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Modern Clinical Applications of Cultured Cells
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
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
General viral structure
production of virus
1. Inoculation of virus in to cell culture
lytic cycle: adsorption, penetration, replication and release
Phase of viral growth in culture
phase1 : adsorption/ penetration
phase2: synthesis
phase3 : assembly
phase4 : release
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
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)
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)
b. Application as therapeutic agent:
i. conjugation of cytotoxin to cancer cell surface
Example: Ricin, extract from castor bean蓖麻子
Ricin
Monoclone Ab target
cancer cells
mAb
Conjugation of monoclone Ab to Ricin
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
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
Viral ingfection blocked by Interferon
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
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
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
coagulation
plasmin( serine proteasea)纖維蛋白溶酶
fibrin( insoluble)
fibrin degradation
produced from CHO-K1 cell by transfection
Structure of tPA:
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
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)
4. Erythropoietin( EPO)
synthesized in kidney
required for red blood cell production
glycoprotein Mr= 30-35kDa
produced by CHO cells
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
Epidermal-equivalent which is layered on the dermal
surface
keratinocyte grow on the surface of dermal equivalent
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
Common cause of neurotropic corneal disease
Corneal nerve injury and disease ( virus infection,
surgery…)
 Trauma to ocular nerves by laser or surgery
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
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
VII. Gene therapy
Transfection of a specific gene into cells isolated
from a patient suffering from a well-characterized
genetic disease.
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
VIII. Risks associated with cell culture products
1. Viruses
retrovirus; tumorgenic
2. Transforming proteins
products of oncogenes, tumorgenic and
growth promoting
3. Residual cellular DNA
reduce cell products to 1pg/ml for safety,
DNA content of , 10pg/dose