BS2510 (2005/6)
Dr D R Davies
Isolation of Viable Cells from Mammalian Tissues and their Use in Metabolic Studies
In the early development of the study of mammalian metabolic biochemistry, there was much use of
intact laboratory animals such as the rat or mouse but over the last 30 year or so there has been the
development of procedures which allow the isolation and use of mammalian cells for such studies. A
major problem with whole animal studies is the biological variation between animals which means
that experiments need to be performed on groups of animals which are as closely matched as possible
in terms of age, weight, sex, diet so that statistically significant results between control and
experimentally treated animals. The smaller the difference in metabolic parameters, the larger the
number of animals that have to be sacrificed to obtain a statistically significant results. Although
there are some aspects of metabolism which require whole animal study, the use of isolated cells has
allowed the possibility of conducting many experiments on a homogeneous population of cells from
a single animal.
Physiological Media
Once an organ or cell is removed from an experimental animal the major problem is to keep it in a
viable state so that it behaves in the same way as it does in the intact animal. Numerous physiological
media have been developed for the study of cells, the most widely used was that developed by Krebs
and Ringer which is an isosmotic medium with an ionic composition similar to that of blood plasma
and designed to prevent cells from shrinking or swelling. Krebs-Ringer-Bicarbonate (KRB) medium
consists of 120mM NaCl, 4.8mM KCl, 1.2 mM MgSO4, 1.2mM KH2PO4, 1.3 mM CaCl2, 24mM
NaHCO3 which is gassed with a mixture of O2/CO2 (95/5 vol/vol). This maintains a good supply of
oxygen to the cell and the CO2 dissolves in the medium to give a carbonic acid/ bicarbonate buffer
mix which maintains the pH at a physiological level (pH 7.4). While this is sufficient for short term
experiments lasting an hour or so, longer term studies require cells to be cultured with a carbon
source (e.g. 5mM glucose), amino acids and vitamin supplements. Often it is necessary to add serum
or plasma, growth factors or hormones and antibiotics such as streptomycin, anti-fungal agents such
as griseofulvin, and anti-mycoplasma agents such as gentimycin.
Erythrocytes
The simplest of all mammalian cell types on which metabolic studies are conducted is the red blood
cell. It is readily available as a suspension of single cells and not made up into a tissue containing
many cell types connected together with connective tissue. The blood is treated with heparin to
prevent clotting and the cells harvested by centrifugation and repeated suspension and washing in
isotonic (0.154 M) NaCl. There is therefore no problem of disrupting the tissue to obtain isolated
cells and it is relatively easy to remove the contaminating white blood cells by passing the blood
sample through a sulphoethyl-cellulose column which binds and removes the white blood cells while
allowing erythrocytes to pass through.
It is possible to prepare many different types of erythrocyte preparations such as 'ghosts', normal
orientation and inside out, and vesicles, normal and inside-out (see handout). Mammalian
erythrocytes do not contain subcellular organelles, such as mitochondria, endoplasmic reticulum and
nuclei .They are therefore not capable of complex metabolism unlike for example liver cells but they
can be used to study the transport of molecules and ions in and out of cells and the requirement for
cofactors such as ATP. They can also be used to examine the location of a protein or protein domains
on either side of the plasma membrane and to identify trans-membrane domains.
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Adipocytes
A metabolically more interesting cell type is the fat cell or adipocyte. The mature fat cell has a
characteristic signet ring shape in which the storage product, triacylgycerol is surrounded by a thin
layer of cytoplasm. Adipocytes can be isolated from fat deposits such as the epidydimal fat pad found
in the abdomen of the male rat. The fat pads are cut into small pieces and incubated with KRB and
collagenase (0.5 mg/ml), gassing with O2/CO2 for 45 minutes in a shaking water bath at 37oC.The
collagen binding the cells together, is disrupted and the incubation mixture with the cell suspension is
forced through a small mesh nylon net to remove the large undigested pieces of tissue. The cell
suspension is then transferred to plastic centrifuge tubes and centrifuged at low speed for about 15
sec. The adipocytes have a very low density and float to the top of the tube while the stromal
vascular cells, erythrocytes and other non-fat cells sediment to the bottom of the tube. The top layer
is aspirated resuspended in more KRB and the centrifugation repeated. Finally the adipocytes are
resuspended cell in KRB with Bovine Serum Albumin added, this protein has a high affinity for fatty
acids which are released from the fat cells and which are potentially damaging to the cells. To
prevent damage to cells polyethylene or polycarbonate tubes and syringes with a wide bore (2-3mm)
are used to transfer cells.
