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Cytogenetics

Optimal Culture Systems

The selection of the appropriate culture system is dictated by the physiological origin of the target cell type. In cytogenetics, cells generally fall into one of two biological categories: hematopoietic cells (blood-derived), which circulate freely and are adapted to grow in suspension, or mesenchymal/epithelial cells (tissue-derived), which exhibit anchorage dependence, meaning they must attach to a solid surface to flatten out, form a cytoskeleton, and undergo mitosis. Choosing the wrong system will result in culture failure (e.g., fibroblasts will not divide if floating in a tube, and lymphocytes will not adhere to a slide)

Suspension Culture Systems

Suspension cultures are used for cells of hematopoietic origin. In this system, cells float freely in the culture medium. They do not attach to the vessel walls, and therefore, enzymatic detachment (trypsinization) is not required during the harvest phase. This method is generally faster and less labor-intensive than monolayer cultures

  • Peripheral Blood (Lymphocytes)
    • Target Cell: T-lymphocytes (stimulated by Phytohemagglutinin - PHA)
    • System: Suspension in sterile, round-bottom or conical tubes (e.g., 15 mL polypropylene tubes)
    • Rationale: Lymphocytes naturally circulate in the bloodstream. In culture, they form “rosettes” or clumps of dividing cells floating in the media. The vertical orientation of the tube allows the cells to settle in close proximity, facilitating the cell-to-cell communication required for blast transformation
  • Bone Marrow (Leukemic Blasts)
    • Target Cell: Myeloid or lymphoid blasts (spontaneous division)
    • System: Suspension in tubes or occasionally loose suspension in flasks
    • Rationale: Like peripheral blood, marrow cells are non-adherent. While stromal cells in the marrow may attach to plastic, the diagnostic cells (the leukemic blasts) remain in the supernatant. Suspension culture allows for easy harvesting by simple centrifugation

Monolayer (Adherent) Culture Systems

Monolayer cultures are required for solid tissues and amniotic fluid. These cells (fibroblasts, amniocytes, trophoblasts) are anchorage-dependent. If they cannot attach to a substrate, they undergo a specialized form of apoptosis (anoikis). There are two primary methodologies for monolayer culture: the Flask Method and the In Situ Method

The Flask Method (Closed System)

In this method, cells are grown on the inner surface of a T-flask (a specialized plastic bottle treated to be hydrophilic). Once the colonies grow and merge (confluence), the cells are detached using a proteolytic enzyme (Trypsin) and harvested as a suspension

  • Solid Tissues (Skin, POC, Solid Tumors)
    • Target Cell: Fibroblasts or tumor cells
    • System: T-25 or T-75 Flasks
    • Rationale: Tissues often require long culture times (2–3 weeks) and yield large volumes of biomass. Flasks provide a large surface area for expansion. Because these specimens are often utilized to detect constitutional abnormalities or tumor clones where mosaicism is less of a concern (or handled differently), the mixing of cells during trypsinization is acceptable
  • Chorionic Villi (Long-Term Culture)
    • Target Cell: Fibroblasts from the mesenchymal core of the villus
    • System: T-25 Flasks or In Situ
    • Rationale: Culturing the mesenchymal core helps confirm results found in the “direct” preparation and rules out confined placental mosaicism

The In Situ Method (Open/Cover-slip System)

In this method, cells are grown directly on a glass coverslip or the bottom of a specialized “chamber slide.” The cells are harvested, fixed, and banded while still attached to the glass. They are never trypsinized or suspended

  • Amniotic Fluid
    • Target Cell: Amniocytes (fetal skin/urinary tract cells)
    • System: 35mm Petri dishes with coverslips or Chamber Slides
    • Rationale (The Mosaicism Advantage): The primary advantage of the in situ method is the ability to analyze distinct colonies. Each colony arises from a single original cell (a clone)
      • True Mosaicism: An abnormality is seen in multiple independent colonies (multiple original cells)
      • Pseudomosaicism: An abnormality is seen in all cells of only one colony (likely a culture artifact arising in vitro)
      • In the flask method, trypsin mixes all cells together, making it impossible to distinguish between a single weird colony (artifact) and true low-level mosaicism. Therefore, in situ is the Gold Standard for prenatal diagnosis

Specialized/Hybrid Systems

Some specimen types utilize unique modifications that blend these concepts to optimize turnaround time

  • Chorionic Villi (Direct/Semi-Direct Preparation)
    • Target Cell: Cytotrophoblast (outer layer of the villus)
    • System: Short-term Suspension
    • Rationale: The cytotrophoblast cells are spontaneously dividing in vivo. They do not need to be cultured long-term to establish a monolayer. The villi are exposed to Colcemid almost immediately (or after 24 hours of incubation) to capture the spontaneous divisions. This mimics a suspension harvest despite the tissue origin, providing rapid results (24–48 hours)
  • Tube Cultures for Amnio (The “Backup”)
    • Some labs maintain a “flask” or “Leighton tube” culture as a backup for amniotic fluid. If the coverslips fail (dry out or break), the backup flask allows the lab to trypsinize the cells and create a new slide without asking for a recollection (second amniocentesis)