FDA-14-223-SOL-1133650_Q A_8.13.14.pdf

FDA-14-223-SOL-1133650

Questions and Answers

Q-1.

Should the testing be done in cryopreserved human hepatocytes only or additionally in 3D spheroid cultures and hepatocyte co-cultures. Can you clarify on the model required both 3D spheroid (which type) as well as co-cultures of which cells?

A-1.

The Government requires cryopreserved primary human hepatocytes to repeat the experiments (first test a set of 100 drugs as training set, then test a second set of 100 drugs as test set). We need to have 3D spheroid of human hepatocytes (hanging drop) and co-cultures with Kupffer cells, neutrophils, macrophage, or eosinophil cells to test the drugs involved in immune mediated drug induced liver injury.

Q-2.

What are the 4 assays to be performed?

A-2.

The assays to be performed include reactive oxygen species: (ROS) level, Adenosine triphosphate (ATP) content, Glutathione (GSH) depletion and Caspase 3/7 activity (or Alanine transaminase (ALT)).

Q-3.

Do all hepatic models listed have to be used? If so, could you clarify the nature of the co-cultures referenced?

A-3.

Three models for the study are listed, 2D (conventional micro-plate based), 3D human (spheroid) hepatocyte models and co-culture model. However, screening 200 drugs with cryopreserved human primary hepatocyte in 2D model is the priority of this study and the results from this will determine which drugs will be tested either in 3D or co-culture models. Therefore, there is no need to test all 200 drugs in either 3D or co-culture models.

Q-4.

Are the 4 assays to be chosen by the contractor?

A-4.

Yes. However, If co-culture model is needed to conduct these 4 assays, the primary human hepatocytes need to be co-cultured with Kupffer cells, neutrophils, macrophage or eosinophil cells

Q-5.

Do contractors have to submit both RIN and 28S/18S ratio, or will one or the other suffice?

A-5.

Yes. Contractors shall submit both RIN and 28S/18S ratio.