Impedance-based cellular assays for regenerative medicine.

Therapies based on regenerative techniques have the potential to radically improve healthcare in the coming years. As a result, there is an emerging need for non-destructive and label-free technologies to assess the quality of engineered tissues and cell-based products prior to their use in the clinic. In parallel, the emerging regenerative medicine industry that aims to produce stem cells and their progeny on a large scale will benefit from moving away from existing destructive biochemical assays towards data-driven automation and control at the industrial scale. Impedance-based cellular assays (IBCA) have emerged as an alternative approach to study stem-cell properties and cumulative studies, reviewed here, have shown their potential to monitor stem-cell renewal, differentiation and maturation. They offer a novel method to non-destructively assess and quality-control stem-cell cultures. In addition, when combined with in vitro disease models they provide complementary insights as label-free phenotypic assays. IBCA provide quantitative and very sensitive results that can easily be automated and up-scaled in multi-well format. When facing the emerging challenge of real-time monitoring of three-dimensional cell culture dielectric spectroscopy and electrical impedance tomography represent viable alternatives to two-dimensional impedance sensing.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Real-time and non-invasive measurements of cell mechanical behaviour with optical coherence phase microscopy.

Cell mechanical behaviour is increasingly recognised as a central biophysical parameter in cancer and stem cell research, and methods of investigating their mechanical behaviour are therefore needed. We have developed a novel qualitative method based on quantitative phase imaging which is capable of investigating cell mechanical behaviour in real-time at cellular resolution using optical coherence phase microscopy (OCPM), and stimulating the cells non-invasively using hydrostatic pressure. The method was exemplified to distinguish between cells with distinct mechanical properties, and transient change induced by Cytochalasin D. We showed the potential of quantitative phase imaging to detect nanoscale intracellular displacement induced by varying hydrostatic pressure in microfluidic channels, reflecting cell mechanical behaviour. Further physical modelling is required to yield quantitative mechanical properties.

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 Ω(1/2)cm for the case cell line and 6.5 ± 0.15 Ω(1/2)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.

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.

Nondestructive online in vitro monitoring of pre-osteoblast cell proliferation within microporous polymer scaffolds.

We present a system for the online, in vitro, nondestructive monitoring of tissue growth within microporous polymer scaffolds. The system is based on measuring the admittance of the sample over a frequency range of 10-200 MHz using an open-ended coaxial probe and impedance analyzer. The sample admittance is related to the sample complex permittivity (CP) by a quasi-static model of the probe's aperture admittance. A modified effective medium approximation is then used to relate the CP to the cell volume fraction. The change of cell volume fraction is used as a measure of tissue growth inside the scaffold. The system detected relative cell concentration differences between microporous polymer scaffolds seeded with 0.4, 0.45, 0.5, and 0.6 x 10(6) pre-osteoblast cells. In addition, the pre-osteoblast proliferation within 56 scaffolds over 14 days was recorded by the system and a concurrent DNA assay. Both techniques produced cell proliferation curves that corresponded to those found in literature. Thus, our data confirmed that the new system can assess relative cell concentration differences in microporous scaffolds enabling online nondestructive tissue growth monitoring.

Chitosan microchannel scaffolds for tendon tissue engineering characterized using optical coherence tomography.

Tendon tissue engineering requires the generation of a uniaxially orientated collagen type I matrix with several organization scales that confer mechanical functionality upon the tendon. A combination of factors in a dose- and time-dependent manner, such as growth factors and mechanical environment, may be the key to an in vitro-engineered tendon. To define the progress of tissue development within a scaffold, on-line systems need to be applied to monitor the newly generated matrix. To address this challenge, we designed a new porous chitosan scaffold with microchannels (diameter: 250 microm), which allows primary porcine tenocytes to proliferate in a bundle-like structure. The cell proliferation and extracellular matrix (ECM) production within the microchannels were successfully assessed under sterile conditions using optical coherence tomography (OCT). A semi-quantitative method that calculated the microchannel occupation ratio (the degree of cell proliferation and tissue turnover based on the total backscattered intensity in the microchannels) was developed. We further investigated the effect of different culture conditions on tendon cell matrix formation. Using a perfusion bioreactor, we demonstrated how fluid flow can increase (p < 1e(3)) ECM production within the microchannels significantly more than static culture. Our study illustrates how using a guiding scaffold in combination with the fast and non-destructive assessment of the microstructure using OCT allows discrimination between the parameters affecting the production and the organization of the ECM.