Primary cell culture by using dissociated cells and explant culture method

Primary cell culture by using dissociated cells and explant culture
method

1. Primary Cell Culture Methods Subtitle: Dissociated Cell Culture & Explant Culture Presented by: Akshata Sutar

2. Introduction to Primary Cell Culture Definition: Primary cell culture refers to the growth of cells directly obtained from living tissue. Primary cell culture refers to the process of isolating cells directly from living organisms and cultivating them in vitro. Unlike immortalized cell lines, primary cells possess a finite lifespan and retain the characteristics of the tissue they were derived from. Types: Dissociated Cell Culture: Isolation of individual cells. Explant Culture: Growth of tissue fragments.

3. Dissociated Cell Culture Definition: Isolation of individual cells from a tissue using enzymatic or mechanical methods. Dissociated cell culture involves separating individual cells from a tissue using enzymatic or mechanical methods. This process creates a suspension of single cells that can then be plated onto a culture dish or flask. The dissociation process liberates the cells from their native extracellular matrix, allowing them to grow and proliferate in a controlled environment

4. Tissue Dissociation Techniques Tissue dissociation involves breaking down the connective tissue and extracellular matrix that hold cells together. This can be achieved through various methods: Enzymatic Digestion Mechanical Dissociation Enzymatic digestion utilizes enzymes like trypsin, collagenase, or dispase to break down specific components of the extracellular matrix. This method is gentle and effective for many tissues, particularly for separating cells with strong adhesion properties. Mechanical dissociation involves physically disrupting tissue using methods like mincing, sieving, or homogenization. This technique is often used for tissues with looser structures or for separating cells that are resistant to enzymatic digestion.

5. Tissue Collection: Obtain tissue samples (e.g., skin, liver) in sterile conditions. Dissociation: Enzymatic Method: Use enzymes (e.g., trypsin, collagenase). Mechanical Method: Mince or grind the tissue. Neutralization: Add serum to stop enzymatic activity. Cell Filtration: Pass through a mesh to remove debris. Centrifugation: Spin to pellet cells and resuspend in culture medium. Plating: Seed cells in appropriate vessels. Procedure for Dissociated Cell Culture

6. Advantages and Disadvantages of Dissociated Cell Culture Advantages : High yield of specific cell types. Allows for detailed studies of cellular processes. Disadvantages: Loss of tissue architecture and intercellular interactions. Limited lifespan of primary cultures

7. Explant Culture Definition : Growing small pieces of tissue on a culture medium . Explant culture involves growing small pieces of tissue directly in a culture dish. This method maintains the tissue's natural structure and cell connections, making it a more realistic model than using separated cells. The main goal is to encourage cells to grow and multiply directly from the tissue, mimicking what happens in the body.

8. Tissue Collection: Acquire tissue samples under sterile conditions. Preparation: Cut tissue into small explants (1-2 mm). Placement: Position explants on culture dishes with media. Incubation: Maintain in a controlled environment (37°C, 5% CO2). Monitoring: Observe for cell outgrowth from the explants. Procedure for Explant Culture

9. Advantages: Maintains tissue structure and cell interactions. Useful for studying tissue behavior in a more natural state. Disadvantages: Slower cell growth compared to dissociated methods. Limited to specific types of tissues. Advantages and Disadvantages of Explant Culture

10. Feature Dissociated Cell Culture Explant Culture Architecture Absent Preserved Growth Rate Higher Slower Complexity More complex Simpler Applications Broad Tissue-specific Comparison of Dissociated and Explant Cultures

11. Method Advantages Limitations Dissociated Culture • High cell yield • Homogeneous cell population • Easy to manipulate • Loss of tissue architecture • Potential changes in cell phenotype Explant Culture • Preservation of tissue structure • More physiologically relevant • Can study cell-cell interactions • Lower cell yield • Heterogeneous cell population • Can be challenging to maintain Dissociated vs. Explant Culture