Cell Culture Techniques

Question 1

Explain the key differences between primary cells and cell lines

Answer 1

Primary cells and cell lines are two distinct types of cells that researchers utilise in laboratory settings

Primary Cells:

• derived directly from living tissue—typically, a biopsy or explant culture—and are minimally manipulated in the lab
• They represent a close model to the in vivo state, retaining many of the physiological properties of cells in the body, including normal morphology, metabolism, and cell cycle controls
• However, primary cells have a limited lifespan and will undergo a finite number of divisions before entering a state of senescence and eventually dying, due to the Hayflick limit
• This limitation reflects the behaviour of most normal cells in the body
• Another characteristic of primary cells is that they maintain their tissue-specific expression patterns, making them highly valuable for studying tissue-specific functions and pathologies

Cell lines:

• a type of cell culture that is subcultured and can proliferate indefinitely in vitro if provided with appropriate conditions
• These cell lines are often derived from tumour cells or have undergone a transformation process, enabling them to bypass the normal controls that limit cell division
• They are genotypically and phenotypically uniform, providing a consistent and reproducible biological system for experimental purposes
• However, these cell lines, due to their altered or transformed nature, often do not completely represent the physiological state of the tissue from which they originated
• They might also accumulate genetic and epigenetic changes over time, leading to cell line drift; may not fully replicate the behaviour of primary cells or tissues in the body

Summary:

In summary, the key differences between primary cells and cell lines include their lifespan (finite for primary cells, potentially infinite for cell lines), their degree of similarity to in vivo conditions (greater for primary cells), their uniformity (greater for cell lines), and the extent of their manipulation and alteration from the original tissue (greater for cell lines)

Question 2

Describe methods of cell isolation and cell immortalisation for the primary and cell lines

Answer 2

Cell Isolation:

Cell isolation, also known as cell extraction, is the process of extracting or collecting cells from tissues

This process can vary significantly based on the tissue source, the cell type to be isolated, and the desired downstream applications

1) Mechanical Disruption:

• This is a simple method used to separate cells from tissues
• It involves physical methods such as mincing, grinding, or shearing tissues to release the cells

2) Enzymatic Disruption:

• In this method, enzymes like trypsin, collagenase, hyaluronidase, or DNase are used to break down the extracellular matrix and intercellular connections
• often leads to a higher yield and viability of isolated cells

3) Density Gradient Centrifugation:

• After mechanical or enzymatic disruption, the cell suspension can be subjected to density gradient centrifugation
• This technique allows separation of cells based on their buoyant density

Cell Immortalisation:

Primary cells have a finite lifespan, while cell lines can proliferate indefinitely due to their ability to bypass the normal controls on cell division - To create these cell lines; a process of cell immortalisation

1) Viral Transformation:

• One common method of immortalising cells is through viral transformation, where cells are infected with a virus that carries genes promoting unlimited cell division

2) Telomerase Expression:

• Primary cells undergo senescence due to the shortening of telomeres after each cell division
• By introducing the telomerase reverse transcriptase (TERT) gene into cells, the enzyme telomerase is expressed, which extends the telomeres and allows cells to divide indefinitely

3) Oncogenic Transformation:

• In this method, oncogenes (genes that have the potential to cause cancer) are introduced into cells
• The expression of these oncogenes can override the normal cellular controls on cell division, leading to immortalisation

Question 3

Discuss differences between 2D and 3D cell culture models and understand their applications in scientific research

Answer 3

2D cell culture:

In a 2D cell culture, cells grow on a flat surface, typically the bottom of a petri dish or a culture flask

Advantages of 2D culture:

1. Simplicity: The 2D system is relatively simple to set up and maintain. It allows for easy observation and manipulation of the cells
2. Cost-effectiveness: 2D culture requires less expensive equipment and reagents compared to 3D culture
3. High throughput: 2D cultures are suitable for large-scale experiments and high-throughput screening; simplicity and cost-effectiveness

Disadvantages of 2D culture:

• Lack of cellular context: Cells in the body exist in a complex 3D environment and interact with other cells around them
• These interactions can dramatically affect cellular behaviour
• In a 2D culture, these interactions are significantly reduced or absent, potentially limiting the physiological relevance of observations

3D cell culture:

3D cell culture is a system where cells are allowed to grow or interact within a surrounding matrix or amongst themselves in all three dimensions

They can be embedded in a gel or matrix, cultivated as spheroids, or grown on special scaffolds

Advantages of 3D culture:

1. Physiological relevance: 3D culture provides a more realistic cellular microenvironment that mimics the in vivo situation which influences cell morphology, behaviour, differentiation, and response to stimuli
2. Disease modelling: 3D cultures can be used to generate organoids or spheroids that recapitulate key structural and functional aspects of tissues or tumours, enhancing the ability to model diseases in vitro

Disadvantages of 3D culture:

1. Technical complexity: Establishing and maintaining a 3D culture, as well as imaging and analysing the samples is more technically challenging and requires specialised equipment
2. Cost and time: 3D cultures often require more time to establish and are more expensive due to the need for additional materials and reagents

Applications in scientific research:

1. Drug discovery and development: Both 2D and 3D cell culture models are used extensively in drug discovery, with 2D models often used for initial high-throughput screening, and 3D models used for more complex testing of drug efficacy and toxicity
2. Basic cell biology research: While 2D cell culture models have been invaluable for studying basic cellular processes. 3D culture models are essential for studying processes that involve complex cell-cell and cell-matrix interactions, such as cell migration, tissue development, and tumour growth
3. Regenerative medicine: 3D cell cultures are key to advancements in tissue engineering and regenerative medicine, where the goal is to create complex, functional tissues that can be used for transplantation or disease modelling.

Question 4

Explain different types of cell transfection methods

Answer 4

Transfection is a method used to introduce foreign DNA or RNA into cells

1) Calcium Phosphate Transfection:

• involves the formation of a calcium phosphate-DNA precipitate that is taken up by cells
• This method can be used for both stable and transient transfection and is most effective in dividing cells
• However, it can be less efficient than other methods and is sensitive to minor changes in pH and temperature

2) Lipid-Mediated Transfection (Lipofection):

• This method uses liposomes, which are vesicles that can enclose the DNA to be transferred
• Liposomes fuse with the cell membrane, delivering the DNA into the cell, used for both stable and transient transfection
• This method is less toxic and more efficient than calcium phosphate transfection

3) Electroporation:

• In this method, an electric field is applied to cells which increases the permeability of the cell membrane, allowing DNA to enter the cell
• However, it can also be more harmful to cells and may result in higher cell death

4) Viral Transduction:

• Here, a virus is used as a vector to deliver the genetic material into the cells
• Viral transduction can be highly efficient and can result in stable integration of the DNA into the host genome
• the production of viral vectors can be laborious and expensive

5) Microinjection:

• involves directly injecting the DNA into the nucleus of the cell using a fine glass microcapillary pipette
• efficient but also very labor-intensive

6) Biolistic particle delivery (gene gun):

• DNA is coated onto microscopic metal particles, which are then “shot” into cells using a device called a gene gun