29. AN INTRODUCTION TO 

 TISSUE CULTURE TECHNIQUES 



Since the technique of tissue culture (i.e. the growth of cells or tissues apart from their 

 normal environment) is receiving accelerated attention, it is proposed here to summarize 

 the essentials of the procedures, followed by a reference list of books and papers which 

 will allow the qualified student to pursue the subject further. 



Tissue culturing developed from experimental embryology, particularly with poikilother- 

 mic forms, although the original study was by Roux in 1885 with chick medullary plate. 

 Harrison (1907) sustained frog medullary tubes in frog lymph. This nutritional medium 

 was supplanted by the plasma clot, then by tissue and embryonic extracts and synthetic 

 media. It was Carrel who sustained chick cells in vitro over many years. The current 

 trend is combining in vitro and in vivo studies, particularly in relation to the develop- 

 ment of cancer. 



To maintain a cell or cells for any length of time, there are a number of basic require- 

 ments. There must be regulation of the temperature, osmotic pressure, pH and other 

 inorganic ion control, adequate and varied metabolites must be available, and vitamins, 

 enzymes, and substrates in proper balance, and a matrix within which the cells can grow. 

 In other words, the living cell must be given an environment approximating that of its 

 natural environment as closely as possible in order to survive. 



The medium for culturing must be very carefully selected with respect to the type of cell 

 or cells to be studied. There is no universal medium satisfactory for all cells or organs, 

 even among the cold-blooded animals. There are, in general, three types of media: the 

 plasma coagulum, biological fluids, and tissue or embryonic extracts. For the plasma 

 coagulum, blood is generally taken from the wing, carotid artery or heart of the hen and 

 prevented from coagulating either with anti-coagulating agents or by the prevention of 

 contamination. Rat-tail collagen has recently been used instead of the plasma clot and 

 agar is sometimes satisfactory. For the biological fluid, serum is the most satisfactory 

 and may be obtained from horse, calf, or human placental cord or even adult blood. The 

 serum is obtained by allowing natural clotting of the whole blood and filtering and steriliz- 

 ing the exuded serum. Bovine amniotic fluid is also used, as is the aqueous humor. For 

 the tissue extracts , the embryo has traditionally been used but this may soon be replaced 

 by synthetic media, for some tissues at least. Autologous and heterologous sources of 

 serum or extract may or may not be toxic, the test must be empirical. Culture media 

 may be chosen for short or long term survival of cells, for slow or rapid growth, or even 

 indefinite growth and also for certain specialized functions. Since there is a variety of 

 combinations of the essential salts which will allow survival for warm or for cold-blooded 

 cells or organ explants, the most frequently used formulae will be presented below. There 

 are many synthetic media which include various organic acids, vitamins, and hormones 

 which are used in special instances (see Paul '60). One of these, for instance, is known 

 as Eagle's medium consisting of 33 different compounds. 



In all tissue culture work the prime requisite is absolute cleanliness because bacteria, 

 fungi and viruses can quickly ruin any culture. Further, chemical contamination is also 

 a constant threat because even some of the so-called cleaning fluids are so tenacious that 

 they also kill the living cells which constitute the culture. The glassware and bottle stop- 

 pers are also a source of contamination. Thus, there is coming into use polystyrene, 

 perspex, cellophane, and other plastics which can be purchased sterile to be discarded 

 after a single use. The sulfur in rubber stoppers and tubing is also toxic and must be 

 washed off completely. Silicone stoppers are now replacing rubber stoppers. Instru- 

 ments must always be sterile, as must the hands and air surrounding the cultures, 



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