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In Vitro ADME-Tox Products and Services FAQs
How long are cryopreserved hepatocytes viable in liquid nitrogen? When stored at the proper storage conditions (< -150° C), the cytochrome P450 enzyme systems remain unchanged after more than 5 years of storage. Can I repeatedly thaw and refreeze cryopreserved hepatocytes if I only need to remove a small aliquot of these products from a vial? No. We have found that repeated freezing and thawing causes extremely low recovery of viable hepatocytes. That is why we pack our cryopreserved hepatocytes in a volume that is convenient for most single-use applications. Is it possible to "plate" cryopreserved human hepatocytes? IVT now offers plateable cryopreserved human hepatocytes. Please refer to the characterization tables to find the lots that show excellent plating efficiency. Contact IVT to inquire about purchasing these cells. What information is available about the human donors from which the hepatocytes, microsomes, and S9 were prepared? We generally provide the following information about each of the donors: AgeGenderRaceTobacco UseAlcohol UsePrescription Medication UseNon-prescription Drug UseSignificant Medical HistoryCause of Death For ethical and privacy reasons we do not provide any information which might identify the donor. Can I store cryopreserved hepatocytes in the cryoshipper? No, the cryoshipper is not intended for storing cryopreserved hepatocytes. Upon receiving your shipment the cryopreserved hepatocyte vials must be removed and stored at temperatures less than -150°C. Where does Celsis IVT have a distributor in Asia? Celsis IVT has the following distributor in Asia: Veritas Corporation Yashima Building 2-7-14 Toranomon Minato-ku Tokyo, 105-0001 Japan T: 81 3 3593 3211 Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it. Website: www.veritastk.co.jp Where does Celsis IVT have a distributor in Spain? Celsis IVT has the following distributor in Spain: CYMIT QUIMICA SL Guillem Tell, 42 08006 Barcelona T. 34-93-2412927 F. 34-93-4144979 This e-mail address is being protected from spambots. You need JavaScript enabled to view it. www.cymitquimica.com Can I plate IVT fresh hepatocyte suspensions and get these to attach? IVT supplies fresh hepatocyte suspensions from human and other species without any guarantee that they will attach. However, we have talked to many of our customers who tell us that they have successfully plated our hepatocyte suspensions using the following technique: Spin cells out of the suspension media at 50 g.Resuspend the cells in IVT Hepatocyte Culture Media (to which you must first add 10% fetal bovine serum).Perform a cell count; dilute to a final concentration of 0.7 million cells/mL in Hepatocyte Culture Media + 5% fetal bovine serum.Add 0.5, 1.0, or 2.5 mL of this cell suspension to individual wells of a 24-, 12-, or 6-well cell culture plate, respectively.Gently shake the plate in all directions (but not in a circular motion) to evenly distribute the cells in each well.Place in a 37 degree Celsius, 5% CO2 incubator for 1–2 hours to allow cells to attach.When cells have attached, wash off any unattached cells using pre-warmed Hepatocyte Culture Media + 5% fetal bovine serum.Incubate overnight.The cells are now ready to be used in your experiments. [Note: This is just one of many possible methods for attempting to plate the IVT Fresh Hepatocyte Suspensions]. Can you plate fresh animal or human hepatocyte suspensions and get them to attach? Celsis In Vitro Technologies supplies fresh hepatocyte suspensions from human and other species without any guarantee that they will attach. Celsis IVT offers plated fresh hepatocytes in a variety of formats. Inquire with Customer Service on which formats are available. Where can I find the fresh animal hepatocyte isolation calendar? Got to http://www.celsis.com/calendar.html to view and fresh animal hepatocyte isolation schedule. Where does Celsis In Vitro Technologies get its human tissues? Celsis In Vitro Technologies has established a network of sources of non-transplantable organs. All of the tissues we receive are considered research grade, and are not transplantable. Celsis In Vitro Technologies only receives liver and other human tissue from approved sources within the United States, and does not accept tissues from any other country. How frequently does IVT receive human tissues? Typically at least once a month, sometimes as frequently as every week. This allows for a prompt start to studies and regular additions to our products. Does IVT charge for consulting? We do not charge anything to consult with our customers in order to help them design the best and most cost-effective protocols to answer their questions. You should talk with us about your needs, whether you have already outlined all of the experimental details of the study that you want to conduct, or would like our input in designing a protocol with you. What applications can be performed on the human renal proximal tubule cells? Metabolism and cytotoxicity assays as well as transport and efficacy studies. Is there characterization data