Cryopreserved Human & Rat Kupffer Cells
Description
Kupffer cells, also known as Browicz-Kupffer cells and stellate macrophages, are specialized macrophages that line the walls of thesinusoids in the liver, which form part of the reticuloendothelial system (RES) (also called mononuclear phagocyte system). They play arole in recycling dead blood cells and helping the liver respond to toxic substances in the blood stream.
ProductCatalog no.

Human Kupffer Cells

HUKCCS

Rat Kupffer Cells

RTKCCS

AmountStorage

1 × 106 viable cells/vial

Store in liquid nitrogen (vapor phase)

Product Use
For Research Use Only. Not for use in diagnostic procedures.

Important Information
Kupffer cell monocultures grow best in medium supplemented
with at least 2% FBS (10% recommended) and NO corticosteroids(e.g. Dexamethasone, Hydrocortisone).

Safety Information
Read the Safety Data Sheets (SDSs) and follow the handling
instructions. Wear appropriate protective eyewear, clothing, andgloves.

Culture Conditions
Media: Kupffer Maintenance Medium and Co-culture MaintenanceMedium for co-culture with hepatocytes, or Kupffer Monoculture
Medium for monoculture.
Culture Type: Adherent monoculture or adherent co-culture
with hepatocytes
Recommended Substrate: Collagen type I
Temperature Range: 36°C to 38°C
Incubator Atmosphere: Humidified atmosphere of 5% CO2

Prepare Media
Prepare Kupffer Thawing/Plating Medium
Kupffer Thawing/Plating Medium consists of Advanced DMEM
supplemented with FBS and a cocktail solution of fetal bovine
serum (FBS), penicillin-streptomycin, human recombinant
insulin, GlutaMAX™-I supplement, and HEPES.
For 500 mL of Kupffer Thawing/Plating Medium, ascepticallymix the following components:
Advanced DMEM
457 mLFBS
25 mL
Thawing/Plating Cocktail – A 18 mL
Note: FBS and Thawing/Plating Cocktail – A are available as part of thePrimary Hepatocyte Thawing and Plating Supplements (Cat. no. CM3000).
Do not add Dexamethasone to the Kupffer Thawing/Plating Medium.

Prepare Kupffer Maintenance Medium
Kupffer Maintenance Medium consists of Advanced DMEM
supplemented with FBS and a cocktail solution of penicillinstreptomycin, ITS+ (insulin, transferrin, selenium complex, BSA,and linoleic acid), GlutaMAX™-I supplement, and HEPES.
For 500 mL of Kupffer Maintenance Medium, asceptically mix
the following components:
Advanced DMEM
455 mLFBS
25 mL
Maintenance Cocktail – B
20 mL
Note: Maintenance Cocktail – B is available as part of the Primary
Hepatocyte Maintenance Supplements (Cat. no. CM4000). Do not add
Dexamethasone to the Kupffer Maintenance Medium.
Publication Number MAN0008963

Prepare Co-culture Maintenance Medium
Hepatocyte-Kupffer Co-culture Maintenance Medium consists ofKupffer Maintenance Medium without FBS.
For 500 mL of Kupffer-hepatocyte Co-culture MaintenanceMedium, asceptically mix the following components:
Advanced DMEM
480 mL
Maintenance Cocktail – B
20 mLPrepare Kupffer Monoculture Medium
Kupffer Monoculture Medium consists of RPMI 1640 Medium
with GlutaMAX™-I supplement and HEPES (Cat. no. 72400), andsupplemented with 10% FBS and 1X penicillin-streptomycin.
For 500 mL of Kupffer Monoculture Medium, asceptically mix
the following components:
RPMI 1640 Medium
445 mLFBS
50 mL
Penicillin-Streptomycin (100X) 5 mL

Co-culture of Kupffer Cells with Hepatocytes
The protocols below assume using 24-well plates for all seeding
densities. Adjust seeding densities for other well formats
appropriately.
Recover Kupffer Cells for Co-culture
1. Remove a vial of frozen Kupffer cells from liquid nitrogenstorage and rapidly thaw in a 37°C water bath until a small
amount of ice remains in the cryovial.
2. Transfer the contents of cryovial into a 15-mL conical tube
containing 9 mL of cold (4°C) Kupffer Thawing/Plating
Medium and place on ice. Alternatively, you may thaw up to
5 cryovials of Kupffer cells in a 50-mL conical tube containing
45 mL of cold medium.
Note: Kupffer cells are very “sticky” at physiological temperature of37°C. If the medium is warmed to 37°C, the Kupffer cells will attach to
any substrate including the walls of the conical tube. Therefore, use of
pre-warmed media is not recommended at this step.

3. Centrifuge the cells at 500 × g for 5 minutes.
4. Resuspend the pelleted cells (note that the pellet will be verysmall) in 1–2 mL of Kupffer Thawing/Plating Medium using aP1000 micropipettor. Serological pipette can also be usedhowever this may lead to clumping of the cells.
5. Count the cells using the trypan blue exclusion assay.
6. Dilute the cells in Kupffer Thawing/Plating Medium to 0.2 × 106
to 0.4 × 106 cells/mL (for inflammatory Kupffer/Hepatocyteco-cultures) or to the desired density per internal protocol.
7. Plate 0.5 mL of cell suspension per well of a 24-well plate
coated with collagen type I substrate.
8. Place the cells in a humidified 37°C/5% CO2 incubator andallow them to attach for 4–6 hours.
9. After 4–6 hours of attachment, replace the medium with fresh
Kupffer Thawing/Plating Medium.
Revision 1.0

10. After 24 hours, replace the medium with Kupffer MaintenanceMedium and proceed with your experiement or co-culture withHepatocytes.
Note: To maintain Kupffer cell cultures, replace spent medium withKupffer Maintenance Medium every 24–48 hours.

