Screening of 12 Gliomata Against Chemotherapeutic Agents in Vitro

Abstract

Using the methods described in detail below, we have attempted to cultivate many types of tumours, and whereas we have had scant success with most carcinomata and sarcomata, our experience with gliomata, comprising astrocytomata, ependymomata, and medulloblastomata, has been almost invariably successful. The results obtained in the testing of 12 gliomata and a culture of HeLa cells are reported here. Methods and MaterialsThe specimens examined were all obtained from Atkinson Morley's Hospital or the Hospital for Sick Children at Great Ormond Street. At operation the, tumours are placed in either a dry sterile screw-capped bottle or into Parker's TC medium 199 and packed in ice in a vacuum flask. They are brought to the Chester Beatty Research Institute and arrive within the hour. Immediately on arrival the specimens are chopped up manually with fine scissors and, being very soft, fragment easily. The "mush" so obtained is transferred to a 0.06% solution of commercial trypsin (" difco ") in Parker's TC medium 199 and agitated in a magnetic stirrer for 30 minutes at 370 C. Thereafter the coarser particles in the suspension are allowed to settle and the supernatant fluid is pipetted off and centrifuged for five minutes at 1,500 r.p.m. The supernatant from this procedure, which contains the trypsin, is removed and the centrifuged cells are resuspended in the final medium, which consists of 20% of the patient's serum +80% Evans N.C.T.C. medium 109, to which folic acid (0.4 mg. /litre) and insulin (438 mg. /litre) have been added. Repeated washings of the cells were found to give less satisfactory results, and it is felt that the trypsin inhibitors in the serum incorporated in the medium are sufficient to neutralize the traces of trypsin remaining after centrifugation.The suspension thus achieved is dispensed in 0.3-ml. quantities into Ambrose tubes (Ambrose et al., 1962) and incubated at 370 C. in a gas phase of 5% CO2 in air. A good monolayer of cells on the base of the tubes has usually formed in 48 hours and the medium is then changed, giving a clear fluid through which observations with an inverted microscope can be made. The monolayer is almost always dense enough to allow counting to be performed with the Chalkley eyepiece (Curtis, 1960). The drugs selected for administration are then added according to the method already described (Ambrose et al., 1962), the same volume-0.08 ml.-of Hanks's saline being added to the control tubes.The drugs chosen for this series were: melphalan (p-di-(2-chloroethyl)amino-L-phenylalanine), thiotepa (triethylene thiophosphoramide), chlorambucil (p-di-(2-chloroethyl)aminophenylbutyric acid), mannitol busul-

Assay of anti-cancer drugs in tissue culture: Relationship of relapse free interval (RFI) and in vitro chemosensitivity in patients with malignant cerebral glioma

“…Attempts to investigate this problem in the laboratory using a variety of in vitro and in vivo techniques (Gazso & Afra, 1969;Mealey et al, 1974;Kornblith & Szypko, 1978;Easty & Wylie, 1963;Saez et al, 1977;Rosenblum et al, 1978Rosenblum et al, , 1983Shapiro et al, 1979;Bullard et al, 1981b) have all demonstrated that human gliomas display considerable variation in chemosensitivity. In this report we present data produced with a rapid 35 S-methionine uptake assay (Darling & Freshney & Dendy, ,1983) where the end-point has been measured by scintillation autofluorography (Thomas et al, 1979;Morgan et al, 1983) which permits a rapid, objective comparison of in vitro and clinical data.…”