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

Conjugation of monoclone Ab to Ricin

Ricin

mAb

Ricin mAb

Monoclone Ab target cancer cells

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

CD3

T cell

OKT3

Organ transplant

OKT3

Infusion of OKT3

Immunosuppressant

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 plasmin( serine proteasea)纖維蛋白溶酶

coagulation 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

Organ Culture techniques in tissue banking

Minimize the risk of disease transmission via tissue graft Use of appropriate methods of storage for tissue

V. Artificial organs

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