Overview of cell culture and harvest
Culture initiation →
Culture maintenance →
Cell harvest
Living cells→
Sterility→
Arrest division
Sterility→
Optimal temperature→
Swell cells
Proper growth medium→
Optimal pH→
Fix cells
± Mitotic stimulant→
Optimal humidity→
Prepare slide
Microbial inhibitors→
Optimal time interval→
Stain/band
The most critical requirement is that living cells capable of cell division be received by the laboratory. The manner in which the sample is collected and subsequently handled will greatly influence whether or not the cells will grow and divide, and the quality of the resulting metaphases. Specimen containers must be sterile and must be labeled with the patient’s name and a second identifier. The laboratory may reject specimens that are improperly labeled or unlabeled.
Specimen Collection and Handling
Sample Requirements
Peripheral Blood Specimens
Peripheral blood samples should be collected in sterile syringes or vacuum tubes containing preservative-free sodium heparin. Vacuum tubes should be discarded if outdated. Peripheral blood cultures can be initiated several days after the blood is drawn; however, for best results, blood samples should be set up within 24 h of collection. Temperature extremes must be avoided if samples are transported or stored. Specimens should be kept at room temperature or refrigerated above 4°C until they can be processed. Culture medium is sometimes added to small blood samples, as these have a tendency to dry up, especially if collected in large containers.
A repeat sample should be requested if these requirements are not met (e.g., the sample is received clotted, on ice, more than 24 h old). It is not always practicable or possible to obtain a new sample, and in such cases, the laboratory should attempt to salvage the original specimen. There may be enough viable cells for a cytogenetic analysis, though the number and quality of cells may be compromised.
Bone Marrow Aspirates
The collection requirements for bone marrow samples are essentially the same as for peripheral blood. Bone marrow aspirates should be collected in sterile syringes or vacuum tubes containing preservative-free sodium heparin and transported at room temperature. The first few milliliters of the bone marrow tap contain the highest proportion of cells and are the best sample for the cytogenetics laboratory. Blood dilutes the bone marrow sample in later taps and reduces the number of actively dividing cells present in the sample. The success of bone marrow culture is dependent on the number of actively dividing cells. Bone marrow specimens should be processed without delay upon receipt to avoid cell death.
Amniotic Fluid Specimens
Amniocentesis can be performed from as early as 10 weeks of gestation until term (see Chap. 12). Fifteen to thirty milliliter of amniotic fluid should be obtained under sterile conditions and collected in a sterile container approved for cell culture. For amniocenteses performed earlier than 15 weeks, 1 mL of fluid is generally drawn for each week of gestation. The first few milliliters of an amniotic tap are the most likely to be contaminated with maternal cells and should not be submitted to the cytogenetics laboratory. Samples should be transported at room temperature. Temperature extremes and long transport times should be avoided.
The amniocentesis procedure has an inherent, albeit small, risk of miscarriage and should not be repeated unless absolutely necessary. Every effort to salvage samples improperly collected or handled should be made to diminish the need for a repeat procedure.
Solid Tissue Biopsies
Solid tissue sources include skin biopsies, chorionic villi, products of conception, lymph node and solid tumor biopsies, and tissue from stillbirths. Products of conception and stillbirths (and in most cases, tumor biopsies) are one-of-a-kind specimens that cannot be recollected, and repeat collection of chorionic villi increases the risk of miscarriage, although subsequent amniocentesis is an option here. Microbial contamination is a common problem for many types of solid tissue samples. Unlike amniotic fluid, blood, bone marrow, and chorionic villi, some solid tissue specimens are not sterile prior to collection. In addition, viable cells may be few or even nonexistent. These factors threaten the integrity of the sample and pose problems for the laboratory.
Small samples should be collected and transported in sterile culture vessels containing growth or tissue culture medium (not formalin). Sterile saline is not optimal for this purpose but should be used if no other option is available. If distance and timing permit the laboratory to receive and process the sample at once, it may be delivered with no liquid added at all. Larger samples may be sent to the laboratory in toto for dissection. Solid tissue samples may be transported and stored on ice until culture is established. Storing tissue specimens on ice slows the action of enzymes that degrade the tissue and slows microbial growth in the event of contamination.
