Modern Clinical Applications of Cultured Cells

1. Modern Clinical Applications of Cultured Cells

2. Mammalian cell products: established and potential Major innovative development of cell culture: 1. Viral vaccine 2. Monoclone antibodies 3. Recombinant proteins 4. Cell and tissue transplant 5. Drug screening and discovery 6. Gene Therapy

3. I. Viral vaccine:  polio viral vaccine in 1954  the basis of vaccination: injection of viral antigen an inactivated pathogenic virus: a disease causing virus which has been chemically inactivated an attenuated live virus: capable of been propagated but has been changed genetically so it cannot produce disease

4. General viral structure

5. production of virus 1. Inoculation of virus in to cell culture lytic cycle: adsorption, penetration, replication and release

6. Phase of viral growth in culture phase1 : adsorption/ penetration phase2: synthesis phase3 : assembly phase4 : release

7. p.f.u. – the quality of virus is usually expressed in plaque forming unit m.o.I. – the virus is added to a cell culture at a multiplicity of infection of 0.1—10 p.f.u./cell with the expectation that this will increase to 103- 104 p.f.u./cell

8. Cell lines for vaccine production  Normal human diploid fibroblast example: WI-38 , MRC-5-- human lung fibroblast lines for poliovaccine production  Vero( African green monkey lines); the first continuous cell lines accepted as substrate for human vaccine production  Dangerous of using human tumorgenic cell line  Using green monkey primary kidney cell lines ( possible contamination SV40)

9. II. Monoclonal antibodies a. For diagnosis Identification of small quantities of specific antigens Example: changes in the level of hormones or enzyme in the blood or urine ( pregnency test by HCG)

10. b. Application as therapeutic agent: i. conjugation of cytotoxin to cancer cell surface Example: Ricin, extract from castor bean蓖麻子 Ricin Ricin Monoclone Ab target cancer cells mAb mAb Conjugation of monoclone Ab to Ricin

11. ii. Preventing immunological response of transplantation Example: OKT3 Immunosuppressant drug during transplantation Recognize surface antigen of CD3 on T- lymphocyte, preventing immunological response of organ transplantation OKT3 OKT3 CD3 Organ transplant Immunosuppressant Infusion of OKT3 T cell

12. III. Recombinant protein Glycoprotein from mammalian cells culture medium taken from the cells supported viral growth could protect cells from viral infection ( later been identified as interferon) 1957 Isaacs and lindenmann

13. Viral ingfection blocked by Interferon

14. 1.Interferon: a. Antiviral activity b. Retard the growth of tumor Interferon  ( 22 subtype) • Isolated from leukocyte from human blood in 1960’s • Isolated from B-lymphoblastoid cell lines( good production by induction by Sendai virus • May be produced from serum free medium used in Leukaemia

15. Interferon ß  synthesized by induction of human fibroblast ( by virus or double strand RNA) minimize the repressor of inducible protein which cause the breakdown of interferon mRNA Interferon   Synthesized by T-lymphocyte • Stimulated by a wide range of mitogens and antigens

16. 2. Plasminogen activators  Thrombosis; deposition of fibrin in the circulatory system and result in the blockage of blood flow t-PA( tissue plasminogen activator) plasminogen plasmin( serine proteasea)纖維蛋白溶酶 coagulation fibrin( insoluble) fibrin degradation

17. produced from CHO-K1 cell by transfection Structure of tPA:

18. 3. Blood clotting factors Haemophilia: a sex – linked genetic disease characterized in an inability to form fibrin due to the absence of factor VIII and IX factor VIII glycoprotein Mr= 265kDa  cloned in 1984  now can be purified by transfection of expression vector into BHK cells

19. factor IX glycoprotein Mr= 57kDa Secreted by hepatocyte Require glycosylation and -carboxylation for full activity Produced by using a rat hepatoma cell line for expression Therapeutic treatment: regular administration of appropriate factor purified from human plasma ( possible contamination of HIV or Hepatitis)

20. 4. Erythropoietin( EPO) synthesized in kidney required for red blood cell production glycoprotein Mr= 30-35kDa produced by CHO cells

21. IV. Cells as a product Artificial skin from two layers derived from human skin  Dermal equivalent formed from fibroblast tissue biopsy, medium, collagen pour in to mould condensation of collagen tissue like matrix formed in 1-2 weeks

22. Epidermal-equivalent which is layered on the dermal surface • keratinocyte grow on the surface of dermal equivalent

23. V. Artificial organs • Organ Culture techniques in tissue banking  Minimize the risk of disease transmission via tissue graft  Use of appropriate methods of storage for tissue

24. Common cause of neurotropic corneal disease Corneal nerve injury and disease ( virus infection, surgery…)  Trauma to ocular nerves by laser or surgery

25. Cornea storage by organ culture • Cleaning of eye • Excision of corneoscleral disc角鞏膜光盤 • Suspension of corneoscleral disc in organ culture medium • Testing medium for bacterial or fungal infection • Examination of corneal endothelium • Reversal of stromal edema before transplantation

26. VI. Drug screening and toxicity tests  Reduced growth rate  Breakdown of membrane permeability  Tissue specific response  Ability to metabolize toxic compound  Stimulated wound healing  Damage repair by use of artificial constructed  Tissue genetic effects/ mutagenecity,  Interaction with DNA

27. VII. Gene therapy Transfection of a specific gene into cells isolated from a patient suffering from a well-characterized genetic disease.

28. example: for sickle cell anemiaor thalassaemia Haemopoietic cells isolated from bone marrow Transfection with normal globin gene by retroviral vector Reintroduce into bone marrow

29. VIII. Risks associated with cell culture products 1. Viruses retrovirus; tumorgenic 2. Transforming proteins products of oncogenes, tumorgenic and growth promoting

30. 3. Residual cellular DNA reduce cell products to 1pg/ml for safety, DNA content of , 10pg/dose