DAIRY BIOTECHNOLOGY

Module 4. Cell culture and fusion technology

Lesson 17
PROTOPLAST / SOMATIC FUSION AND HYBRIDOMA TECHNOLOGY

17.1 Definition

Fusion of one cell with another cell to form a hybrid cell is called protoplast or cell fusion. Protoplasts are prepared by removing the cell wall and are commonly used in bacteria and plants. In animal cell culture, it is known as cell fusion.

17.2 Methods

Several methods/protocols which include chemical, mechanical and electrical are used to fuse dissimilar cells. The methods involve presence or absence of a known fusogen and the most common one is the use of polyethylene glycol (PEG) besides electrofusion, virus mediated fusion (e.g., Epstein–Barr Virus or Sendai virus), liposome-mediated fusion, micro-orifice etc. which are described below:

17.2.1 Polyethylene glycol

Polyethylene glycol (PEG) induced cell fusion has become a standard technique particularly in the hybridoma technology. PEG induced fusions are easy to perform and a high number of cells can be fused in a shorter time. However, the technique suffers from low fusion efficiency and the many unwanted fusion products are created.

17.2.2 Liposome mediated fusion

Liposomes are small lipid molecules in which large number of plasmids are enclosed. They can be induced to fuse with cell cultures using PEG, and therefore have been used for gene transfer. They offer protection to DNA/RNA from nuclease digestion, low cell toxicity, stability and storage of nucleic acids due to encapsulation in liposomes besides having high degree of reproducibility and applicability to a wide range of cell types.

17.2.3 Electrofusion or E fusion

Electrofusion was first given by Zimmerman in early 1980’s which led to higher fusion efficiencies under controlled conditions. Electrofusion is the process of combining two cell types (similar or dissimilar) resulting in fusion of cytoplasmic contents of both cells with the help of an electric pulse. These protocols ensure higher viability and cell fusion efficiency. The two cell types are initially kept in a chamber with low conductivity medium. It is important that cell fusion medium has a low conductivity i.e. high resistivity. The cells are then aligned by Dielectrophoresis (application of non-uniform alternating electric fields) and electric pulse is applied. Cell electrofusion begins at the time of application of the high electric field pulses and proceeds for some time after the pulses are applied. After cell membrane fusion maturation, the cells are placed in tissue culture medium to promote cell viability and growth.

17.2.4 Dielectrophoretic cell trapping

Recently, micro-orifice based cell fusion has also been used which assures high-yield fusion without compromising the cell viability. Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. Dielectrophoresis (DEP) assisted cell trapping method was used for parallel fusion with a micro-orifice array. The method involves construction of a microfluidic chip that contains a chamber and partition. The partition divides the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generates an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment move towards the micro-orifice array by hydrostatic pressure. The cells are kept in micro-orifice using DEP assisted trapping to establish cell to cell contact through orifice. Cell fusion occurs by application of a pulse and the fused pair immobilizes at micro-orifice. The unfused cells are removed and the chip is monitored for time lapse imaging of the selected fusants.

17.2.5 Laser induced cell fusion

Initially, laser-induced cell fusion was used to fuse two embryonic cells. Later on, this technique was also applied to fuse B lymphocytes with myeloma cells in suspension as well as to fuse plant protoplasts. The mammalian cells, however, could only be fused by a UV laser microbeam after binding them together via an avidin- biotin bridge. Although, the avidin-biotin bridge created a very specific bond between selected cells, it limited the fusion yield and was time consuming too. This technique was improved by combination of an optical trap with a pulsed UV laser (laser cell fusion trap) which does not depend on any natural cell contact or specific cell receptors. The two cells are selected by simply dragging one cell towards a second cell with the aid of the optical trap. Fusion efficiency can still be enhanced by addition of PEG.

17.2.6 Virus mediated fusion

Cells growing in culture are induced by some of the viruses such as 'Sendai virus' to fuse the cells to form hybrids. The virus induces two different cells first to form heterokaryon and finally the two nuclei fuse together to form fusants. The cells including fusants are plated on a selective medium e.g. HAT which allows the multiplication of hybrid cells only.

17.3 Applications

1. For production of therapeutic hybrids by combining a tumor cell with a dendritic cell to be used in immunotherapy.

2. Cell fusion is extensively used for production of hybridoma for manufacture of monoclonal antibodies.

3. Used for Nuclear Transfer – for fertility treatment and animal propagation.

4. Hybrid cells are used to study gene expression.

17.4 Definition of Hybridoma Technology

The formation of hybrid cell line produced by fusing a specific antibody producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture is called hybridoma technology.

17.4.1 History

Hybridoma technology was discovered in 1975 by Georges J. F. Kohler (West Germany) and Cesar Milstein of Argentina (now working in U.K.). They shared the Noble prize for physiology and medicine in 1984 with Niels Kaj Jerne of Denmark (now working in Germany) who made several other contributions in the area of immunology.

