The use, in diabetic rats and monkeys, of artificial capillary units containing cultured islets of Langerhans (artificial endocrine pancreas).

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.

Karyology: vaccine manufacturers' views.

Chromosomal analyses and life span studies of WI-38 cell cultures used for vaccine production at the Connaught Laboratories Ltd will be described. Twenty cultures derived from the same seed lot and 25 cultures derived from different starter cultures were analyzed. Karyology tests at population doublings 31 and higher were within acceptable limits. The WI-38 cells reached a finite lifespan of approximately 65 population doublings. Normal growth characteristics were observed within 2/3 of the finite lifespan, i.e. until population doubling 40. The finite lifespan of MRC-5 was found to be 75 population doublings, with normal growth pattern until passage 50. The karyology test results, cell yields and the finite lifespans indicated that these cells could be used as substrates for vaccine production at higher passage levels than previously suggested. Using serially subcultivated cells at higher passage levels would make possible an increase in the volume of vaccine from existing cell seed and would also result in significant reduction in production costs.

The use of foetal, calf and adult bovine sera for the growth of serially subcultivated diploid cells.

Medium CMRL-1969 supplemented with either foetal calf, calf or adult bovine sera, or a combination of the various sera, was used to subcultivate diploid cells for the preparation of substrates for vaccine production. Supplementation with calf or adult bovine sera gave satisfactory cell yields for at least 3-4 population doublings. The finite lifespan of the diploid cells grown in media supplemented with foetal calf was always longer than with calf or adult bovine sera. However, a combined supplement of adult serum plus a low concentration of foetal calf serum gave results equally as good as foetal calf serum alone. Sera obtained aseptically from adult animals kept in isolation and monitored constantly for health are more desirable than sera obtained from abattoirs. Selection of donor animals and pooling of certain serum batches were found to be important for the preparation of serum supplements. The final protein concentration of the cell culture medium should not exceed 0.8% when adult bovine sera are used. For the growth of WI-38 or MRC-5 cells the use of foetal calf serum can be reduced or eliminated.

Large-scale cultivation of cells in closed systems: stationary and moving.

Basic metabolic differences have been observed for cell cultures propagated in conventional stationary or rotating systems. The influence of these differences on virus characteristics is largely unknown but could be significant in maintenance of genetic stability of attenuated strains. Current requirements for good manufacturing procedures prompted the development of closed systems for both stationary and rotating cultures. Both are suitable for large-scale virus production. The methods will be described along with data on yields of a number of vaccine viruses.

Improved cell dispersal method by low-temperature incubation of tissue in a trypsin-citrate solution.

Tissues soaked at low temperature in a trypsin-citrate solution, washed to remove enzymes, and disrupted by rapid mechanical vibrations gave yields of viable cells 1.6 to 5 times those from conventional procedures.

Improved chemically defined basal medium (CMRL-1969) for primary monkey kidney and human diploid cells.

An improved tissue culture basal medium, CMRL-1969, supplemented with serum, has been evaluated by measuring the growth responses of primary cultures of trypsin-dispersed monkey kidney cells (PMKC) and of an established culture of a human diploid cell strain (HDCS). Medium H597, an early modification of medium 199 which has been used successfully in the preparation of poliomyelitis vaccine for 15 years, was used for comparison. In addition, parallel testing was done with Basal Medium Eagle (BME) widely used for the growth of HDCS. The improvements in basal medium CMRL-1969 are attributed to changes in amino acid concentrations, in vitamin composition, and, in particular, to enhanced buffering capacity. The latter has been achieved by the use of free-base amino acids and by increasing the dibasic sodium phosphate. The new medium has already been used to advantage for the production of polioviruses in PMKC where equivalent titers were obtained from cultures initiated with 70% of the number of cells required with earlier media. The population-doubling time was reduced in this system. Also, with small inocula of HDCS, the time required to obtain maximum cell yield was shorter with CMRL-1969 than with BME. Both media were supplemented with 10% calf serum. Maximum cell yields after repeated subcultivation in the new basal medium were greatly increased and the stability of the strain, as shown by chromosomal analysis, was not affected. Basal medium CMRL-1969 can be prepared easily in liquid or powdered form.

Effects of Tween 80 and freon 113 on measles virus.

Measles vaccines were prepared from the same virus fluids by inactivation with formaldehyde or by extraction with ether, ethyl acetate, or Freon 113 in the presence of Tween 80. Tests of antigenic potency, based on antibody levels in guinea pigs, showed that the formaldehyde-inactivated vaccines were more potent than the solvent-inactivated preparations and had the additional advantage of long shelf life. Residual Tween 80 in the solvent-extracted vaccines resulted in marked loss of immunogenic potency without significant loss of hemagglutinating activity. Neither extraction with organic solvents nor exhaustive dialysis efficiently removed Tween 80 from aqueous solutions.