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The tissue, eg. kidney, testis, thyroid, is made into a single cell suspension by limited digestion with a proteolytic enzyme such as trypsin. The single cells are grown in glass or plastic bottles in buffered medium consisting of physiologic salts, an energy source (eg glucose), amino acids, antibiotics and usually animal serum. The cells adhere to glass & plastic surfaces, divide and form a confluent monolayers. After several days these cells must be split into 4-12 flasks by disrupting the monolayer back into single cells using trypsin with EDTA. This is called "passaging the cells". Cultures after the first passage from a tissue are called primary cell cultures, at the second pass, secondary cell cultures.