BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 3. CELL ENGINEERING

Cell Cultivation. Cell Lines

One of the Fundamental properties of Cell cultures is their limited lifespan. After 50 — 100 divisions, the cultured Cells die. The younger the Organism from which the cells were originally harvested for cultivation, the more times they are able to divide before dying. For instance, fetal cells can undergo up to 50 divisions in culture, whereas newborn cells can divide 30 times. Occasionally, mutant cells emerge in culture that are capable of dividing an unlimited number of times. Such cells can give rise to a cell line.

A cell line consists of cells that can be cultivated outside the organism for an indefinitely long period of time. Many cell lines originate from tumors. Since 1951, an aneuploid tumor cell line (with 60 — 70 Chromosomes) known as HeLa has been continuously maintained in culture. These cells were derived from uterine tumor tissue of Henrietta Lacks, an African American woman who died of Cancer (USA). Today, under laboratory conditions, this cell line outperforms other later-established cancer cell lines derived from Caucasian patients in terms of growth rates. The most common cell lines are listed in Table 3.1.

Class="center">Table 3.1

Most Common Cell Lines

The majority of cell lines are cultivated on a solid surface, although some (such as BHK 21, HeLa, and SP 2) can grow in suspension. Substrate-attached cells are typically flattened and exhibit an elongated, spindle-like Morphology. Conversely, cells growing in suspension generally have a spherical shape.

Cell cultivation is carried out in plastic or Glass vessels whose surfaces are specially treated to promote Cell Adhesion. To ensure sterility, disposable sterile glassware and plasticware are used. After seeding onto the nutrient medium, cell cultures are incubated at 37°C and a pH of 6.8 — 7.5 in an atmosphere containing 5% CO₂ and 95% air. Even a slight pH fluctuation of 0.2 — 0.4 leads to cell death.

To maintain cell cultures in a functionally active state, standard nutrient media are employed, with mineral salts, Amino Acids, Vitamins, and Antibiotics serving as their primary components.

Table 3.2

Composition of Eagle's Medium

In 1955, H. Eagle proposed a multicomponent mixture for cell cultivation, which became known as Eagle's medium (Table 3.2). Various modifications of Eagle's nutrient medium composition have since been developed by other researchers.

To stimulate Cell Division, mitogens are added to the culture medium. Mitogens encompass a group of substances known as growth factors. These substances are Proteins that share hormone-like properties. Nerve growth factor (NGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) have all been identified. Lectins also exhibit mitogenic activity. Legumes serve as the primary source of lectins, accounting for 1.5 — 3% of their total protein content.

Cell mass for cultivation is obtained by treating the target tissue with Proteolytic Enzymes (such as Trypsin or collagenase) along with compounds that bind Ca²⁺ ions (such as EDTA). The tissue is then subjected to gentle mechanical dissociation. To isolate individual cells, the suspension is fractionated. Larger cells are separated from smaller ones via sedimentation or centrifugation. Modern cell identification techniques have been developed with remarkably high throughput—capable of Processing 5,000 cells per second. The isolated cells are transferred to a nutrient medium for cultivation. Because isolated single Eukaryotic cells rapidly perish, the cells selected for culture are plated onto a feeder layer of cells that have lost their capacity to divide due to irradiation while temporarily retaining their metabolic activity.

Liquid nitrogen is used for long-term storage of cell cultures. To prevent freeze damage, glycerol is added to the culture. A major advantage of cell lines is that, following thawing, they retain their proliferative capacity over extended periods.