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Cell culture refers to the removal of cells from an animal or any other live tissue and their subsequent growth in a favorable artificial environment.
Enzymatic Disaggregation Mechanical Disaggregation
CELLS
Primary culture: Cell line Cell strain Continuous cell line
Culture
Production/Fermentation
VAQTA
PART 1
VACCINES
This accelerated the use of cell culture in vaccine industry. First vaccine created with the use of human cell strains was the Rubella vaccine developed by Stanley Plotkin
Serum: source of nutrients, hormones and growth factors. But has many disadvantages: potential to induce hypersensitivity, batch variability, possibility of introducing contaminants such as bovine viruses, prions, mycoplamas, difficulty in downstream processing Thus serum free media is preffered in case of vaccine production.
Methodology
MDCK cells in the 125 mL spinner flask
Formalin-inactivation
PART 2
HORMONES
Hormone Production
Hormone crucial part of the body metabolism Required in small amount Deficiency or imbalance significant adverse effects. In such cases, hormones supplied externally Different approaches in hormone production: chemical synthesis, rDNA methods, animal pharming Example in study: Insulin Islets of Langerhans are micro-organs scattered throughout the pancreas, and responsible for synthesizing and secreting pancreatic hormones cells produce insulin
Insulin production
Insulin can be obtained by Chemical synthesis Bovine and porcine insulin rDNA methods, cellular transformation Somatic gene transfer into pancreatic progenitors to develop beta cell line
Ralph A. Rosenberg demonstrated production on insulin producing cell lines (US patent no. US004332893)
Influenced by temperature sensitive lesion Stored at about -60oC to -70 oC for at least 6 months until ready for use
PART 2
BLOOD PRODUCTS
Blood Products
Blood products are any component of the blood which is collected from a donor for use in a blood transfusion. These blood products used for biological processes are mostly from in vivo sources Problems:
Availability Patient specific requirements Risk of infection and transmission of blood borne diseases
Ex vivo blood production is limited by both biological and engineering challenges significant volumes required
Blood Products
Uses of blood products Platelets are used clinically in prophylaxis and treatment of thrombocytopenic hemorrhage Red blood cells are transfused to support the transport of oxygen Specific lymphocytes find application in the treatment of various immunodeficiency diseases Blood products also may be used for rescue from high dose cancer chemotherapy Human hemoglobin has been packaged in liposomes for administration as neo-erythrocytes Perfluorochemicals have been tested as hemoglobin substitutes, but these perfluorocarbons contain a potentially toxic surfactant
Methodology
Step 1: Establishing hESC cell line (hESCs) may be derived from the inner cell mass of a blastocyst stage human embryo or an established cell line may be used Step 2: Cell selection The most robust selection method for hESCs and various TA intermediates to date employs positive selection-the use of cell surface markers specific to a desired cell type (negative selection can also be used) Fluorescent activated cell sorting (FACS) using mABs or other ligand directly or indirectly fluorescently labelled and allowed to bind to cells within a heterogeneous cell population.
Methodology
Step 3: Forced Aggregation of hESCs Aggregation of the known concentration of hESCs may be forced by placement of the hESCs into low-attachment holding vessels which are shaped to better capture the cells
Step 4: Bioreactors designed to allow for the production of high density cultures of a single cell type "smart surface a surface in a bioreactor that has been modified to comprise a ligand that binds differentially to a certain cell type According to the specified media inducers, specific type of blood cells can thus be generated
CONCLUSION
The use of mammalian cell culture thus proves to be a promising approach for manufacture of therapeutics This being very specific and efficient makes it safe for use; which translates into a huge demand for large scale manufacturing capacity To further enhance these techniques,
intensive research in cell line engineering, media development, feeding strategies, cell metabolism, better process understanding and their impact on product quality and scale-up
References
www.actip.org/pages/library/AnimalCell http://www.historyofvaccines.org/content/articles/human-cell-strains-vaccinedevelopment www.expressionsystems.com/ Schroeder IS, Rolletschek A, Blyszczuk P, Kania G, Wobus AM; Differentiation of mouse embryonic stem cells to insulin-producing cells; Nat Protoc. 2006;1(2):495-507 Armen H. Tashjian Jr, Animal cell cultures as a source of hormones; Biotechnology and Bioengineering Volume 11, Issue 2, pages 109126, March 1969 Feng Li, Natarajan Vijayasankaran, Amy (Yijuan) Shen, Robert Kiss and Ashraf Amanullah; Cell culture processes for monoclonal antibody production; 466-477; September/October 2010; 2010 Landes Bioscience Latha Muniappan and Sabire zcan; Induction of insulin secretion in engineered liver cells by nitric oxide; BMC Physiology 2007, 7:11