ORGAN CULTURE IN VITRO 



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ORGAN CULTURE IN VITRO 



Sci., 1912, 155, 1191), Thomson, G. D. 

 (Proc. Roy. Soc. Med., 1914, 7 and 

 Marcus Beck Lab. Reports, 21) , Chlopin, 

 N. (Arch. f. Mikr. Anat., 1922, 96, 

 435-493) and Maximow, A. (Contrib. 

 Embry. Carnegie Inst., 1925, 16, 49- 

 115), showed that small fragments of 

 chicken and mammalian embryos would 

 continue to develop when cultivated 

 in vitro. It has since been found that 

 isolated organ rudiments have a sur- 

 prising capacity for growth and differ- 

 entiation in culture. Thus the eye 

 rudiment of a 3-day check embryo forms 

 a retina of almost adult type during 

 cultivation (Strangeways, T. S. P., and 

 Fell, H. B., Proc. Roy. Soc, B, 1926, 

 100, 273-283); the rudiments of the 

 long-bones may increase to four times 

 their original length in 11 days, ossify 

 and even undergo considerable anatom- 

 ical development (Fell, H. B., and Robi- 

 son, R., Biochem. J., 1929, 23, 767-784, 

 and Fell, unpublished results) ; the early 

 stages of joint-formation take place 

 in vitro (Fell, H. B. and Canti, R., 

 Proc. Roy. Soc, B, 1934, 116, 316-351); 

 the thyroid rudiment forms vesicles 

 and secretion (Carpenter, E., J. Exper. 

 Zool., 1942, 89, 407-431); embryonic 

 ovaries produce sex cords and ova 

 (Martinovitch, P. N., Proc. Roy. Soc, 

 B, 1938, 125, 232-249, Ibid, 1939, 128, 

 138-143; Gaillard, P. J., Proc. Roy. 

 Neth. Acad, of Sci., 1950, 53). Some 

 organs which are already largely or 

 completely differentiated when ex- 

 planted, can be maintained in a healthy 

 state for various periods in vitro, e.g. 

 the prostate gland of infant rats (Price, 

 D., Ann. Rep. Strangeways Res. Lab., 



1949, 13) and mice (Lasnitzki, Z., Brit. 

 J. Cancer, in press), late foetal mouse 

 bones (Fell, H. B. and Mellanby, E., 

 Brit. Med. J., 1950, 2, 535-539), rabbit 

 lymph glands (Trowell, A. O., Exp. 

 Cell Research, in press), parathyroid 

 (Kooreman, P. J., and Gaillard, P. J., 

 Arch. Chir. Nederl., 1950, 2, 326), 

 pituitary (Martinovitch, P. N., Nature, 



1950, 165, 33-34). 



It is possible to practice organ culture 

 under very simple conditions. The 

 writer works on an open bench in a 

 small, clean culture room with a sealed 

 window and no outside ventilation; 

 the door of the culture room which 

 opens into a larger laboratory, is left 

 open. A separate culture room, though 

 an advantage, is not necessary, how- 

 ever. No mask or cap is worn. The 

 culture bench is covered with a large 

 sheet of plate glass; a beaker of distilled 

 water is kept boiling on a tripod beside 

 the operator and instruments, pipettes, 

 etc. are frequently rinsed in it. Direct 



sunlight is excluded by a green, trans- 

 lucent plastic curtain which makes a 

 very pleasant diffused light. Under 

 these conditions the writer often works 

 for weeks without losing a single ex- 

 plant from contamination. If occa- 

 sional colonies should appear, the 

 culture room is thoroughly steamed by 

 boiling a bucket of water over a gas 

 ring for some hours and the walls, 

 ceiling, floor and benches are then 

 wiped with a cloth impregnated with 

 lysol. This procedure usually abolishes 

 any airborne infection. 



It may well be that in a city, or in 

 a hot dusty atmosphere, much more 

 stringent precautions may be necessary 

 than these simple arrangements, but 

 it is advisable to try the simple organ- 

 isation first and only elaborate when 

 strictly necessary. 



1. Preparation of tissue. In experi- 

 ments with early organ rudiments, the 

 most difficult and also the most im- 

 portant part of the technique is to 

 dissect and handle the delicate tissues 

 without damaging the cells. For this 

 it is essential to prepare adequately 

 fine instruments. The writer uses a 

 narrow Graefe's cataract knife and an 

 ordinary sewing needle broken in half 

 and mounted in a thin glass rod. 



A No. 1 knife is best but it is much 

 too thick and coarse as it comes from 

 the makers, and for the dissection of 

 early embryos it must be ground to a 

 suitable shape by the worker himself. 

 It is first rubbed on a fairly coarse car- 

 borundum stone to render the distal 

 third of the blade as thin and flexible 

 as possible, and is then smoothed and 

 shaped on an Arkansas stone. Through- 

 out the sharpening process the knife is 

 repeatedly examined under a dissect- 

 ing binocular microscope and when 

 finished, the end third of the blade 

 should be narrow, very flexible and have 

 a perfect point; the flexibility should 

 be carefully graded or the end will snap. 

 In the writer's experience beginners 

 have great difficulty in learning to pre- 

 pare knives properly. 



The needle is mounted by melting 

 the tip of a glass rod and pushing the 

 broken end of the needle into it. The 

 needle should not be stuck too far into 

 the rod or the glass may crack during 

 sterilisation. To sharpen the point, 

 the needle is rotated with the finger and 

 thumb and, at the same time it is rubbed 

 on the end of an Arkansas stone. It 

 should be remembered that where 

 needles are sharpened the stone is 

 spoilt for knives. 



When the knives and needles are 

 ready, they are carefully wiped with 



