An important definition is provided. Subconfluency is approximately 80% confluency and is at the end of the logarithmic phase of growth.
Sample preparation
The test should be run on the test product itself or extracts. Sample preparation should be according to 10993-12. And positive and negative controls should be part of each test. The conditions should simulate or exaggerate clinical use without deforming or degrading the test article. But, it can be expected of some test articles, such as biodegradables.
Suggested vehicles are cell culture media with or physiologic saline.
Both polar and non-polar should be considered. Cell culture medium with serum is the preferred media because it supports cell growth and extracts both polar and non-polar. Different serum’s can be used and should be cell line appropriate.
Conditions of not less than 24 +/-2 hours at 37 +/-1oC, 50 +/-2oC for 72 +/- 2 hours, 70oC +/-2 for 24 +/-2 hours or 121 +/-2oC for 1 +/-0.2 hours. Special circumstances might dictate extractions at 37oC for a period less than 24 hours but this should not be less than 4 hours.
Cell culture with medium should not be used at temperatures above 37 +/-1oC.
The extract should not be pH adjusted, filtered, centrifuged or manipulated in any way as it could affect the results. If these do occur they must be reported in the report.
Preparation of materials for direct contact tests
The sample should have a flat surface to contact. Sterility needs to be considered. If sterile care should be taken to minimize contamination.
If clinically the product is sterilized prior to use, it should be sterilized that way for the testing.
If the product is used unsterilized, it should be tested that way and care should be handled aseptically.
Liquids should be used directly or put onto/into an inert carrier such as filter paper.
If the test article is absorbent it should be soaked in media before extraction.
Cell Lines
A list of ATCC cell lines is provided that includes the 929, V79, and Balb/3t3 amongst others with a disclaimer that other cell lines may be used if they yield similar results as the cell lines listed.
Primary cell lines should only be used if reproducibility and accuracy can be demonstrated.
The absence of mycoplasma should be confirmed.
The cell culture medium should be sterile and should be appropriate for the cell line. Antibiotics can be used if they don’t affect the assay with a pH between 7.2 and 7.4.
Tests and controls should be conducted in triplicate.
Test on Extracts
Pipette an unspecified amount into an unspecified vessel and distribute the cells evenly. Then incubate at 37 degrees with or without carbon dioxide, whatever is appropriate for the medium being used. It should be a subconfluent layer or freshly suspended. Use the neat extract or prepare dilutions. If the test is on a monolayer, replace the media with the extract. Incubate for at least 24 hours and determine the cytotoxic effects.
Test by direct contact
Start by pipetting an unspecified amount into an unspecified size vessel and incubating the cultures at 37oC with or without carbon dioxide whatever is appropriate for the media. Grow the cell layer to sub confluency, the test article is to be placed directly on the cell layer in the center and to be a size of 10%. Incubate for at least 24 hours and determine the cytotoxic effects.
Test by indirect contact
Agar diffusion
This is not for leachates that cannot diffuse through the agar. The cells are to be grown at 37oC with or without carbon dioxide whatever is appropriate for the media to a confluent layer and verify the sub confluency. The culture media is removed and the cells are covered with an appropriate agar serum mix of 0.5 to 2% agar for the cell line. The test article is to be placed on the agar above the cells the size of 1/10th the cell surface and incubated for 24 to 72 hours.
Filter diffusion
Using a 0.45 micron filter evenly disperse the cells over the filter and incubate at 37oC with or without carbon dioxide until confluence and verify sub confluence. Remove filter at the end of the growth curve and confluence established and place cell side down on agar. Place the test article on the filter, if a liquid contain it with an inert ring,and incubate for 2 hours plus or minus 10 minutes. Remove the test article and the filter and use appropriate staining methods to determine cytotoxicity.
Cytotoxicity scoring
Qualitative scoring on extracts is a 5 point range from 0 to 4 with classifications running thru none, slight, mild, moderate, and severe and descriptors of cell condition and overall survival rate.
Qualitative scoring on direct and diffusion tests is a 5 point range from 0 to 4 with classifications running thru none, slight, mild, moderate, and severe and descriptors of area affected.
A quantitative scoring description suggests quantifying cell death, inhibition, proliferation or colony formation with the use of vital dye. Establishes a 30% cell death threshold for the determination of cytotoxicity.
Annex A (informative) (very informative)
Neutral red uptake cytotoxicity
Balb/c 3t3 in 96 well plates for 24 hours exposed to a range of the test article for 24 hours at which point the up take of neutral red will be determined by measuring at 540 nm.
Annex B (informative) (very informative)
Colony forming cytotoxicity
V79 cells in 6 well plates for 24 hours are then exposed to a range of the test article for 6 days and the number of colonies are then counted.
Annex C (informative) (very informative)
MTT Cytotoxicity
L929 cells in 96 well plates for 24 hours are then exposed to a range of the test article for 24 hours at which point the formazan formation is determined with MTT and evaluation at 570 nm.
Annex D (informative) (very informative)
XTT Cytotoxicity-vital dye
L929 cells in 96 well plates for 24 hours are then exposed to a range of the test article for 24 hours at which point the formazan formation is determined with XTT and evaluation at 450 nm.