Studies on Adipocytes - Insulin-Stimulated Glucose Transport
As with other hydrophilic substrates, glucose does not enter cells by simple diffusion but rather by a
process of facilitated diffusion which involves a transmembrane protein called a Glucose Transporter
(GLUT) which speeds up the uptake of the sugar (see Lodish p 638, 908-909). This is a saturable
process because the transporter has a particular affinity for glucose process but unlike an enzyme it
does not transform the substrate, but rather moves it from one side of the plasma membrane to the
other. Glucose enters the cell because of the concentration gradient until the Glucose concentration
inside is the same as that outside. In order to study this phenomenon in isolation from the further
metabolism of glucose non-metabolizable analogues, such as 2-deoxyglucose or 3-O-Methyl
Glucose, are used which are taken up by cells but not further metabolised. You can thus measure
glucose transport independent of its metabolism, which complicates the interpretation of the data .
This was how glucose transporters which facilitate the transport of the sugar were initially
characterized in red blood cells and since then a family of similar proteins have been discovered
which are closely related but sufficiently different to have different properties and functions in
different cell types. (See handout)
 Glut1 - is the erythrocyte-type transporter, which is also found in brain and kidney. It is an
integral plasma membrane with 12 transmembrane helices. It is a 50 kDa protein with 492 amino
acids with a Km value for glucose of 1-2 mM.
 Glut4 - is found in insulin responsive tissues, muscle, heart and adipose tissue. It is similar
but slightly larger with 509 amino acids and a high Km of 4 - 15mM for glucose.
One of the major mechanisms by which insulin exerts its hypoglycemic effect is via increasing the
uptake of the sugar by muscle, heart and adipose tissue and thus lowering blood glucose.
Isolated adipocytes can be used as a model system to study this effect. Adipocytes can be incubated
in KRB at 37o in the presence of [14C] deoxyglucose and the effect of various effectors on glucose
uptake can be examined. After a fixed incubation time an aliquot of the cell suspension is taken,
inhibitors such as cytochalasin B and phloretin are added to block further uptake or loss of glucose,
the mixture layered on to silicone oil in a microcentrifuge tube and rapidly centrifuged. The cells
float to the top of the oil while the incubation medium sinks to the bottom of the tube. The
radioactivity associated with the cells is an indication of the uptake of deoxyglucose at that time
point. Insulin can be shown to cause a 20-30 fold increase in the glucose transport and the effect can
be mimicked by the addition of the protein phosphatase inhibitor, okadaic acid,. This suggests that
the effect of insulin involves the phosphorylation of protein(s) on specific serine residues leading to
the activation of the protein. Unusually the activation involves a change in the location of the protein
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from a compartment within the cell to the plasma membrane. As the result of its mechanism of
action, the protein can only be active when located in the plasma membrane. Using histological
techniques with monoclonal antibodies raised against Glut4, it is possible to show that the protein is
located at some intracellular sites associated with the trans-Golgi reticulum, recycling endosomes and
with 'Glut4 storage vesicles' (GSV) but when the cells are stimulated with insulin the protein is
translocated to the plasma membrane. This can be confirmed by isolating organelle fractions (see
previous lectures) from the adipocytes, separating the low density ER from the plasma membrane
and showing a shift in the location of the Glut4 after insulin treatment. The movement of the
transporter in the intact cell in response to added insulin can now be visualised under a confocal
microscope if the Glut4 is tagged at its N-terminus with GFP (green fluorescent protein) by fusing
the two genes. Glut4-GFP appears to move to the plasma membrane along cytoskeleton microtubule
structures - disruption of the microtubule cytokeleton with vinblastine or colchicine, leads to the
inhibition of the insulin-stmulated glucose uptake (see Fletcher et al. Biochem J. (2000) 352, 267-76)
Other metabolic features which can be studied with adipocytes:
 Function of the insulin receptor
 Fatty acid and triacylglycerol synthesis
 Hormonal stimulation of triacylglycerol hydrolysis to Free Fatty Acids and glycerol
 Function of Hormone sensitive lipase
Hepatocytes
The mammalian liver consists primarily of hepatocytes (parenchymal cells) which are very large
cells compared to the other cell types (such as the endothelial and Kupfer Cells) which make up 35%
of the number of cells but only 5.8 % of the total volume of the liver as opposed to 72 % of the total
volume for hepatocytes. The hepatocyte has been subject to intensive research since the liver is very
interesting research in metabolic terms, the cells are easily isolated and a suspension of the cells has
been regarded as a homogenous preparation of identical cells. (this is not entirely true as there are
some differences in metabolic function between periportal and perivenous hepatocytes)
Preparation of isolated hepatocytes ( You do not need to know the details of the isolation just the
general principles involved)
 The rat is anaesthetised with Nembutal or phenobarbital, a laparotomy is performed and
the intestine moved aside to reveal the hepatic portal vein.