Prepare Hepatocytes for Co-culture
Fresh Hepatocytes: Prior to plating, subject fresh hepatocytes toa Percoll® purification step by using 50:50 v/v of 90% Isotonic
Percoll®:Kupffer Thawing/Plating Medium. Centrifuge the cells
at 100 × g for 10 minutes. If the cell recovery is too low, you may
increase the g force to 120 × g. Expect to see an increase inviability and a decrease in yield. This step is required to remove
residual Kupffer cells.
Cryopreserved Hepatocytes: For cryopreserved hepatocytes, you
may omit the Percoll® step if high quality platable human or rat
hepatocytes (e.g., Gibco® Transporter or Induction Qualified
Hepatocytes) are used. To enhance viability prior to plating, werecommend thawing hepatocytes in Hepatocyte Thaw Medium(Cat. no. CM7500).
90% Isotonic Percoll® Medium: Dilute Percoll® Medium (GEHealthcare Lifesciences, Cat. no. 17-0891-01) with 10% Dulbecco’s
Phosphate Buffer Solution (10X) (Cat. no. 14200-075).
Recommended Rat and Human Hepatocyte Seeding Density:
Freshly isolated hepatocytes: 24-well plate, 0.6 × 106 cells/mL
Cryopreserved hepatocytes: 24-well plate, 0.8 × 106 cells/mL
Co-culture with Hepatocytes
Plate Kupffer cells 24 hours prior to plating hepatocytes for best
results. However, you may also plate hepatocytes 4–48 hours after
Kupffer cells are plated.
1. Seed hepatocytes on top of Kupffer cells following standard
protocol for plating hepatocytes using Kupffer
Thawing/Plating Medium.
2. After allowing hepatocytes to attach for 4–6 hours, replace themedium with Co-culture Maintenance Medium.
3. Allow the hepatocytes to culture for 24 hours prior to
conducting experiments. Do not apply a matrix overlay (e.g.,Geltrex® matrix) in co-cultures.

5. Count the cells using the trypan blue exclusion assay.
6. Dilute the cells in Kupffer Monoculture Medium to 0.2 × 106 to0.4 × 106 cells/mL or to the desired density per internal
protocol.
7. Plate 0.5 mL of cell suspension per well of a 24-well plate
coated with collagen type I substrate.
8. Place the cells in a humidified 37°C/5% CO2 incubator andallow them to attach for 4–6 hours.
9. After 4–6 hours of attachment, replace the medium with freshKupffer Monoculture Medium.
10. After 24 hours, replace the medium with Kupffer Monoculture
Medium and proceed with your experiement.
Note: To maintain Kupffer cell cultures for longer (1–2 weeks), replacespent medium with Kupffer Maintenance Medium every 24–48 hours.

Kupffer Cell Activation
Add lipopolysaccharide (LPS, 1 µg/mL) to culture medium toactivate Kupffer cells for 2–24 hours (depending on experimentaldesign) prior to experiment in either Kupffer cell monoculture or
co-culture to mimic liver inflammation.
LPS activation changes happen fairly quickly, and morphologicalchanges in the cells can be observed in less than 2 hours.
1–4 hours: Cells start out darker and more square-like (rat) or
elongated (human), then become spindle-like with longer, thincytoplasmic projections and may appear dendritic-like. Cells exhibit
high level of motility by migrating around the plate.
4–8 hours: Cells return to a rounder morphology.
8–24 hours: Cells flatten and their cytoplasm enlarges, creatinglarge round cells with many vacuoles inside (macrophage-like).
Cells exhibit lower motility.

Related Products
ProductCat. no.

Advanced DMEM

12491

Primary Hepatocyte Thawing and Plating Supplements

CM3000Primary Hepatocyte Maintenance Supplements

CM4000Fetal Bovine Serum, Certified, US Origin

16000

Kupffer Cell Monoculture

RPMI 1640 Medium, GlutaMAX™, HEPES

72400

Recover Kupffer Cells for Monoculture
1. Remove a vial of frozen Kupffer cells from liquid nitrogenstorage and rapidly thaw in a 37°C water bath until a small
amount of ice remains in the cryovial.
2. Transfer the contents of cryovial into a 15-mL conical tube
containing 9 mL of cold (4°C) Kupffer Monoculture Medium
and place on ice. Alternatively, you may thaw up to 5 cryovialsof Kupffer cells in a 50-mL conical tube containing 45 mL ofcold medium.

Penicillin-Streptomycin (5000 U/mL)

15070

Note: Kupffer cells are very “sticky” at physiological temperature of37°C. If the medium is warmed to 37°C, the Kupffer cells will attach to
any substrate including the walls of the conical tube. Therefore, use of
pre-warmed media is not recommended at this step.

3. Centrifuge the cells at 500 × g for 5 minutes.
4. Resuspend the pelleted cells (note that the pellet will be verysmall) in 1–2 mL of Kupffer Monoculture Medium using a
P1000 micropipettor. Serological pipette can also be usedhowever this may lead to clumping of the cells.

DPBS (10X), no Calcium, no Magnesium

14200

Collagen I, Coated Plate 24 Well

A1142802

Explanation of Symbols and Warnings
The symbols present on the product label are explained below:

Temperaturelimitation

Use byBatch code

Catalog number

Manufacturer

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