Sample preparation
The test should be run on the test product itself or extracts. Sample preparation should be according to 10993-12. And positive and negative controls should be part of each test. The conditions should simulate or exaggerate clinical use without deforming or degrading the test article. But, it can be expected of some test articles, such as biodegradables.
Suggested vehicles are cell culture media with or physiologic saline.
Both polar and non-polar should be considered. Cell culture medium with serum is the preferred media because it supports cell growth and extracts both polar and non-polar. Different serum’s can be used and should be cell line appropriate.
Conditions of not less than 24 +/-2 hours at 37 +/-1oC, 50 +/-2oC for 72 +/- 2 hours, 70oC +/-2 for 24 +/-2 hours or 121 +/-2oC for 1 +/-0.2 hours. Special circumstances might dictate extractions at 37oC for a period less than 24 hours but this should not be less than 4 hours.
Cell culture with medium should not be used at temperatures above 37 +/-1oC.
The extract should not be pH adjusted, filtered, centrifuged or manipulated in any way as it could affect the results. If these do occur they must be reported in the report.
Preparation of materials for direct contact tests
The sample should have a flat surface to contact. Sterility needs to be considered. If sterile care should be taken to minimize contamination.
If clinically the product is sterilized prior to use, it should be sterilized that way for the testing.
If the product is used unsterilized, it should be tested that way and care should be handled aseptically.
Liquids should be used directly or put onto/into an inert carrier such as filter paper.
If the test article is absorbent it should be soaked in media before extraction.
Cell Lines
A list of ATCC cell lines is provided that includes the 929, V79, and Balb/3t3 amongst others with a disclaimer that other cell lines may be used if they yield similar results as the cell lines listed.
Primary cell lines should only be used if reproducibility and accuracy can be demonstrated.
The absence of mycoplasma should be confirmed.
The cell culture medium should be sterile and should be appropriate for the cell line. Antibiotics can be used if they don’t affect the assay with a pH between 7.2 and 7.4.
Tests and controls should be conducted in triplicate.
Test on Extracts
Pipette an unspecified amount into an unspecified vessel and distribute the cells evenly. Then incubate at 37 degrees with or without carbon dioxide, whatever is appropriate for the medium being used. It should be a subconfluent layer or freshly suspended. Use the neat extract or prepare dilutions. If the test is on a monolayer, replace the media with the extract. Incubate for at least 24 hours and determine the cytotoxic effects.
Test by direct contact
Start by pipetting an unspecified amount into an unspecified size vessel and incubating the cultures at 37oC with or without carbon dioxide whatever is appropriate for the media. Grow the cell layer to sub confluency, the test article is to be placed directly on the cell layer in the center and to be a size of 10%. Incubate for at least 24 hours and determine the cytotoxic effects.
Test by indirect contact
Agar diffusion
This is not for leachates that cannot diffuse through the agar. The cells are to be grown at 37oC with or without carbon dioxide whatever is appropriate for the media to a confluent layer and verify the sub confluency. The culture media is removed and the cells are covered with an appropriate agar serum mix of 0.5 to 2% agar for the cell line. The test article is to be placed on the agar above the cells the size of 1/10th the cell surface and incubated for 24 to 72 hours.
Filter diffusion
Using a 0.45 micron filter evenly disperse the cells over the filter and incubate at 37oC with or without carbon dioxide until confluence and verify sub confluence. Remove filter at the end of the growth curve and confluence established and place cell side down on agar. Place the test article on the filter, if a liquid contain it with an inert ring,and incubate for 2 hours plus or minus 10 minutes. Remove the test article and the filter and use appropriate staining methods to determine cytotoxicity.
Cytotoxicity scoring
Qualitative scoring on extracts is a 5 point range from 0 to 4 with classifications running thru none, slight, mild, moderate, and severe and descriptors of cell condition and overall survival rate.
Qualitative scoring on direct and diffusion tests is a 5 point range from 0 to 4 with classifications running thru none, slight, mild, moderate, and severe and descriptors of area affected.
A quantitative scoring description suggests quantifying cell death, inhibition, proliferation or colony formation with the use of vital dye. Establishes a 30% cell death threshold for the determination of cytotoxicity.
Annex A (informative) (very informative)
Neutral red uptake cytotoxicity
Balb/c 3t3 in 96 well plates for 24 hours exposed to a range of the test article for 24 hours at which point the up take of neutral red will be determined by measuring at 540 nm.
Annex B (informative) (very informative)
Colony forming cytotoxicity
V79 cells in 6 well plates for 24 hours are then exposed to a range of the test article for 6 days and the number of colonies are then counted.
Annex C (informative) (very informative)
MTT Cytotoxicity
L929 cells in 96 well plates for 24 hours are then exposed to a range of the test article for 24 hours at which point the formazan formation is determined with MTT and evaluation at 570 nm.
Annex D (informative) (very informative)
XTT Cytotoxicity-vital dye
L929 cells in 96 well plates for 24 hours are then exposed to a range of the test article for 24 hours at which point the formazan formation is determined with XTT and evaluation at 450 nm.
