Animal Cell, Tissue and Organ Culture

Content
Requirements for animal cell, tissue and organ culture
  Substrates for cell culture
  Substrate treatment
  Culture media
    Natural media
    Synthetic media
  Sterilization of glassware, equipments and culture media
  Isolation of animal material (tissue)
    Disaggregation of tissue
    Establishment of cell culture
Cultivation of animal cell en masse in bioreactor
Immobilized cell culture
Insect cell culture
Somatic cell culture
Organ culture
  Organ culture on plasma clots
  Organ culture on agar
  Organ culture in liquid medium
  Whole embryo culture
Valuable products from cell cultures
  Monoclonal antibodies
  Production of commercial products from insect culture

Valuable Products From Cell Culture
From cultured animal cells several valuable products such as human monoclonal antibodies, and biochemicals can be produced on a large scale. Several million dollars have been earned from this industry in Europe, America, Africa, Japan and India. This industry has better future. More interestingly, the genetically engineered cells have revolutionized the cell culture industry. Several specific promoters of human origin are utilized for high expression of foreign genes.
For large scale production of certain biochemicals, the genetically engineered baculovirus-infected animal cells are also in use in a bioreactor. To fulfil the process several 'perfusion systems1 have been developed that retain the cells in .the bioreactor at the time of replacement of conditioned medium with fresh medium. This results in increase in cell density and in turn cell productivity. For commercial production of products a large scale cell culture system and scaling up of process are required. Therefore, 'master cell banks' (MCBs) are established to meet out the demand. The MCBs are used to develop master working cell bank (MWCB) which meets the demand of production system. After several subculturing, the MWCB is regularly checked for any kind of changes occurring in cells. Thus, the large scale cultures are the source of all valuable products which are produced in a bioreactor. Diagram of a compact bioreactor is given in Fig. 6.6.
Some of the important products which are produced from animal cell cultures are : (i) enzymes (asperagenase, collagenase, urokinase, pepsin, hyaluronidase, rennin, trypsm, tyrosin hydroxylase), (ii) hormones (leutinizing hormone, follicle stimulating hormone, chorionic hormone and erythropoietin), (iii) vaccines (foot and mouth disease vaccine, vaccines for influenza, measles and mumps, rubella and rabies), (iv) monoclonal antibodies, (v) interferons, etc. (Table 6.7). Tolbert et al. (1982) got success in producing large quantities of human interleukin-2 or T-cell growth factor by culturing a permanent lymphoblastoid T-cell line in a large batch suspension culture in a bioreactor.


Table 6.7. Some products of medical use derived from animal cell cultures.
 
Products
Application
Erythropoietins
 
Erythropoietin-a
Anaemia resulting from cancer and chemothcraoy
 
Erythropoietin-p
Anaemia secondary to kindney disease
Human growth hormones
 
hGH
Human growth deficiency in children, renal cell carcinoma
 
Somatotropin
Chronic renal insufficiency, Turners' syndrom
Monoclonal antibodies (therapeutic)
 
Anti-lipopolysacharide
Treatment of sepsis
 
Murine anti-idiotype/human

 
B-cell lymphoma
B-cell lymphoma
Monoclonal antibodies (diagnostics)
 
Anti-fibrin 99
Blood clot
 
99 Tcm-FAb (breast)
Blood cancer
 
PR-356CYT-356-in-lll
Prostate adinocarcinoma
Plasminogen activator
 
Urokinase type plasminogen activator
Acute myocardial infarction, acute stroke, pulmonary embolism, deep vein thrombosis
 
Tissue type plasminogen activator
 
Recombinant plasminogen activator
Vaccines
 
HIV vaccines (gpl20)
AIDS prophylaxis and treatment
 
Malaria vaccine
Malaria prophylaxis
 
Polio vaccines
Poliomyelitis prophylaxis
Source: based on Feichter (1996).
Monoclonal Antibodies
Production of monoclonal antibodies (MoAb) and hybridoma technology has been described in Genetic Engineering for Human Welfare. The mouse MoAb have revolutionized the field of biology and more specifically immunology. The mouse MoAb have been used in human patients with varying level of success who were suffering from leukemia, lymphoma, melanoma and colorectal cancer. Clinical trials have indicated several limiting factors such as : (i) heterogenecity of tumour cells (not all malignant cells carry relevant antigen), circulating free antigens (they bind Fab on antibody molecules and thus block MoAb from binding to the target cells), (ii) antigenic modulation (antigen modulated off the cell surface as a consequence of binding of MoAb to the cancer cell as in leukemin. This problem can be overcome by applying monovalent MoAb) (Balasubramanian et al, 1996).
Therefore, several strategies have been developed for the production of human MoAb. These are (i) human-human hybrids (sensitized human B-cells from an individual are exposed naturally or by vaccination and then fused with human immortal cells obtained from tumours of lymphocytic cells.
The first human MoAb have been produced through this technique but the process is slow), (ii) interspecific hybrids (human B-cells isolated from bone marrow, periplasmal blood, spleen, lymph node, etc. are fused with non-secretary mouse or rat myeloma cells. From the hybrid cells human chromosomes were eliminated. The interspecific hybridoma produces MoAb which are of human type as expressed by human B-cells), (iii) EBV-transformation (The Epstein Ban virus transforms sensitized B-cells and results in production of immortal line of B-cells; the transformed B-cells secrete low amount of antibodies).
Table 6.8. Biotechnological applications of some insect cell culture systems.
Insects
Application
Aedes aegypti (yellow fever mosquito)
Arbovirus antigens, vaccines, diagnostics
Autographa californica (alfalfa looper)
Bioinsecticides
Bombax mori (silkworm)
Bioinsecticides
Heliothes virescens (tobacco bollworm)
Bioinsecticides
Spodoptera frugiperda (fall armyworm)
Recombinant proteins
Trichoplusia ni (cabbage looper)
Recombinant proteins
Source: Agathos (1991).
Production of Commercial Products from Insect Cell Cultures
Commercially desired proteins may be produced in vitro by using a susceptible continuous cell line of insect in a bioreactor. The fully grown cells are allowed to be infected by genetically engineered baculovirus. Baculovirus infects the cell line and lyse them resulting in release of protein products in the medium. Thereafter, protein is purified.
Adopting the same method bioinsecticides (occlusion bodies) can also be produced by using wild type virus. The occlusion bodies are then isolated from the bioreactor and used for the management of crops against the attack of insects (Agathos, 1991). Biotechnological application of some products derived from insect cell culture are given in Table 6.8.