BIO 2101

Comprehensive Biology Laboratory

Exercise #3a

Culture of Animal and Plant Cells

In vitro cell culture systems enable the study of:

Cell growth and division

Cell differentiation

Genetic manipulations for gene structure / function studies

Biotechnology

Cells used may be either:

-Primary cultures or

-Immortalized cell lines

Isolation of cells

Cells can be released from soft tissues by enzymatic digestion with proteases and collagenases

OR

Pieces of tissue can be placed in growth media, and the cells that grow out are available for culture (Explant Culture)

Primary cells vs. Immortalized cells

Cells that are cultured directly from a subject are known as primary cells.

With the exception of some derived from tumors, most primary cell cultures have limited lifespan.

An established or immortalized cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification.

When a primary culture is sub-cultured, it is known as secondary culture or cell line or sub-clone. Sub-culturing of primary cells to different divisions leads to the generation of cell lines.

1. Cell lines derived from primary cultures of normal cells are finite cell lines.

2. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes an established or immortalized cell line.

Preparation of cell cultures from tissues

Most researchers “obtain” cell lines: ATCC or from other researchers

Transformation of cultured cells implies a spontaneous or induced permanent phenotypic change resulting from a heritable change in DNA and gene expression.

Transformation often involves the deletion or mutation of the p53 gene, which would normally arrest cell cycle progression if DNA were to become mutated, and overexpression of the telomerase gene.

Cell lines

 HL-60 (Human leukemia cells)

 NG108-15 (Mouse neuronal cells)

 SWSIS 3T3 (Mouse embryonic fibroblast)

 HEK293T (Human embryonic kidney fibroblast)

 MG63 (Human bone fibroblast)

 more …

Fibroblasts are the most common cells of connective tissue in animals. It synthesizes the extracellular matrix and collagen, the structural framework (stroma) for animal tissues.

General culture requirements

 Appropriate temperature and gas mixture (typically, 37oC, 5% CO2 ) in a cell incubator

 pH: 7.2-7.5 (buffering by sodium bicarbonate)

 Humidity is required

 Glucose, growth factors, and the presence of other nutrient components

 Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

 Manipulate in a biological safety cabinet (“Tissue culture hoods”): prevent contamination

• Tissue culture experiments are typically carried out in special work stations called “Tissue culture hoods”

• Such hoods provide personal protection from harmful agents within the cabinet

• Reduce the risk of microbial contamination

• Environmental Protection from contaminants contained within the cabinet.

Class 2 Biological Safety Cabinets

The Class 2 Biological Safety cabinet must meet the requirements for personnel, environmental and product protection. Used when working with low to moderate risk biological agents (Biosafety level 1, 2 and 3 agents). Examples include Salmonellae, Hepatitis B virus and Measles virus.

When you are working with very high risk biological agents, Class 3 cabinets should be used.