Protocol for use: No 8 CYP inhibition assay using upcyte Hepatocytes (v )

Transcription

1 Description of protocol This PFU describes how to culture upcyte s for use in CYP inhibition assays. Required products upcyte s (cryopreserved) Each vial contains ~5x10 6 upcyte s (upcyte; upregulated hepatocytes ) frozen per vial from which at least 50% recovery is expected after thawing. Thawing Medium This is ready-to-use for thawing upcyte s. No additional supplements are required. containing 0.5% Dimethyl sulfoxide (DMSO) Use this medium to pre-culture upcyte s in a T-flask directly after thawing and for seeding cells into 48-well plates. In order to obtain this containing 0.5% DMSO, add the entire contents of supplement A to the basal medium. In addition, add L-Glutamine to a final concentration of 2mM and supplement the medium with DMSO to a final concentration of 0.5% [v/v], i.e. 2.5mL. containing 0.1% DMSO containing 0.1% DMSO is designed for optimal enzyme activities in upcyte s. In order to obtain this containing 0.1% DMSO add the entire content of Supplement A to the basal medium. In addition, the medium needs to be supplemented with L-Glutamine to a final concentration of 2mM and supplemented with DMSO to a final concentration of 0.1% [v/v], i.e. 0.5mL. The shelf life of the fully supplemented media is 6 weeks. Once fully supplemented do not freeze the upcyte Media. Add antibiotics if preferred. Cell culture vessels (T150 flasks, 48-well plates) These are not provided by upcyte technologies. Either use pre-coated plates or prepare them as follows: dilute collagen type I with 0.02M acetic acid to a final concentration of 50µg/mL. Add 0.1mL/cm 2 of the diluted collagen solution to the culture dishes and incubate for 1h at RT. Wash the plate twice with PBS and use directly or air dry before storing at 4 C. Storage upcyte s can be stored in liquid or vapour phase nitrogen. They should not be stored at -70 C. Store media protected from light at 2-8 C. Do not freeze. The expiration date of the medium is indicated on the label. Store Supplement A and B at -20 C. The solutions may arrive partially thawed but this does not affect their performance. Due to the manufacturing process the medium may appear opaque but this does not affect the performance of the cells Hamburg, Germany Page 1

2 Thawing of cryopreserved upcyte s 1. Thaw the upcyte s according to the PFU No. 12. Note: We recommend thawing on a Monday so that sufficient cells can be generated before reseeding them onto 48-well plates the following Friday. Pre-culture of upcyte s 7 days ( plus 0,5%DMSO) 2. Dilute the cells to cells/ml prewarmed High Performance Medium containing 0.5% DMSO and transfer 30ml into each T150 flask. The resulting seeding density will be 5000 cells/cm². 3. Culture the upcyte s in a humidified incubator under an atmosphere of 95% air / 5% CO Change the medium the next day. 5. Continue to culture the cells 3-4 days (Friday) or until the monolayer reaches 70 80% confluence. Seeding of upcyte s into 48-well plates 6. Trypsinize the cells according the protocol described in PFU No Re-suspend and pool the cells in 10mL prewarmed High Performance Medium containing 0.5% DMSO. It may be necessary to remove cell clumps by gently pipetting the cells up and down using a 1mL pipette. 9. Seed upcyte s at a density of 150,000 cells/cm² (i.e. confluence) in containing 0.5% DMSO in 48-well plates (0.5mL/well) or on an appropriate collagen Type I-coated multi-well plate format. 10. The next Monday or after 7 days replace the medium with High Performance Medium containing 0.1% DMSO (0.5mL/well). Conditioning culture of upcyte 3 days ( plus 0,1%DMSO) 11. If higher CYP1A2 activities are required, the cells can be pre-induced with M omeprazole. Other CYPs do not require preinduction. 12. Refresh the medium with High (with omeprazole for CYP1A2 pre-induction) daily for a total of 3 days. 13. After 3 day culture, the cells can be used in inhibition assays. 14. Aspirate the medium and wash the cells twice with PBS (containing Ca 2+ and Mg 2+ ). 15. Pre-incubate the cells with 0.1mL of an appropriate CYP-selective inhibitor dissolved in Krebs Henseleit buffer (KHB) for 5-30min (See Table 1 for concentrations and preincubation periods). 16. After the pre-incubation, add 0.1mL of the CYP-selective substrate (See Table 1 for concentrations and incubation periods). 17. Analyze the production of metabolites using appropriate analytical methods e.g. HPLC. 8. Determine cell number and viability by e.g. Trypan blue exclusion or a cell counter Hamburg, Germany Page 2

