A unit was constructed that consisted of a core of hollow fibers through which low-molecular-weight substances, such as glucose and insulin, could pass freely but were impermeable to high-molecular-weight proteins, such as antibodies. Islets of Langerhans from normal rats were planted in the space surrounding the fibers, and either blood or nutrient medium was circulated through the fibers themselves. In experiments with animals, the units were attached to the vascular system of diabetic rats and monkeys. Blood glucose concentrations in the rats were reduced to nondiabetic levels within one hour and were maintained for the duration of the experiments. In monkeys the blood glucose level declined from 210 mg./100 ml. to 90 mg./100 ml. in four hours and insulin in the serum rose to 93 muU./ml. in one-half hour. Also, we have found that islets from monkeys cultivated in the artificial endocrine pancreas (AEP) continue to release insulin into circulating tissue culture medium for over eight months.

An improved chemically defined basal medium (CMRL-1415) has been used to advantage in studying the effects on trypsinized, newly explanted mouse embryo cells of certain glycoproteins from plasma and serum, certain nonprotein macromolecules, and various combinations of these, in stationary cultures. When protein and nonprotein fractions were separated from fetal calf serum, the entire growth activity was found to be associated with the protein. When 100 mg % of dialyzed, freeze-dried, supernatant solution of Cohn's fraction V (method 6) from human plasma was used as a supplement for CMRL-1415, there was considerable improvement in the cultures; and seromucoid prepared from calf serum had a similar effect. Supernatant V was further fractionated by gel filtration to give a threefold concentration of growth activity in a single, highly purified o~x-acid glycoprotein (orosomucoid). Starch gel electrophoresis of horse serum that was used to supplement the basal medium revealed a decrease of both oq-acid glycoprotein and ~-macroglobulin during the cultivation of mouse embryo cells. When horse serum was fractionated on DEAEcellulose columns, the only fraction that showed growth activity was a slow as-globulin. When the ~2-macroglobulin of Schultze was prepared from horse serum by salt precipitation, it was equally effective. When the ~2-macroglobulin from horse serum was tested (at 100 mg %) in combination with otl-acid glycoprotein from Supernatant V, seromucoid from calf serum, or unfractionated Supernatant V, the growth response was greatly in excess of that produced by any of these supplements tested separately. The ~2-macroglobufin from horse serum could be replaced by certain nonprotein macromolecules (e.g., dextran or Ficoll). ~hus, dextran (mol. wt. 100,000 to 200,000) had no visible effect on the cells when used alone at 0.1 or 1%. But when these levels of dextran were used in combination with low molecular weight glycoproteins (e.g., unfractionated Supernatant V at 100 mg %), the cultures remained active and healthy for unusually long periods.

An improved chemically defined medium, CMRL-1415, has been devised by testing the response of trypsinized, newly explanted mouse embryo cells in stationary cultures to various modifications of an earlier medium, CMRL-1066. The improvements are attributed to changes in amino acid levels, in the vitamin-coenzyme composition, and to an enhanced buffering capacity resulting from the use of free base amino acids, galactose and pyruvate, together with greatly reduced levels of glucose and sodium bicarbonate and the inclusion of both monobasic and dibasic sodium phosphate in the ratio 1 "4. When the medium is equilibrated with 5% COs in air, an initial pH of 7.2-7.4 is achieved, with excellent buffering capacity. CMRL-1415 contains 50 ingredients (9 fewer than CMRL-1066) and is prepared from six stable stock concentrates. By omitting sodium bicarbonate (to give CMRL-1415-ATM), the medium may be used in unsealed cultures in free gas exchange with air.

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Previous reports from this laboratory described a synthetic medium, solution 199, that contained 60 ingredients and supported cell life for an average period of four to five weeks. The assays were carried out in roller tubes in which chick embryo mesenchyme tissues were cultivated directly on the glass. More recently, the synthetic media have been improved very considerably, and Earle's replicate culture assay procedures have been adapted for use in testing them. By means of these techniques, it has been possible to make accurate quantitative experiments with new synthetic solutions some of which are capable of yielding, in one week, five- and sixfold increases in the population of replicate cultures prepared from washed suspensions of Earle's L strain mouse cells. This was acommplished by omitting the purines and pyrimidines from solution 199, by adding greatly increased levels of cysteine, ascorbic acid, and glutathione, and by the addition of certain purified coenzymes. In solution 703, which is described in detail, cultures of L strain cells have been maintained in a healthy, active state for over five months, with renewal of the medium twice a week.