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August 10, 2022 Resources

Cell Proliferation

Definition

Cell proliferation is an increase in the number of cells due to cellular growth and cell division. When cells increase their mass and volume without increasing in number this is cell growth. In contrast cell division is the formation of two daughter cells from a parent cell. Thus, cell proliferation is a composite of two cell behaviours—growth and division. Cell proliferation represents a balance between the loss of cells due to death or differentiation and an increase in cell number due to cell division. For multicellular organisms, cell proliferation is important for growth of the organisms and wound healing. In contrast uncontrolled cell proliferation is a hallmark of cancer. A list of the latest reviews and research on cell proliferation can be found here and here.

cell proliferation

Figure 1: Cell division, growth, proliferation, and differentiation. Filled blue ovals represent nucleus.

Cell proliferation and differentiation

As mentioned above, cell proliferation is increasing the number of cells whereas cell differentiation is acquiring a special function and/or shape (morphology) by a less specialised cell. Differentiation is often achieved in stages and terminal differentiation is commonly associated with permanent exit from the cell cycle. Thus, fully differentiated cells cannot divide further. During normal development, there is an inverse relationship between proliferation and differentiation. This is important for maintaining tissue homeostasis and cell replacement. Figure 2 depicts differentiation where single celled myoblasts differentiate into myotubes (green) containing multiple nuclei.

Figure 2: Cell differentiation: Myoblast cells forming multinuclear myotubes. Image sourced from CRBS cell mage library

Examples of cell proliferation

Examples of proliferating cells in adult humans include skin fibroblasts, intestinal epithelial cells, and liver regeneration following surgery or massive cell loss. The formation of different blood cells encompasses both proliferation and differentiation of hematopoietic stem cells. A detailed discussion on proliferation can be found here.

Measuring cell proliferation

Proliferating cells have active metabolism and DNA synthesis preceding cell division. These features are exploited to obtain rates of cell proliferation. The following table lists the reagents that are commonly used to measure cell proliferation.

Table 1: The nucleotide analogues used to detect cell proliferation through DNA replication.

   
Reagent   
   
Principle   
   
Method of assay   
   
Advantage   
   
Disadvantages   
   
BrdU (5-bromo-2′-deoxy-uridine assay)
   

   
   

   

   

   

   
Thymidine analogue incorporated into the newly replicated   DNA of proliferating cells
   
   

   
BrDU incorporated cells are stained with anti BrDU antibody
   

   
Can be measured
   
with flow cytometry
   

   
Imaging including high throughput methods like MCS plate   and HCS   

   
Applicable to cells and tissue
   

   
   

   
Requires additional antibody and DNA denaturation
   

   
End point assay   

   
EdU (5-ethynyl-2´-deoxyuridine)   
   
Applicable to cells and tissue
   

   
no additional antibodies or DNA denaturation steps required   

   

   

   
End point assay   

Table 2: Nuclear proteins used to measure cell proliferation.

   
Reagent   
   
Principle   
   
Method of assay   
   
Advantage   
   
Disadvantage   
   
Ki-67   
   
Present in S, G2, and M phase cells   
   
Immunohistochemistry and flow cytometry-based   detection using monoclonal antibody against the Ki-67 protein   
   
Useful for measuring proliferation of tumours   
   
End point assay requires tissue fixation
   

   
Present in all cells except G0 stage   quiescent; cells with variable amounts in each stage contributing to   variations in staining   

   
Phosphorylated histone 3 (PH3)   
   
Present in G2 and M phase cells, marking condensed   chromatin prior to chromosomal segregation   
   
Immunohistochemistry using specific molecular   antibodies
   

   
   

   
Proliferating tumours, more specific   
   
End point assay   

Table 3 Reagents measuring cell proliferation through metabolic activity

   
Reagent   
   
Principle   
   
Method of Assay   
   
Advantages   
   
Disadvantage   
   
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium   bromide)
   
   
   

   
Measurement of the redox potential of a cell   is indicative of its proliferative abilities
   

   
Soluble tetrazolium is reduced into insoluble   purple formazan crystals by NADPH dependent oxidoreductase. It can also be   spontaneously reduced to Formazan in lipidic bilayers of cells still   reporting the reduction potential of a cell.
   
Formazan is solubilised and its absorbance is   measured at 5000-600nm    

   

   

   

   

   

   
Colorimetric based measurement amenable to   microplate reading    

   
Easy to perform
   

   
Can be employed in high throughput studies
   

   

   
   

   
End point assay
   

   
MTT is insoluble in culture medium
   

   

   

   

   
   

   
XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)
   

   

   

   
   

   
MTS
   

   
   

   
WTS
   

   
   

   
Alamar blue   
   
Soluble in medium    
   
Positive reaction requires presence of at least   100 cells per dish   

Table 4 Measuring cell proliferation dynamically

   
Reagent   
   
Principle   
   
Method of Assay   
   
Advantages   
   
Disadvantages   
   
Carboxy fluorescein diacetate succinimidyl   ester (CFSE   
   
CFSE by itself is non fluorescent, once it is   inside viable cells it is cleaved by cytoplasmic esterase enzyme to generate fluorescent   dyes which react with amine groups of protein forming stable conjugates. Upon   division the fluorescence is halved in daughter cells and thus proliferation   can be followed through generations.   
   
Cells are incubated in PBS containing CFSE   and then washed to remove lingering extracellular stain. Cells are then   followed either by flow cytometry or in microplate reader   
   
Cell viability is not affected, dynamic assay,   can follow cells through multiple generation.
   

   
Easy to use
   

   
Applicable to both tissues and cells   

   
Cells can be followed till a specific number   of generations beyond which background noise affects detection.
   

   
   

Cell proliferation rate calculation

Cell proliferation is generally measured by estimating the increase in the number of cells (cell count) over time starting with a specific number of parental cells. The cell count is obtained after staining the cells (with fluorescent markers, antibodies, or colorimetric reagents) to select dividing or metabolically active cells. Cell counts at different time points are utilized to calculate proliferation. The proliferation rate is expressed as a proliferation index. Different software utilize different formulae to calculate this proliferation index.  For example, proliferation measurements using tracking dyes often use the following formula to obtain the proliferative index:

Cell proliferation assay

Features such as DNA replication and high metabolic activity are often used as endpoint measurements of proliferation, meaning the cells used for measurement once cannot be reused for other experiments. This is due to requirement of fixation of the cells/tissue or prolonged staining for carrying out the measurement. Even techniques employing live cells using fluorescent markers may be restricted by the number of generations that can be followed with the labelling agent. Depending on the choice of reagents used, some information regarding the dynamic progression of cell proliferation over time may be lost. Similarly, to test the effects of chemicals/drugs that modulate proliferation, multiple parallel cultures might be necessary if such end point measurements are done throughout different time points. Measuring proliferation of live cells non-invasively over time takes care of these issues and provides the missing temporal information. Moreover, cells in a population might exist as an ensemble with different proliferating abilities across time and this information will only be captured if the proliferation of live cells is tracked across different timepoints. Adapting non-invasive proliferation measurements in a multi-well format allows simultaneous comparison of different drugs.

 

 

Bibliography

Munson M. An improved technique for calculating relative response in cellular proliferation experiments. Cytometry Part A. 2010;77A(10):909-910. doi:10.1002/cyto.a.20935

Roederer M. Interpretation of cellular proliferation data: Avoid the panglossian. Cytometry Part A. 2011;79A(2):95-101. doi:10.1002/cyto.a.21010