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The architectural structure of cells is essential for the cells' function, which becomes especially apparent in the highly "structure functionally" tuned skeletal muscle cells. Here, structural changes in the microstructure can have a direct impact on performance parameters, such as isometric or tetanic force production. The microarchitecture of the actin-myosin lattice in muscle cells can be detected noninvasively in living cells and in 3D by using second harmonic generation (SHG) microscopy, forgoing the need to alter samples by introducing fluorescent probes into them. Here, we provide tools and step-by-step protocols to guide the processes of obtaining SHG microscopy image data from samples, as well as extracting characteristic values from the image data to quantify the cellular microarchitecture using characteristic patterns of myofibrillar lattice alignments., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell viability of many cell types strongly relies on their ability to adjust to mechanical conditions and alterations. Cellular mechanisms for sensing and responding to mechanical forces and pathophysiological variations in these processes have become an emerging research field in recent years. An important signaling molecule involved in mechanotransduction as in many cellular processes is Ca 2+ . New experimental methods to probe cellular Ca 2+ signaling live under conditions of mechanical stimulation facilitate new insights into previously overlooked aspects of mechanical regulation of cells.Here, we describe a protocol for using Ca 2+ imaging in combination with a cell stretching device, the IsoStretcher. Cells grown on elastic membranes can be isotopically stretched in-plane, and their intracellular Ca 2+ level can be accessed online on the single cell level using fluorescent calcium indicator dyes. We show a protocol for functional screening of mechanosensitive ion channels and related drug screenings using BJ cells, a foreskin fibroblast cell line that strongly reacts to acute mechanical stimulation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Muscle cells (i.e. skeletal muscle fibers) are fully viable and functional when their excitation-contraction (EC) coupling machinery is intact. This involves intact membrane integrity with polarized membrane, functional ion channels for action potential generation and conduction, an intact electro-chemical interface at the level of the fiber's triad, followed by sarcoplasmic reticulum Ca 2+ release, and subsequent activation of the chemico-mechanical interface at the level of the contractile apparatus. The ultimate end result is then a visible twitch contraction upon a brief electrical pulse stimulation. For many biomedical studies involving single muscle cells, intact and viable myofibers are of utmost importance. Thus, a simple global screening method that involves a brief electrical stimulus applied to single muscle fibers and assessment of visible contraction would be of high value. In this chapter, we describe step-by-step protocols to (i) obtain intact single muscle fibers from freshly dissected muscle tissue using an enzymatic digestion procedure and (ii) provide a workflow for the assessment of twitch response of single fibers that can be ultimately classified as viable. For this, we have prepared a unique stimulation pen for which we provide the fabrication guide for do-it-yourself rapid prototyping to eliminate the need for expensive specialized commercial equipment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The cultivation of cells in 3D systems is commonly regarded to be more physiological than in 2D as it comes much closer to the natural situation in tissues in many different aspects. However, 3D cell culture is much more complex. Cells within the pores of a printed 3D scaffold face a special situation concerning cell-material interaction and cell adhesion, cell proliferation, and supply of medium and oxygen into the core of the scaffolds. Biological assays (for cell proliferation, viability, and activity) have been validated primarily for 2D cell cultures and need to be adapted for 3D cultures. Likewise, in imaging, a number of points need to be taken into account in order to get a clear picture of the cells in 3D scaffolds, preferably with the method of multiphoton microscopy. Here, we describe a method for pretreatment and cell seeding of porous inorganic composite scaffolds (α-TCP/HA) for bone tissue engineering and for cultivation of the cell-scaffold constructs. The analytical methods described are the cell proliferation assay and the ALP activity assay. A step-by-step protocol is provided here that safely tackles typical difficulties that arise with this 3D cell-scaffold setting. In addition, MPM imaging of cells is described both with and without labeling. The combination of biochemical assays and imaging provides valuable insights into the possibilities of analysis with this 3D cell-scaffold system., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Two-dimensional in vitro culture models are widely being employed for assessing a vast variety of biological questions in different scientific fields. Common in vitro culture models are typically maintained under static conditions, where the surrounding culture medium is replaced every few days-typically every 48 to 72 h-with the aim to remove metabolites and to replenish nutrients. Although this approach is sufficient for supporting cellular survival and proliferation, static culture conditions do mostly not reflect the in vivo situation where cells are continuously being perfused by extracellular fluid, and thus, create a less-physiological environment. In order to evaluate whether the proliferation characteristics of cells in 2D culture maintained under static conditions differ from cells kept in a dynamic environment, in this chapter, we provide a protocol for differential analysis of cellular growth under static versus pulsed-perfused conditions, mimicking continuous replacement of extracellular fluid in the physiological environment. The protocol involves long-term life-cell high-content time-lapse imaging of fluorescent cells at 37 °C and ambient CO 2 concentration using multi-parametric biochips applicable for microphysiological analysis of cellular vitality. We provide instructions and useful information for (i) the culturing of cells in biochips, (ii) setup of cell-laden biochips for culturing cells under static and pulsed-perfused conditions, (iii) long-term life-cell high-content time-lapse imaging of fluorescent cells in biochips, and (iv) quantification of cellular proliferation from image series generated from imaging of differentially cultured cells., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Viability of cells is strongly related to their Ca 2+ homeostasis. Ca 2+ signal fluctuations can be on a slow time scale, e.g., in non-excitable cells, but also in the range of tens of milliseconds for excitable cells, such as nerve and muscle. Muscle fibers respond to electrical stimulation with Ca 2+ transients that exceed their resting basal level about 100 times. Fluorescent Ca 2+ dyes have become an indispensable means to monitor Ca 2+ fluctuations in living cells online. Fluorescence intensity of such "environmental dyes" relies on a buffer-ligand interaction which is not only governed by laws of mass action but also by binding and unbinding kinetics that have to be considered for proper Ca 2+ kinetics and amplitude validation. The concept of Ca 2+ dyes including the different approaches using ratiometric and non-ratiometric dyes, the way to correctly choose dyes according to their low-/high-affinity properties and kinetics as well as staining techniques, and in situ calibration are reviewed and explained. We provide detailed protocols to apply ratiometric Fura-2 imaging of resting Ca 2+ and Ca 2+ fluctuations during field-stimulation in single isolated skeletal muscle cells and how to translate fluorescence intensities into absolute Ca 2+ concentrations using appropriate calibration techniques.

Multiphoton microscopy allows continuous depth-resolved, nondestructive imaging of scaffold-seeded cells during cell or tissue culture. Spectrally separated images in high resolution can be provided while cells are conserved in their native state. Here we describe the seeding of mesenchymal stem cells to bacterial nanocellulose hydropolymer scaffolds followed by 2-channel imaging of cellular autofluorescence (AF) and collagen-I formation using second harmonic generation (SHG) signals. With this approach the simultaneous observation of the progression of cell morphology and production of extracellular matrix as hallmarks of viability and cell fitness is possible.