Culture Initiation
Growth Media
All specimens for chromosome preparation are grown and maintained in an aqueous growth medium. Some media are formulated for specific cell types (e.g., AmnioMAX™, Chang Medium®, or Amniochrome™ for amniocytes, giant cell tumor-conditioned medium for malignancies, PANDIS for breast tumors), while others are appropriate for a broad spectrum of cell types (e.g., RPMI 1640, MEM). All culture media are balanced salt solutions with a variety of additives including salts, glucose, and a buffering system to maintain the proper pH. Phenol red is often used as a pH indicator in many media. If the medium becomes too acidic, it will turn yellow, while medium that is too basic becomes pink or purple.
Commercial media are available either in powder forms that must be rehydrated, or as ready-to-use aqueous solutions. Both complete and incomplete media are commercially available, but most commercial media are incomplete. Incomplete media do not contain all of the nutrients and additives necessary for cell growth. Incomplete culture medium must be supplemented with one or more additives before being used for cell culture:
l-Glutamine
l-Glutamine is an amino acid essential for cell growth. l-Glutamine is unstable and breaks down on storage to d-glutamine, a form that cannot be used by cells. l-Glutamine must therefore be stored frozen to retain its stability, and it is optimal to add it to the culture medium just prior to use. There are some commercially available complete media that contain l-glutamine.
Serum
Serum is essential for good cell growth. Too little does not allow for maximum cell growth, but too much can have a detrimental effect. Fetal bovine serum (FBS) is preferred; culture medium is generally supplemented with 10–30% FBS.
Antibiotics
Microbial inhibitors are added to culture media to retard the growth of microorganisms. This is a stopgap measure at best, and should never be relied upon to compensate for sloppy technique. Good sterile technique is always the best defense against contamination.
Penicillin/streptomycin, kanamycin, and gentamicin are bacterial inhibitors commonly used in tissue culture. Fungicides routinely used include nystatin and amphotericin B. Fungicides can adversely affect cell growth and generally are only used when the potential for contamination outweighs this potentially negative effect.
Bacterial contamination of cultures imparts a cloudy appearance to the culture medium. Fungal contamination presents to the unaided eye as “woolly” masses in the medium, or when observed under an inverted microscope, as branching hyphae. Mycoplasma and viral contamination can be hard to detect and treat. Mycoplasma should be suspected if the background level of chromosome breaks and rearrangements is higher than usual.
Mitotic Stimulants (Mitogens)
Some cells, particularly mature lymphocytes, do not spontaneously undergo cell division and must be stimulated to divide by the addition of an appropriate mitogen to the cell culture.
Phytohemagglutinin (PHA) is an extract of red kidney beans that stimulates division primarily of T-lymphocytes. Cell division starts 48 h after the addition of PHA, with additional waves of division at 24-h intervals. The culture period for blood specimens is based on this knowledge. For routine peripheral blood cultures, 72 h is usually optimal. Blood specimens from newborns may require a shorter culture period. T-cell mitogens may also be indicated for bone marrow samples when some chronic lymphoproliferative disorders (depending upon the immunophenotype), or well-differentiated T-cell disorders are suspected.
Some hematopoietic studies require stimulation of B-lymphocytes, and B-cell mitogens are indicated for bone marrow samples when some chronic lymphoproliferative disorders (depending upon the immunophenotype) or mature B-cell disorders are suspected. There are a number of B-cell mitogens available, including Epstein–Barr virus, LPS (lipopolysaccharide from E. coli), protein A, TPA (12-0-tetradecanoylphorbol-13-acetate), and pokeweed. A cocktail including PHA and interleukin-2 (IL2) has proven successful as a lymphoid mitogen for bone marrow samples. The synthetic oligonucleotide DSP-30 has been shown to improve detection of abnormalities in patients with CLL, often together with IL2, and may be useful for other B-cell neoplasms as well [1–3].
