Hybridoma cell

Hybridoma cell as Industrial Cell

1. Hybridoma cell Siti hamidah sead | evy mastura shukri 1

2. What is hybridoma cell? What is contribution of the cell? 2

3. introduction Hydridoma cell is fusion of 2 cells in order to obtain or express a specific monoclonal antibodies. B-cells Tumor cell HYBRIDOMA B-lymphocytes myeloma 3

4. What is monoclonal antibodies? Monoclonal antibodies artificially produced against a specific antigen in order to bind to their target antigens. Uses of monoclonal antibodies are: CANCER RHEUMATOID ARTHRITIS CARDIOVASCULAR DISEASES ULCERATIVE COLITIS 4

5. Method of culturing hybridoma cells Immunisation of mouse Isolation of B cells from spleen Cultivation of myeloma cells Fusion of myeloma and B cells Separation of cell lines Screening suitable cell lines In vitro / in vivo Harvesting p 5

6. 1. Immunisation of mouse 2-4 weeks old mice are immunized with antigen against which to raised monoclonal antibodies by subcutaneous injection. Series of injections of the antigen over the course of several weeks. 2.Isolation of B cells from spleen B cells are isolated from the spleen of immunized mice. p 6

7. 3. Cultivation of myeloma cells Myeloma cells are isolated from bone marrow. The myeloma cells used are HGPRT ( Hypoxanthine-guanine phosphoribosyl transferase) mutant cells which raised by mutation using 8-azaguanine. 4. Fusion of myeloma and B cells Using electrofusion: cells are allowed to fused with the application of electric field. By using PEG medium (Polyethylene Glycol) p 7

8. 5. Separation of cell lines HAT ( Hyphoxanthine Aminopterine Thymidine) medium is used for the selection of hybrid cells. .B cells are HGPRT+ and can survive in the HAT medium but the undergo normal cell death after some division. Myeloma cells are HGPRT deficient so the cells could not survive in HAT medium since Aminopterin blocks the pathway that allows for nucleotide synthesis. Hybrid cells has HGPRT enzyme from B cells as well as they have the ability to multiply repeatedly as myeloma cells. So, only hybrid cells can survive in HAT medium. p 8

9. 6. Screening suitable cell lines Screening technique used is called ELISA. The hybridoma culture supernatant, secondary enzyme labeled conjugate, and chromogenic substrate. Then it is incubated and the formation of a colored product indicates a positive hybridoma . Alternatively, immunocytochemical screening can also be used. p 9

10. 7.Methods multiplying hybridoma cell IN VIVO VS. I N VITRO Introduce of hybridoma cells into peritoneal cavity of mice. Ascetic fluid is isolated from the mice. Isolate antibodies from ascetic fluid. Culturing of hybridoma cells in suitable culture media ( Antibodies is isolated and purified. p 10

11. 8. Harvesting Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics and fetal bovine serum) and produce antibodies. Multiwell plates are used initially to grow the hybridomas . After selection, they are changed to tissue culture flasks to provides enough cells for cryopreservation and supernatant for subsequent investigations. p 11

12. ADVANTAGES OF HYBRIDOMA 12

13. Disadvantage of hybridoma 13

14. conclusion Hybridoma is fusion of two cell between tumor cell and Beta cell which specifically beta lymphocytes cell from mice. The hydridoma is used to produce monoclonal antibodies to treat cancer disease such as cardiovascular disease and ulcerative diseases Hybridoma can be cultured either in vitro or in vivo . 14

15. references National Research Council (US) Committee on Methods of Producing Monoclonal Antibodies. Monoclonal Antibody Production. Washington (DC): National Academies Press (US); 1999. 4, Summary of Advantages and Disadvantages of In Vitro and In Vivo Methods.Available from: https://www.ncbi.nlm.nih.gov/books/NBK100200/ Prospecs Protein Cloning, https:// www.prospecbio.com/monoclonal_antibodies Arya George . 2018. Hybridoma Technology. Slide. Mar Ivanios College Trivandrum.Linkedln 15