Effect of Lactogenic Hormone on Embryonic Tissues Cultivated in vitro.

Since lactogenic hormone∗ is known to produce a specific effect on epithelial cells of the pigeon crop 1 in vivo, experiments were designed to determine whether it would stimulate the growth of embryonic pigeon crop epithelium cultivated in vitro. If stimulation could be observed in this manner such a test might be more delicate and superior to the present methods employed for determining hormonal potency.

Preliminary to testing the hormone on the specific crop epithelium, which reacts to it in vivo, experiments were carried out on the following non-specific tissues cultivated in vitro: (1) Embryonic chick connective tissue, (2) embryonic chick epidermis, (3) embryonic pigeon oesophageal epithelium. Finally the hormone was tested on cultures of embryonic epithelium taken from the crop sac areas known to be reactive in vivo in the hatched pigeons.

In all cases the dilutions were performed in triplicate. Also, every experiment was repeated 3 times before the results were reported. Each flask contained about 12 fragments of tissue.

(1) Embryonic Chick Connective Tissue. Chick heart tissue from 10- to 14-day-old embryos was used as the source of fibroblasts. The tissues were finely cut into fragments of about 1 mm. in diameter and immersed in Tyrode solution until ready for use.

The embryonic fluid was prepared by mincing 14-day-old chick embryos in a tissue grinder and suspending the pulp in 4 parts of Tyrode solution. The suspension was centrifugated at high speed to throw down all cellular material after which the clear supernatant fluid was carefully removed with a pipette. This clear liquid was used for coagulating the plasma as well as a nutrient for the tissue cells.

Guinea pig plasma was used in all experiments. This was collected by first drawing 1.0 cc. of a 1-1,000 solution of heparin into a 10.0 cc. syringe and then heart blood to the 10.0 cc. mark. The mixture was quickly centrifugated, the clear plasma removed with a pipette and kept in ice water until ready for use.