3 Inhibition assay Culture Conditioning period Re-seeding Pre-culture period Thawing Protocol for use: No 8 Inhibition method at a glance Thaw the cells according to PFU No. 12. Seed cells in a T150 flask at 5,000 cells/cm 2 (30mL/flask) in High Performance Medium containing 0.5% DMSO Pre-culture for 4-5 days or until cells are 70-80% confluent. Change the medium every 2-3 days Tryspinize the cells according to PFU No. 12. Re-seed the cells in 48-well plates at 150,000 cells/well, 0.5mL/well, in containing 0.5% DMSO Replace the medium with High (7days after thawing). Replace the medium with High daily for 3 days Wash the cells with PBS before pre-incubating with CYP inhibitors Incubate the cells CYP substrates and analyze the samples by HPLC Hamburg, Germany Page 3

4 Table 1. Recommended concentrations of inhibitors and CYP substrates for upcyte inhibition studies CYP CYP1A2 CYP2B6 CYP2C9 CYP3A4 Inhibitor (concentration range) -Naphthoflavone Ticlopidine Sulfaphanazole ) Ketoconazole Pre-incubation Substrate time (min) (concentration) 5 Phenacetin (26 M) 30 Bupropion (mechanistic inhibitor) (500 M) 5 Tolbutamide (75 M) 5 Testosterone (250 M). Incubation duration for substrate (min) 30 Results from inhibition assays using upcyte s Figure 1 shows the concentration-dependent inhibition of CYP1A2, CYP2B6, CYP2C9 and CYP3A4 in upcyte s from Donor by CYP-selective inhibitors. Ticlopidine is a mechanistic inhibitor of CYP2B6 and requires a longer pre-incubation period in order to produce sufficient reactive metabolite to inhibit the enzyme. Figure Hamburg, Germany Page 4

5 Product information Product Supplements/Components Product number upcyte s Frozen vial (1mL) CHE002 (cryopreserved) Thawing Medium ready-to-use (50mL) MEH001 containing 0.5% DMSO containing 0.1% DMSO Basal Medium (500mL) Supplement A (5mL) L-Glutamine (5mL) DMSO (not in Kit 2.5mL) Basal Medium (500mL) Supplement A (5mL) L-Glutamine (5mL) DMSO (not in Kit 0.5mL) MEH003 MEH003 Purchaser Notification Limited Use Label License (upcyte technologies) This cellular product (upcyte cells) generated using the upcyte technology is provided under an intellectual property license from. The transfer of the upcyte cells is conditioned on the buyer using the purchased product solely in research conducted by the buyer, and the buyer must not use, sell or otherwise transfer this product, its components or any data generated with the product (a) for diagnostic, therapeutic or prophylactic purposes; or (b) for resale; or (c) for the production of therapeutic, diagnostic, prophylactic or any other products; or (d) to provide a service to deliver information or materials to a third party. This intellectual property license is timely limited and granted to the buyer for 12 month after delivery of the upcyte cells. After this time the buyer has to destroy remaining upcyte cells and populations thereof or obtain a follow-up license. For information on purchasing a license to this upcyte technologies product for other purposes, for an extended time frame and/or for commercial purposes, contact,, Hamburg, Germany or. During the generation of these upcyte cells a product licensed from Life Technologies Corporation containing the following Limited Use Label License was used: Limited Use Label License (Life Technologies Corporation) This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research conducted by the buyer, and the buyer must not use, sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; or (b) resale, whether or not resold for use in research or (c) or for the commercial production of therapeutic, diagnostic, or prophylactic products or other products not intended for research use; or (d) to provide a service to deliver information or materials to a customer other than for research purposes of such customer. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA USA or outlicensing@lifetech.com Hamburg, Germany Page 5