Improved sensitivity and decreased sample size in a cytotoxicity test for biomaterials: A modified colony microassay using a microplate and crystal violet staining

Tsuchiya, Toshie; Ikarashi, Yoshiaki; Arai, Tadashi; Ohhashi, Jyunichi; Nakamura, Akitada

doi:10.1002/jab.770050412

Cited by 9 publications

(6 citation statements)

References 5 publications

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“…Where a specific sensitivity is required, primary cell cultures and cell lines obtained directly from living tissues should only be used if reproducibility and accuracy of the response can be demonstrated 14-16. However, several problems, such as the preparation of test materials (in the case of the extract method), choice of cell type, the test procedure and method used to quantify results, etc., have been encountered in vitro cytotoxicity tests 17-19. In this study, the cytotoxicity of latex gloves to cultured L-929 cells was determined under various extraction conditions by using the extract dilution method.…”

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confidence: 99%

Evaluation of the Extraction Method for the Cytotoxicity Testing of Latex Gloves

et al. 2005

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In this study, the cytotoxicity of medical latex gloves to cultured L-929 cells was determined using various extraction conditions. According to the extraction time and temperature, three types of extraction conditions were used: 1) 24 h at 37℃; 2) 72 h at 37℃; 3) 72 h at 50℃. Also, four different extraction vehicles were used, namely, distilled water (DW), 9 g/l sodium chloride (saline) in DW, and culture media with or without serum. Under the above-mentioned conditions, the samples were extracted and then 2-fold serially diluted in the concentration range 3.13 - 50%. When extracted with either DW or saline for 24 h or 72 h at 37℃, only 50% diluted samples showed distinct cytotoxicity to L-929 cells. Moreover, no cytotoxic potentials were observed when gloves were extracted with DW or saline at 50℃ for 72 h. Cytotoxicity was markedly greater when gloves were extracted with culture medium, irrespective of the presence of serum in the medium. These results suggest that optimal extraction conditions should be established for the cytotoxicity evaluations of biomaterials and medical devices.

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confidence: 99%

Evaluation of the Extraction Method for the Cytotoxicity Testing of Latex Gloves

et al. 2005

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“…Cell proliferation was quantitatively estimated by crystal violet (Wako Pure Chemical Industries, Osaka, Japan) staining, as previously described 28. After the culture period, cells were fixed with 100% methanol at room temperature, followed by application of 0.1% crystal violet in methanol.…”

Section: Methodsmentioning

confidence: 99%

“…Cell proliferation was quantitatively estimated by crystal violet (Wako Pure Chemical Industries, Osaka, Japan) staining, as previously described. 28 After the culture period, cells were fixed with 100% methanol at room temperature, followed by application of 0.1% crystal violet in methanol. After a proper wash, cells were again incubated in methanol; 100 L from each well was transferred to a new 96-well plate, and the absorbance was measured at a wavelength of 590 nm, using an ELISA reader (Bio-Tek Instruments, Winooski, VT).…”

Section: Cell Proliferation Studymentioning

confidence: 99%

Effects of a biodegradable polymer synthesized with inorganic tin on the chondrogenesis of human articular chondrocytes

Banu¹,

Tsuchiya²,

Sawada³

2005

J Biomedical Materials Res

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Recent study has shown that biodegradable polymers are attractive candidates for chondrocyte fixation and further transplantation in cartilage tissue engineering. Poly (glycolic acid) (PGA), a polymer of glycolic acid, is widely used in orthopedic applications as a biodegradable polymer. Organotin, lead, antimony, and zinc are catalysts commonly used in synthesizing PGA. Here, we investigated the biocompatibility of PGA, synthesized with and without inorganic tin as a catalyst in chondrogenesis of human articular chondrocytes in a micromass culture system. Significant enhancement of chondrocyte proliferation and expression of the collagen type II protein gene were observed in cultures treated with PGA synthesized with a tin catalyst. However, aggrecan gene expression was very similar to the control culture. Amount of collagen type II protein was also increased in the same group of cultured chondrocytes. In contrast, PGA without a catalyst caused overall inhibition of chondrogenesis. Despite several positive findings, extensive investigations are essential for the feasibility of this PGA(Sn) in future clinical practice.

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“…39 Briefly, medium from all wells in the micromass culture was discarded after the culture period, and cells were fixed with 100% methanol at room temperature. After fixation, cells were stained with 0.1% crystal violet in methanol for 20 min.…”

Section: Crystal Violet Stainingmentioning

confidence: 99%

Markedly different effects of hyaluronic acid and chondroitin sulfate‐A on the differentiation of human articular chondrocytes in micromass and 3‐D honeycomb rotation cultures

Banu

Tsuchiya

2006

J Biomedical Materials Res

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A source of morphologically and functionally available human cartilagenous tissue for implantation is required in the field of tissue engineering. To achieve this goal, we evaluated the effects of hyaluronic acid (HA-810 and 1680 kDa), and chondroitin sulfate (CS-A 16 and C-34 kDa) on human articular chondrocytes (HC) in micromass and rotation culture conditions. Cell proliferation was increased by CS-A 16 kDa under micromass and rotation cultures, while cell differentiation was increased under rotation but not micromass conditions. Proliferation and differentiation due to CS-C 34 kDa were very similar to the control under both culture conditions. With HA, cell proliferation was increased depending on the molecular weight under micromass and rotation conditions. In contrast, chondrocyte differentiation was enhanced under rotation conditions, but decreased under micromass conditions depending on the molecular weight of HA. In both culture conditions, aggrecan gene was continuously expressed. However, the collagen type II gene was more weakly expressed in rotation than the micromass culture conditions. Thus, the chemical structures of polysaccharides, and the culture condition, rotation or micromass, caused differences in chondrogenesis.

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Improved sensitivity and decreased sample size in a cytotoxicity test for biomaterials: A modified colony microassay using a microplate and crystal violet staining

Cited by 9 publications

References 5 publications

Evaluation of the Extraction Method for the Cytotoxicity Testing of Latex Gloves

Evaluation of the Extraction Method for the Cytotoxicity Testing of Latex Gloves

Effects of a biodegradable polymer synthesized with inorganic tin on the chondrogenesis of human articular chondrocytes

Markedly different effects of hyaluronic acid and chondroitin sulfate‐A on the differentiation of human articular chondrocytes in micromass and 3‐D honeycomb rotation cultures

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