Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Direct contact method with new essential technical steps to measure cytotoxicity of PFO sample. ( A ) The culture medium (non-volatile liquid) over PFO sample (transparent volatile liquid) retained the quantity of PFO sample in the well by blocking its evaporation. (A , left) Initial quantity of PFO and culture medium the wells. (A , middle and right) Loss in quantity of PFO sample over time in wells without a layer of culture medium to block evaporation in comparison to the other well (right). Arrow indicates upper surface of PFO sample. ( B , left) Phase contrast microscopy of ARPE-19 cells incubated for 1 hour after exposure to a control PFO sample. The cells retained confluent organization, and after 3 hours of culture ( C , left) did not stain with Trypan blue. In contrast, ARPE-19 cells incubated for 1 hour after exposure to a toxic PFO sample ( B , right) showed a loss of confluency, and after 3 hours of culture ( C , right) they were heavily stained with Trypan blue. The letter N and word TOX1 in figure 1B are lab internal codings only.

Article Snippet: Each 96-well plate was partitioned into columns in the following way: (1) culture media only, i.e., no cells; (2) cells incubated in culture medium alone; (3) positive control cells incubated in culture medium containing a cytotoxic ISO-recommended product, i.e., liquefied phenol meeting USP testing specifications (P9346, Sigma-Aldrich); (4) negative control cells incubated in culture medium with PFO, provided by non-Alamedics manufacturers and commercial companies and not associated with any toxic clinical cases; and (5) test cell cultures incubated in culture media with the suspected toxic PFO samples from Alamedics.

Techniques: Blocking Assay, Evaporation, Microscopy, Incubation, Staining

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Direct contact method to test viability of L929 and ARPE-19 cultures exposed for 30 minutes to culture medium, non-toxic PFO, and suspected toxic PFO. After exposure, the cultures were grown for 24 hours. Cytotoxicity was determined by the MTT assay. Both cell lines responded similarly to the different samples. Cells exposed to culture medium alone and to culture medium with non-toxic PFO (the non-Alamedics PFO and the Alamedics PFO lot # 210715) had viabilities that exceeded 70%, the cut off for designating the medium as non-toxic or toxic according to ISO norms. Cultures exposed to suspected toxic Alamedics PFO lots # 050514 and # 061014 had viabilities below 70%.

Article Snippet: Each 96-well plate was partitioned into columns in the following way: (1) culture media only, i.e., no cells; (2) cells incubated in culture medium alone; (3) positive control cells incubated in culture medium containing a cytotoxic ISO-recommended product, i.e., liquefied phenol meeting USP testing specifications (P9346, Sigma-Aldrich); (4) negative control cells incubated in culture medium with PFO, provided by non-Alamedics manufacturers and commercial companies and not associated with any toxic clinical cases; and (5) test cell cultures incubated in culture media with the suspected toxic PFO samples from Alamedics.

Techniques: MTT Assay

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Comparison of ARPE-19 cell culture viability at 24 and 72 hours after 30 and 60 minutes of PFO exposure. After 30 minutes of exposure to Alamedics PFO lots # 180214 and # 080714, the viabilities were <70% and considered to be toxic at 24 hours of culture. However at 72 hours, the viabilities were >70% and not considered to be toxic. After 60 minutes of suspected toxic PFO exposure, cell viabilities at both 24 and 72 hours were <70%. Hence selection of parameters such as exposure times and post-exposure growth periods are important for applying the direct contact method.

Article Snippet: Each 96-well plate was partitioned into columns in the following way: (1) culture media only, i.e., no cells; (2) cells incubated in culture medium alone; (3) positive control cells incubated in culture medium containing a cytotoxic ISO-recommended product, i.e., liquefied phenol meeting USP testing specifications (P9346, Sigma-Aldrich); (4) negative control cells incubated in culture medium with PFO, provided by non-Alamedics manufacturers and commercial companies and not associated with any toxic clinical cases; and (5) test cell cultures incubated in culture media with the suspected toxic PFO samples from Alamedics.

Techniques: Cell Culture, Selection

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Summary of PFO lot sources and comparison of results obtained by the direct contact and extract dilution exposure methods.

Article Snippet: The Alamedics suspected toxic PFO sample lots # 180214 and #150414 were taken from Container 1 (Table ).

