Multicellular spheroids are three dimensional in vitro microscale tissue analogs. The current article examines the suitability of spheroids as an in vitro platform for testing drug delivery systems. Spheroids model critical physiologic parameters present in vivo, including complex multicellular architecture, barriers to mass transport, and extracellular matrix deposition. Relative to two-dimensional cultures, spheroids also provide better target cells for drug testing and are appropriate in vitro model for studies of drug penetration. Key challenges associated with creation of uniformly sized spheroids, spheroids with small number of cells and co-culture spheroids are emphasized in the article. Moreover, the assay techniques required for the characterization of drug delivery and efficacy in spheroids and the challenges associated with such studies are discussed. Examples for the use of spheroids in drug delivery and testing are also emphasized. With these challenges and the possible solutions, multicellular spheroids are becoming an increasingly useful in vitro tool for drug screening and delivery to pathological tissues and organs.

Growth patterns of a number of human tumor cell lines that from three-dimensional structures of various architectures when cultured without carrier beads in a NASA rotary cell culture system are described and illustrated. The culture system, which was designed to mimic microgravity, maintained cells in suspension under very low-shear stress throughout culture. Spheroid (particulate) production occurred within a few hours after culture was started, and spheroids increased in size by cell division and fusion of small spheroids, usually stabilizing at a spheroid diameter of about 0.5 mm. Architecture of spheroids varied with cell type. Cellular interactions that occurred in spheroids resulted in conformation and shape changes of cells, and some cell lines produced complex, epithelial-like architectures. Expression of the cell adhesion molecules, CD44 and E cadherin, was upregulated in the three-dimensional constructs. Coculture of fibroblast spheroids with PC3 prostate cancer cells induced tenascin expression by the fibroblasts underlying the adherent prostate epithelial cells. Invasion of the fibroblast spheroids by the malignant epithelium was also demonstrated.

No abstract

No abstract

The effects of three beta-lactam antibiotics on Escherichia coli were studied by means of flow cytometry. Since these agents block bacterial cell wall synthesis in such manner as to prevent septa1 formation without appreciably affecting nucleic acid synthesis, the resulting cell elongation caused by these agents can be assessed by nucleic acid fluorescent staining. It was shown by this technique that the somatic effects of cefazolin, cefamandole and moxalactam were related both to the antibiotic concentration and time of exposure to the drugs and were observable within 30 minutes of the initial exposure of the cultures to these agents. These results demonstrate that fluorescent cytometry can provide accurate assessment of the effects of compounds that inhibit cell wall formation. This technology could be a useful tool for comparing antibiotic somatic effects on bacteria and for rapidly and reliably determining their sensitivity and resistance to these agents. Key terms: Sub-inhibitory antibiotics, betalactams, bacterial filaments, bacterial division P-Lactam antibiotics and other agents which inhibit cell wall synthesis are known to produce a variety of morphological alterations in susceptible organisms. A common response of gram negative bacilli to the effects of these agents is an abnormal elongation of the individual cells with subsequent formation of long filamentous forms (5-8, 13). This type of aberrant morphological change is the result of the selective binding of the antibiotics to cellular surface protein components responsible for cell wall septum formation and the separation of two divided organisms. In this process nucleic acid synthesis is not significantly affected and in exponentially growing cells, DNA replication, RNA synthesis, and other metabolic processes continue throughout the cell division cycle.Cell elongation and filament formation have been found to occur at a range of antibiotic concentrations both above and below the minimal inhibitory dose for the agent. (8) Furthermore, different antibiotics produce cell filamentation at a different spectrum of antibiotic levels with respect to their minimal inhibitory concentration (MIC). For example, studies by Greenwood and O'Grady showed that Escherichia coli exhibited cell elongation after 1 hr exposure to cephalosporin C at concentrations ranging from 1/2 to 1/4 MIC, while a ' This study was supported in part by the Office of Naval Research, Washington, DC, and by a grant from Coulter Electronics, Inc. in support of the Institute for Cell Analysis. similar effect was observed after the same exposure to cephalexin a t 1/32 to 4 MIC. (5) As part of the evaluation of new antibiotics which act as inhibitors of cell wall synthesis, it is useful to be able to compare the relative effectiveness of different agents in interrupting the normal cell wall replication of susceptible bacteria. Present techniques for studying the morphological alterations produced by these different agents invariably involve the direct examination of the cells, either by lig...