available for the human renal proximal tubule cells? Yes, our web site has the up to date characterization information for our human renal proximal tubule cells as well as other products offered by In Vitro Technologies. Do I need to use any special medium for these cells? Yes, we recommend that you use our InVitroGROTM PT medium which has been optimized and tested with the human renal proximal tubule cells. What is the expiration date of the InVitroGROTM PT medium? The InVitroGROTM PT medium will last 60 days from the day of receipt. How should I store the cryopreserved human proximal tubule cells? The cells should be stored in liquid nitrogen. What is in the InVitroGROtm PT medium? The formulation of InVitroGROTM PT medium is proprietary. Will the human renal proximal tubule cells attach to a plate/flask? Yes, the human renal proximal tubule cells will attach to untreated plastic as well as collagen coated plates. What InVitroGROTM hepatocyte medium should I use for an induction assay? Our InVitroGROTM HI medium is optimized and formulated for use in induction assays. Go to http://www.celsis.com/file_library.html and click on protocols to find our induction assay protocol. How do I store microsomes, S9, hepatocytes or media? Media: (2° to 8° Celsius) Do not freeze media. Storing media refrigerated maximizes shelf life. Antibiotics: (-15° to -25° Celsius) Do not store in refrigerator. Avoid storage in "Frost-Free" freezer as temperature cycles can damage product. Microsomes/S9: (-70° to -90° Celsius) Store in mechanical cryogenic-freezer. Limit number of freeze/thaw cycles. Hepatocytes: (< -150° Celsius) Store in vapour phase of liquid nitrogen freezer. Do not allow product to thaw until ready for use. Do not store in liquid phase of liquid nitrogen freezer or Dewar type flask. I have never used microsomes or S9 before. How do I use them? You can find detailed instructions for use of microsomes and S9 right on our web site. Check the Product Sheets and Protocols in the File Library. How long can I store microsomes and S9? These products should always be stored in an ultracold freezer at temperatures < -70° C. Stored under these conditions the cytochrome P450 enzyme systems are remarkably stable, showing little, if any, loss of activity after storage for more than 5 years. However, some of the phase II enzyme systems, particularly those in S9 such as N-acetyltransferase, are not stable even at -70° C, and lose significant activity within a matter of weeks or months. Can I measure phase II metabolism using microsomes or S9 fractions? Yes, but with microsomes and S9 you must add the necessary phase II cofactors. For example, if you want to measure glucuronidation (UDPGT) in a microsomal incubation, you will need to add uridine diphosphoglucuronic acid (UDPGA) in addition to the normally required NADPH regenerating system. As an alternative, consider using cryopreserved hepatocytes, since these contain all of the cofactors necessary to perform coupled phase I/II metabolism. Can I repeatedly thaw and refreeze microsomes and S9 if I only need to remove a small aliquot of these products from a vial? Yes, as long as you carefully thaw these products on ice, remove what you need, and then quickly refreeze the vial to -70° C. As an alternative to repeated thawing and freezing, you can thaw the vial once and distribute the contents into single-use vials for future use. That way, you only have to thaw the microsomes/S9 one more time. How many animals are used to make each lot of microsomes and S9? The number of animal livers used to make a batch of microsomes or S9 varies, depending on the species. For small animals such as mice and rats, we always use at least 25 livers to make each lot of product. For larger animals such as monkeys and dogs the number is much smaller, typically 4 to 7 livers per lot of product. What is the difference between M-class and H-class microsomes? H-class microsomes are formulated to have high activity for use in inhibition studies. M-class microsomes are formulated to have average activity for use in metabolism studies. How are the M-class and H-class microsomes manufactured? M-class and H-class microsomes are purposed-pooled using a proprietary algorithm to ensure consistent and reproducible product across the two classes. How are the InVitroCYP microsomes different from other microsomes? The pooled microsome products that are available in the market today offer "average" cytochrome P450 (CYP) activity, which may not provide sufficient enzyme activity levels in some assays. The InVitroCYP classes provide differentiation so that researchers get the results they need. How are the CYP enzyme levels reported? InVitroCYPs are the only microsomes on the market with substrate data reported at both Km and Vmax, providing you with maximum visibility for predictive performance and quality. Because some studies look for specific interactions, InVitroCYP C-class microsomes are designed by you to fit your research criteria, e.g. microsomes prepared for specific donor demographics such as age, gender, race, CMV status etc. What's the difference between Km and Vmax levels? Km