ORGAN CULTURE IN VITRO 



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ORGAN CULTURE IN VITRO 



for explantation, the writer washes 

 them in fresh Tyrode which is then 

 removed and replaced by embryo ex- 

 tract of about half the concentration 

 of that used for the culture medium 

 and lacking the extra glucose. They 

 are sucked into a pipette and deposited 

 on the surface of the clot in the watch- 

 glass. The Petri dish is then placed 

 under the shade on the dissecting 

 binocular microscope, the lid removed 

 and the surplus extract carefully sucked 

 off with an extremely fine pipette, care 

 being taken not to damage the clot. 

 Organ cultures do not grow well under 

 a fluid phase, but they quickly become 

 surrounded by a shallow pool of fluid 

 exuded from the clot. 



The Petri dishes are not sealed, but 

 under the conditions described, the 

 medium does not dry. If the dishes 

 are laid directly on the incubator shelf, 

 drops of moisture form on the inner 

 surface of the lid, but this can easily 

 be prevented by placing the vessels on 

 planks of wood which are left perma- 

 nently in the incubator. Should drops 

 form in spite of this precaution, it is 

 probable that the temperature of the 

 incubator is fluctuating unduly. 



Many tissues grow and develop well 

 at normal body temperature, but others 

 do better if the temperature is lowered 

 to 34°C. Martinovitch has kept rat 

 and mouse ovarian explants and rat 

 pituitaries in a healthy differentiated 

 state for several weeks at 34°C. 



The writer transfers explants to fresh 

 medium three, or in the case of large, 

 rapidly growing avian rudiments, four 

 times a week. To make the fourth 

 transplantation, she prepares a double 

 set of watch-glasses on the Friday of 

 each week; one set is employed the same 

 day and the other placed in the refriger- 

 ator for use on the Saturday when 

 the explants are again subcultivated. 

 They are then left undisturbed until 

 the Monday. This routine gives excel- 

 lent results for cultures of avian long 

 bone rudiments. 



During incubation, some types of 

 explant become firmly anchored to the 

 clot by migrating cells. To detach the 

 tissue without damage, the clot is 

 pulled away on one side so as to rupture 

 the zone of outgrowth; the explant is 

 then loosened from the underlying 

 clot with the knife and needle, after 

 which the zone of growth on the other 

 side is ruptured, so that the explant 

 now lies freely in the pool of exuded 

 fluid. It is advisable not to rupture 

 both sides of the zone of growth before 

 detaching the explant from the clot; 

 none of the old clot should be left at- 



tached to the tissue. The explant is 

 sucked into a pipette containing a little 

 Tyrode and deposited in a hollow- 

 ground slide in a Petri dish. The 

 Tyrode is replaced by the more dilute 

 extract mentioned above and the tissue 

 is placed on a fresh clot in the manner 

 already described. 



3. Gaillard's method. Professor P. J. 

 Gaillard (personal communication) uses 

 a rather different technique which has 

 given excellent results in the cultiva- 

 tion of the parathyroid from human 

 infants and of the human fetal ovary. 



The explants are cultivated in em- 

 bryological watch glasses sealed with 

 a small glass plate. They are grown 

 on a very soft clot composed of a mix- 

 ture of (a) 5-15% adult human blood 

 plasma containing 0.5 cc. of a 0.1% 

 solution of heparin for 10 cc. of blood, 

 (b) 10% placental vein serum, (c) 

 65-75% of Gey's balanced saline solu- 

 tion, (d) human fetal brain press juice. 

 To make the brain press juice, frag- 

 ments of fetal brain are placed in a 

 Petri dish at 4°C. for 24 hrs. which 

 facilitates the separation of the tissue 

 fluid; the tissue is then minced with a 

 tissue press, an equal quantity of Gey's 

 saline solution is added and the mix- 

 ture is centrifuged for 15 min. at 6,000- 

 8,000 r.p.m. The supernatant fluid is 

 decanted into Pyrex glass tubes and 

 stored at — 20°C. The explants are 

 transferred to fresh medium twice a 

 week. 



Parathyroid explants completely 

 liquefy the clot in 3 days. During the 

 first few days in vitro the peripheral 

 zone degenerates and is washed away 

 during transplantation, leaving a 

 healthy ball of tissue which enlarges 

 during subsequent cultivation and may 

 survive for as long as 60 days. The 

 central part of the ovarian cultures 

 degenerates, but the necrotic matter 

 is later resorbed and replaced by 

 healthy tissue growing in from the 

 periphery. 



4. Other methods. Recently Trowell 

 has devised a modification of the watch- 

 glass method which can be used for 

 metabolic studies and which enables 

 organized tissue to be grown in a fluid 

 medium in any type of gaseous at- 

 mosphere required. He has success- 

 fully grown rabbit lymph glands by 

 this technique which should be ap- 

 plicable to many physiological prob- 

 lems. 



Some rudiments develop well in 

 tubes (Strangeways, T. S. P. and Fell, 

 H. B., Proc. Roy. Soc, B, 1926, 100, 

 273-283). The medium is placed in a 

 small test tube (2" x ^") and allowed to 



