TISSUE CULTURE 



249 



TISSUE CULTURE 



apparently no satisfactory chemically 

 defined culture medium for the healthy 

 survival and active growth of any mam- 

 malian tissue in vitro for an extended 

 period of time, although there is a great 

 (.leal of research in progress with the aim 

 of obtaining such a medium (White, 

 P. R., Anat. Rec, 1946, 94, 61). 



6. Only small amounts of tissue can 

 be cultured by present methods. How- 

 ever, by using thin, strip-shaped ex- 

 plants, cultures of 25 or 30 mm. diam- 

 eter may be more or less routinely 

 grown from at least several cell types 

 (Earle, W. R., Arch. Path., 1939, 27, 

 88). Workers with roller tube and 

 Carrel flask cultures frequently have 

 been impressed with the extensive 

 sheets of cells arising from proliferation 

 of isolated cells which have scattered 

 over the surface of the medium by lique- 

 faction of the plasma around the parent 

 culture. Probably more extended 

 study of the explant, the cell support 

 and the nutrient medium will permit 

 growing routine cultures of far larger 

 tissue masses, and allow further uti- 

 lization of the use of tissue culture tech- 

 niques in research on vaccines and endo- 

 crine products. 



7. When a fragment of animal tissue 

 is first explanted to culture the proc- 

 esses of adaptation to the new condi- 

 tions are often very complicated. 

 Some cell types die rapidly, others more 

 slowly while still others are finally 

 eliminated apparently by overgrowth. 

 Meanwhile surviving cells are being 

 affected by products of necrosis and by 

 residual tissue substances brought over 

 from the parent organism by the ex- 

 planted tissue. The cycle of adapta- 

 tion and final dominance of a cell strain 

 able to survive under the culture condi- 

 tions may last for months. 



For many purposes short-term cul- 

 tures of freshly e.xplanted tissues are 

 entirely adequate and satisfactory. 

 For other purooses, however, the study 

 of such cultures carried only a few hrs., 

 or a few days, may be ineffective and 

 misleading in reflecting only the initial 

 reaction of the cells to the new culture 

 conditions. Where this is the case re- 

 sort must be made to the use of more 

 exacting culture methods which permit 

 longer experimental periods and the 

 use of relatively stable cell strains iso- 

 lated and maintained in vitro. 



8. The technique for long-term tissue 

 cultures under even incompletely con- 

 trolled experimental conditions requires 

 consideraole equipment and technical 

 assistance and an extensive period of 

 training both in operation of equip- 

 ment and in the maintenance of rigidly 



aseptic techniques. It is necessary to 

 establish a fundamental working knowl- 

 edge of the behavior of cells iu vitro. 

 Therefore much valuable time and dis- 

 appointment will be saved if, in the 

 initiation of any extensive tissue culture 

 program, adequate training of the 

 worker in an established tissue culture 

 laboratory is first insured. 



Measurement. 1. The most direct 

 method is to measure growth by in- 

 crease in weight of the culture; but 

 without a fibrin matrix growth of the 

 culture is often erratic, while if the 

 cells are grown in such a matrix extreme 

 difficulty is experienced in routinely 

 separating the cells from the matrix. 

 The data relate to cell mass, not to cell 

 number. 



2. Another way is to estimate change 

 in area of culture, the width being used 

 as an index. This is satisfactory for 

 experts observing long-term cultures, 

 but interpretation is difficult. An in- 

 crease in culture diameter may be due 

 to increase in mitosis or in cell migra- 

 tion. In some cases measurement of 

 growth by determination of surface 

 area is not feasible. The action of 20- 

 methylcholanthrene on mouse sub- 

 cutaneous fibroblasts so alters the archi- 

 tecture and cell density of the cultures, 

 the rate of cell migration that use of the 

 culture width or area as an index of cell 

 proliferation is grossly misleading. 



3. By determining the number of cells 

 seen in mitosis in a culture area relative 

 to the total number of cells seen in that 

 area an estimate may be made of the 

 relative frequency of cell mitotic pro- 

 liferation. This method is valid only 

 if it is clearly shown that the experi- 

 mental conditions do not disturb the 

 duration of mitosis, or if correction is 

 made for such disturbance. Possible 

 complication from a diurnal variation 

 in the rate of cell mitosis must also be 

 eliminated. The value of the method 

 may be increased by development of 

 comparative microcinematographic 

 techniques and also by use of Chalkey's 

 methods for statistical analysis of tissue 

 elements (Chalkey, H. W., J. Nat. 

 Cancer Inst., 1943,4, 47). 



4. Chemical methods estimating cul- 

 ture growth by determination of in- 

 crease in ash content, aerobic or an- 

 aerobic oxygen consumption, glycolysis, 

 etc. are at present not entirely satis- 

 factory but measurement of intake of 

 radioactive isotopes may prove helpful. 

 See in this connection Cohn, W. E., and 

 Bones, A. M., J. Gen. Physiol., 1945, 

 28, 449. 



Many factors, often unknown, influ- 

 ence results. Sometimes these are 



