Pierre O. Bagnaninchi

Real-time label-free monitoring of adipose-derived stem cell differentiation with electric cell-substrate impedance sensing

Real-time monitoring of stem cells (SCs) differentiation will be critical to scale-up SC technologies, while label-free techniques will be desirable to quality-control SCs without precluding their therapeutic potential. We cultured adipose-derived stem cells (ADSCs) on top of multielectrode arrays and measured variations in the complex impedance Z* throughout induction of ADSCs toward osteoblasts and adipocytes. Z* was measured up to 17 d, every 180 s, over a 62.5–64kHz frequency range with an ECIS Zθ instrument. We found that osteogenesis and adipogenesis were characterized by distinct Z* time-courses. Significant differences were found (P = 0.007) as soon as 12 h post induction. An increase in the barrier resistance (Rb) up to 1.7 ohm·cm2 was associated with early osteo-induction, whereas Rb peaked at 0.63 ohm·cm2 for adipo-induced cells before falling to zero at t = 129 h. Dissimilarities in Z* throughout early induction (<24 h) were essentially attributed to variations in the cell-substrate parameter α. Four days after induction, cell membrane capacitance (Cm) of osteo-induced cells (Cm = 1.72 ± 0.10 μF/cm2) was significantly different from that of adipo-induced cells (Cm = 2.25 ± 0.27 μF/cm2), indicating that Cm could be used as an early marker of differentiation. Finally, we demonstrated long-term monitoring and measured a shift in the complex plane in the middle frequency range (1 kHz to 8 kHz) between early (t = 100 h) and late induction (t = 380 h). This study demonstrated that the osteoblast and adipocyte lineages have distinct dielectric properties and that such differences can be used to perform real-time label-free quantitative monitoring of adult stem cell differentiation with impedance sensing.

A novel optical coherence tomography-based micro-indentation technique for mechanical characterization of hydrogels

Depth-sensing micro-indentation has been well recognized as a powerful tool for characterizing mechanical properties of solid materials due to its non-destructive approach. Based on the depth-sensing principle, we have developed a new indentation method combined with a high-resolution imaging technique, optical coherence tomography, which can accurately measure the deformation of hydrogels under a spherical indenter at constant force. The Hertz contact theory has been applied for quantitatively correlating the indentation force and the deformation with the mechanical properties of the materials. Youngs moduli of hydrogels estimated by the new method are comparable with those measured by conventional depth-sensing micro-indentation. The advantages of this new method include its capability to characterize mechanical properties of bulk soft materials and amenability to perform creeping tests. More importantly, the measurement can be performed under sterile conditions allowing non-destructive, in situ and real-time investigations on the changes in mechanical properties of soft materials (e.g. hydrogel). This unique character can be applied for various biomechanical investigations such as monitoring reconstruction of engineered tissues.

Online monitoring of collagen fibre alignment in tissue‐engineered tendon by PSOCT

One of the major challenges in engineering a functional tendon is to be able to monitor the evolving arrangement of collagen fibres, which leads to the formation of a complex extracellular matrix. Polarization‐sensitive optical coherence tomography (PSOCT) is a non‐destructive imaging technique capable of examining the organization of tissues online. In this study, PSOCT was used for the first time to visualize the evolution of the collagen fibre alignment in tissue‐engineered tendons in response to varying growth environments. The tendon constructs consisted of rat tenocytes embedded in collagen hydrogels and cultured in Flexcell Tissue Train Culture plates. They were subjected to cyclical loading for 1 h at 1 Hz every day, using the Flexcell system. Different strain level and cell seeding densities were examined at different time points over a 14 day period. The birefringence, a characteristic of the growing tendon, was found to increase over culture time and with the increase of cell‐seeding densities. In addition, it was revealed that the effect of contraction of tenocytes on the fibre alignment was greater than the application of external forces during stretching. These results demonstrate that PSOCT can be a powerful tool for monitoring the change in collagen organization online and non‐destructively at different time points in growing engineered tendons. Copyright

Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

Abstract. Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell’s morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

