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Hepatocytes
Microsomes & S9 Human Renal Proximal Tubule Cells
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Frequently Asked Questions
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Cryopreserved hepatocytes
When stored at the proper storage conditions (< -150° C), the cytochrome P450 enzyme systems remain unchanged after more than 5 years of storage. |
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.
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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.
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We generally provide the following information about each of the donors: - Age
- Gender
- Race
- Tobacco Use
- Alcohol Use
- Prescription Medication Use
- Non-prescription Drug Use
- Significant Medical History
- Cause of Death
For ethical and privacy reasons we do not provide any information which might identify the donor.
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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.
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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.
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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.
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Distributors
Celsis IVT has the following distributor in Asia:
Charles River Japan
INNOTECH Bldg. 11F
3-17-6 Shin-Yokohama,
Kohoku-ku, Yokohama,
Kanagawa 222-0033
Japan
T: 81 454 749 336
F: 81 454 749 341
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Celsis IVT has the following distributor in Spain:
CYMIT QUIMICA SL
Guillem Tell, 42
08006 Barcelona
T. 34-93-2412927
F. 34-93-4144979
info@cymitquimica.com
www.cymitquimica.com
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Fresh hepatocytes
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]. |
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.
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Got to http://www.celsis.com/calendar.html to view and fresh animal hepatocyte isolation schedule.
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General information
Human renal cells
Metabolism and cytotoxicity assays as well as transport and efficacy studies.
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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.
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Yes, we recommend that you use our InVitroGROTM PT medium which has been optimized and tested with the human renal proximal tubule cells. |
The InVitroGROTM PT medium will last 60 days from the day of receipt. |
The cells should be stored in liquid nitrogen. |
The formulation of InVitroGROTM PT medium is proprietary. |
Yes, the human renal proximal tubule cells will attach to untreated plastic as well as collagen coated plates. |
InVitroGRO media
LiverPool
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.
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Microsomes
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.
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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. |
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. |
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. |
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.
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We generally provide the following information about each of the donors: - Age
- Gender
- Race
- Tobacco Use
- Alcohol Use
- Prescription Medication Use
- Non-prescription Drug Use
- Significant Medical History
- Cause of Death
For ethical and privacy reasons we do not provide any information which might identify the donor.
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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.
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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.
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M-class and H-class microsomes are purposed-pooled using a proprietary algorithm to ensure consistent and reproducible product across the two classes.
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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.
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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.
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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.
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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
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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.
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S9
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.
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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. |
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. |
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. |
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.
|
We generally provide the following information about each of the donors: - Age
- Gender
- Race
- Tobacco Use
- Alcohol Use
- Prescription Medication Use
- Non-prescription Drug Use
- Significant Medical History
- Cause of Death
For ethical and privacy reasons we do not provide any information which might identify the donor.
|
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.
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