A new methodology for plant cell viability assessment using intracellular esterase activity

Area of Science:

• Plant biotechnology
• Cell biology
• Bioprocess engineering

Background:

• Assessing plant cell viability is crucial for optimizing cell culture conditions in bioreactors.
• Traditional methods like the trypan blue exclusion test can be labor-intensive and prone to errors due to cell counting.
• There is a need for more efficient and reliable methods for determining cell viability in plant cell suspension cultures.

Purpose of the Study:

• To develop and validate a novel method for assessing Alfalfa (Medicago sativa L.) cell viability using cytoplasmic esterase activity.
• To correlate esterase activity with cell viability determined by membrane integrity.
• To establish a convenient, simple, and reproducible method for monitoring cell viability during plant cell culture in bioreactors.

Main Methods:

• Culturing Alfalfa (Medicago sativa L.) cells in a bioreactor using a batch procedure.
• Extracting and measuring cytoplasmic esterase activity (EC 3.1) using fluorescein diacetate as a fluorogenic substrate.
• Assessing cell viability using the membrane integrity test with trypan blue dye for comparison.

Main Results:

• Cytoplasmic esterase activity was found to be conclusively correlated with cell viability assessed by membrane integrity.
• The esterase activity method avoids the difficult step of cell counting required in the trypan blue exclusion method.
• Esterase activity per viable cell is dependent on cell viability but independent of cellular metabolism, ensuring reliability.

Conclusions:

• Cytoplasmic esterase activity provides a convenient, simple, and reproducible method for determining Alfalfa cell viability in bioreactors.
• This enzymatic assay offers a valuable alternative to traditional viability tests, streamlining bioprocess monitoring.
• The esterase activity method enhances the efficiency and accuracy of viability assessments in plant cell culture.