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Dear All,
I am having a query about the uptake of drug in the cell. If i want to
study the uptake of doxorubicin in the HepG2 cell, is it necessary
that the cell should adhere to the tissue culture plate. Will it be
possible to study the uptake across the cells in suspension or freshly
seeded cells. Especially when there is no report about the
paracellular absorption, is it necessary to form the confluent
monolayer when my interest is transcellular uptake. Please guide me in
these regards.
Thanking you all in anticipation
Wishes,
Devang P. Shah.
Senior Research Fellow (PhD Tech Fellow),
Dept of Pharmaceutical Sciences and Technology,
Pharmacology Lab II,
Institute of Chemical Technology,
N. P. Marg,
Matunga, Mumbai 400 019.
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Below data is from ATCC: Are you following each of the steps? Also,
there
may be a windows of passages. Too few and the cells do not respond, too
many and the cells do not respond. Search the literature for the
correct
number of passages.
ATCC complete growth medium: The base medium for this cell line is
ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003.
To
make the complete growth medium, add the following components to the
base
medium: fetal bovine serum to a final concentration of 10%.
Temperature: 37.0*C
Subculturing: Protocol:
1.Remove and discard culture medium.
2.Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA
solution to remove all traces of serum that contains trypsin inhibitor.
3.Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells
under an inverted microscope until cell layer is dispersed (usually
within 5
to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking
the
flask while waiting for the cells to detach. Cells that are difficult to
detach may be placed at 37*C to facilitate dispersal.
4.Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by
gently
pipetting.
5.Add appropriate aliquots of the cell suspension to new culture
vessels.
6.Incubate cultures at 37*C.
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Devang,
You can study the cells before they adhere, however the results will
be one
step further from the in vivo situation.
In this case, the reason for developing the monolayer is not to be
concerned
with paracellular movement or tight junctions (since I assume you are
not
using a transwell system, and just measuring internalization), but
rather
having the cells in a steady state of surface protein expression.
Thus, the confluent state will be closer to actual tumor tissue, than
the
freshly seeded or suspension cells.
Will there be any discernable difference in the kinetics or extent of
drug
uptake in either system?
That's a good question.
Hopefully, you will study both, and let us know when you are done.
Good luck,
-Shawn
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Hi Devang,
You can study the uptake of drug in the cell suspension by two methods
and it is not necessary to from confluent monolayer but only thing is
that you have to measure cell density in suspension. The methods are
as follows:
Method 1: Oil-spin method: Incubate a vial containing cell suspension
at 37 *C for 5 min as well as vial containing drug in suspension
buffer. Initiate the reaction by mixing equal volume of cell
suspension and drug in suspension buffer. Aliqouts 75 ul of sample
from the mixture at 15, 30, 45 and 60 sec. and centrifuge at 8000 g
for 40 sec through 200 ul of silicon oil. (You may need to adjust the
density). After centrifugation step cells will settle down at the
bottom of centrifuge tube. Freez each centrifuge tube in liquid
nitrogen and cut it after freezing for the collection of cell pellet.
The extract the entrapped drug form cell pellet by crushing it with
methanol or Acetonitrile
Method 2: Media-loss Method: Incubate a vial containing cell
suspension at 37 *C for 5 min as well as vial containing drug in
suspension buffer. Initiate the reaction by mixing equal volume of
cell suspension and drug in suspension buffer. Aliqouts 75 ul of
sample from the mixture at 0, 0.5, 4,6, 12, 20, 30 and 45 min and
centrifuge at 8000 g for 40 sec. Collect the supernatant and mix it
with methanol or Acetonitrile and estimate the concentration in
supernatant
Method 1 gives direct measurement of uptake of drug in cell line
whereas method 2 is an indirect way of measuring the uptake by
monitring loss of drug form media and is more convenient and than
method 1
Thanks
Rhishikesh Thakare
Advinus Therapeutics, Pune, India
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If you do not adhere to the published recommendations for a cell line be
prepared to substantiate your use.
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Hello,
As other people suggested there is no need for the cells to adhere to
study the cellular uptake of a drug. But if you can plate them, you
can go for that as it can give good reproducibility.
However, if the absorption of your drug involves a carrier mediated
pathway (transporter/receptor), and you are gonna work with freshly
seeded cells I suggest you to ensure that your cells express that
protein using a positive control.
Coming to paracellular diffusion, you won't be able to delineate that
in an uptake study unless you use a transwell plate.
Thanks
Ravi Talluri
Sai Advantium
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In high-resolution autoradiographic studies with cell lines:
uptake-retention of radio-labeled estradiol, we found considerable
variations, perhaps linked to the (rapid) changes of growth and to
varying degrees of crowding - with related intracellular alterations.
