Equipment Required for Fusion and Growth of Hybridomas

The production of hybridomas requires the following equipment:

(1) Biohazard or laminar flow hood. This is not absolutely essential, and a 'still air' box to protect from airborne organisms will suffice. While there is no documented evidence that the procedures for the production of mouse hybridomas are harmful to humans, it is now considered preferable to use biohazard hoods which do not blow air over the cultures and into the face of the operator.

The use of biohazard hoods should be regarded as mandatory for any tissue culture work using human cells or any other material of human origin. As a general rule, the use of hypodermic needles should be avoided when using human material, and great care must be taken to guard against infection with hepatitis or human immunodeficiency viruses.

(2) C02 incubator. It is standard practice to buffer the cultures with C02 (7-10%) and sodium bicarbonate (2.0-3.7 g/1). The C02 regulation must be reliable, but need not be complex or expensive. The older simple balltype metering devices are inexpensive and trouble-free, but are now regarded as very old-fashioned. The more complex and expensive electronic C02 regulators that are now standard are becoming more reliable.

In my opinion, it is best to avoid incubators with inbuilt fans, although most modern incubators have them. The problem with fans is that they are usually inaccessible and impossible to clean, although some manufacturers have made the fans both accessible and autoclavable. If an incubator with a fan becomes contaminated with moulds, the spores are blown into the cultures. Incubators with smooth unobstructed interiors that are easily dismantled and cleaned are preferable.

If there is an air intake at the top of the incubator, it is a good idea to cover it with fine gauze or permeable surgical dressing to prevent moulds and dust from dropping into the incubator. The tissue culture room should have an ultraviolet germicidal lamp, and it should be switched on whenever no one is in the room. Care should be taken to ensure that the ultraviolet light cannot be switched on when the room is occupied, as ultraviolet light can cause damage to the eyes and skin.

Temperature regulation and uniformity are important. The temperature gauge on most incubators cannot be trusted, and the internal temperature should be measured frequently with a thermometer in a beaker of water. It should be 37 ± 0.5°C. It is important that the incubator be adequately humidified to prevent drying of cultures.

(3) Liquid nitrogen facility. This is absolutely essential for storage of cells, and must be organized so that there is no possibility of the nitrogen running out.

(4) Inverted microscope, preferably with phase optics. This is absolutely essential for monitoring growth of cells.

(5) 37°C Water bath. This must be cleaned frequently, as it is a potent source of infection for cultures.

(6) Centrifuge. This does not need to be refrigerated.

(7) Sterile pipettes. Disposable plastic pipettes are convenient, but very expensive. Alternatively, if glass pipettes are used, a hot-air sterilizing oven will be required. The tissue culture room should be kept clean, tidy and uncluttered to minimize the chance of contamination.

(8) Gamma counter. This is desirable for screening assays, but not absolutely essential, because it can be replaced by autoradiography or nonisotopic assays such as enzyme-linked immunoassay.

(9) Others. Animal holding facilities, small forceps, scissors, tissue culture ware, standard microscope, haemocytometers.

(10) A note concerning yeast contamination. It has been observed that laboratory workers who make their own bread or pastry sometimes have great difficulty with contamination of tissue culture by yeasts, presumably owing to carry-over from the kitchen to the laboratory.

0 0

Post a comment