Non‐Invasive Monitoring of Osteogenic Differentiation on Microtissue Arrays under Physiological Conditions Using Scanning Electrochemical Microscopy

Abstract

In this paper, we present a non‐invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP‐2), C2C12 microtissues differentiate and express alkaline phosphatase (ALP), which is indirectly detected through an enzymatic assay producing an electroactive species. The latter is detected using SECM by scanning at constant height over live microtissues at physiological pH (7.5) as well as more alkaline pH (8.5). As a control, expression of ALP is confirmed using a standard colorimetric assay. Detecting differentiation on live samples at physiological conditions represents a significant improvement for continuous monitoring of tissue differentiation or further use of the microtissues for, e.g., regenerative medicine.

Soft Probes for Bio-electrochemical Imaging

“…[18] In their approach, microtissues were grown on a Petri dish in situ and the oxygen consumption or the osteogenic differentiation were monitored. [17] Nevertheless, the growing conditions of microtissues and real tissue inside animal bodies are very different. Compared to microtissues, tissues directly from patients are representative and provide a higher reliability for clinical studies.…”

“…[8] Over the years, SECM has been employed for various applications, for instance to map the topography and surface reactivity of various materials, probe enzymatic activities in living cells, screen electrocatalytic materials for energy research, track oxygen consumption in microtissues and in embryos, and investigate kinetics at the liquid/liquid interface. [9][10][11][12][13][14][15][16][17][18][19][20] In order to satisfy various types of applications and experiments, several methods for the fabrication of different types of SECM probes have been developed. For instance, submicrometer glass-encapsulated microelectrodes and self-assembled spherical gold microelectrodes.…”

“…Similarly, non-invasive SECM assays for detecting osteogenic differentiation on arrays of microtissues have been also reported. [17] Although numerous approaches have been applied to the study of tissues, such as colorimetric and fluorometric assays, [60,61] SECM can provide a more sensitive spatial detection of biomarkers in comparison to colorimetric methodologies, and a wider range of information can be extracted since not only the fluorescent tagged species can be detected. Moreover, when studying living cells by optical methods, genetic modifications or the addition of fluorescent tags are required affecting not only their energy status, but also their whole metabolic response.…”

“…[54,[174][175][176] For instance, the enzymatic activity and oxygen production/consumption in plant tissues and micro-tissues have been monitored. [17,18,[177][178][179] SECM has also been applied to study the molecular transport through skin samples. [157,180] The lateral dimensions of tissue samples can approach square centimeters and height differences can be in the critical domain of the SECM probe.…”

Micro/nanoelectrochemical probe and chip devices for evaluation of three-dimensional cultured cells

“…14,25,26 Additionally, SECM was applied in the non-invasive monitoring of osteogenic differentiation of C2C12 microtissues. 27 Differentiation was assessed by measuring the activity of ALP, which is known to be highly expressed during differentiation. SECM was applied to the evaluation of senescence-associated β-galactosidase (SA-β-Gal), which is expressed only in senescent cells 28 in MCF-7 spheroids (Fig.…”