For Research Use Only. Not for use in diagnostic procedures.
Cryopreserved Pooled Plateable Human Hepatocytes
(5 Donor Pool)
Catalog Number HMCPP5
Pub. No. MAN0016011
Rev. 2.0
Product description
Pooled plateable human hepatocytes are a mixture of plateable primary hepatocytes produced by combining cells from 5 individual
donors in a single vial. Pooling of donors helps to minimize lot-to-lot variation and improves predictability of drug metabolism
studies and other biological assays. The cells are provided in a cryopreserved format similar to single donor plateable hepatocytes and
have been shown to have representation of each donor when plated. Additionally, every lot of cells is tested for Phase I enzyme
activity, cell viability, plating efficiency, and morphology.
Product Catalog No. Amount Shipping Storage
Pooled Plateable Hepatocytes (5 Donor) HMCPP5 1.5 mL (>6 × 10
6
cells)
Liquid nitrogen (vapor
phase) in Dewar
Liquid nitrogen vapor
phase.
*Shelf life duration is determined from the Date of Manufacture.
Product use
For Research Use Only. Not for use in diagnostic procedures.
Important information
Cryopreserved primary hepatocytes should be kept at all times in the vapor phase of liquid nitrogen. The product shall only be removed
when it is needed and thawed immediately after removal from liquid nitrogen container.
Safety information
Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Product specification
Form: Cryopreserved
Species: Human
Quantity: 1 Vial
Cell type: Hepatocytes
Donor source: 5 donor
Product size: 1.5 mL
Number of cells: >6 × 10
6
cells
2 Cryopreserved Pooled Plateable Human Hepatocytes (5 Donor Pool)
Procedural guidelines
• If plating hepatocytes with an overlay, refer to the specification sheet for Geltrex
™
LDEV-Free Reduced Growth Factor Basement
Membrane Matrix (Cat. No. A1413201), which will provide the lot concentration and technical tips.
• Geltrex
™
Matrix should be thawed on ice for 2–3 hours before application, or overnight at 4°C, and kept ice-cold to prevent gelling.
• Read the directions supplied with the Hepatocyte Maintenance and Plating Supplement Packs (Cat. No. CM4000 and CM3000) and
prepare Maintenance and Plating Media using Williamʹ s Medium E.
• If using HepExtend
™
Supplement: Prepare 1X HepExtend
™
Supplement in Hepatocyte Maintenance Media.
• Once thawed, cryopreserved hepatocytes must be used immediately and will not retain metabolic activities if refrozen.
Thaw, centrifuge, and resupend
1. Warm all media to 37°C in a water bath:
Hepatocyte Thaw Medium (HTM, Cat. No. CM7500)
Plating medium (Williamʹ s Medium E supplemented with Hepatocyte Plating Supplement Pack, Serum-Containing)
2. Remove the vial of hepatocytes to be thawed from liquid nitrogen and rapidly thaw by placing at 37°C in a water bath with gentle
agitation for 1–2 minutes (or once a sliver of ice is left in the tube). Complete thawing can be detrimental to the cell viability.
3. Wipe the vial with 70% alcohol in hood; pour or use wide-bore pipette tip to transfer hepatocytes into HTM, then centrifuge at 100 × g
for 10 minutes at room temperature.
4. Pour supernatant off into waste bottle and invert completely. Do not shake. Add ~1 mL of plating media per 1 × 10
6
total cells.
Count, plate, and incubate
1. Determine cell viability and yield.
2. Dilute to correct seeding density with Plating Medium, see Table 1.
3. Transfer hepatocytes to multi-well plate (collagen coated).
4. For a 24-well plate: Human hepatocytes, 500 μL. The density is dependent on assay application, see Table 1.
5. Place plate in incubator, and with hand on top of lid disperse cells by shaking.
6. Incubate plate at 37°C for 4–6 hours.
Do not move plate during this time, as the cells are forming a monolayer.
If planning to overlay hepatocytes, use this time to calculate the amount of Geltrex
™
Matrix needed.
7. After incubation, agitate plates to loosen debris and aspirate medium.
8. If using an overlay, proceed to the next step. If not using an overlay, replace medium with warm Maintenance Medium, or alternative
medium, depending on your application.
Note: Do not let the hepatocytes dry out. Replace the medium quickly.
Overlay
Important: Geltrex
™
Matrix and the Maintenance Medium used for its dilution must be kept ice cold to prevent premature gelling. Keep
Geltrex
™
Matrix and Incubation Medium on ice; preferably use cold pipettes when mixing.
1. Calculate the amount of incubation medium needed to feed the plated hepatocytes and place this volume on ice.
2. Find the protein concentration of Geltrex
™
Matrix on its specification sheet - each lot is slightly different.
3. Calculate the amount of Geltrex
™
Matrix that needs to be added to the incubation medium. If using HepExtend
™
Supplement, prepare
Hepatocyte Maintenance Media with 1X concentration of HepExtend
™
Supplement.
4. Add Geltrex
™
Matrix to the cold Maintenance Medium (with or without HepExtend
™
Supplement) on ice. Mix well by pipeting
several times.
5. Apply overlay to plated hepatocytes and incubate at least two hours or up to 24 hours before use.
Note: The gel layer will settle out of the media over the top of the hepatocytes.
6. Replace the Maintenance Medium daily.
Cryopreserved Pooled Plateable Human Hepatocytes (5 Donor Pool) 3
Table 1 General Seeding Density Guide for Cyropreserved Hepatocytes (12 mL media per plate)
Plate size Quantity*
6-well 0.9 × 10
6
– 1.1 × 10
6
cells/mL
12-well 0.8 × 10
6
– 1.0 × 10
6
cells/mL
24-well 0.7 × 10
6
– 0.9 × 10
6
cells/mL
48-well 0.6 × 10
6
– 0.8 × 10
6
cells/mL
96-well 0.5 × 10
6
– 0.7 × 10
6
cells/mL
* Each lot may require slight adjustments in seeding density to form optimal monolayer.
Related products
Product Catalog No.
HepExtend
™
Supplement (50X) A2737501
Williamʹ s Medium E (1X), no phenol red A1217601
Primary Hepatocyte Maintenance Supplements CM4000
Primary Hepatocyte Thawing and Plating Supplements CM3000
Geltrex
™
LDEV-Free Reduced Growth Factor Basement
Membrane Matrix
A1413201
Collagen I, Coated Plate A11428
Hepatocyte Thaw Medium CM7500
Human Plateable Hepatocytes, Transporter Qualified HMCPTS
Human Plateable Hepatocytes, Induction Qualified HMCPIS
Human Plateable Hepatocytes, Metabolism Qualified HMCPMS
Trypan Blue Solution, 0.4% 15250061
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