Growth Factors
A variety of additional growth factors are commercially available and are used by some laboratories to achieve optimal cell growth for different sample types. These include giant cell tumor extract (GCT) for bone marrow culture and specially formulated amniotic fluid culture media.
Culture Vessels
Choice of culture vessel depends in part on the growth needs of the sample and in part on the individual preference of the laboratory. Blood and bone marrow samples consist of single free-floating cells. For such suspension cultures, sterile centrifuge tubes or tissue culture flasks (T-flasks) may be used. The cells from samples such as amniotic fluid, chorionic villi, skin biopsies, and other solid tissues need to attach to a surface to grow. Such samples may be grown in T-flasks or with an in situ method.
Flask Method
Cells are grown on the inner surface of T-flasks until adequate numbers of dividing cells are present. Cell growth is monitored using an inverted microscope. To remove the cells from the surface of the culture flask where they have been growing, the cultures are treated with an enzyme such as trypsin. This enzymatic treatment releases the individual cells into the fluid environment and permits their collection, harvest, or subculture, as needed.
In Situ Method
Amniotic fluid, CVS, and other tissue samples can be grown directly on coverslips in small petri dishes, in “flaskettes,” or in slide chambers. Growth of these cultures is also monitored with an inverted microscope. They are harvested as “primary” cultures (those that have not been sub-cultured) when adequate numbers of dividing cells are present, and cells do not have to be enzymatically removed prior to harvest. The cells can therefore be analyzed as they grew in situ.
Advantages of the In Situ Method Over the Flask Method
The primary advantage of using the in situ method is that it provides information about the colony of origin of a cell. This is important when deciding whether an abnormality seen in some but not all cells represents true mosaicism (constitutional mosaicism) or an artifact of tissue culture (pseudomosaicism). True mosaicism is said to be present when there are multiple colonies from more than one culture with the same chromosomal abnormality. Pseudomosaicism is suggested if a single colony with all or some cells exhibiting a chromosomal abnormality is found. In such cases, all available colonies should be studied to rule out the possibility of true mosaicism. If only a single colony with a potentially viable abnormality is found, it may result in an equivocal diagnosis. Low-level mosaicism cannot be completely ruled out in such cases. Clinical correlation may help clarify the picture. A repeat amniocentesis may confirm the presence of true mosaicism but cannot, of course, eliminate the results of the first study.
No inference can be made about the origin of cells when using the flask method, since cells from all colonies are mixed together after they are released from the growing surface. It is impossible to tell if multiple cells exhibiting the same chromosomal abnormality arose from one or multiple colonies. Thus, two or more cells exhibiting the same structural abnormality or having the same extra chromosome or three or more cells lacking the same chromosome must be treated as potential true mosaics if the flask method is used. However, it should be noted that the presence of multiple abnormal colonies in the same in situ culture might also represent artifact. Guidelines for interpretation of mosaicism are available for both methods.
Another advantage of the in situ method is that there is usually a shorter turnaround time (TAT), since only primary cultures are harvested. Flask cultures are often sub-cultured, adding days to the culture time.
Preparation of Specimens for Culture
Amniotic fluid specimens, whole blood, and bone marrow samples arrive in the laboratory as single cells in a fluid environment. Whole blood or bone marrow can be added directly to the culture medium, or the white blood cells can be separated from the other blood elements and used to inoculate the culture medium. Separation of the white blood cells is easily accomplished by centrifuging the sample or allowing it to rest undisturbed until the blood settles into three distinct layers. The lowest layer consists of the heavier red blood cells, the top layer consists of plasma, and the narrow middle layer—the buffy coat—consists of the desired white blood cells. The buffy coat can be removed and used to establish the suspension culture.
