Equipments Required for Animal Cell Culture
Laminar Flow Cabinets
LAF hoods are the aseptic working table for inoculation of animal cells. The basic purpose of using a LAF hood is to provide protection from contamination from any organism like fungi or bacterial cells under aseptic conditions, and to protect the operator from potential infection risk of infection from the cultured cells.
The working area of LAF hood is first made sterile by using 70% ethanol. When the LAF is kept in “ON” position, the sterile air flows inside the cabinet which maintains the sterile conditions required for the transfer of cultured cells.Depending on the nature of the cells and organisms being handled, tissue culture hoods can be grouped as follows :
a) Class I hoods are found with in specially designed sterile work areas and give good protection to the operator and, to a lesser degree, the cell culture. There is an open front from which the air is drawn over the cell culture and goes out through the top of the hood.
b) Class II hoods offer protection to both operator and the cell culture and is the most common type found in a tissue culture laboratory. The cell culture is protected in a stream of sterile air and the operator is protected from contamination by the inflow of air into the base of the work area. The inflow of stream of sterile air into the base of work area protects the culture and operator from contamination.
c) Class III hoods contains a full physical barrier which screens the worker, and is mainly used for working with highly pathogenic organisms. In this, a physical barrier separates the operator from the inoculation work. The open front is replaced with glass or Perspex with a pair of heavy duty gloves attached to it. All the work is assessed from this glass.
The CO2 incubators provide the suitable environmental conditions to the growing animal cells. Generally CO2 incubators are used in animal cell cultures. This is
a) to maintain the sterility of the chamber for which filtered High Efficiency Particulate Air (HEPA) is used.
b) to maintain constant temperature the incubators is made airtight using a silicon gasket on the inner door.
c) to keep an atmosphere with a fixed level of CO2 and high relative humidity which prevents the dessication of the medium and maintains the osmolality?
This type of microscope is used for visualizing cell cultures in situ.The cells in culture vessel remain at the bottom of the vessel and the medium floats above the growing cells. It is impossible to observe these cells under the ordinary microscope, therefore, the inverted microscope is used for such purposes. The inverted microscope has the optical system at the bottom and the light source at the top, this arrangement helps to observe the cultured cells in the plates.
Only low speed centrifuges are used generally at 20oC to avoid disruption of the separated bands of cells. The motor releases the heat which leads to the increase in temperature. Therefore, use of low temperature for centrifugation is recommended so that cells are not exposed to high temperature.
Besides these conditions, the culture rooms should have light (diffused light and darkness each for a period of 12 hours) and temperature maintained at 25+/- 20C, with relative humidity at 98% and uniform air ventilation. The cultures should be monitored at regular intervals under aseptic conditions.
Sterilized Glassware, culture media and other equipments
The glassware are thoroughly washed and all the equipment sterilized by heat, steam, or Millipore filter paper. The glassware like glass coverslips, instruments, Pasteur pipettes, test tubes etc are sterilized by dry heat. Apparatus containing glass and silicon tubing, disposable tips for micropipettes, screw caps, Millipore filters etc are sterilized by autoclaving.
Isolation of animal material (Tissue)
The culture animal material is washed in balanced salt solution to avoid contamination. The tissue to be cultured should be properly sterilized with 70% ethanol and removed surgically under aseptic conditions.
Disaggregation of tissue – To obtain the cell suspension for primary cell culture, the tissue is disintegrated either mechanically or by using enzymes.
(i) Physical or mechanical disaggregation- After removing the tissue under aseptic conditions, it is pressed through a sieve of 100 micrometer. It is then kept in a sterile Petri dish containing buffered medium with balanced salt solution. The cells are then alternately passed through the sieve of decreasing pore size (50 micrometer and 20 micrometer mesh). The debris which remains on the sieve is discarded and the medium containing cells is collected and cells are counted by using haemocytometer. This method is cheap and quick but it damages a lot of cells.
(ii) Enzymatic disaggregation- In this method, enzymes are used for dislodging the cells of tissues. The two important enzymes used in tissue disaggregation are-collagenase and trypsin. -a) Collagenase- The intracellular matrix contains collagen therefore collagenase is used for disaggregation of embryonic, normal as well as malignant tissues. The tissues are kept in medium containing antibiotics and then dissected into pieces in basal salt solution. After washing the chopped tissue with distilled water, it is transferred to complete medium containing collagenase. After a few days (around 5 days), the mixture is pipetted so that the medium gets dispersed. The whole treatment is left for sometimes during which the epithelial cells settle on bottom of test tubes. The enzyme collagenase is removed by centrifugation. Suspension consists of cells which are then plated out on the medium. (b) Trypsin- Use of trypsin for disaggregation is called trypsinization. On the basis of role of temperature on trypsin, the activity of trypsin is of two types- Cold trypsinization and warm trypsinization.
Cold trypsinization- The sample tissue to be disaggregated is chopped into 2-3 small pieces and kept in sterile glass vial. The tissues are subsequently washed with sterile water and dissected and then kept in BSS. The whole content is then placed on ice and soaked in cold trypsin for 4-6 hours to allow the penetration of enzymes in tissue. After this the trypsin is removed and the tissue is incubated at 36.50C for 20-30 minutes. About 10 ml of medium containing serum is added to the vials containing the cells and the cells are dispersed by repeated pipetting. The cells are counted by haemocytometer and are plated and incubated for 48-72 hours for cell growth.
Warm trypsinization- The initial steps are the same as in cold trypsinization however, in this case the tissue pieces are treated with warm trypsin (36.50C). The tissues are stirred for 4 hours and then pieces are allowed to settle down. The disassociated cells are collected at every 30 minutes. The process is repeated by adding fresh trypsin back to pieces and incubating the contents. The trypsin is removed by centrifugation after 3-4 hours during which the complete disaggregation of tissues takes place. The glass vials containing dispersed cells are then placed on ice. The cells are counted using haemocytometer and cell density is maintained at an appropriate number. The cells are then plated on medium and incubated for 48-72 hours for cell growth.
(iii) Treatment with chelating agents- The tissues like epithelium (which needs Ca2+ and Mg2+ ions for it’s integrity are treated with chelating agents such as citrate and ethylene-diamine-tetra-acetic acid (EDTA). Chelating agents are mainly used for production of cell suspensions from established cultures of epithelial type.