A new method for the 3‐D in vitro growth of human RT112bladder carcinoma cells using the alginate culture technique

Abstract

We studied the response to different in vitro culture conditions and the ability for polarization in three-dimensional (3-D) and two-dimensional (2-D) cell culture systems of the commonly used human bladder carcinoma cell line RT112. In the case of 2-D culture the cells were grown on glass or plastic coverslips, filter membranes, or bovine lens capsules. The alginate culture technique (ACT) was used to test the effects of 3-D cell culture on the polarization of the RT112 epithelial cells. Our studies show clear differences in the arrangement of the cells depending on the cultivation procedure. The RT112 cells cultured on glass and plastic supports were irregular and flattened in shape whereas the cells grown on filters or on lens capsules developed into 2-3 layers consisting of markedly polarized cells. However, ACT was superior to cell culture on either artificial supports or even on lens capsule. During the 3-D cultivation the transformed epithelial cells regenerated multicellular spheroids which maintained a tissue-like, geometrically well-ordered and highly prismatic organization. This ACT-induced morphogenesis is novel and distinct from that reported with conventional culture conditions. Microscopic investigations showed that RT112 cells grown in alginate were both much more tightly packed and more regularly organized compared to 2-D cultures. In addition, the spheroidal organized cells exhibited well developed cell-cell contacts, a distinct endoplasmatic reticulum, and a marked Golgi apparatus. In summary, ACT can be used for 3-D in vitro growth of the transformed human epithelial cell line RT112 that offers substantial advantages over conventional cell culture methods.

The lysosomal/phagosomal membrane protein h‐lamp‐1 is a target of the IgA1 protease of Neisseria gonorrhoeae

“…Chang human conjunctiva cells (ATCC CCL20.2) and RT112 human bladder carcinoma cells [16] were cultured as monolayers in DMEM medium supplemented with 10% fetal calf serum (FCS) at 37°C in 5% CO2. The human myelomonocytic cell line JOSK-M and myelocytic HL60 cells, obtained from the German Collection for Microorganisms, Braunschweig (DSM ACC30 and DSM ACC3), were grown as suspensions in RPMI 1640 (Gibco BRL, Paisley, UK) supplemented with 10% FCS (Boehringer Mannheim, Germany), 2 mM L-glutamine at 37°C, 5% C0 2 .…”

“…To test whether the ability of the IgAl protease to cleave phagosomal h-lamp-1 molecules could promote the intracellular accommodation and survival of N. gonorrhoeae, we investigated the survival of isogenic IgAl protease mutants in Chang conjunctiva epithelial cells, a well-characterised in vitro infection model [17,26], and RT112 bladder carcinoma cells [16]. For this purpose, we infected the cells with N. gonorrhoeae N303 (Opa 50 + , IgAl protease+) or with N876 (Opa 50 + , IgAl protease") at a bacteriaxell ratio of 30:1 for 6 h (Chang) or 12 h (RT112), respectively.…”

Isolating and maintaining highly polarized primary epithelial cells from normal human duodenum for growth as spheroid-like vesicles

“…Following the concept that a change in cell shape may initiate a corresponding change in cell function, the principal advantage of the 3-D technique appears to be the striking improvement in both cell morphology and polarity over that of conventional cultures. Recent investigations demonstrated that epithelial cell differentiation in vitro was greater when the cells were grown in 3-D cultures (Walling et al, 1991: Chevillard et al, 1993Boxberger and Meyer, 1994;Yamanari et al, 1994;Boxberger et al, 1995). Our in vitro system, which combines the advantages of cell lines and organ cultures, features the use of small vesicles of cultured epithelial ceils which appear to possess the original tissue architecture.…”

A Salmonella fim Homologue in Citrobacter freundii Mediates Invasion In Vitro and Crossing of the Blood-Brain Barrier in the Rat Pup Model

“…Sequence analysis was performed with the Genetics Computer Group and CLUSTALW programs and tools available at the website of the National Center for Biotechnology Information. Nucleotide and protein sequence homology (8). The T24 cell line was cultivated in McCoy's 5A medium supplemented with 2 mM glutamine, nonessential amino acids, and 10% fetal calf serum.…”