This project is financially supported by the Cancer Pilot Award from UF Health Cancer Center (X. T. and D. S.) and the Gatorade Award Start-up Package (X. T.). The authors sincerely appreciate the intellectual discussions with and the technical supports from Dr. Jonathan Licht (UFHCC), Dr. Rolf Renne (UFHCC), Dr. Ji-Hyun Lee (Biostatistics, UF), Dr. Hugh Fan (MAE, UF), Dr. Warren Dixon (MAE, UF), Dr. Ghatu Subhash (MAE, UF), Dr. Mark Sheplak (MAE &#38; ECE, UF), Dr. Malisa Sarntinoranont (MAE, UF), Dr. Scott Banks (MAE, UF), Dr. Matthew Traum (MAE, UF), Dr. David Hahn (University of Arizona), Dr. Weihong Wang (Oracle Corporation), Dr. Youhua Tan (Hong Kong Polytechnic University), and the Support Team of Nikon (Drs. Jose Serrano-Velez, Larry Kordon, and Jon Ekman). The authors are deeply grateful for the generous and effective support from all members of Tang&#39;s, Siemann&#39;s, and Guan&#39;s research laboratories and all staff members of the MAE &#38; ECE &#38; Physics &#38; Radiation Oncology Departments, UF.

<ol>
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There are no conflicts of interest to declare.

The imaging process (step 6.3) is critical to ensure that the fluorescence images are of sufficiently good quality to yield valid quantification results. The z-stack images of fluorescent protein or beads should have a z-range that is large enough to include the in-focus images for all the Z-positions that the sample spans. Another critical step is collecting the reference images of fluorescent beads after dissolving the cells (step 6.5). Because the reference images need to be taken at the same positions in step 6.3, no relative displacement should be induced between the Petri dish, environment chamber, and the microscope. The investigators performing the dissolving step must be careful to remove the lid of the Petri dish and ensure that the applied mechanical perturbation is not large enough to alter the location of the dish in the environment chamber.
Solutions are provided below to resolve some errors that might occur during experiments. If no macro is activated after clicking Enter in step 6.4, it is most likely because the left bottom area of the screen is occupied by a non-Element window. In such a case, the left bottom area of the window needs to be cleared so that macros can be activated in Elements. Another common error is that the bright-field images appear black. This issue is caused by an insufficient time interval between the acquisitions of fluorescence and bright-field images. Slight delays in the fluorescence imaging time counting can accumulate over time and cause considerable delays and interfere with the bright-field imaging. One solution is to adjust the duration of one imaging cycle of all the positions to be less than (not equal to) the time interval between the start of consecutive motions. This operation refreshes the time counting and eliminates the cumulative error at the beginning of each imaging cycle.
This all-optical interrogation technology supports (1) a wide range of hardware/software, including but not limited to Nikon, (2) diverse types of validated hydrogel systems, including gelatin, PEG, Matrigel, and collagen I gels, and (3) the programmable customization based on different needs of researchers. However, if any of the bottom-level control functions are not available from a commercial microscope, customization of the functions using AMFIP becomes challenging. Another limitation of this technique is the spatial drift of the sample in both the XY and focus (Z) planes. Although this limitation can be overcome during the post-processing of the images, it is essential to improve the auto-focus function to correct the real-time drift of the samples. This improvement will increase the throughput of the imaging process and reduce the potential error caused by the drift during experiments.
Mechanotransducers, such as YAP, may serve as new therapeutic targets for the development of promising cancer therapies25,26,27. Emerging data suggest that YAP promotes the proliferation and invasion of cancer cells. Mechanics-induced YAP translocation from the cytoplasm to the nucleus activates the transcription of genes related to cell migration, proliferation, invasion, and apoptosis, leading to aberrant cell behaviors28,29,30,31. This work aimed to explore the potential correlation of the YAP N/C ratio and cell mechanics in two typical human lung cancer and normal cell lines. During the 10 h cell spreading period, PC9 cells show similar YAP concentrations in the nucleus and cytoplasm (Figure 3D and Figure 5A). B2B cells show a higher YAP concentration in the nucleus than in the cytoplasm (Figure 3C and Figure 5A). This relationship found during the early spreading stage is different from the majority of published findings that compare YAP concentration in the nucleus between normal and cancer cells. Although not necessarily at the early spreading stage, most published findings show that YAP is more concentrated in the nucleus of cancer cells than in the nucleus of normal cells27,28. Only one study on breast cancer reported an exception32 that shows YAP is more concentrated in the cytoplasm, which agrees with our current observations made in lung cancer PC9 cells. To the best of the authors&#39; knowledge, this work is the first to show a lower YAP N/C ratio in a human lung cancer cell line. The authors hypothesize that the reason for a stable YAP N/C ratio in PC9 cells might be due to the low variation in the cell/nucleus spread area and traction in PC9 cells at the early spreading stage. The dissection of the underpinning molecular mechanisms of low YAP N/C ratio in PC9 and B2B cells is ongoing.
