UNIT 3: Secondary Cell Culture, Cell Fusion, and Organ Culture Technique

Exam Focus: Distinguish clearly between finite and continuous cell lines. Cell fusion, especially the chemical method using **PEG**, is a high-yield question, usually asked in the context of Hybridoma technology.

Table of Contents

1. Secondary Cell Culture
   • Sub-culturing (Passaging)
   • Finite Cell Line
   • Continuous Cell Line
   • Growth Kinetics of Cells in Culture
2. Cell Fusion
   • Virus-mediated Fusion
   • Electrofusion
   • Chemical Fusion
3. Organ Culture Technique
   • Hanging Drop Technique
   • Watch Glass Technique

1. Secondary Cell Culture

Sub-culturing (Passaging)

The process of transferring cells from a confluent primary culture or an existing cell line to fresh medium and a new culture vessel. This step is essential to prevent growth arrest due to contact inhibition or nutrient depletion.

Finite Cell Line (Cell Strain)

These cell lines, typically derived from normal tissue, have a **limited lifespan** in vitro, undergoing a fixed number of cell divisions before they stop dividing (senescence).

Continuous Cell Line

These cell lines are **immortal** and can be cultured indefinitely. They are usually derived from malignant tumors or result from spontaneous or induced transformation of a finite cell line. They are genetically unstable and often used in industrial production (e.g., therapeutic proteins).

Growth Kinetics of Cells in Culture

Cell growth in culture follows four phases when plotted as cell number versus time:

1. **Lag Phase:** Cells adapt to the new environment.
2. **Log (Exponential) Phase:** Cells divide rapidly and regularly; maximum growth rate.
3. **Plateau (Stationary) Phase:** Growth rate slows as cell death balances cell division (due to nutrient limitation or contact inhibition).
4. **Decline Phase:** Cell death rate exceeds cell division.

2. Cell Fusion

The process of joining two or more cells to form a single, hybrid cell (heterokaryon), often essential for creating hybridomas in antibody production.

Virus-mediated Fusion

Uses inactivated **Sendai virus** (or other enveloped viruses) whose surface glycoproteins induce the fusion of cell membranes.

Electrofusion

Uses a brief, high-voltage electric pulse to create transient pores in adjacent cell membranes, leading to their fusion.

Chemical Fusion

Uses chemical agents to destabilize the cell membranes. The most common agent is **Polyethylene Glycol (PEG)**, which facilitates fusion and is widely used in hybridoma technology.

3. Organ Culture Technique

Methods designed to culture tissue fragments or whole organs while retaining their physiological structure and function.

Hanging Drop Technique

Tissue fragments are suspended in a drop of medium from a coverslip and inverted over a hollow slide, allowing nutrient access while promoting gaseous exchange.

Watch Glass Technique

Tissue explants are supported on a suitable platform (e.g., lens paper) inside a watch glass, with the medium maintained just below the tissue, often used for embryonic organs.