TISSUE CULTURE 



Mi 



TISSUE CULTURE 



culture fluid and limited access of the 

 cells to adequate oxygenation. 



In Roller Tube Cultures (Gey.G.O.and 

 M. K., Am. J. Cancer, 1936, 27, 45) 

 the culture vessel is a test tube usually 

 about 15 X 150 mm., or in some cases a 

 larger container is used. A thin layer 

 of plasma and nutrient medium is 

 coated over the inner surface of the 

 tube to within 5 cm. of the mouth, and 

 while this plasma layer is still liquid 

 numerous small tissue fragments are 

 embedded in it. After the plasma has 

 clotted, about 1 ml. of nutrient solution 

 is added and the tube sealed with a 

 rubber stopper. In the incubator the 

 tube is slipped into a hole in the front 

 end of a drum-shaped frame rotating 

 at about 7-10 r.p.h., so that as the 

 drum rotates about its axis the super- 

 natant culture fluid slowly washes over 

 the clumps of cells embedded in the 

 plasma lining the tube. The fluid is 

 changed every 2 to 4 days. At periods 

 of 9 to 15 days, colonies of cells may be 

 dissected loose from the plasma film 

 lining the culture tube. They are then 

 removed from the culture tube by 

 means of the pipette, cut to convenient 

 size, and subplanted to new cultures. 



This type of culture is better adapted 

 than is the coverslip preparation for 

 routine growing of large numbers of 

 cell clumps since each test tube can 

 accommodate 5 to 20 cell clumps, each 

 of them as large as that in a coverslip 

 culture. The fluid can be readily 

 changed with only minimal disturbance 

 of the embedded cultures. Where an 

 extensive series of cultures is carried 

 bacterial infection is usually less 

 troublesome than with slide cultures. 

 Since the tube may be sealed with a 

 rubber stopper, there is less gas (CO2 

 and O2) leakage than in the slide prepa- 

 ration (Earle, W. R., U. S. Pub. Health 

 Reports, 1931, 46, 199S). Moreover the 

 rotating mechanism for the roller-tube 

 unit is cheaply and easily constructed 

 and the cost of routine culture tubes 

 (pyrex test tubes) is only a few cents. 



But the use of "roller-tube" cultures 

 is not without limitations. The thin 

 layer of plasma clot used is often eroded 

 by the cells so that frequent patching 

 of the clot by fresh additions of plasma 

 becomes necessary. This patching in- 

 terferes with accuracy in control of 

 conditions of the culture and the cul- 

 tures themselves are not infrequently 

 lost by eroding entirely out of the clot. 

 The curved tube surface, the thick tube 

 wall, and the separation which the 

 tube makes necessary between micro- 

 scope objective and condenser all com- 

 plicate microscopic examination and 



limit it to low magnifications. This 

 handicap can be partially overcome by 

 subculturing to coverslip preparations 

 for detailed microscopic study, but this 

 is objectionable because of disturbance 

 of the culture and because of the poor 

 control of culture conditions in the 

 coverslip preparations. Although each 

 roller tube may contain a number of 

 implants, each of them is usually small 

 so that the total volume of explanted 

 tissue is not large. The consequent 

 necessity of handling many cell clumps 

 makes the initial planting of the cul- 

 tures relatively slow. 



With some cell types, following lique- 

 faction of the plasma film in the roller 

 tube, loose cells scatter over the inner 

 surface of the tube and proliferate 

 luxuriantly. This gives roller tube 

 cultures which can be handled and 

 transferred by scraping and shaking 

 cells loose, as described below for the 

 cellophane substrate cultures. 



Attempts have been made to adapt 

 the cellophane substrate to roller tubes 

 by inserting a loose sleeve of perforated 

 cellophane in the roller tube on top of 

 the cell clumps. Some workers have 

 reported that the cellophane sleeve has 

 a tendency to rotate within the tube 

 and so scrape cells off. Some method 

 must be used to prevent this. Other 

 workers have apparently had extremely 

 satisfactory results with cellophane 

 substrate roller tube cultures. Too 

 little information is at present available 

 to evaluate the method. 



Flasks for Carrel Flask Cultures 

 (Carrel, A., J. Exp. Med., 1923, 38, 407) 

 are made in several sizes. A well made 

 "D" 3.5 type flask, as currently used is 

 disc shaped, 3.5 cm. in diameter, with 

 top and bottom blown plane and paral- 

 lel, each about ^ mm. in thickness. 

 The sides of the flask are vertical, so 

 that the total separation from top to 

 bottom of the flask is 10.0 to 11.0 mm. 

 A side neck of 10.3-10.8 mm. internal 

 diameter, 1.0-1.3 mm. wall thickness, 

 32-34 mm. length, projects from the 

 side wall of the flask and slopes upward 

 to form an angle of 140° with the top 

 surface of the flask. The end of the 

 throat is fire-polished and free from 

 bead or overhang. The entire flask is 

 made of Pyrex glass, and is oven an- 

 nealed to be strain-free under polarized 

 light. 



The cell clump is planted on the bot- 

 tom of the flask in a layer of medium 

 which consists of 0.6 cc. of chicken 

 plasma and 0.7 cc. of some fluid culture 

 medium (20% chick embryo extract, 

 40% horse serum and 40% physiological 

 saline). After this has clotted, 1 to 2 