17.4.2 Monoclonal antibody production using Hybridoma technology

Monoclonal antibodies (Mab) are nowadays widely used as a therapeutic means to treat cancer, chronic and autoimmune diseases as well as for diagnostic purposes like blood group typing and disease testing/diagnostics. Hybridoma technology is widely used for the production of hybridoma cells. Hybridomas are the cells that have been engineered to produce monoclonal antibodies of consistent quality, high specificity and in large amounts. The various steps for production of monoclonal antibodies using hybridoma technology have been shown in Fig 17.1

• The first step to make a hybridoma is to generate antibody producing B cells. The mice are injected by intraperitoneal (IP) route with the antigen against which monoclonal antibodies are to be raised over a course of several weeks until an appropriate antibody titre is achieved.

• Blood is taken and examined for the presence of antibodies. Some B cells will produce antibodies that bind specifically to epitopes on the antigen of interest while there will be some non-specific antibodies also that will not bind.

• Splenocytes are then isolated from the mouse spleen and fused with immortalized myeloma cell (that can grow indefinitely).

• The myeloma cells should be selected which do not produce any antibody and also lack hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene that makes them sensitive to the HAT medium (hypoxanthine aminopterin thymidine medium).

• Both the B cells and myeloma cells are fused using polyethylene glycol or Sendai virus by making the cell membranes more permeable.

• Next step is to separate fused hybridoma cells from unfused B cells and myeloma cells.

• Fused cells are then incubated in the HAT (Hypoxanthine Aminopetrin Thymidine) medium. Aminopterin in the medium blocks the pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the de novo or salvage pathways.

• Unfused B cells die as they have a short life span. Only the hybridoma (hybrid of B cells and myeloma cells) cells survive since HGPRT gene coming from B cells is functional.

• These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The medium is then diluted in multiwell plates to such an extent that each well contains only one cell. The supernatant from each well is checked for the desired antibody. Since the antibodies in the well are produced by the same B cell, they will be directed towards the same epitope, and hence are known as monoclonal antibodies.

• Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics and foetal bovine serum) and produce antibody. The hybridomas are then screened for the antibody specificity. The desired clones are then transferred to large tissue culture flasks and are cryopreserved. The monoclonal antibodies thus produced are then checked for any cross reactivity.

17.4.3 Large scale production and purification of monoclonal antibodies

Hybridomas are cultured to high densities in culture flasks or roller tubes/bottles. The antibody containing medium is then filter, sterilized and frozen. Alternatively, hybridoma cells can also be grown in dialysis based mini-fermentors which leads to high density cultures. The antibodies are purified from cell homogenate or cell debris obtained from the medium using ion-exchange chromatography or antigen affinity chromatography.

17.4.4 Further advancements in monoclonal antibody production

Monoclonal antibodies can be produced using mouse cell lines. However, these antibodies can cause adverse immune reaction with repeated use. With the advent of recombinant DNA technology, the xenogenic portions of mouse MAb (mouse monoclonal antibody) is replaced with human immunoglobulin to construct humanized monoclonal antibody which still requires refinement or by the use of transgenic mice which contains segments of human gene coding for antibodies. Several new products with MAb for pharmaceutical use are in the market after its approval in 1986 and many more products are under Phase I and II clinical trials. The production of MAbs has been at a fast pace as a result of advent of recombinant DNA technology, new analytical techniques and screening methods.

17.4.5 Applications

1. Monoclonal antibodies are widely used for the treatment of a number of diseases like cancers, allergies etc.

2. Monoclonal antibody therapy can be used to destroy malignant tumor cells and prevent tumor growth by blocking specific cell receptors (immuno-therapy).

3. Find application in diagnostics including ELISA (Enzyme Linked Immuno-Sorbant Assays) based assays.

4. Pregnancy can be detected by assaying of hormones using monoclonal antibodies.

5. Used for detection of pathogens by immunological assays including ELISA.

6. Monoclonal antibodies have been in vogue to immunize against certain diseases in humans and cattle. The most promising outcome is the prospect of developing anti-malarial vaccine.

Books

Culture of Animal Cells: A Manual of Basic Technique, 4^th Edition R. Ian Freshney (Editor), Wiley-Liss Publishers, ISBN: 0471348899

Animal Cell Culture and Technology: The Basics Michael J. Butler (Editor), Irl Pr Publishers, ISBN: 0199634165

Animal cell culture : A practical approach, John R.W. Masters (Ed.) Oxford University Press, ISBN13: 978-0-19-963796-6

Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, R Ian Freshney, John Wiley and Sons, ISBN: 978-0-470-52812-9

Internet Resources

http://en.wikipedia.org/wiki/Hybridoma_technology

http://globalresearchonline.net/volume1issue2/Article%20017.pdf

http://en.wikipedia.org/wiki/Monoclonal_antibodies

http://www.cultek.com/inf/otros/perfil proveedores/Perfil%20CYTOPULSE/Electrofusion.pdf

http://www.btxonline.com/content/PDFS/Electrofusion_vs_PEG.pdf