There is an
abundance of information available detailing cell culturing techniques.
Hepatocyte cells are widely used in the study of the physiology and pathology
of the liver. These cultures are often used in the study of drug interactions
and drug metabolising enzymes. Studies in animal models such as rats have
differing enzyme activity and regulation. It is for this reason that human
hepatocytes are needed when studying a human model.

Below is a sample
protocol for a long-term media deriving hepatic cells from liver tissue. The
steps involve a two-step collagenase perfusion. This will determine the quality
of the hepatocyte cells. Tissue samples last for up to one month.

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Protocol Culture Media: (Pritchard et al)

1)     
Dissolve
one 5L Williams E media in 4L deionised water. Add 11.9 g HEPES and 11g NAHCO3
adjust pH to 7.2. Adjust volume to 5 litres.

2)     
Sterilize
by passing through a0.22 ?m
filter. Refrigerate.

3)     
Add the
complementary additives. Add 250ml of Williams E media. 10mlBSA
linoleic-linolenic solution, 5ml insulin(50mg), 1ml transferin(25mg),50 ?l selenium acetate solution (64 ?l g), 200 ?l dexamethasone solution(0.2mg), 100 ?l liver growth factor (100 ?g), 250 ?l cAMP solution (12.25
?l), 5ml glucagon solution(5mg), 250 ul
epidermal growth factor (250mg),500mg prolactin(50UI),100ul ethamolamine(0.3
?g), 50ml glutamine solution(1.46g),
50mg penicillin/streptomycin. Sterilize by passing through a 0.22 ?m filter.

4)     
Add 15ml
fungizone. The mixture is then aliquoted in 25ml fractions and stored at -80°C

5)     
The
final media is made prior to use by supplementing 250ml of Williams E media
with 25ml of the mixture.

Collagenase Solution (Pritcherd et al)

1)     
1L of
HEPES buffer with antibiotics, fungizone, and 10 mL of 70 mM CaCl2 added to the
solution. Divide the solution of 250 and 750 mL.

2)     
 Dissolve 500 mg collagenase in the 250 mL and
sterilize by passing through a filter. Because of the cost of collagenase, this
solution should be prepared only when perfusion of the tissue has been shown to
proceed correctly (see 3.2., step 4).

3)     
Add to
the 750-mL aliquot of HEPES buffer. This solution is dissociated the liver
tissue.

Perfusion step:

1.)   
The
liver sample is placed in the perfusion vessel and the edge is examined in
order to locate the veins and arteries that will be perfused.

2.)   
All
solutions and buffers are kept at 37°C, except for the albumin-HEPES solution
used for hepatocyte washings. Do not expose the solution to oxygen solutions
and buffers before perfusion, this will cause oxidative stress in the tissue.

3.)   
The
tissue is rinsed with HEPES buffer. Antibiotics and fungizone are added. Blood
is washed away; the tissue is heated to 37°C. A cannula is inserted into the
veins and arteries.

4.)   
The
collagen solution should be prepped at this stage.

5.)   
The
tissue is then perfused with 1 L of EGTA solution.

6.)   
The tissue
is then perfused with 1 L supplemented HEPES buffer in order to remove EGTA. The
reservoir of the perfusion vessel is emptied and washed several times with
water.

7.)   
 The tissue is then perfused with the
collagenase solution. For approximately 20 minutes. The tissues soften and
start to dissociate.

Hepatocyte Isolation:

1.      
The
tissue is transferred a steel container and examined for tumours and
impurities.

2.      
Gently disrupt
the cells.

3.      
Add 1-2
L of BSA-HEPES buffer.

4.      
Filtered
using a nylon filter (250 mesh) and the filtrate is added into 150 mL
centrifuge tubes. The filter is washed with BSA- HEPES solution to gather any
hepatocytes that are trapped.

5.      
Tubes
are centrifuged for 5 min.

6.      
The
supernatant is discarded and the pellet, containing the hepatocyte cells, is
resuspended in 200 mL of BSA-HEPES solution.

7.      
Repeat
steps 5 and 6 twice.

8.      
The
pellet is resuspended in an equal volume of BSA-HEPES solution.

9.      
500 µL
of hepatocyte suspension is added to 9.5 mL of the culture medium.

 

Hepatocyte Plating and Culture:

1.      
After
cell count is determined the culture medium is complemented with FCS (5% in
volume).

2.      
The
hepatocyte suspension is diluted in this medium to 3.5 or 10×106 viable
cells/mL for plating in 60 or 100 mm diameter culture dish.

3.      
Culture
dishes are distributed on stainless steel trays and 2 or 7 mL of culture medium
are added per 60 or 100 mm dish.4. Then, 1 mL of an appropriately diluted
suspension of cells (3.5 or 10×106 viable cells) is added per dish. This number
of cells per dish corresponds approximately to a cell density of 12.5×104 cells
per cm2 for a confluent monolayer. Cell suspension should be homogenized by
agitation while adding solution to the trays.

4.      
.
Culture dishes are then placed in an incubator, in a humid atmosphere of air 5%
CO2 at 37°C.

5.      
After 4 hours,
the serum is removed and replaced by new serum

6.      
The
culture medium is changed every 24 hours for short-term cultures and every
48-72 hours for long-term cultures.

 

Hepatocyte Transfection:

Once the hepatocytes
are prepared using the above methods the transfection can take place.
Transfection involves altering the pores of the cell membrane in order allow
the uptake of bacterial DNA. This is done in order for the cell to express
foreign DNA. There is a variety of ways of carrying out a transfection. The
process can be done via physical, viral or chemical means. These methods
include cell squeezing, electrophoresis, by using a viral vector or by exposing
the tissue to calcium phosphate.

