Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Abstract

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell−1 s−1, and 5 and 35 amol cell−1 s−1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.

Real‐time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side‐entry reactor (μSER) shows potential for pH control

“…The importance of online monitoring has been described among the development needs for microbioreactors [40,41], and timecourse profiles of process parameters have been shown for fermentation [42] and stem cell culture [43]. Therefore, further applications of this approach are not only of interest for analytical and synthetic enzymatic reactions, enzyme stability studies or bioprocess development, but for a variety of biotechnological applications where continuous flow microreactors can be envisioned.…”

Long-Term Retinal Differentiation of Human Induced Pluripotent Stem Cells in a Continuously Perfused Microfluidic Culture Device

“…The microfluidic culture device had been designed specifically for regenerative medicine process development and enabled greater control over the cellular microenvironment with regards to shear stress and transport of soluble factors. The MFCD has previously been used to successfully expand hESCs and mESCs meaning that this was the first attempt to undertake a lengthy directed differentiation process. The modular design of the MFCD was advantageous for the simple implementation of stepwise differentiation protocols.…”

“…The microfluidic perfusion culture device was based on previously published designs . In addition to the previously described design, an aluminium frame was placed around each of the two inter‐connects to reinforce the top plate in order to prevent liquid from leaking (Figure A–D).…”

“…This is especially important during directed differentiation protocols, the majority of which rely upon seeding aggregates of PSCs (known as embryoid bodies, EBs) onto 2D surfaces for extended periods with complete medium exchanges every 1‐2 days. The device can be multiplexed and configured for non‐invasive real‐time measurements of confluency and oxygen uptake …”

Effects of Flow Rate on Mesenchymal Stem Cell Oxygen Consumption Rates in 3D Bone-Tissue-Engineered Constructs Cultured in Perfusion Bioreactor Systems

“…Oxygen monitoring as an indicator of metabolic activity, cell proliferation, and culture progression is a promising method for the on-line measurement of tissue-engineered construct characteristics. Many 3D perfusion systems use a fluorescent material to monitor oxygen entering and exiting the construct [1][2][3][4][5][6]. Flow perfusion systems that measure oxygen uptake have successfully measured metabolic activity and cell proliferation in a 3D construct, allowing for the non-destructive monitoring of cells in vitro [3,5,7].…”