Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates (original) (raw)
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Microencapsulation of human cells: Its effects on growth of normal and tumour cells in vitro
British Journal of Cancer, 1991
The growth kinetics of established human colorectal tumour cell lines (HT29, HTI 15 and COLO 320DM) and human diploid fibroblasts (Flow 2002) were studied in conventional culture and in microcapsules formed from alginatepoly(L-lysine) -alginate membranes. The tumour lines grew rapidly in microcapsules but, in the case of the substrate-adherent lines HT29 and HTl 15, only after a prolonged lag phase. This phase was reduced by serial passage in microcapsules. The anchorage-independent line COLO 320DM showed no lengthening in lag phase. Microencapsulated fibroblasts underwent negligible growth but remained viable. Some evidence for functional differentiation (microvilli, cell-cell junctions) of the tumour line HTl 15 within the microcapsules was observed. We conclude that the use of microcapsules provides an alternative system with some advantages for the study of human cancer and its metastases in vitro.
in Vitro Cellular & Developmental Biology-plant, 1987
Human colonic adenocarcinoma cells have been successfully grown on polystyrene microcarriers by modifying the culture conditions used in monolayer culture. The method can be divided into two culture phases: a) a phase of spreading, wherein cells were seeded in presence of serum-supplemented medium; b) a phase of active growth wherein spread cells on the beads were allowed to grow in a serum-free medium. Under these conditions, optimal spreading and growth of HT 29 and HRT 18 cells on the microcarriers were obtained. A differential propagation was observed between HT 29-D4 and HT 29-D9 cells (both clonal populations derived from HT 29 cells) on the microcarriers that is tentatively related to the discrepancy observed in the spreading efficiency of these clonal cells on serum-coated culture flasks. An index of spreading efficiency (IS index) has been defined to quantify the efficiency of spreading of each cell line on microcarriers. These data gave the opportunity to develop serum-free, scale-up methods to culture cells like HT 29 which release potentially useful products.
Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests
Cytotechnology, 2000
We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 +/- 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 +/- 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at -20 degrees C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (-85 degrees C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically - (4 degrees C for 2 days) or cryogenically - (-85 degrees C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison ...
New chemotherapeutic drug sensitivity assay for colon carcinomas in monolayer culture
Cancer research, 1988
Ten previously untreated colon carcinomas were tested for chemotherapeutic drug sensitivity in primary monolayer culture. Colon carcinomas were partly digested to groups of epithelial cells which plated with a mean efficiency of 42 +/- 9% (SE) on a collagen I-bovine serum albumin substrate in serum-free medium, producing patches of tightly adherent epithelial cells. The cultured cells were judged epithelial by the presence of cytokeratins, an epithelial cell surface epitope, junctional complexes, and brush borders. Each carcinoma was plated in 40 to 60 Petri dishes (35 mm), yielding a mean of 28 +/- 8 (SE) colonies per dish (6832 +/- 1952 cells). Drugs tested in duplicate plates were mitomycin C, cisplatin, streptozotocin, and 5-fluorouracil at 0.1, 1, 10, and 100 micrograms/ml, and at 0.1, 1, and 2x the peak tolerated drug concentration in serum. Twenty-four h after plating, any nonadherent cells were removed, and the adherent tumor cells were continuously exposed to the drugs for ...
Cancer Research, 2018
The tumor microenvironment plays an important role on tumor drug sensitivity; thus the incorporation of microenvironment features on cancer models is expected to improve their predictive power. Patient-Derived Explants (PDE) have been proposed as potential models; however there are typically short term cultures. Our goal was to improve culture longevity and vitality to evaluate efficacy of repeated drug treatments, taking advantage of dynamic culture systems. Fresh ovarian and colorectal cancer (OC and CRC, respectively) samples were mechanically dissociated into PDE and cultured in dynamic conditions. The cell population dynamics, cell viability and proliferation were assessed by a panel of readouts, e.g. immunohistochemistry, rezasurin reduction capacity, PDE concentration and morphometric measurements. To this date, 20 OC and 18 CRC were successfully cultured as PDE, retaining the original tumor architecture and main cellular components: epithelial cells, fibroblasts and immune c...
