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The Process of Mammalian Cell Culturing

BY: Gayathri Raghavan | Category: Biotech-Research | Submitted: 2013-03-12 04:26:51
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Article Summary: "In order to improve the performance of immobilized- cell cultures, modern-day scientists primarily focused on two objectives: (1) provide cells with optimum- growth conditions; (2) obtain large quantities of products of high concentration at low cost. In majority of the cases, optimal conditions for product formation and cell gr.."


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Introduction

In order to improve the performance of immobilized- cell cultures, modern-day scientists primarily focused on two objectives:

(1) provide cells with optimum- growth conditions;
(2) obtain large quantities of products of high concentration at low cost. In majority of the cases, optimal conditions for product formation and cell growth differ from each other. That is, usually the generation of a product is not associated with growth.

The first objective of optimal providing optimal growth conditions should be prioritized in the initial culture phase, while the second objective of obtaining high-quality products should precede the later stages and may involve in subjecting cells to nutritional or environmental stress.

Alternatively, in there is an increase in the rate of the specific product production from such stress, then the overall product yield may not improve due to the decrease in cell density. The primary objectives of hollow- fiber mammalian cell culture can be achieved via a variety of interdependent design, optical parameters, and biochemical, biological, and physical phenomena. The important factors, given below, aid in understanding the complexity involved in a HFBR system, modeling and operational challenges, and the protein transport's role in HFBR operations.

1. Cell Types

Animal cells that are used are either anchorage-dependent (surface substratum required for growth and attachment) or anchorage-independent (suspension growth). Epithelial cells and fibroblast are the most commonly used anchorage-dependent types, while, hematopoietic cells and lymphoblast are the commonly used anchorage- independent types. The frequently studied epithelial cells are CHO-K1 (Chinese

Hamster Ovary Cells) used for producing recombinant proteins; and HeLa (Human Cervical Cancer Cells) widely used for research purposes. Popular fibroblast cells include BHK-21 cell line (Baby Hamster Kidney Cells) used for producing veterinary vaccines and clotting factors; MRC-5, used for producing human vaccines; and Vero cells (African Green Monkey Kidney Cells) used for producing polio vaccine.

2. Extracellular Matrix

Many mammalian cell types in culture medium synthesize and deposit extracellular matrix (ECM). The ECM primarily constitutes collagens, glycoproteins, and proteoglycans. Ryll observed the ECM effect on the convective flow inside the hollow-fiber reactor. The ECM resistance offers the fluid flow to rise in the reactor.

3. Inoculation

After the mammalian cells are propagated in batch culture, the inoculum is suspended inside the growth medium and introduced in the HFBR's ECS. The inoculation is carried out by either countercurrent arrangement (cells added to downstream part of the reactor) where the ECS is perfused through lumen recycle flow or by dead-end arrangement where cell concentration and high-molecular weight (high-MW) growth factors are important.

4. Cell Growth Inside HFBR

After the inoculation phase, there exists a lag phase during which mammalian cells try adapting to the HFBR culture conditions. Within 3 to 4 weeks of the growth phase, the ECS is filled with cells. The cell density of ECS reaches a steady state and is packed to about 50 percent porosity. When the cell densities reach higher magnitude, the HFBR system exhibits sensitivity to variations in culture parameters thereby inviting strict control over culture conditions.

5. Impact of Metabolites and Nutrients

The key factor that influences the growth of the cell inside the HFCR bioreactor is the nutrient availability to cells. The required nutrients should be equal to the consumption rate of the cells, and the optimal nutrient level for cell growth differs from the optimal nutrient level for product generation. The primary nutrient is the oxygen that is supplied via an external unit (a hollow-fiber module) and the other nutrients required by mammalian cells in the culture include vitamins, carbohydrates, salts, amino acids, fatty acids, lipids, and trace elements. Glutamine and glucose are also important for cell growth. Mammalian cell growth largely depends on medium composition and cell line.

6. Serum Supplementation

Traditionally, mammalian cells have been cultivated in a medium that contains fetal bovine serum, (FBS) rich source of proteins; attachment factors; growth factors; and hormones. The serum offered protection against pH fluctuations; neutralization of toxins; and protease inhibition. Today, serum-free media are used in the successful production mono clonal antibodies in hollow-fiber reactors.

Although began as small scale production, mammalian cell cultures are now produced on large scale. Scientists are conducting various experiments in order to achieve high- cell densities and high-product concentrations. The scope of mammalian cell culturing in HFBR has not only garnered international attention but has also demonstrated the presence of another bioprocess tool.

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