Modern Clinical Applications of Cultured Cells

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Modern Clinical Applications of Cultured Cells
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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
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General viral structure
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production of virus 1. Inoculation of virus
in to cell culture lytic cycle
adsorption, penetration, replication and release
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?Phase of viral growth in culture phase1
adsorption/ penetration phase2 synthesis
phase3 assembly phase4 release
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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 0.110 p.f.u./cell
with the expectation that this
will increase to 103- 104
p.f.u./cell
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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)
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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)
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b. Application as therapeutic agent i.
conjugation of cytotoxin to cancer cell surface
Example Ricin, extract from castor
bean???


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

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• 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

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2. Plasminogen activators ? Thrombosis
deposition of fibrin in the circulatory
system and result in the blockage of blood flow


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?produced from CHO-K1 cell by transfection
Structure of tPA
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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
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?Therapeutic treatment regular administration
of appropriate factor purified from human
plasma ( possible contamination of HIV
or Hepatitis)
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4. Erythropoietin( EPO) synthesized in
kidney required for red blood cell
production glycoprotein Mr 30-35kDa
produced by CHO cells
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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

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

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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

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Common cause of neurotropic corneal
disease ?Corneal nerve injury and disease ( virus
infection, surgery) ? Trauma to ocular nerves
by laser or surgery
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• Cornea storage by organ culture
• Cleaning of eye
• Excision of corneoscleral disc?????
• 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

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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
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VII. Gene therapy Transfection of a
specific gene into cells isolated from a
patient suffering from a well-characterized
genetic disease.
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example for sickle cell anemia or thalassaemia
Haemopoietic cells isolated from bone
marrow Transfection with normal
globin gene by retroviral vector
Reintroduce into bone marrow
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VIII. Risks associated with cell culture
products 1. Viruses retrovirus
tumorgenic 2. Transforming proteins
products of oncogenes, tumorgenic and
growth promoting
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3. Residual cellular DNA reduce cell
products to 1pg/ml for safety, DNA content
of , 10pg/dose