 The portal vein is cannulated just above the splenic vein and a ligature tied to secure the
cannula.
 A second ligature is tied loosely around the inferior vena cava and this blood vessel is
then cut.
 KRB (without Ca2+) gassed with O2/CO2 is pumped into the liver via the portal vein and
exits via the inferior vena cava. All of the liver lobes are cleared of blood visibly changing in
colour from dark red to light brown. The second ligature is then tied to prevent any further loss
of fluid through this blood vessel.
 The thoracic cavity is opened and the vena cava is cannulated through the atrium of the
heart and this cannula secured with a ligature. A closed perfusion system can then be set up to
recirculate buffer through the liver.
 Ca2+ and collagenase is then added to the recirculating KRB and the liver is perfused with
the enzyme for 15 - 20min after which time the liver structure starts to disintegrate.
 The capsule surrounding each lobe is carefully teased away and the cells squeezed out in
with a plastic spatula.
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 The disintegrated tissue is resuspended in KRB gassed with O2/CO2, filtered through 6
layers of muslin, and the filtrate is centrifuged at very low speed (50 x g) to harvest the
hepatocytes in a pellet and to separate out the smaller cells.
 The hepatocyte pellet is resuspended in fresh buffer and re-pelleted by low speed
centrifugation- this is done twice.
 It is possible to divide the pellet into 40 x 1ml aliquots, which can then be re-gassed with
O2/CO2 for 15 min to allow them to recover. In this way it is possible to perform 40 different
experiments on the same liver sample which enables much more information to be obtained in a
single study using a single animal whereas previously over a hundred animals would have been
used.
Criteria for Cell Viability
The collagenase digestion method involving periods of ischaemia (lack of oxygen) is a fairly severe
way of producing isolated cells and may result in damage to the plasma membrane and/or organelles.
How do we know that the cells are metabolically similar to those in the intact animal?
 The integrity of the plasma membrane can be tested by staining the cells with Trypan Blue
- this dye is only taken up by damaged cells. >90% unstained cells is a good indication of viable
cells. The cells may also be checked for leakage of cytoplasmic components such as LDH or K+.
The level of K+ in the cytosol , normally > 100mM, is maintained by the plasma membrane
Na+K+ATPase and thus slow leakage of K+ implies damage to the plasma membrane and low
levels of cellular ATP
 A good indicator of cell viability is a high [ATP]/[ADP] ratio. The cells may become
damaged due to periodic anoxia during the isolation procedure, the mitochondria may not
function and [ATP] may become depleted. This is easily restored in the viable cell by the
provision of glucose and oxygen. One of the best measures of anoxia in the liver cell is the
[AMP]. If the mitochondria are not functioning ATP is not made from ADP, the adenine
nucleotides are in equilibrium in the cytoplasm {approx. concentrations shown below in brackets
[ ]} and as the result of the activity of Adenylate Kinase the following occurs:
2 ADP
AMP
[2mM]
[0.2mM]
+
ATP
[10mM]
This enzyme has an equilibrium constant of about 1. If the [ATP] concentration drops by about 15 %
the [AMP] increases by about 3 fold to maintain the equilibrium, hence the AMP concentration is a
very sensitive indicator of cell anoxia and viability. These changes in [AMP] have an important
function in that they have a profound effect on metabolism. An increase in AMP stimulates
glycolysis and inhibits gluconeogenesis (why do you think this is?) - the general effect is to switch on
a pathway which generates ATP and to switch off pathways which consume ATP.
 The NADH/NAD+ ratio is another good indicator of the state of anoxia of the cell but quite
difficult to measure directly but can be estimated indirectly from the Lactate/Pyruvate ratio since
the reaction catalysed by LDH is freely reversible. An L/P ratio of 10 or less is considered to
indicate that the cells are viable and well oxygenated but rises to 100 in ischaemic conditions.
 The rate of gluconeogenesis is another good indicator of liver cell function - this may be
measured by studying the conversion of [14C] Lactate into [14C] Glucose by the hepatocytes
incubated glucagon. The hormone should stimulate gluconeogenesis 2-4 fold. Gluconeogenesis
requires active cytoplasmic enzymes (e.g. Fructose 1,6-bisphosphatase), functional mitochondria
(PEP carboxylase), a supply of ATP and low [AMP] and an intact endoplasmic reticulum (Glu-6Pase) and the hormonal stimulation requires a functional plasma membrane (Glucagon receptor,
G-protein and adenylate cyclase), hence this is a very good measure of hepatocyte viability.
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