Monitoring Local Cell Viability in Engineered Tissues: A Fast, Quantitative, and Nondestructive Approach

Abstract

Assessment of cell viability is a key issue in monitoring in vitro engineered tissue constructs. In this study we describe a fully automated, quantitative, and nondestructive approach, which is particularly suitable for tissue engineering. The approach offers several advantages above existing methods. Living and dead cell numbers can be separately determined for both isolated cells and cells that form networks during tissue formation. Moreover, viability can be locally monitored in time throughout the three-dimensional tissue. The viability assay is based on a dual fluorescent staining technique using CellTracker Green (CTG) for detection of living cells and propidium iodide (PI) for dead cells. CTG and PI images are created with a confocal laser scanning microscope. To determine the number of living cells, CTG fluorescence intensity is determined from the CTG image. Thereby, novel image-processing techniques have been developed, normalizing for various undesired influences that alter measurements of absolute CTG fluorescence intensities. Dead cell numbers are determined from the PI image, using an improved computerized counting method. The approach was first evaluated on C2C12 monolayers, of which images were taken directly after probe addition and 24 h later. Results show that at both times, computed living and dead cell numbers highly correlate with manually counted cell numbers (r > 0.996). Next, the approach was applied for monitoring viability in three-dimensional engineered skeletal muscle tissue constructs, which were subjected to unfavorable environmental conditions. This example illustrated that local viability can be quantitatively, nondestructively, and locally monitored in three-dimensional tissue constructs, making it a promising tool in the field of tissue engineering.

Decellularized homologous tissue-engineered heart valves as off-the-shelf alternatives to xeno- and homografts

“…After simulation of the crimping procedure cell viability was assessed and compared to the non-crimped control (n=1) by whole-mount staining with cell tracker green CMFDA (CTG; Molecular Probes, the Netherlands) and propidium iodine (PI; Molecular Probes, the Netherlands), as described previously [38]. Visualization was performed with a 2-photon confocal microscope (Laser Scanning Microscope 510 META NLO, Zeiss, Germany) to observe living cells in green and dead cells in red.…”

Stored Human Septal Chondrocyte Viability Analyzed by Confocal Microscopy

“…Several methods have been used in the past, including the trypan blue exclusion test, flow cytometry, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), and fluorescent staining for light microscopy. 17 However, limitations with these techniques led many authors to adopt CLSM for assessing the viability of a wide variety of tissues, including bacteria, 18 microbes in food products, 19 and brain. 20 Confocal laser scanning microscopy has already been used effectively to assess the viability of cultured human nasal septal chondrocytes seeded onto polymer fleeces.…”

Compression Induced Cell Damage in Engineered Muscle Tissue: An In Vitro Model to Study Pressure Ulcer Aetiology

“…In all experiments an optimum probe concentration of 10 M ͑CTG͒ and 5 M ͑PI͒ was used. 7 The constructs were loaded with CTG for 5 min, rinsed twice with poly butene sulfone, and incubated for 30 min in a humidified incubator. After rinsing again, PI dissolved in fresh growth medium was added to each well ͑3 ml per well͒ after which the six-well plate was mounted onto the microscope stage.…”

“…For each strain regime, images were taken from the central horizontal section of the constructs at tϭ0, 1, 2, 4, 6, and 8 h. The four corner tiles which were situated only partly below the circular indenter were excluded from data analyses. Dead cell numbers were quantified by automated counting of nuclei from PI images using previously developed image analysis software 7 written in Matlab ͑The Matworks, Natick, MA͒. Since the constructs contain multinucleated myotubes, this is not exactly equal to the number of dead cells, since myotubes may be only partly damaged.…”

“…For this purpose, a novel compression device was developed. Finally, by quantification and localization of viable and dead cells using previously developed image analysis techniques 7 it is possible to test the hypothesis that cell deformation forms an additional mechanism for cell death, which may play a role in the onset of pressure ulcers.…”