Medical Device Testing Guide - Toxikon Corporation

CYTOTOXICITY TESTS—ISO 10993-5
Rev. May 2011
PURPOSE: Cytotoxicity in-vitro screening assays are used to assess – in a fast and sensitive way – the biocompatibility of the
test material (extract) when in contact with a specific cell culture.
AA01-1
Agar Diffusion-Sample needed: 3 cm 2 or 1mL
This qualitative assay determines the biological reactivity of a monolayer of the L929 mammalian cell in response to a test
material. The test is designed for a variety of solid and liquid test materials. The cells are allowed to grow to approximately
80% confluency in cell culture dishes and then overlaid with an agarose layer. The test material is placed over the agar layer,
which protects the cells from mechanical damage while allowing the diffusion of leachables from the test material onto the cell
layer. The plates are incubated for forty-eight hours at 37°C in a 5% CO 2 incubator and scored for reactivity at twenty-four
and forty-eight hours on a scale from Grade 0 (no reactivity) to Grade 4 (severe reactivity). The test item is considered noncytotoxic
if none of the cultures exposed to the test item shows greater than mild reactivity (Grade 2). This assay is
appropriate for screening purposes.
Approximate turnaround time is two to three weeks.
AA02-1
L929 MEM Elution-Sample needed: 120 cm² ≤ 0.5 mm thick, 60 cm² ≥ 0.5 mm thick, or 4 g
The ―in-vitro‖ biological reactivity of the L929 mouse fibroblast cell culture is qualitatively determined in response to an
extract of the test material in triplicate. The cells are allowed to grow to sub-confluency in tissue culture plates. An extract of
the test material is prepared in Minimum Essential Media (MEM), which is transferred onto the cell layer in triplicate. The
plates are incubated for forty-eight hours at 37°C in a 5% CO 2 incubator, and scored for reactivity at twenty-four and fortyeight
hours on a scale from Grade 0 (no reactivity) to Grade 4 (severe reactivity). The test item is considered non-cytotoxic if
none of the cultures exposed to the test item shows greater than mild reactivity (Grade 2). This assay is appropriate for
screening purposes.
Approximate turnaround time is two to three weeks.
AA03-1
AA03-2
L929 MTT Cytotoxicity (1 Concentration)-Sample needed: 30 cm² ≤ 0.5 mm thick, 15 cm² ≥ 0.5 mm thick, or 1 g
L929 MTT Cytotoxicity (4 Concentrations)-Sample needed: 30 cm² ≤ 0.5 mm thick, 15 cm² ≥ 0.5 mm thick, or 1 g
The ―in-vitro‖ biological reactivity of the L929 mouse fibroblast cell culture is quantitatively determined in response to an
extract of the test material in 6 replicates. The cells are allowed to grow to semi-confluency in tissue culture plates. An extract
of the test material is prepared in Minimum Essential Media (MEM), which is transferred onto the cell layer. The plates are
incubated for forty-eight hours at 37°C in a 5% CO 2 incubator, and quantitatively analyzed for viability via the reduction of the
yellow water-soluble MTT by the mitochondrial reductases enzymes into a blue-violet insoluble formazan. The number of
viable cells correlates to the color intensity determined by photometric measurements after dissolving the formazan. The test
item is considered non-cytotoxic if the percentage of viable cell is equal to or greater than 70% of the untreated control. This
assay is appropriate for biocompatibility testing.
Approximate turnaround time is two to three weeks.
AA04-1
AA04-2
Neutral Red Uptake (NRU) Cytotoxicity (1 Concentration)
Sample needed: 30 cm² ≤ 0.5 mm thick, 15 cm² ≥ 0.5 mm thick, or 1 g
Neutral Red Uptake (NRU) Cytotoxicity (8 Concentrations)
Sample needed: 30 cm² ≤ 0.5 mm thick, 15 cm² ≥ 0.5 mm thick, or 1 g
The ―in-vitro‖ biological reactivity of the Balb/c 3T3 mouse fibroblast cell culture is quantitatively determined in response to
an extract of the test material in 6 replicates. The cells are allowed to grow to semi-confluency in tissue culture plates. An
extract of the test material is prepared in Dulbecco Modified Eagle Media (DMEM), which is transferred onto the cell layer.
The plates are incubated for forty-eight hours at 37°C in a 5% CO 2 incubator, and quantitatively analyzed for viability via the
incorporation of Neutral Red (NR) by the lyzosomes of the live cells. The number of viable cells correlates to the color
intensity determined by photometric measurements after desorbtion of the NR. The test item is considered non-cytotoxic if the
percentage of viable cell is equal to or greater than 70% of the untreated control. This assay is appropriate for biocompatibility
testing.
Approximate turnaround time is two to three weeks.
SENSITIZATION TESTS—ISO 10993-10
PURPOSE: The sensitization tests are used to determine the allergic or sensitizing capacity to the repeated or prolonged
exposure of a test material. Sensitization is characterized by the fact that reactions are delayed, not localized, and independent
of dose.
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