On the bottom of the flask is spread the medium which must supply the tissues with a scaffold for their growth and with the substances necessary for the synthesis of new protoplasm. This medium is composed of two parts, one solid and the other fluid (fig. I). The solid part is made of coagulated blood plasma, that is, of a clot of blood from which the red and white corpuscles have been removed. Such a plasma is obtained by freeing blood of its corpuscles by centrifugation. It then becomes a clear, citrin fluid which is introduced into the flask with a pipette. The tissue cells are next deposited by a spatula in this medium which coagu lates after a few minutes. Thus they become embedded in a trans parent jelly, which they use as a scaffold, but not as a nutrient medium. The fluid medium is placed on the surface of the coagu lum. Its role is to remove the waste products of the culture, and to supply the tissues with the necessary nutrient substances.
Every two or three days, the fluid is removed and replaced by a large amount of a saline solution composed of sodium, potassium and calcium chlorides, sodium phosphate and bicarbonate, and glucose, which is called Tyrode solution. After the tissues have been thoroughly washed, the fluid is removed and replaced by a small amount of nutrient medium. This medium ordinarily con sists of embryo proteins, or proteoses and peptones, or blood serum, according to the nature of the tissues. The tissues and fluids are handled with sterile instruments in rooms which are kept strictly clean. It is most important that no bacteria be al lowed to penetrate into the flasks. If infection of the medium takes place, death of the tissues soon occurs. This accident is almost completely prevented by the recently developed techniques. After the preparation of the culture is completed, the flasks are placed in an incubator and maintained at body temperature.
Several cell types have so far been obtained in pure state : con nective tissue cells or fibroblasts, epithelial cells, blood and tissue macrophages, and the malignant elements of a few tumors.
Strains of fibroblasts are cultivated easily from almost every em bryo, or adult tissue of human beings, dogs, rats, mice, fowls, etc.
They remain packed together while macrophages wander through the medium. In addition, they multiply more rapidly than epithe lial cells. Therefore, it is easy to obtain colonies composed exclus ively of them. Fibroblasts are either polygonal or spindle shaped with long, sharp processes, and with an oval nucleus which generally contains two smaller nucleoli. They aggregate in a dense tissue which doubles in volume every 48 hours when kept at body temperature in a medium composed chiefly of embryo proteins. They grow indefinitely. A strain of chicken fibroblasts isolated 16 years ago is multiplying to-day at the same rate as at the begin ning of its life in vitro. When the flasks are taken from the incubator and kept at room temperature, the cells stop multi plying and remain in this condition for three or four weeks without dying. Therefore, living culture cells can be sent for long dis
tances without inconvenience. Strains of tissue cells have been exchanged between laboratories located in New York and Berlin and after this long journey have promptly regained their usual rate of growth. Epithelial cells are more difficult to obtain in pure culture. Nevertheless, since Fischer isolated a strain of epi thelium from the few cells of the iris which adhere to the lens of the chick embryo, pure cultures of thyroid gland, Malpighian epithelium, lens epithelium and several types of carcinomatous cells have been obtained by Ebeling, Kirby, Fischer and Carrel. Their rate of growth is less than that of the connective tissue cells, but they can be maintained in a condition of active life for sev eral years. They keep their normal characteristics indefinitely. They grow in a thin layer at the surface of the solid medium, and rarely form a dense tissue. They readily digest the coagulum. Leucocytes from the blood and wandering cells from the tissues have also been obtained in pure cultures. They do not aggregate as a tissue, but remain isolated from one another and scatter through the medium. When they accumulate in a large number or are packed together, they generally die or become transformed into fibroblasts. Their growth is slow and they are less resistant than epithelial or connective tissue cells. However, they can live in flasks for several months. The malignant elements of sarcomata and carcinomata have also been isolated and maintained alive indefinitely outside of the body.
The rate of growth of the colonies of epithelial cells and of fibroblasts can easily be ascertained. The measurements are made by placing the flasks on a projectoscope and drawing the outline of the colony immediately after the preparation of the culture and subsequently every two days. The area of the colony at the dif ferent stages of its development is measured with a planimeter, and the ratio of the area of new growth to that of the original fragment is calculated. The relative increase of the colony in 48 hours expresses the rate of growth. The curve representing the continuous growth of a colony of epithelial cells or of fibroblasts in a flask containing a nutrient medium is a parabola. If the medium is non-nutrient, the curve is S-shaped. Modifications in the form of this curve indicate whether the medium contains substances that activate or depress the rate of cell multiplication. This method cannot be used for measuring the activity of wander ing cells, because they never grow as a tissue, but remain at cer tain distances from one another and scatter on several planes. Their rate of multiplication can be approximately reckoned by the time taken by the cells to invade the entire area of the flask.