A. Preparation of primary cultures of rat hepatocytes (or freshly
isolated hepatocytes)
1. Method
Rat hepatocytes will be isolated aseptically by a two-step collagenase
perfusion of liver according to methods described (Moldeus, P.,
J. Hogberg, and S. Orrenius. Isolation and use of liver cells.
Methods Enzymol. 51:60-70, 1978). Hepatocytes will be sedimented
at 70 g for 2 minutes, resuspended and sedimented twice more for
a total of three times. Cells will then be resuspended in culture
medium and plated onto plates precoated with rat tail collagen.
The average cell viability is typically > 88%. With
this isolation technique, it is anticipated that there will be
~5-10% contamination with non-parenchymal cells.
2. Availability and Cost
This will be done ~ three times per week (Monday, Tuesday and
Thursday or other days if needed) to serve ~ 14 investigators.
PI's that need cells need to notify the Core (Core technician
Xiu-Yan or Shelly Lu, M.D.) no later than 24 hrs prior to the
scheduled day of cell preparation. A BLACK BOARD WILL BE POSTED
OUTSIDE OF THE CORE LAB MMR 415 FOR SIGN UP. There will be a
limit on the number of investigators one cell preparation can
serve - up to three if plating is required, but no limit as long
as there are enough cells if plating is not required. Cells will
be plated according to instructions provided by the PIs. Cells
will be attached ~ 2 hrs after plating which is the time that
they should be picked up from the Core (unless prior arrangement
have been made). medium change will be done by individual labs.
Cell isolation will be done only if there is at least one
request for cells. The cost for one prep is $100. Thus,
the cost to each PI will depend on the number of investigators
sharing these cells.
B. Human hepatocytes
Cultured or freshly isolated human hepatocytes will be prepared
according to published methods. Anticipated availability is ~
3-4 times/month. Cultured human hepatocytes will be provided
after the first change of medium (4 hours after plating). PIs
that need cells should notify the Core as early as possible.
Cells will be made available to PIs on a first come first serve
basis. The cost is $120 for each isolation. The cost to each
investigator depends on the number of investigators sharing these
cells.
C. Cell lines
The Cell Culture Core will provide cell line banking to PIs.
Cell lines will be thawed and plated out as per request and the
Core will also freeze and store cell lines for PIs. Protocols
for caring of the cells will be provided, mostly according to
ATCC. At the present time, we have the following cell lines already
available:
Alex (PLC/PRF/5): human hepatoma
BHK: Baby hamster kidney
C2C12: mouse myoblast
CaC0-2: human colon carcinoma
CHO: Chinese hamster ovary cells
COS-7: transformed CV1
CV1: monkey kidney
FM3A: mouse fibroblast
HepG2: human hepatoblastoma
HeLa: cervical carcinoma
HT-29: human intestinal
HuH-7: human hepatoma
Mahlavu: human hepatoma
MDCK: distal monkey kidney tubule
NIH3T3: mouse fibroblast
Vero: monkey kidney
ts85: E1 enzyme ts mutant of FM3A
2.2.15: HBV producing cell lines derived from HepG2
sf9: insect cells
Charge for freezing+storage will be $6 each time
Charge for thawing will be $14 each time
D. Other services to be provided free of charge to investigators
include:
1. Quality Assurance
a. For all cultured cells, mycoplasma testing will be
done on a regular basis.
b. For cell lines, isoenzyme analysis will be performed regularly to ensure there is not cross-contamination with other cells.
2. Training
For individuals interested in learning rat and human hepatocyte
isolation or tissue culture techniques, the Core will provide
training.
3. Development of other techniques
If there is demand, the Core will develop important or useful
new techniques, e.g. isolation and culturing of periportal and
perivenous rat hepatocytes, mouse hepatocytes and intestinal cells.
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