is measure of how easily the enzyme can be saturated by the substrate. Vmax is the maximum rate of an enzyme catalysed reaction or when the enzyme is saturated by the substrate Why does Celsis provide both Km and Vmax reports? Celsis IVT used to only report Km values. However, many of our customers prefer Vmax values over Km. Km and Vmax values will be reported on all lot specific data sheets. I have never used microsomes or S9 before. How do I use them? You can find detailed instructions for use of microsomes and S9 right on our web site. Check the Product Sheets and Protocols in the File Library. How long can I store microsomes and S9? These products should always be stored in an ultracold freezer at temperatures < -70° C. Stored under these conditions the cytochrome P450 enzyme systems are remarkably stable, showing little, if any, loss of activity after storage for more than 5 years. However, some of the phase II enzyme systems, particularly those in S9 such as N-acetyltransferase, are not stable even at -70° C, and lose significant activity within a matter of weeks or months. Can I measure phase II metabolism using microsomes or S9 fractions? Yes, but with microsomes and S9 you must add the necessary phase II cofactors. For example, if you want to measure glucuronidation (UDPGT) in a microsomal incubation, you will need to add uridine diphosphoglucuronic acid (UDPGA) in addition to the normally required NADPH regenerating system. As an alternative, consider using cryopreserved hepatocytes, since these contain all of the cofactors necessary to perform coupled phase I/II metabolism Can I repeatedly thaw and refreeze microsomes and S9 if I only need to remove a small aliquot of these products from a vial? Yes, as long as you carefully thaw these products on ice, remove what you need, and then quickly refreeze the vial to -70° C. As an alternative to repeated thawing and freezing, you can thaw the vial once and distribute the contents into single-use vials for future use. That way, you only have to thaw the microsomes/S9 one more time. How many animals are used to make each lot of microsomes and S9? The number of animal livers used to make a batch of microsomes or S9 varies, depending on the species. For small animals such as mice and rats, we always use at least 25 livers to make each lot of product. For larger animals such as monkeys and dogs the number is much smaller, typically 4 to 7 livers per lot of product. What information is available about the human donors from which the hepatocytes, microsomes, and S9 were prepared? We generally provide the following information about each of the donors: AgeGenderRaceTobacco UseAlcohol UsePrescription Medication UseNon-prescription Drug UseSignificant Medical HistoryCause of Death For ethical and privacy reasons, we do not provide any information which might identify the donor. How do I plate the cryo-plateable hepatocytes for use with the Promega ADME-Tox assay kits? The cryo-plateable hepatocytes should be plated following the Celsis IVT's plating method. For CYP450 gene induction studies, cells are typically exposed for 1–3 days to inducers (Optimal length of treatment time should be determined empirically, though maximal inductions are typically reached after 48 hours of exposure to an inducer.). For determining metabolism with P450-Glo substrate, perform the P450-Glo™ Assay as described in Promega's Technical Bulletin 325. With the P450-Glo assay kit, what plate format should be used to plate the cryo-plateable hepatocytes? The cryo-plateable hepatocytes may be plated in any format with a collagen I coating. However, when measuring the luminescence a white opaque polystyrene non-treated flat-bottom 96- or 384-well plates is preferred. (Do not use treated plates, black plates or clear plates.) Sample may need to be transferred from cell culture plate to an opaque, untreated plate depending on your assay design. Make sure that the plate used is compatible with your luminometer. How quickly can I get results from the P450-Glo kit vs conventional LC/MS methods? From starting the P450-Glo reaction to the measurement of luminescence taks from 1 - 4 hours, depenindg on the substrate used. See P450-Glo Technical Bulletin 325 for optimal times. What does "pre-qualified hepatocyte lots" mean? Celsis IVT is testing our extensive inventory of cryopreserved hepatocytes for use with Promega's ADME-Tox assay kits. Confirmed lots that work well with the Promega assays will be indicated on the Celsis IVT characterization tables. Coming Soon! Or contact IVT customer service at +1 410 455 1242 for product suggestions from our currently validated lots. What seeding density should be used for a 96-well or 384-well plate? Optimal seeding densities for human and most animal species are as follows: 96-well plate: 50,000 cells per well 384-well plate: 10,000 cells per well Please note: we recommend mouse hepatocytes be seeded at lower densities.
What concentration of the P450-Glo substrate should I use? Recommended substrate concentrations can be found in Promega's P450-Glo Technical Bulletin 325. |
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