Amniotic fluid contains a variety of cells that arise from the fetal skin, urinary and gastrointestinal tracts, and the amnion. These are collectively referred to as amniocytes. Most of the cells in an amniotic fluid sample are dead or dying and are not suitable for cytogenetic analysis. Amniotic fluids are centrifuged at low speed (800–1,000 rpm) to retrieve the small number of viable cells. The cell pellet is then used to establish the cultures. The supernatant may be used for a variety of biochemical tests including α-fetoprotein (AFP) and acetylcholinesterase (AChE) assays for open fetal defects.
Solid tissue samples received in the cytogenetics laboratory are usually too large to culture directly and must be disaggregated before use. To obtain single cells, the sample must be finely minced using sterile scissors or scalpels, or alternately, cell dispersion can be achieved by enzymatic digestion of the sample using collagenase and/or trypsin.
Culture Maintenance
After cultures have been initiated, they are allowed to grow under specific conditions of temperature, humidity, and pH until adequate numbers of dividing cells are present. The optimal temperature for human cell growth is 37°C, and it is essential that incubators be maintained at this temperature. Cultures are maintained either “open” or “closed” systems, depending upon the type of incubator used.
Open systems are those that allow the free exchange of gases between the atmosphere inside the culture vessel and the surrounding environment of the incubator. To facilitate the exchange of gases, the tops or caps of tissue culture vessels are loosely applied. A CO2 incubator is required for open systems to maintain the 5% CO2 level necessary to sustain the ideal pH of 7.2–7.4. A humidity level of 97% should be maintained to prevent cell death due to cultures drying out. This can be accomplished by placing pans of sterile water in the bottom of the incubator. A major disadvantage of open systems is that they are susceptible to microbial contamination, especially fungi, due to the moist warm surfaces in the incubator. An open system is required for samples grown on coverslips using the in situ method.
Closed systems are those in which the culture vessels are tightly capped to prevent exchange of gases. Humidification is self-maintained, and CO2 incubators are not required. Commercial media are buffered to the appropriate pH necessary to sustain short-term cultures such as those from blood and bone marrow samples. Long-term cultures from amniotic fluid and solid tissue specimens require the use of additional buffering systems to maintain the proper pH over the longer culture period. Microbial contamination is not as great a risk with closed systems.
In the final analysis, the decision to use an open or closed system, or a combination of both, involves the type of sample being processed and the preference of the laboratory.
Culture Maintenance and Growth Interval
Once the culture requirements are met, the cells must be allowed time to grow and divide. The time in culture varies depending upon the cell type involved.
Peripheral blood cultures require little maintenance once the growth requirements have been met. The culture vessels are placed in an incubator for a specified period of time, usually 72 h.
Likewise, bone marrow cultures need little attention once the culture has been initiated. Bone marrow contains actively dividing cells and therefore can be harvested directly, without any time in culture, or a 24- to 48-h culture time may be used to increase the mitotic index. Longer culture periods are generally not advised since the abnormal cancerous cells may be lost over time or be diluted out by normal precursor cells that may be present. A short growth period usually provides a more accurate reflection of makeup of the tumor; however, there are exceptions, as some tumor cells are slow growing, and some mitogens require longer culture times.
Amniotic fluid and solid tissue specimens require longer culture periods and do not grow at predictable rates. Cell growth is monitored periodically until there are sufficient numbers of dividing cells present, indicating that the culture is ready for harvest. An inverted phase-contrast microscope is used to visualize the mitotic cells that appear as small, refractile spheres. In situ amniotic fluid cultures are generally harvested at 6–10 days, sometimes earlier. For amniotic fluid and solid tissue specimens grown using the flask method, the culture interval may be 2 weeks or more.
Amniotic fluid and solid tissue specimens cultured with either the in situ or flask method become depleted of required nutrients and additives during the culture period. Depleted medium must be removed and replenished with fresh medium. This process is called “feeding” the culture and is done on a regular basis throughout the culture maintenance period dependent upon the number of cells growing, the length of time in culture, and the protocol of the laboratory. Exhausted medium becomes acidic and will appear yellow if the medium contains a pH indicator such as phenol red.
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