In-depth imaging and quantification of degenerative changes associated with Achilles ruptured tendons by polarization-sensitive optical coherence tomography

The objective of this study was to develop a method based on polarization-sensitive optical coherent tomography (PSOCT) for the imaging and quantification of degenerative changes associated with Achilles tendon rupture. Ex vivo PSOCT examinations were performed in 24 patients. The study involved samples from 14 ruptured Achilles tendons, 4 tendinopathic Achilles tendons and 6 patellar tendons (collected during total knee replacement) as non-ruptured controls. The samples were imaged in both intensity and phase retardation modes within 24 h after surgery, and birefringence was quantified. The samples were fixed and processed for histology immediately after imaging. Slides were assessed twice in a blind manner to provide a semi-quantitative histological score of degeneration. In-depth micro structural imaging was demonstrated. Collagen disorganization and high cellularity were observable by PSOCT as the main markers associated with pathological features. Quantitative assessment of birefringence and penetration depth found significant differences between non-ruptured and ruptured tendons. Microstructure abnormalities were observed in the microstructure of two out of four tendinopathic samples. PSOCT has the potential to explore in situ and in-depth pathological change associated with Achilles tendon rupture, and could help to delineate abnormalities in tendinopathic samples in vivo.

Polyelectrolyte Multilayer Coating of 3D Scaffolds Enhances Tissue Growth and Gene Delivery: Non‐Invasive and Label‐Free Assessment

Layer-by-layer (LbL) deposition is a versatile technique which is beginning to be be explored for inductive tissue engineering applications. Here, it is demonstrated that a polyelectrolyte multilayer film system composed of glycol-chitosan (Glyc-CHI) and hyaluronic acid (HA) can be used to coat 3D micro-fabricated polymeric tissue engineering scaffolds. In order to overcome many of the limitations associated with conventional techniques for assessing cell growth and viability within 3D scaffolds, two novel, real-time, label-free techniques are introduced: impedance monitoring and optical coherence phase microscopy. Using these methods, it is shown that LbL-coated scaffolds support in vitro cell growth and viability for a period of at least two weeks at levels higher than uncoated controls. These polyelectrolyte multilayer coatings are then further adapted for non-viral gene delivery applications via incorporation of DNA carrier lipoplexes. Scaffold-based delivery of the enhanced green fluorescent protein (EGFP) marker gene from these coatings is successfully demonstrated in vitro, achieving a two-fold increase in transfection efficiency compared with control scaffolds. These results show the great potential of Glyc-CHI/HA polyelectrolyte multilayer films for a variety of gene delivery and inductive tissue engineering applications.

Two-dimensional and three-dimensional viability measurements of adult stem cells with optical coherence phase microscopy

Cell viability assays are essential tools for cell biology. They assess healthy cells in a sample and enable the quantification of cellular responses to reagents of interest. Noninvasive and label-free assays are desirable in two-dimensional (2D) and three-dimensional (3D) cell culture to facilitate time-course viability studies. Cellular micromotion, emanating from cell to substrate distance variations, has been demonstrated as a marker of cell viability with electric cell-substrate impedance sensing (ECIS). In this study we investigated if optical coherence phase microscopy (OCPM) was able to report phase fluctuations of adult stem cells in 2D and 3D that could be associated with cellular micromotion. An OCPM has been developed around a Thorlabs engine (λo = 930 nm) and integrated in an inverted microscope with a custom scanning head. Human adipose derived stem cells (ADSCs, Invitrogen) were cultured in Mesenpro RS medium and seeded either on ECIS arrays, 2D cell culture dishes, or in 3D highly porous microplotted polymeric scaffolds. ADSC micromotion was confirmed by ECIS analysis. Live and fixed ADSCs were then investigated in 2D and 3D with OCPM. Significant differences were found in phase fluctuations between the different conditions. This study indicated that OCPM could potentially assess cell vitality in 2D and in 3D microstructures.

Real-time quantitative monitoring of hiPSC-based model of macular degeneration on Electric Cell-substrate Impedance Sensing microelectrodes.

Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. Humanized disease models are required to develop new therapies for currently incurable forms of AMD. In this work, a tissue-on-a-chip approach was developed through combining human induced pluripotent stem cells, Electric Cell–substrate Impedance Sensing (ECIS) and reproducible electrical wounding assays to model and quantitatively study AMD. Retinal Pigment Epithelium (RPE) cells generated from a patient with an inherited macular degeneration and from an unaffected sibling were used to test the model platform on which a reproducible electrical wounding assay was conducted to model RPE damage. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on the microelectrode arrays. A spatially controlled RPE layer damage that mimicked cell loss in AMD disease was then initiated. Post recovery, significant differences (P<0.01) in migration rates were found between case (8.6±0.46 μm/h) and control cell lines (10.69±0.21 μm/h). Quantitative data analysis suggested this was achieved due to lower cell–substrate adhesion in the control cell line. The ECIS cell–substrate adhesion parameter (α) was found to be 7.8±0.28 Ω1/2 cm for the case cell line and 6.5±0.15 Ω1/2 cm for the control. These findings were confirmed using cell adhesion biochemical assays. The developed disease model-on-a-chip is a powerful platform for translational studies with considerable potential to investigate novel therapies by enabling real-time, quantitative and reproducible patient-specific RPE cell repair studies.

Cellular micromotion monitored by long-range surface plasmon resonance with optical fluctuation analysis.

Long-range surface plasmon resonance (LRSPR) is a powerful biosensing technology due to a substantially larger probing depth into the medium and sensitivity, compared with conventional SPR. We demonstrate here that LRSPR can provide sensitive noninvasive measurement of the dynamic fluctuation of adherent cells, often referred to as the cellular micromotion. Proof of concept was achieved using confluent layers of 3T3 fibroblast cells and MDA-MB-231 cancer cells. The slope of the power spectral density (PSD) of the optical fluctuations was calculated to determine the micromotion index, and significant differences were measured between live and fixed cell layers. Furthermore, the performances of LRSPR and conventional surface plasmon resonance (cSPR) were compared with respect to micromotion monitoring. Our study showed that the micromotion index of cells measured by LRSPR sensors was higher than when measured with cSPR, suggesting a higher sensitivity of LRSPR to the micromotion of cells. To investigate further this finding, simulations were conducted to establish the relative sensitivities of LRSPR and cSPR to membrane fluctuations. Increased signal intensity was predicted for LRSPR in comparison to cSPR, suggesting that membrane fluctuations play a significant role in the optical micromotion measured in LRSPR. Analogous to cellular micromotion measured using impedance techniques, LRSPR micromotion has the potential to provide important biological information on the metabolic activity and viability of adherent cells.

Motility imaging via optical coherence phase microscopy enables label-free monitoring of tissue growth and viability in 3D tissue-engineering scaffolds

As the field of tissue engineering continues to progress, there is a deep need for non‐invasive, label‐free imaging technologies that can monitor tissue growth and health within thick three‐dimensional (3D) constructs. Amongst the many imaging modalities under investigation, optical coherence tomography (OCT) has emerged as a promising tool, enabling non‐destructive in situ characterization of scaffolds and engineered tissues. However, the lack of optical contrast between cells and scaffold materials using this technique remains a challenge. In this communication, we show that mapping the optical phase fluctuations resulting from cellular viability and motility allows for the distinction of live cells from their surrounding scaffold environment. Motility imaging was performed via a common‐path optical coherence phase microscope (OCPM), an OCT modality that has been shown to be sensitive to nanometer‐level fluctuations. More specifically, we examined the development of human adipose‐derived stem cells and/or murine pre‐osteoblasts within two distinct scaffold systems, commercially available alginate sponges and custom‐microfabricated poly(d, l‐lactic‐co‐glycolic acid) fibrous scaffolds. Cellular motility is demonstrated as an endogenous source of contrast for OCPM, enabling real‐time, label‐free monitoring of 3D engineered tissue development. Copyright