Description Primary hepatocyte culture is a valuable tool that has been broadly used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 and, since then, has undergone many modifications. The most commonly used technique was described by Seglen in 1976 .
Product
Catalog no.
Amount
Storage
Rat Primary Hepatocytes
RHC001
1X10^6/vial
in liquid nitrogen
Product Use For Research Use Only.Not for use in diagnostic procedures
Important Information Hepatocyte cell grow best medium supplemneted with 10%FBS in M199 Media
Culture Conditions Culture Type:Adherent
Temperature Range:36℃ to 38℃
Incubator Atmosphere:Humidified atmosphere of 5% CO2
Size:The typical hepatocyte is cubical with sides of 20-30 µm
Passaging Adherent Cells All solutions and equipment that come in contact with the cells must be sterile.
Always use proper sterile technique and work in a laminar flow hood.
1. Remove and discard the spent cell culture media from the culture vessel.
2. Wash cells using a balanced salt solution without calcium and magnesium
(approximately 2 mL per 10 cm2 culture surface area). Gently add wash solution to
the side of the vessel opposite the attached cell layer to avoid disturbing the cell layer,
and rock the vessel back and forth several times.
Note: The wash step removes any traces of serum, calcium, and magnesium that
would inhibit the action of the dissociation reagent.
3. Remove and discard the wash solution from the culture vessel
4. Add the pre-warmed dissociation reagent such as trypsin or TrypLE™ to the side of
the flask; use enough reagent to cover the cell layer (approximately 0.5 mL per 10
cm2). Gently rock the container to get complete coverage of the cell layer.
5. Incubate the culture vessel at room temperature for approximately 2 minutes. Note
that the actual incubation time varies with the cell line used.
6. Observe the cells under the microscope for detachment. If cells are less than 90%
detached, increase the incubation time a few more minutes, checking for dissociation
every 30 seconds. You may also tap the vessel to expedite cell detachment.
32 | Cell Culture Basics
Part 4. Methods
7. When ≥ 90% of the cells have detached, tilt the vessel for a minimal length of time
to allow the cells to drain. Add the equivalent of 2 volumes (twice the volume used
for the dissociation reagent) of pre-warmed complete growth medium. Disperse the
medium by pipetting over the cell layer surface several times.
8. Transfer the cells to a 15-mL conical tube and centrifuge then at 200 × g for 5 to 10
minutes. Note that the centrifuge speed and time vary based on the cell type.
9. Resuspend the cell pellet in a minimal volume of pre-warmed complete growth
medium and remove a sample for counting.
10. Determine the total number of cells and percent viability using a hemacytometer,
cell counter and Trypan Blue exclusion, or the Countess® Automated Cell Counter. If
necessary, add growth media to the cells to achieve the desired cell concentration and
recount the cells.