UNIT 2: Cell Culture Techniques and Primary Cell Culture

Exam Focus: The distinction between Primary and Secondary Culture is vital. Focus on the two main methods for isolating cells (mechanical vs. enzymatic disaggregation) and understanding the difference between adherent and suspension cultures.

Cell Culture Techniques
Primary Cell Culture

1. Cell Culture Techniques

Sterilization

Maintaining an **aseptic environment** is critical. All media, reagents, and equipment must be sterile to prevent contamination by microorganisms.

**Methods:** **Autoclaving** (for glassware), **Dry Heat**, and **Filtration** (for heat-labile components like serum and certain media) are commonly used.

Isolation of Cells and Tissues

The first step involves obtaining a tissue sample and separating the cells for culturing.

Mechanical and Enzyme Disaggregation

Methods used to separate the tissue into individual cells.

Method	Mechanism	Pros and Cons
Mechanical Disaggregation	Physical disruption (e.g., mincing, forcing tissue through a syringe).	Pros: Gentle. Cons: Low cell yield.
Enzyme Disaggregation	Use of proteolytic enzymes like Trypsin and Collagenase to digest the extracellular matrix.	Pros: High cell yield, faster. Cons: Enzymes can damage cell surface receptors.

Primary and Secondary Culture

**Primary Cell Culture:** Cells taken directly from a tissue and established for growth in vitro.
**Secondary Cell Culture (Sub-culturing):** The process of transferring cells from a primary culture or an existing cell line into fresh medium and vessels once they reach confluence.

2. Primary Cell Culture

Anchorage Dependent Growth

Most normal cells (e.g., epithelial cells, fibroblasts) are **anchorage-dependent**; they require a solid surface (substrate) to attach to and spread out before they can proliferate.

Non-anchorage Dependent Growth

Cells like blood cells and many transformed cell lines are non-anchorage dependent and can proliferate while suspended in the culture medium.

Adherent vs. Suspension Culture

Feature	Adherent Culture	Suspension Culture
Growth Type	Cells attach and form a monolayer.	Cells grow freely floating in the medium.
Cells	Most primary and epithelial cells.	Haematopoietic cells, some continuous/transformed cell lines.
Passaging	Requires trypsinization (detachment).	Simple dilution is required.
Application	Morphological and attachment studies.	Large-scale production in bioreactors.