These changes seem to render such in vitro conditions quite variable,
difficult to select a defined point of time with sufficiently stable
and reproducible conditions. In vivo systems appear to be much more
stable and homeostatic-reproducible. Such was our impression from
albeit limited pilot experiments. Perhaps the in vitro conditions can
be sufficiently controlled (?). The question still remains, as Shawn
Spencer states, how representative are the data. And I wonder too how
much confidence one can put in results from drug uptake and binding in
cultured cells, i.e. whether such data can be considered representative
for in vivo conditions. - Are there correlative controls available with
in vivo confirmation?
Walter E Stumpf
--
University of North Carolina
2612 Damascus Church Rd
Chapel Hill, NC 27516
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Along that same thread, the use of transformed cells vis a vis primary
cells
in vitro may be a confounding (or the ultimate?) factor.
We have tried to infect a number of transformed lymphoblastoid cell
lines
with herpes virus, (the viruses require expression of a cell-surface
receptor for entry), however depending on which cell line used (e.g.,
HSB-2
vs. SUP-T1 vs. MOLT-3), there will be a large variance in the tropism
of the
cells (i.e., number of viral DNA copies found). So now the question
becomes,
does each cancer patient express different or varying degrees of cell
surface receptors? Apparently, they do.
How would that impact the in vitro or population efficacy and
efficiency of
an apparently transported drug like doxorubicin? In which experimental
situations does the choice of primary cells or in situ work become most
relevant? Do we have a tendency to just pick in vitro cell lines
based on
what we think will show a positive result? If so, how is that a
scientifically valid finding?
In the end, the decision to use in vitro cells (e.g., choosing hepG2
versus
liver slices), the timing (confluent or not), and the choice of cell
line,
all seem to come at a price; perhaps we are getting ripped off.
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Dear Walter,
I haven't performed any autoradiographic studies earlier.
However, I worked on uptake of various radioactive labelled drugs for
screening their interactions and affinities towards various influx and
efflux transporters. I used to observe very low variability (SD and CV
% for n=4 -6 wells). If we plate the same number of cells and maintain
the similar culture conditions, data should be very tight with in a
passage. There could some variability between passage to passage due
to changes in some intracellular conditions. I observed this kind of
variability in primary culture more than a cell line especially when I
screened the compounds for transporter proteins. We assumed that could
be due to changes in the pattern of expression ( In some studies, we
confirmed this hypothesis by RT-PCR and WB)
Coming to the query in IVIVC. That's a big question. It varies from
study to study. I feel that in vitro can help you in getting a trend
for a set of compounds (high to low..). Say like your compound is
highly cleared by CYP metabolism in body (e.g Midazolam). Then there
are high chances to get a good correlation between your in vitro
micorsomal clearance vs in vivo clearance.
Some studies also have shown very good correlation between Caco-2
permeability and bioavailability (If the absorption is the limiting
factor)..The problem with in vivo is, being a complex system (so many
processes happening parallely), it's really difficult to control the
system to explain the mechanisms. And we all no well about interanimal
variability..
I feel that say we have 1000 compounds, In vitro studies can get the
rank order very quickly and we can take the compounds with better
properties to in-vivo which is the ultimate to get the realistic
picture.
This are just my thoughts based on my experience in this area.
Thanks
Ravi Talluri
Sai Advantium
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Dear Ravi
Thank you for your response. Yes, we believe there must be utility to
the in vitro systens (which are complex and variable, more so than it
seems). And we agree, for the meaningful interpretation of data from
in vitro experiments, correlative in vivo controls need to provide and
establish evidence for the in vivo meaning of the approach.
Walter __
Walter E. Stumpf
University of North Carolina
Chapel Hill, NC 27516
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Dear All,
Thank you very much for the kind inputs to my query. So the take home
message will be to allow the cells to adhere so as to facilitate the
expressions of various proteins and try to do some in vivo
biodistribution studies so that we can detect the maximal
concentration of drug in the liver so as to endorse the site specific
drug delivery. This will also establish some in vitro in vivo
correlationship.
Thank you all once again.
Regards and wishes,
Devang P. Shah.
Senior Research Fellow (PhD Tech Fellow),
Dept of Pharmaceutical Sciences and Technology,
Pharmacology Lab II,
Institute of Chemical Technology,
N. P. Marg,
Matunga, Mumbai 400 019.
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Yes,
The take home message even constitute the void of the work in this
field. So, it is one of the factors that play ultimate role in the
trend observed in the IVIVC. So, distinction, identification and
mimicking of the various protein expressions in the adherent cells
plus checking back iv vivo is the best way out.
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