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Density gradient centrifugation of cells separated from multicellular tumor spheroids.

Cells from a murine fibrosarcome (FSa) have been grown in vitro as multicell tumor spheroids (MTS). The growth rate of these MTS was determined. Following selected periods of growth, MTS were made into a single cell suspension and separated on linear density gradients of Renografin. While only 1 population of cells were separated from small spheriods (400 mum diameter), at least 3 subpopulations of tumor cells were separated and isolated from large spheroids (800 mum in diameter).

Animals↗

The VM model of glioma: preparation of multicellular tumour spheroids (MTS) and their response to chemotherapy.

A cell line, 497-P(1), derived from the VM spontaneous murine astrocytoma has been used to develop an in vitro therapeutic model of human glioma. In this study we describe the preparation of MTS from this cell line. The in vitro chemosensitivity of 497-P(1) MTS has been examined and compared to the sensitivity of the monolayer culture. BCNU and CCNU both produced growth delay in MTS at doses below the ID50 of the monolayer culture. MTS, however, were considerably more resistant to vincristine and procarbazine when compared to the monolayer culture.

Animals↗

Growth and characterization of multicellular tumor spheroids of human bladder carcinoma origin.

We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGH-U1 cells placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and and consistently than cells derived from monolayer culture. Spheroid diameter increased at a rapid rate of approximately 100 microns/d in multiwell dishes, and necrosis occurred only in spheroids of diameter greater than 1 mm. Spheroids placed in spinner culture at a higher concentration (approximately 1.5 spheroids/ml) grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture was maintained at approximately 190 microns over a wide range of spheroid diameters (400 to 1000 microns). Sequential trypsinization of spheroids, which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids. This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human bladder biopsies to form spheroids.

Anthraquinones↗

Multicellular recordings of cultured brainstem neurons in microelectrode arrays.

Several vital systemic functions are controlled by the brainstem, which has been studied in a variety of experimental preparations and by various techniques, including in-vitro electrophysiological preparations. Although these in-vitro approaches have greatly advanced the understanding of brainstem neurons, most recording methods with microelectrodes and patch pipettes are invasive. To take advantage of in-vitro approaches but avoid their potential problems, we have studied brainstem neurons in microelectrode arrays (MEA). Neurons were isolated from the medulla oblongata and cultured in DMEM. Extracellular recordings were performed with no evident perturbations to the cellular environment. Neurons started firing after 24-48 h in culture, reached stable activity in 3-4 weeks, and retained this activity for at least 3 months. From their firing patterns, these neurons could be divided into tonic and bursting units. The latter could be further divided into regular and irregular bursters based on their burst intervals. Cells were stimulated or inhibited by exposure to 10% CO2. The stimulatory effect of CO2, though smaller, was still seen after selective ablation of serotonergic neurons or with low Ca++ and high Mg++ in the extracellular medium. Similar treatments had no significant effect on CO2-inhibited units. The abundance of units with respect to their firing patterns and CO2 responses, together with the long-term stable non-invasive recordings with no evident perturbation to cellular environments, suggests that MEA represent another promising in-vitro approach for studying brainstem neurons.

Animals↗

Spore coat protein synthesis during development of Dictyostelium discoideum requires a low-molecular-weight inducer and continued multicellularity.

The major spore coat proteins of Dictyostelium discoideum are synthesized during the culmination stage of development. In an attempt to examine the regulatory mechanisms involved, spore coat protein synthesis by pseudoplasmodia harvested prior to culmination and incubated in submerged culture under various environmental conditions has been monitored. It is reported that the synthesis of spore coat proteins SP170, SP103, SP94, SP82, SP76, and SP55 is dependent upon the presence of a low-molecular-weight (Mr approx 100), heat-stable factor secreted by cells incubated at high density in buffer. Previous studies have implicated cyclic AMP, ammonia, and amino acids in spore cell differentiation. Partial purification of the spore coat protein inducing factor (SPIF), together with attempts to mimic its activity, indicate that SPIF is not identical with any of these molecules and it is probably also distinct from DIF and "fruit juice," two other factors which regulate the spore-stalk decision and the initiation of culmination, respectively, in D. discoideum. In addition to SPIF, the continued expression of the spore coat protein genes also requires that the integrity of the pseudoplasmodium be maintained. Unlike the expression of many other genes after aggregation, this latter requirement cannot be replaced by exogenous cyclic AMP. Termination of spore coat protein gene expression occurs despite the presence of excess exogenous SPIF and hence involves mechanisms other than the destruction or depletion of SPIF.

Age Factors↗