rises, pH drops (acidic)

Mitogens (Stimulating Division)

Most mature human cells (like the T-lymphocytes in a blood tube) are in the G0 “resting” phase. To see chromosomes, we must force them to divide. We add Mitogens

  • Phytohemagglutinin (PHA)
    • Target: T-Lymphocytes
    • Use: Constitutional blood karyotypes. It mimics an immune response, triggering T-cells to blast transform and divide
  • Lipopolysaccharide (LPS) / DSP-30
    • Target: B-Lymphocytes
    • Use: To reveal abnormalities in CLL (Chronic Lymphocytic Leukemia) or other B-cell disorders that might be missed by unstimulated culture
  • Pokeweed Mitogen (PWM): Stimulates both B and T cells (less common)
  • No Mitogen (Unstimulated)
    • Target: Spontaneously dividing cells (Leukemic blasts, Bone Marrow, Amniocytes, Solid Tumors). Adding PHA to a bone marrow sample is dangerous because the T-cells will overgrow the leukemic blasts, hiding the cancer

Environmental Conditions

Once the media is prepped and inoculated, the physical environment controls the reaction

  • Temperature: Strictly controlled at \(37.0^\circ\text{C} \pm 0.5^\circ\text{C}\)
    • \(>38^\circ\text{C}\): Cells die (protein denaturation)
    • \(<36^\circ\text{C}\): Cell cycle slows (mitotic index drops)
  • CO2 Concentration: Typically 5% CO2
    • This is required to balance the Sodium Bicarbonate buffer in the media. Without CO2, the media turns purple (alkaline), and cells die
  • Humidity: Maintained at \(>90\%\)
    • Prevents the evaporation of the media. If media evaporates, the osmolarity (salt concentration) increases, causing hypertonic shock

Preparation Workflow (QC)

  • Sterility Check: Before committing patient cells, aliquots of new media batches are often incubated for 24–48 hours to ensure no bacteria grow
  • Expiration: “Complete” media (with serum/glutamine) has a short shelf life (typically 2–4 weeks). It should be made in small batches to ensure freshness