Techniques: Comparison, Cytotoxicity Assay, Cell Culture, Control

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Direct contact method with new essential technical steps to measure cytotoxicity of PFO sample. ( A ) The culture medium (non-volatile liquid) over PFO sample (transparent volatile liquid) retained the quantity of PFO sample in the well by blocking its evaporation. (A , left) Initial quantity of PFO and culture medium the wells. (A , middle and right) Loss in quantity of PFO sample over time in wells without a layer of culture medium to block evaporation in comparison to the other well (right). Arrow indicates upper surface of PFO sample. ( B , left) Phase contrast microscopy of ARPE-19 cells incubated for 1 hour after exposure to a control PFO sample. The cells retained confluent organization, and after 3 hours of culture ( C , left) did not stain with Trypan blue. In contrast, ARPE-19 cells incubated for 1 hour after exposure to a toxic PFO sample ( B , right) showed a loss of confluency, and after 3 hours of culture ( C , right) they were heavily stained with Trypan blue. The letter N and word TOX1 in figure 1B are lab internal codings only.

Article Snippet: The Alamedics suspected toxic PFO sample lots # 180214 and #150414 were taken from Container 1 (Table ).

Techniques: Blocking Assay, Evaporation, Comparison, Microscopy, Incubation, Control, Staining

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Direct contact method to test viability of L929 and ARPE-19 cultures exposed for 30 minutes to culture medium, non-toxic PFO, and suspected toxic PFO. After exposure, the cultures were grown for 24 hours. Cytotoxicity was determined by the MTT assay. Both cell lines responded similarly to the different samples. Cells exposed to culture medium alone and to culture medium with non-toxic PFO (the non-Alamedics PFO and the Alamedics PFO lot # 210715) had viabilities that exceeded 70%, the cut off for designating the medium as non-toxic or toxic according to ISO norms. Cultures exposed to suspected toxic Alamedics PFO lots # 050514 and # 061014 had viabilities below 70%.

Article Snippet: The Alamedics suspected toxic PFO sample lots # 180214 and #150414 were taken from Container 1 (Table ).

Techniques: MTT Assay

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Comparison of ARPE-19 cell culture viability at 24 and 72 hours after 30 and 60 minutes of PFO exposure. After 30 minutes of exposure to Alamedics PFO lots # 180214 and # 080714, the viabilities were <70% and considered to be toxic at 24 hours of culture. However at 72 hours, the viabilities were >70% and not considered to be toxic. After 60 minutes of suspected toxic PFO exposure, cell viabilities at both 24 and 72 hours were <70%. Hence selection of parameters such as exposure times and post-exposure growth periods are important for applying the direct contact method.

Article Snippet: The Alamedics suspected toxic PFO sample lots # 180214 and #150414 were taken from Container 1 (Table ).

Techniques: Comparison, Cell Culture, Selection

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Direct contact method to test viability of L929 and ARPE-19 cultures exposed for 30 minutes to culture medium, non-toxic PFO, and suspected toxic PFO. After exposure, the cultures were grown for 24 hours. Cytotoxicity was determined by the MTT assay. Both cell lines responded similarly to the different samples. Cells exposed to culture medium alone and to culture medium with non-toxic PFO (the non-Alamedics PFO and the Alamedics PFO lot # 210715) had viabilities that exceeded 70%, the cut off for designating the medium as non-toxic or toxic according to ISO norms. Cultures exposed to suspected toxic Alamedics PFO lots # 050514 and # 061014 had viabilities below 70%.

Article Snippet: However, the viabilities of cell cultures exposed to the suspected toxic Alamedics PFO lot # 180214 for 30 min were 59% and 89% after 24 and 72 hours growth in culture medium.

Techniques: MTT Assay

Journal: Scientific Reports

Article Title: Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation

doi: 10.1038/s41598-018-19428-5

Figure Lengend Snippet: Comparison of ARPE-19 cell culture viability at 24 and 72 hours after 30 and 60 minutes of PFO exposure. After 30 minutes of exposure to Alamedics PFO lots # 180214 and # 080714, the viabilities were <70% and considered to be toxic at 24 hours of culture. However at 72 hours, the viabilities were >70% and not considered to be toxic. After 60 minutes of suspected toxic PFO exposure, cell viabilities at both 24 and 72 hours were <70%. Hence selection of parameters such as exposure times and post-exposure growth periods are important for applying the direct contact method.

Article Snippet: However, the viabilities of cell cultures exposed to the suspected toxic Alamedics PFO lot # 180214 for 30 min were 59% and 89% after 24 and 72 hours growth in culture medium.

Techniques: Comparison, Cell Culture, Selection