TISSUE CULTURE 



247 



TISSUE CULTURE 



15 X 180 mm. Using the tube as an 

 example, a thin laj'er of plasma and 

 nutrient medium is placed over the 

 inner surface to within 5 cm. of the 

 mouth, and while this plasma layer is 

 still liquid numerous small cell dumps 

 to be grown are embedded in it. After 

 the plasma has clotted about 1 cc. 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 end of a slowly rotating drum so 

 that as the drum rotates about its axis 

 the supernatant culture fluid is slowly 

 washed over the clumps of cells em- 

 bedded 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 are separated from the plasma 

 mass lining the culture tube by pushing 

 them loose with a pipette tip, removed 

 from the culture tube by means of the 

 pipette, cut to convenient size, and sub- 

 planted to new cultures. 



This type of culture is better adapted 

 than the slide culture for routine grow- 

 ing of large numbers of cell clumps, 

 since every test tube can accommodate 

 20 or more clumps, each of them at least 

 as large as that in a slide 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 probably 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 preparation. Moreover the 

 rotating mechanism for the roller-tube 

 unit is cheaply and easily constructed 

 while the cost of routine culture tubes 

 (pyrex test tubes) is only a few cents. 



But the use of "roller-tube" cultures 

 is not without its limitations. The thin 

 layer of plasma clot used is often eroded 

 through by the cells so that frequent 

 patching of the clot by fresh addition 

 of plasma becomes necessary. This 

 patching interferes with accuracy in 

 control of conditions of the culture and 

 the cultures themselves are not infre- 

 quently lost by eroding entirely out of 

 the clot. The curved tube surface, the 

 thick tube wall, and the distance of 

 separation v/hich they make necessary 

 between microscopic objective and con- 

 denser complicate examination and limit 

 it to low magnifications. Thishandicap 

 can be only partiallj' overcome by sub- 

 culturing to slide cultures to facilitate 

 detailed microscopic study. The sub- 

 culturing is objectionable because the 

 orientation of cells is interfered with 

 and new conditions require control. 

 Though numerous explants are pro- 



vided in roller tube cultures each of 

 them is usually small so that the total 

 volume of explanted tissue is not large. 

 The necessity of handling many cell 

 clumps makes the initial planting of the 

 cultures relatively slow. 



Carrel Flask Cultures, Carrel, A., J. 

 Exper. Med., 1923, 38, 407. These flasks 

 are made in many sizes of which the 

 "D" 3.5 flask will be described. It is 

 disc shaped, 3.5 cm. in diameter, with 

 topand bottom blown plane and parallel, 

 each about § mm. in thickness. The 

 sides of the flask are vertical, about 10 

 mm. high, so that the total separation 

 from top to bottom of the flask is about 

 10 mm. A side neck of 10 mm. internal 

 diameter, and 25 to 35 mm. long pro- 

 jects out from the side wall of the flask 

 and slopes upward at about 35° to the 

 bottom of tlie flask. 



The cell clump is planted on the bot- 

 tom of the flask in a layer of solid 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). Sometime after this has 

 clotted 1 to 2 cc. of the same fluid cul- 

 ture medium is added, the flask sealed 

 with a rubber stopper and incubated as 

 usual . About 3 times weekly the prepa- 

 ration is unsealed, the old culture me- 

 dium removed, the solid clot with its 

 contained cells soaked for about 15 min. 

 in isotonic saline, this saline removed, 

 fresh nutrient fluid added and the flask 

 resealed. At intervals of, say, 28 days, 

 the whole sheet of plasma may be 

 slipped loose from the floor of the flask, 

 poured out of the flask and the cell 

 sheet cut into explants of suitable size 

 and these reinoculated to make new 

 cultures in other flasks. 



This type of culture, like the "roller 

 tube" culture is well suited for carrying 

 relatively large numbers of cultures 

 over extended periods. Washing of the 

 culture and renewal of the culture fluid 

 can be done quickly. As routinely car- 

 ried out at the National Cancer In- 

 stitute, the actual time required for 2 

 relatively new operators to wash and 

 renew the nutrient medium on 142 cul- 

 tures is 70 min. from the time they enter 

 the sterile room to the sealing of the 

 last culture a substantial part of which 

 time is spent in setting up of apparatus 

 and preparation of solutions. Each 

 culture flask receives one explant of 

 about 4 to 4.5 mm. width and 15 mm. 

 length, while the thickness of the ex- 

 plant is only the thickness of the culture 

 sheet of the previous culture generation. 

 Since only one culture is made in each 

 culture flask, transplantation is rapid 



