Workshop February 2006

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1 Theoretical and practical approaches of Hepatocyte primary culture Workshop February 2006 Lecture (2) Disaggregation & purification of target cells Coarse organizer Dr. Abo bakr Mohamed Eltayeb

2 General characteristics of lipid bilayer

3 General characteristics of protein molecules

4 Plasma membrane

5 Disaggregation and purification of cells * Cells in tissues are connected by each others with different types of junctions besides, extracellular matrix that held cells together. * To have cell culture even primary or indefinite cells, you should obtain cells isolated properly from each others. * By this way you will not obtain a specific cell type but you have to purify your target cell type * According to the individual variations, you should make a clonal selection of your specific cell type, by one cell cloning.

6 The interaction between cell adhesion receptors and their ligands are relatively weak A lot of weak interactions make a strong bond Principal classes of cell-adhesion receptors Cadherins Ig-family of cell adhesion molecules Integrins Selectins Others such as Mucins Connexins

7 Cells attach via cell adhesion molecules (CAM) found in extracelluar matrix, usually are glycoproteins. And other proteins called connexins.

8 Cells attach at specialized cell junctions Tight junctions Desmosomes Gap junctions

9 Connective tissue is one of the cement agents that hold tissues together, in addition the liver is a reticular organ that hepatic chords are built on the bases of reticular CT skeleton.

10 Cell-cell junctions require divalent cations as Ca+2 for their integrity. Cell to cell adhesion is mediated by a variety of cell adhesion molecules, the connections between cells and extracellular matrix have to be broken.to break calcium dependent adhesion (cadherins and selectins) we use EDTA or EGTA (both calcium chelators). Extracellular matrix proteins such as fibronectin and laminin are protease sensitive Proteoglycans can be partially degraded by hyaluronidase or heparinase. Thus for tissue dissociation or for releasing cells from monolayers various proteases solutions are used, sometimes with association with chelating agents. Besides this enzymatic disaggregation there are many types for disaggregation of cells as homogenization, agetation etc. but the most used and safety method is the enzymatic.disaggregation

11 Enzymes used in enzymatic disaggregation Trypsin Collagenase II, II, IV and V Elastase Hyaluronidase DNase Pronase (bacterial protease) You should know the following points about using these enzymes Usually a combination of enzymes is used The purer is the less toxic The cruder the more effective due to contamination with other proteases.

12 enzymatic procedures guide Some tissues such as fibrous connective tissue are resistant to trypsin Collagenase particularly connective tissue and muscle Hyaluronidase to dissolve proteoglycans DNase to dissolve DNA aggregates from damaged cells

13 Target cell purification After disaggregation, you have a variety of cells; hence it is difficult to find a single cell type in one organ or a piece of tissue. So it is necessary to purify your target cell type. There are many methods used in cell purification, all methods depends on different characters of cells as shape, size density, phagocytic capacity and resistance against some chemical compounds. The most commonly used method are depending on cell size by using different types of column chromatography and cell density by using density gradient centrifugation.

14 .Cells can be fused together to form hybrid cells It is possible to fuse one cell with another from the same type or different type to form a combined cell with two separate nuclei, called heterocaryon. There are some reagents used to enhance cell fusion, the most commonly used is Polyethylene glycol. It alters the plasma membranes of the cells in a way that induces them to fuse together. Further processing make the two nuclei of the heterocaryon to fuse to get one nucleus, in such case the result cell is called hybrid cell. Cell fusion is now a standing separate branch of cell biology that has many progresses as production of monoclonal antibodies, RBCs microinjection, and.many others As example of the benefits of cell fusion, the following is a simple description for monoclonal antibody production

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16 Practical session Purification of hepatocytes and cell fusion

17 Hepatocyte purification 1 ml of liver cell suspension layered on ml of Ficol 30% centrifugation for 1 min at 1000 RPM, all hepatocytes will be accumulated on the bottom of the tube, collect them and wash three times with PBS, by this way you will obtain pure hepatocytes 98%, other cell will be on the higher phase.

18 Fusion of hepatocytes together 1 ml of 44% PEG (prewarmed to 37 o C) in 20 mm Tris/0.15 M NaC1 was gently added to cell 10 6 after aspiration of PBS (washed two times at least by PBS), shake gently for 30 seconds and centrifuge 2000 rpms, room temperature. and leave it at room temperature for I min, then add 3 ml of PBS, shake for 30 seconds and leave for 1 min as previous The PEG was removed by repeated washings with PBS, also you can use different PEG concentrations %. hematoxyline. Observe under the microscope, watch the formation Smear, fix by drying and stain by promophenol blue or of heterocaryons as depicted below.

19 Heterocaryons and polyheterocaryon formation induced by PEG method

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