ORGAN CULTURE IN VITRO 



245 



ORGAN CULTURE IN VITRO 



a clean duster, then rinsed in two 

 changes of fresh absolute alcohol and 

 thoroughly dried on a glass cloth. It 

 is convenient to sterilize a knife and 

 a needle together in a single tube. A 

 very firm, deep wad of cotton wool is 

 ranimed into the bottom of a test tube, 

 the instruments are slid gently into the 

 tube which is corked with a cotton wool 

 bung and sterilized by dry heat. It 

 is advisable to sterilize the knives and 

 needles point downwards, otherwise 

 moisture may condense on the steel 

 and corrode it. 



After use, the instruments should be 

 very carefully wiped to avoid rusting 

 and re-sharpened before being sterilized 

 again. When once the knives have 

 been properly ground, it takes only a 

 few minutes to clean and sharpen them 

 on the Arkansas stone. 



The explants are prepared under a 

 dissecting binocular, the stage of which 

 is fitted with a small glass shade to pre- 

 vent airborne contamination during 

 the manipulation. It is important to 

 select the right magnification and it is 

 often convenient to make the gross dis- 

 section (e.g. the removal of a limb or 

 eye from the body) under a low power 

 which provides ample depth of focus, 

 and to complete the process under a 

 higher magnification. The writer uses 

 transillumination for the dissection 

 which is done in a large hollow ground 

 slide containing Tyrode; the tissue 

 should not be left in Tyrode for longer 

 than is absolutely necessary. 



It is important to remove from the 

 rudiments as much of the epidermis 

 as possible, otherwise the epithelium 

 envelopes the explant, keratinizes and 

 the imprisoned cells degenerate. To 

 detach the epidermis the very thin, 

 sharp knife blade is slid beneath it and 

 the tissue is then cut by gently stroking 

 the edge of the knife with the point of 

 the needle. It is essential to reduce 

 trauma to a minimum, or the explants 

 will not prosper during subsequent 

 cultivation. 



There is an optimum size of explant. 

 If it is very minute it may not thrive 

 in vitro, though sometimes this difficulty 

 may be overcome by placing it in con- 

 tact with some other tissue (Borghese, 

 E., J. Anat., 1950, 84, 303). If the 

 explant is too large the interior be- 

 comes necrotic. There may also be 

 an optimum stage of development at 

 which a given tissue should be ex- 

 planted. If taken too early it may not 

 differentiate in complete isolation, 

 while if removed from the embryo a 

 few hours later it may attain an almost 

 adult structure. On the other hand, 



if explanted when differentiation is 

 already well advanced, the tissue may 

 be unable to adapt itself to life in vitro 

 and either degenerate or lose its charac- 

 teristic structure. 



2. Watch-glass method. In the 

 Strangeways Research Laboratory a 

 simple form of moist chamber is em- 

 ployed for organ culture (Fell, H. B. 

 and Robison, R., Biochem. J., 1929, 

 23, 767-784). It consists of a Petri 

 dish 80 mm. in diameter and 10 mm. 

 deep, carpeted with a thin layer of ab- 

 sorbent cotton wool in the center of 

 which a round hole is cut. A watch- 

 glass 40 mm. in diameter is laid over the 

 hole and the dish is sterilized by dry 

 heat. Ten cubic centimeters of sterile 

 aq. dest. is pipetted into the Petri 

 dish. It is not essential to use exactly 

 the size of watch-glass and Petri dish 

 mentioned; but it is important that the 

 diameter of the Petri dish should be 

 fairly large relative to that of the watch- 

 glass for otherwise the culture medium 

 will tend to dry. 



The culture medium is dropped into 

 the watch-glass with a pipette; the 

 first drop is always deposited on the 

 cotton wool as this is found to reduce 

 the risk of infection. The type of 

 medium depends on the tissue to be 

 cultivated. Many avian and mam- 

 malian rudiments grow well in a mixture 

 of fowl plasma and the extract of an old 

 (12-14 day) chick embryo. Bone rudi- 

 ments grow very well in a mixture of 

 3 pts. of plasma: 1 pt. of concentrated 

 extract of a 14-day chick embryo made 

 with Tyrode containing 1% glucose, 

 so that the final medium contains 0.25% 

 glucose. It is important to see that 

 the tissue has a suitable depth of me- 

 dium beneath it, as large explants soon 

 exhaust and partly liquefy the clot in 

 their neighborhood. For chick bone 

 rudiments of about 1.25 mm. in length 

 the writer uses 12 drops of medium, 

 but by the time they have attained a 

 length of about 5 mm. she adds 20 drops 

 to each watch-glass. Organ cultures 

 are often more fastidious about their 

 culture medium than unorganised tis- 

 sues. It is therefore advisable to use 

 freshly made embrj-o extract and 

 plasma which are not more than a week 

 old. Antibiotics are unnecessary. 



For many mammalian tissues an ad- 

 mixture of homologous plasma is desir- 

 able. Martinovitch, P. N. (Nature, 1950, 

 165, 33-34) grows the anterior pituitary 

 of young rats in a mixture of 6 drops of 

 heparinized rat plasma, 3 drops of 

 chicken plasma and 3 drops of concen- 

 trated chick embryo extract. 



When the organ rudiments are ready 



