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.

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 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.
  • 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 15min 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+ATP

[2mM][0.2mM][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-6-Pase) 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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