Charles M. Pomerat

QUANTITATIVE CINE ANALYSIS OF CELL ORGANOID ACTIVITY

Recent improvements in the technique for managing cells in vilro, together with a growing appreciation of the value of phase-contrast, time-lapse cinematography in the development of dynamic concepts regarding human elements, invite precise studies on drug action. Such operations can be facilitated with the use of perfusion chambers of the types described by Pomerat,’ Christiansen, Danes, Allen and Leinfelder? and Buchsbaum, Wertman, Dwyer, Kuntz, and Gillette.3 In his “Cell Physiology and Pharmacology”, Danielli4 has presented important suggestions for attacks on the problem of the mechanism of drug action a t the cellular level. For the tissue culturist concerned with behavioral activities which can be recorded with time-lapse photography, it becomes necessary not only to be thoroughly familiar with the mobile structures themselves, but also to evolve techniques by which the data available in the dynamic film projection can be converted to the static printed page by using reproductions of selected film frames. Of even greater importance is the necessity for evolving simple, reliable methods for converting positional changes of cell organoids into graphs from which generalized formulas or constants may be extracted. A method which would make possible an accurate evaluation of membrane activity associated with pinocylosis (cell drinking) might be of inestimable value in describing the action of surface-active agents such as detergents. A better appreciation of factors regulating the speed of cell intake could prove of practical value in reducing or increasing the ingestion rate of components introduced into the culture medium. Such information would enrich our knowledge of cell nutrition and of drug action. Frederic5 and his associate have made important inroads in the analysis of changes in mitochondria1 movement and form. Because their contributions in this field are represented elsewhere in this monograph we have omitted discussion of mitochondria per se in this article. However, some overlap of statements will be found with those in a report6 made a t the Lisbon meetings of the SociCtC Internationale de Chirurgie where some of the same ground was covered and certain essential bibliographical references were noted. The present paper is concerned particularly with the mechanics of analysis and the reproduction of new data.

CELLULAR CHANGES INDUCED BY RADIATION

At a recent Ciba Foundation symposium, van Bekkuml stated: “Biochemical changes that can be demonstrated before structural damage to the cells becomes apparent are of the greatest interest, because these may be expected to be closely related to or even identical with the primary biochemical lesion.” For more than a decade our tissue culture laboratory has been guided by such a point of view. To achieve our purposes, an eclectic position has been assumed in the use of a wide variety of methods for cell cultivation, but with special attention given to phase-contrast microscopy and the development of cinematographic devices, including instrumentation for the simultaneous recording of the time, temperature, and action potentials. More than 250,000 feet of 16-mm. film records have been accumulated that serve as a reference library regarding the behavior of various species of cells i.n vitro. We have been especially concerned with activities such as membrane movement, pinocytosis, the mobility of mitochondria, the motion of aggregated bodies in the juxtanuclear zone, nuclear rotation, contractions of the nuclear membrane, and the rhythmic pulsation of whole cells or their proceses. These records have invited quantitative analyses in relation to metabolic activity. While an immense amount of work remains unfinished, preliminary inroads have been made in the description of some activity manifestations of the cell with a group of techniques designed to convert dynamic cinematographic records into graphical summaries. The analysis of nuclear rotation in the epithelium of adult human nasal mucosa (PomeraP) and the ascent of vacuoles in the axoplasm from the growth cones of regenerating neurons from chick dorsal root ganglia as a result of pinocytosis (Nakai3) are representative of this effort. Cinematographic records have been particularly useful in the study of events taking place in the course of mitosis. Occasionally, film sequences accumulated with other goals in view have made invaluable data available for the study of cell division (Hsu,~ Hsu and Moorhead,6 Moorhead and Hsufi). In attempting to apply in vitro findings regarding biochemical mechanisms involved in ionizing radiations to the injury observed in irradiated cells, it is hoped that the analysis of time lapse, phase cinematographic records may prove useful. The review of the literature on the use of tissue cultures in the study of ionizing radiations by Stroud and Brues’ is a valuable key to the contributions in this field. A summary statement regarding our own efforts was presented a t the Conference on Research in Radiology held a t Highland Park, Ill., on

Reticulo-Endothelial Immune Serum (REIS). VI. Production of Potent Serum by Anamnestic Reaction.

Summary In the preparation of antiorgan sera the administration of small amounts of antigen (spleen and bone marrow) was found more effective than the rapid immunization of rabbits with massive antigen dosage. A single injection of the antigen 30-40 days after the last dose gives rise to an anamnestic response of the host and to a serum of higher potency. This can be evaluated by complement fixation titres and by the outgrowth of tissue explants exposed to the action of the “anamnestic” serum.

IDENTIFICATION OF THE INHIBITORY FACTOR OF RETICULO-ENDOTHELIAL IMMUNE SERUM (REIS) IN A GLOBULIN FRACTION.

SUCCESSFUL demonstration of the inhibitory properties of strong concentrations of reticulo-endothelial immune serum (REIS) by in vitro (Pomerat and Anigstein2) and in vivo methods (Anigstein and Pomerat3) as well as evidence for its stimulating action at high dilution (Pomerat4) have initiated a search for the active principle involved. Isolation of albumin and globulin fractions of REIS have been carried out according to the following procedure. To 5.0 ml of serum was added, with constant stirring, an equal volume of saturated ammonium sulfate. The suspension was allowed to stand for one hour at 370, following which the precipitate was separated by centrifugation. The supernafant albumin fraction was removed with a capillary pipette, placed in a Cellophane bag (10 mm diameter Cellophane tubing was found to be satisfactory) and dialyzed against cold Tyrodes solution until the external fluid was sulfate-free. The albumin solution was then passed through a micro-Seitz filter and tested for its REIS effect. In our first experiments, the.precipitated globulins were redissolved in 5 per cent. saline and dialyzed against distilled water until free of chlorides. This procedure apparently resulted in some denaturation, since the precipitated globulins were no longer completely soluble in dilute salt solutions. The procedure finally adopted was to redissolve the precipitate from the original ammonium sulfate treatment in cold Tyrode and then to dialyze the solution against Tyrode for the same length of time as used in the treatment of the albumin fraction. The solution remained completely clear. Sterilization and testing of the globulin fraction were accomplished in the same manner as described for the albumin solution. REIS fractions were added at various concentrations to tissue culture media in which heart fragments from chicks incubated for 6 days were grown. In some experiments the spleen of 18-day incubated chicks was used. Twelve per cent. embryonic extract and 50 per cent. heparinized rooster plasma served as the basic medium. Several preparations were used for each concentration tested.