TISSUE CULTURE 



346 



TISSUE CULTURE 



ml. of the same fluid culture medium 

 is added; the flask sealed with a rubber 

 stopper, and incubated as usual. 

 About 3 times weekly the preparation 

 is unsealed, the old culture medium re- 

 moved, the solid clot with its contained 

 cells soaked for about 15 min. in iso- 

 tonic balanced saline, this saline re- 

 moved, fresh nutrient fluid added, and 

 the flask resealed. At intervals of 

 for instance 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. These may be reinocu- 

 lated 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 

 carried out at the National Cancer In- 

 stitute, the actual time required for 2 

 operators to set up apparatus and solu- 

 tions, wash and renew the nutrient 

 medium on 200 plasma substrate cul- 

 tures is about 90-110 min. In plant- 

 ing, each culture flask receives one ex- 

 plant of about 3.0 to 4.5 mm. width 

 and 15 mm. length, while the thickness 

 of the explant is only the thickness of 

 the culture sheet of the previous cul- 

 ture generation. Since only one cul- 

 ture is used in each culture flask, trans- 

 plantation is rapid. Growth from this 

 type explant of a rapidly growing cell 

 strain will often routinely cover the 

 floor of the flask at 28 days. 



There are several advantages over 

 the roller tube culture. The plasma 

 clot is usually thicker (though a thin 

 clot can be used) so that there is less 

 trouble from clot erosion; with many 

 cell strains "patching" of the clot with 

 fresh plasma is not necessary. The 

 clot is of such thickness and texture that 

 it can be slipped loose from the flask 

 as a sheet, and slid out onto a sterile 

 glass plate, where the culture can be 

 easily and accurately cut up for sub- 

 inoculation by means of a Bard-Parker 

 ^11 blade attached to a j^7 handle. 

 Because a single very thin strip-shaped 

 explant is placed in each flask, actual 

 subplanting of cultures is much more 

 rapid than with the roller tube prepara- 

 tions in general use and the actual 

 amount of tissue is probably greater. 

 If desired, flask cultures may be in- 

 cubated on slowly rocking shelves but 

 this is necessary only in studies in 

 which the whole surface of the culture 

 must be washed with a moving film of 

 fluid. Cultures can be routinely photo- 



graphed at magnifications of 200 to 400 

 diameters and can be examined regu- 

 larly with up to a 4 nmi., 0.65 N.A. 

 achromatic objective. The 5.5 mm. 

 Bausch and Lomb objective of 0.65 

 N.A. is extremely useful. For higher 

 numerical aperture photographs sub- 

 inoculation must be made to slide 

 cultures. 



The general use of the Carrel flask has 

 been limited. Since the flasks require 

 a high quality of precision glassblowing, 

 thej^ are expensive. D3.5 flasks cur- 

 rently sell for $2.25 each. Apparently 

 the dimensional and fabrication speci- 

 fications of the flask were never pub- 

 lished so that many of the flasks sold 

 have been unsatisfactory in use, or 

 have been so fragile as to make their 

 use prohibitively expensive due to 

 breakage. (Satisfactory flasks are now 

 available from Mr. Otto Hopf, Upper 

 Black Eddy, Penn.) In many points 

 their manner of use and accessory ap- 

 paratus used with them has been in- 

 adequately described in text and papers 

 on tissue culture. The writer considers 

 the Carrel flask as the most satisfactory 

 of the three culture methods described 

 for routine qualitative work, and as 

 a method which warrants greater at- 

 tention than it has received. 



The preceding types of tissue culture 

 preparations have all allowed the 

 growth of a very small mass of tissue, 

 or of a very thin sheet of cells adherent 

 to a more or less plane substrate. An- 

 other type of three dimensional substrate 

 culture has recently been reported. 

 (Earle, W. R., SchilUng, E. L., Shan- 

 non, J. E., Jr., 1951 Annual Meeting 

 of the Tissue Culture Association, De- 

 troit; J. Nat. Cancer Inst., in press). 

 In some instances this was built from 

 folded perforated cellophane sheets; 

 in others it consisted of a mass of com- 

 mercially available Pyrex glass chem- 

 ical absorption tower packing helices 

 of I inch lumen, | mm. rod size. The 

 mouse strain L cells studied were im- 

 planted as a cell suspension and ad- 

 hered to the surfaces of the matrix, 

 while nutrient fluid was periodically 

 circulated through the interstices of 

 the substrate mass. Cell proliferation 

 of 4 X to 8.5 X the original inoculum 

 was obtained with both types of sub- 

 strates. In one instance an estimated 

 1145 mg. (wet weight) of cells was ob- 

 tained. In a number of instances the 

 weight exceeded 500 mg. per culture. 



It appears that the general methods 

 and principles involved in this type 

 culture may already be extrapolated to 

 make practical the growth of far larger 

 masses of tissue cells. Even this type 



