Assess Cell Proliferation Using Pressure as a Tool

Enhancing Cell Proliferation Monitoring with Pressure Controllers  

Why Measure Cell Proliferation? 

Cell proliferation serves as a crucial indicator of cell health and viability. Monitoring the proliferation rate is essential for assessing whether cells are actively dividing and growing as expected. Variations in proliferation rates may signify potential issues such as cell death, cellular stress, or the presence of toxins.  

Additionally, insights into the kinetics of cell division, factors influencing cell growth, and the regulation of cellular processes are essential for understanding normal development, tissue regeneration, and disease progression. 

Common Methods for Evaluating Cell Growth

Several widely used methods exist for evaluating cell proliferation: 

• Cell Counting: 

Manual counting using a hemocytometer or automated cell counters is a basic yet effective method. However, it can be time-consuming and subjective. 

• DNA Synthesis Detection: 

Since DNA replication is a fundamental event in cell multiplication, techniques that measure DNA synthesis can indirectly assess cell proliferation. 

• Flow Cytometry: 

Flow cytometry is a versatile technique involving labeling cells with fluorescent dyes like carboxyfluorescein succinimidyl ester (CFSE), which dilute as cells divide. Flow cytometry can measure DNA content using DNA-binding dyes (e.g., propidium iodide) to determine cell cycle phases. 

 The choice of method depends on factors such as cell type, research goals, and available resources. Researchers often combine these techniques for a comprehensive understanding of cell proliferation. 

Innovative Approach: 

We propose an alternative method for real-time cell growth monitoring, coupled with precise flow rate control within a microfluidic chip. This method relies on calculating hydrodynamic resistance and necessitates the use of pressure controllers alongside flow sensors to regulate the flow rate. This novel approach enhances the accuracy and efficiency of cell proliferation assessments in diverse research contexts. 

Partial results

Determining cell proliferation within the biochip in real-time 

We followed the protocol to find the correlation between cell proliferation and the pressure increase for maintaining a steady flow rate. The experiment was repeated on 5 biochips to increase statistical significance. The pressure applied to maintain a flow rate of 10 µL/min as a function of the number of cells (estimated after injection and counted after 3 days of perfusion) is shown in the figure. 

We can observe a correlation between the pressure applied and cell number for cells cultured for 3 days and counted afterwards. The slope from the curve was determined and lead to a linear function making it possible to estimate the number of cells, and therefore, cell growth, as a function of the applied pressure under identical conditions. 

Cell viability under steady and dynamic flow conditions 

To assess the influence of flow rate on cells, cell morphology of cells cultured under dynamic and static conditions were compared (images on the right). 
We observe that the actin network is more developed under dynamic conditions compared to static conditions. In fact, under flowing (dynamic) conditions, a low shear stress is applied on cells.

This shear stress tends to elongate cells, and as consequence, 2-dimensional cell proliferation is favored. Under static conditions, 3-dimensional cell growth is favored as no shear is applied. Cells growing 3-dimensionally could lead to increased cellular heterogeneity as they do not have access to the same amount of nutrients or oxygen within the microfluidic chamber. Under dynamic conditions, cells are in a favorable growth environment that is a continuous and homogeneous perfusion culture with steady and low shear stress. 

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