Introduction to cell culture

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

From cell lines

Applications of cell culture

General production of normal and recombinant proteins

Genetically modified cells and cell lines Master cell bank

Culture

Production/Fermentation

Purification Pharmaceutical formulation and bottling

Industrial scale production using cell culture

Key barriers to large-scale mammalian culture:
oxygen supply limitations, waste product accumulation, need for more sophisticated process control, shear sensitivity of animal cells, the challenges of growing adherent cell lines the control of carbon dioxide concentrations on scale-up, and the minimization of the risk of contamination

Industrial scale production using cell culture

Class of compounds industrially produced on large scale by cell culture are:
Recombinant therapeutics Vaccines, Hormones Monoclonal antibodies
Commercial name ReFacto Enbrel Herceptin RotaShield RabAvert Used for Antihemophilic factor Treatment of Rheumatoid arthritis Treatment of metastatic breast cancer Vacciniation against Rotavirus Immunization for rabies

VAQTA

Immunization for hepatitis A

PART 1

VACCINES

Vaccine production using cell culture

Earlier, vaccine production was from live animals and chicken eggs drawbacks: Research animals are costly and require extensive monitoring, may be carrying other bacteria or viruses that could contaminate the eventual vaccine, Dependence on availability of the animal products, Difficulty during epidemics

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

Production of Influenza H5N1 Vaccines

Influenza highly contagious disease affects the respiratory system Avian influenza caused by H5N1 viruses have been common Most popular method of H5N1 vaccine production was egg based method But due to its disadvantages, virus propagation in mammalian cell lines was promoted Advantages:
cell lines characterized & in compliance with regulatory guidelines; raw materials for production defined & easily produced in a short period; no dependence on embryonic eggs

Production of Influenza H5N1 Vaccines

2 regulatory-approved continuous cell lines being used for influenza vaccine production:
1. 2. MDCK (Madin-Darby canine kidney) cells Vero (African green monkey kidney) cells

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

Infected with H5N1 viruses strain NIBRG14

Observe the cytopathic effect (CPE)

Select high HA-titre H5N1 cells

Estimate virus titers (HA and plaque assay)

Formalin-inactivation

H5N1 viruses - purified by sucrose gradient zonal centrifugation

Formulated with alum adjuvant

Production of Influenza H5N1 Vaccines

Optaflu commercially available H5N1 vaccine
Other vaccines commercially available: Hepatitis A vaccines VAQTA Rubella vaccine MERUVAX II Varicella (chickenpox) vaccine Varivax Zoster (shingles) vaccine Zostavax

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)

Insulin production using cell culture

Step 1: Preparation of a suitable culture of insulin-producing pancreatic beta cells Islets of Langerhans are scarce and scattered in a pancreatic tissue Rate zonal sedimentation used to separate these islet cells. Digested for about 20 to 30 minutes at about 37C, in a solution of collagenase and later trypsinized. Final preparation in a calcium and magnesium-free phosphate buffered saline solution Then cells are centrifuged and resuspended in M199 media

Insulin production using cell culture

Step 2: Preparation of the transforming virus stocks Strain of Rous sarcoma virus (RSV) used: sarc-SR-RSV-D mutant Mutation in the transforming gene caused the expression of this gene to be sensitive to the environmental temperature.
permissive temperature mutant viral transforming gene is stable and can function to transform the cells nonpermissive temperature mutant viral transforming gene is inactivated and incapable of transformation

Influenced by temperature sensitive lesion Stored at about -60oC to -70 oC for at least 6 months until ready for use

Insulin production using cell culture

Step 3: Infection and conditional transformation Should preferably take place within about four hours after the pancreatic cells prepared virion-containing supernatant is generally sufficiently acid to kill at least a portion of the pancreatic cells. Thus pH adjustment done. The virus-infected pancreatic cells are then cultured at the permissive temperature which is that temperature at which the transforming gene is expressed (33oC to 34oC) Subject to a glucose challenge which will induce production of insulin Storage condition: at nonpermissive temperature (cryo temperature)

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

Human embryonic stem cells -pluripotent

Production strategy for blood products

1. Immortal pluripotent cells (e.g., hESCs) are cultured in the presence of combinations of maintenance-, proliferation- and growth- and/or maturation (Eg: cytokines, lymphokines, colony stimulating factors, mitogens etc.) 2. Later a populations of single cell species may be produced. 3. Exposing the pluripotent cells to culture conditions that produce TA cells 4. proliferating the TA cells in a proliferation reactor 5. differentiating the TA cells in a differentiation reactor to produce a population of mature blood cells

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