Advances in automated real‐time flow cytometry for monitoring of bioreactor processes

Abstract

Flow cytometry and its technological possibilities have greatly advanced in the past decade as analysis tool for single cell properties and population distributions of different cell types in bioreactors. Along the way, some solutions for automated real‐time flow cytometry (ART‐FCM) were developed for monitoring of bioreactor processes without operator interference over extended periods with variable sampling frequency. However, there is still great potential for ART‐FCM to evolve and possibly become a standard application in bioprocess monitoring and process control. This review first addresses different components of an ART‐FCM, including the sampling device, the sample‐processing unit, the unit for sample delivery to the flow cytometer and the settings for measurement of pre‐processed samples. Also, available algorithms are presented for automated data analysis of multi‐parameter fluorescence datasets derived from ART‐FCM experiments. Furthermore, challenges are discussed for integration of fluorescence‐activated cell sorting into an ART‐FCM setup for isolation and separation of interesting subpopulations that can be further characterized by for instance omics‐methods. As the application of ART‐FCM is especially of interest for bioreactor process monitoring, including investigation of population heterogeneity and automated process control, a summary of already existing setups for these purposes is given. Additionally, the general future potential of ART‐FCM is addressed.

Application of an Escherichia coli triple reporter strain for at‐line monitoring of single‐cell physiology during L‐phenylalanine production

“…Therefore, the triggering event for their expression can be monitored by measurement of fluorescence levels of the reporter strain. These fluorescence levels are almost exclusively measured with flow cytometry as it allows at‐line high‐throughput single‐cell data acquisition during cultivations [ 12 , 13 ]. For the design of reporter strains, a great diversity of fluorescent proteins is available which differ in characteristics such as brightness, maturation times, and oligomeric state [ 14 ].…”

“…For the design of reporter strains, a great diversity of fluorescent proteins is available which differ in characteristics such as brightness, maturation times, and oligomeric state [ 14 ]. However, mostly fast maturating fluorescent proteins with bright detectability and no cytotoxic effects on the host strain are desirable [ 13 , 14 ]. There are various single‐reporter strains already available, allowing monitoring of different single‐cell characteristics such as growth, stress responses of different kind and cellular fitness [ 15 , 16 , 17 , 18 ].…”

A knowledge integration strategy for the selection of a robust multi-stress biomarkers panel for Bacillus subtilis

“…By measuring the expression of a few key biomarker genes using amplicon panels [ 10 , 11 ] or qPCR [ 12 , 13 ], instead of characterising the global transcription using genome-scale RNA-seq or Microarray, the cellular states can be assayed at reduced costs. Moreover, while these sequencing measures are not easily carried out in real-time, having cellular state biomarkers is particularly attractive for single-cell measurement technologies that seek to develop ‘live cell’ biosensors using flow cytometry [ 14 , 15 ] or microfluidics systems [ [16] , [17] , [18] ]. These ‘live cell’ biosensors require the biomarker panels to be small, consisting of only a few key genes.…”

Determination of stem cell pluripotency in spent cultivation media by Raman spectroscopy

“…Unfortunately, operator variation in the FCM results are commonplace 35 . The source of variation is two‐fold: In the sample, the cell clusters are not homogeneously distributed, and when analyzing the FCM data, important decisions about the gating must be made, 36 and these choices are prone to variation 37 . This drastically reduces the comparative usefulness of these across a process development pipeline and in turn makes it harder to accurately assess the efficacy of newly implemented protocol steps.…”