Heterogeneous responses of human colon carcinomas to hexamethylene bisacetamide
PubMed, 1988
Primary cultures of resected human colon carcinoma were used to study differentiation agents directly on the biologically relevant cancer cells rather than on highly selected established cell lines. To achieve primary cultures which remained viable and replicating for several days, carcinomas were partly digested to epithelial organoids, which were selectively plated with high efficiency on collagen I-bovine serum albumin films in specially formulated serum-free medium. A monoclonal antibody, 29-15, was identified which binds to a cell surface epitope expressed on 16 of 21 invasive colon carcinomas of the Dukes' B2, C, or D histopathology classes, but not expressed on any of 11 noninvasive benign tumors (adenomas) at identical antibody titer. Noncytotoxic concentrations of the differentiation agent, hexamethylene bisacetamide (HMBA), induced the loss of the 29-15 epitope from HT29 colon carcinoma cells. HMBA also induced HT29 cells to lose the capacity for anchorage-independent growth with a similar dose-response curve and time course to the loss of 29-15 epitope. Twelve primary cultured human colon carcinomas exhibited differential responses when exposed to 1 to 7 mM HMBA for 7 days. Four moderately to well-differentiated carcinomas lost expression of the 29-15 epitope at each HMBA concentration. The tumor growth fraction was decreased in each tumor, with a mean decrease of 76% at 5 mM HMBA. A dose-dependent induction of nonproliferating tumor colonies, lacking [3H]thymidine labeling, occurred in three of the four carcinomas. In six other tumors, including those at less differentiated stages, HMBA induced the opposite effect: a two- to threefold increase in the tumor growth fraction at the optimal value of 5 mM HMBA, an increase in mean colony size, and no loss of the 29-15 malignancy epitope. No effects were observed in the two other carcinomas tested. Thus HMBA was able to induce growth arrest and loss of the malignancy epitope 29-15 in those carcinomas already at an advanced stage of differentiation, and to exert a growth stimulating effect on those carcinomas apparently at more immature stages.
In Vitro Cellular & …, 1988
Using innovative approaches, we addressed several problems often associated with in vitro chemosensitivity testing of individual human tumors: 1) obtaining a high rate of evahability; 2) excluding participation of nonmalignant stromal and vascular components usually present in tumor specimens; 3) preserving cell-to-cell interactions present in the original tumor; 4} assessing drug-induced cytotoxicity without sacrificing the tumor culture. To circumvent these problems, tumor specimens were processed as follows: i) tissue tfresh or cryopreserved} was mechanically or enzymatically dissociated under mild conditions into cellular clusters (termed micro-organs); if) large micro-organs were separated by a brief decantation, resuspended, and then exposed to fluorescein acetate to visualize (under naked eye) viable micro-organs; iii) fluorescent (i.e., viable} micro-organs were collected using a Pasteur pipette, and then planted on a solid support made of cellulose fibers impregnated with collagen. Since tumor micro-organs have been previously shown to consist solely of malignant cells, the procedure described here not only preserves a critical portion of the tumor architecture but eliminates at the onset necrotic tissue and nonmalignant cellular components that could interfere with the chemosensitivity testing. Drug-induced cytotoxicity was measured by "fluorescent cytoprinting", a novel, nondestructive procedure for assessing micro-organ viability in situ. The key feature of fluorescent cytoprinting is that cytotoxic effects are no...tt measured against control cultures but against a baseline provided by a cytoprint of the same culture before drug addition. Using three experimental designs, we tested the potential of the method for clinical applications. The results using 469 human malignant tumors showed that tbe micro-organ culture assay can distinguish individual tumor chemosensitivity profiles with an overall success rate of 96%. For three commonly used chemotherapeutic drugs, the observed frequency of responding tumors was found to be comparable to previously reported clinical results using single agents.
Differentiation of a colon cancer cell line on a reconstituted basement membrane in vitro
International Journal of Experimental Pathology, 2002
Basement membrane, a thin extracellular matrix, functions as a tissue stabilizer that promotes tissue integrity and differentiated phenotype. We studied a human colon cancer cell line, SNU 61, to evaluate its ability to differentiate on basement membrane. Cells were cultured on plastic, reconstituted basement membrane (Matrigel) or polyhydroxyethyl methacrylate (poly HEMA) for 72 h and evaluated by light and electron microscopy. On Matrigel, the cells showed gland formation with highly polarized cells containing basal nuclei and well developed brush border microvilli on the luminal surface. Apoptosis was noted mainly at the luminal side. On electron microscopic examination, numerous long microvilli, abundant cytoplasmic organelles and intercellular junctions were noted in the Matrigel-cultured cells. Intermediate cytoskeletons were scattered in the cytoplasm and existed on the axes of microvilli. Junctional complexes and desmosomes were frequently formed along intercellular spaces. The cells cultured on poly HEMA, on the other hand, were poorly differentiated and contained a few glandular structures with small lumens. Brush border microvilli, characteristic of enterocytic differentiation, were few in number and were developed on the basal surface. Intermediate filaments and microtubules were fewer than in the Matrigel-cultured cells. Carcinoembryonic antigen was expressed on the luminal surface of the Matrigel-cultured cells and in the cytoplasm of the poly HEMA cultured cells. CD44 stained the basolateral surface in the Matrigelcultured cells, but the basal side was not stained in the poly HEMA cultured cells. These results are consistent with the different localization of microvilli in the Matrigel and in the poly HEMA cultured cells. Our observations suggest that human colon cancer cells on basement membrane can undergo glandular differentiation and that extracellular matrix is an important factor in morphogenesis.