For this
transfection electrophoresis will be used. Many gene transfers have been
carried out on liver tissue using this method. An electric field is produced
which then increases the permeability of the cell. The voltage passes through
the cells at a distance of 1-2 millimetres. Electrophoresis is known to be a
highly successful method. Is capable of transfecting adherent cells without the
need for trypsinsing before treatment.

 

Electroporation
(using a BioRad Gene Pulser):

1.      
Inspect cuvettes for damage prior to use. Cool
cuvettes via refrigeration or by ice. Cool the cuvette chamber slide, cells,
and DNA on ice.

2.      
Add 1-3 µl DNA to 40 µl electrocompetent cells
and gently mix.

3.      
For 0.2 mm cuvettes, set the Gene Pulser to 25
µF and 2.5 kV. (1.5 kV should be used for 0.1 mm cuvettes.) Set the Pulse controller
should be set to 200x.

4.      
Transfer the sample to a cuvette and tap gently
to insure even distribution of solution. Insert the cuvette into the cold
chamber slide and push the chamber slide into the chamber until the cuvette is
seated between the contacts in the base of the chamber.

5.      
Pulse once by pressing the 2 red buttons at the
same time until the Gene Pulser beeps. The time constant should be about 4.0
msec.

6.      
Add 1ml plasmid broth to a cuvette then transferring
to a test tube. (This must be done rapidly after the cells are shocked as cell ability
to uptake foreign DNA will decrease over time.) Incubate for 30-60 min on a
37°C on a shaker to allow the cells to recover and for expression of protein to
occur.

7.      
To turn off the Gene Pulser, flip the on-off
switch off, on, then off again to fully discharge the capacitors.

 

Image 14: Image
showing the BioRad Gene Pulser.( www.bio-rad.com
2017)

Note that a number
of factors may influence the quality of the result.

1.)   
Bubbles
in the sample and poorly mixed DNA.

2.)   
Damaged
cuvettes.

3.)   
Wet
cuvettes. Dry before usage.

4.)   
The
sample must have contact with both sides of the cuvette.

5.)   
High
salt content in the DNA sample. Drop dialysis can be used to remove excess
salt.

 

Expected
Results of Transfection:

After the
transfection protocol has taken place all that remains to do is to confirm the
success of the procedure. There is a number of ways in which this can be
carried out.

Green Fluorescent Protein

The presence of green fluorescent protein which was included in the
Clontech plasmid vector could be used to confirm the presence of the gene of
interest in the newly transfected hepatocyte cells. A floe cytometer is used to
determine the amount of successfully transfected cells. Depending on the
quality of the transfection the fluorescence will vary. The results will vary
on the condition of the cells, their activity, if the cells have aggregated.

It is highly likely
that the cells producing fluorescence contain the gene of interest (FMO3) due
to the fact that both genes were introduced on the same plasmid. GFP does not
alter the expression of other genes. It only requires the use of blue light to
visualise the GFP. This makes it a relatively simple confirmation method.

SDS-Page

One way to determine the protein content of the cell culture
is by using a discontinuous polyacrylamide gel combined with sodium dodecyl
sulfate (SDS) to denature the proteins. This is known as sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). The standard SDS-PAGE protocol
is also called the laemmli method, name after the scientist that refined the
method.

SDS PAGE negatively charge the proteins. The proteins are
then run through a gel toward a positively charged anode. This separates the
proteins by length and size. If the size of the expected protein is previously
known the protein can then be determined by comparing the gel to a protein
ladder.

Western-Blotting
Technique

Western blotting is often used
separate and identify proteins. The proteins are separated by molecular weight
via gel electrophoresis. This produces a series of bands representing the
proteins. The results of this are labelled with antibodies specific to the
protein of interest.

The unbound
antibody is washed off leaving only the bound antibody to the protein of
interest. The antibodies bound to the protein can then be detected. Only the
protein in which the antibody has bind to will show a visible band. The size
and thickness of the band indicates the amount of protein present. A secondary
antibody can be used for chemiluminescent visualization horseradish peroxidase
is often used for this purpose. 

Controls
for Experiment:

Both SDS-PAGE and Western Blotting
rely on protein ladder to compare the presence and size of proteins examined.
The use of negative controls where the wells are not filled with a protein
sample should be used. They should be treated in the same way that the protein
samples are tested. This should account for any experimental variability
present. A positive control with a defined protein sample should be used to
confirm what a positive result looks like under the particular experimental
conditions.

In Western Blotting loading
control antibodies should be used. Loading controls are essential for the
correct interpretation the experimental data. They confirm the equal protein
loading between the samples tested.

 

Size
Estimation:

From the results gathered from
SDS-Page and Western Blotting the size of the protein can be ascertained. This
can be further confirmed by using resources such as Uniprot and Pubmed. As
mentioned earlier in the paper the protein of interest has a molecular mass of 60kDa.

Summary and Possible Treatment:

After
carrying out the above protocol and transfecting the foreign DNA into the cell.
Assuming the cells have survived the transfection and the protein has been
confirmed present. All that is left is to ascertain the quality of the cells
and the effect that the newly transfected gene will have on the cell type.

Knowing
that the cell type is hepatocytic and the protein of interest is FMO3(flavin-containing oxygenases) which is an enzyme
found in the liver it is highly likely that cell will accept it readily and
will not have any negative effects associated with the transfection.

These transfected
hepatocyte cells may have therapeutic applications. Much research has been
carried out on the applications of liver directed gene therapy and
transplantations. Both normal and altered genes have been introduced into
animal and human subjects in the case of inherited disorders. (Guha et al 2001)

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