During the first 10 h of spreading, these two cell lines show a distinct relationship between the YAP N/C ratio, cell traction, and spread area (Figure 5). For B2B cells, a higher YAP N/C ratio is correlated with a higher cell and nucleus spread area (Figure 6A,B), which is consistent with the reported data of other normal cells33. Interestingly, although the developmental trend of this relationship is generally found in all B2B cells recorded, two different degrees (high and low) of this relationship are found. B2B cells that spread and migrate simultaneously show lower traction and higher cell and nucleus spread area with a higher YAP N/C ratio (2.05 &#177; 0.32). For B2B cells that spread and remain at the same location, they show higher traction and lower cell and nucleus spread area with a lower YAP N/C ratio (1.74 &#177; 0.21). These two degrees of relationships are demonstrated in the bifurcated scattered data groups (Figure 6C,D). As reported in the literature, stationary normal cells, such as embryonic fibroblast NIH 3T3 cells, have higher traction than migratory cells34. The data reported in this paper suggest that the spreading and non-migrating B2B cells applied higher traction than spreading and migrating B2B cells, likely suggesting that high traction is needed for non-migrating cells to stabilize on the substrate.
In addition, these data show that stationary normal B2B cells generate a higher peri-nuclear force, whereas previous research conducted by other researchers reported only higher cell traction generated at the periphery of stationary cells34,35,36,37. The authors think that the difference in the intrinsic tendency of migration in the experiments might cause these contradictory results. In the published experiments, square-shaped micropatterning had been used to confine single cells from spreading and inhibit migration; whether the cells had the tendency to migrate is unknown. As migratory cells often show high traction force at the periphery of the cells38, it is likely that cells with the tendency to migrate will still maintain high periphery traction even though their migration is restricted. In this present study, the stationary cells are not restricted by any micropattern but do not migrate, indicating that the cells tend to maintain their non-migrating state. Another possibility is that the cell shape defined by the micropattern may affect the distribution of focal adhesions and traction forces39. The results in this study were generated without any confining micropatterning and represent the force distribution of stationary cells in their original shape.
To the best of the authors&#39; knowledge, only one publication to date specifically reported the finding of peri-nuclear forces in normal cells (mouse embryonic fibroblasts), potentially caused by the actin cap spanning across the nucleus40. YAP cytoplasm-to-nucleus translocation is correlated with the increase in peri-nuclear force40. A thorough search of the relevant literature did not yield any publicationsthat report a peri-nuclear force or the actin cap in cancer cells. An indirect study on melanoma cancer cells demonstrated that the actin rim (another peri-nuclear actin organization located around but not covering the nucleus) reduces cell migration rates41, indirectly suggesting the existence of a peri-nuclear force. However, no direct experimental data are reported. In this study, the authors found that both PC9 and B2B cells show peri-nuclear displacement and traction. The mechanisms of the generation of the peri-nuclear forces and their effects remain controversial. In normal cells, the actin cap was reported to play a role in regulating nucleus morphology and chromatin organization42, transmitting mechanical signals from focal adhesions into the nucleus through linkers of nucleoskeleton and cytoskeleton (LINC) complex43, and regulating cell migration44. Lamin A/C is related to the formation and disruption of the actin cap40,41,42,43,44. However, the report that claimed that the actin cap generates a peri-nuclear force did not consider the potential role of the actin rim40. In cancer cells, overexpression of Lamin A facilitates the formation of an actin rim and restricts cancer cell migration. Overexpression of Lamin B reduces actin rim formation and promotes migration. The peri-nuclear force might be involved in this process due to the existence of peri-nuclear actin organization and the effect of Lamin A. However, the results of this study did not show any evidence of measured peri-nuclear forces or the behavior of the actin cap. Therefore, the discovery of peri-nuclear forces in PC9 cells in this present study is the first report showing peri-nuclear forces and displacements in lung cancer cells. The authors are currently investigating the molecular mechanisms and functions of peri-nuclear forces in CRISPR/Cas9-engineered PC9 and B2B cells.
Beyond the all-optical mechanobiology interrogation that is demonstrated in this paper, the integrated multi-functional system can be applied to optically probe a myriad of other essential physiological and pathobiological signals in living systems. For example, the authors&#39; laboratory has recently established multiple stably transduced human cancer cell lines that co-express three light-responsive membrane proteins: membrane voltage indicator QuasAr2 (excitation: 640 nm; emission: 660 nm-740 nm), membrane voltage depolarizer CheRiff (excitation: 488 nm), and membrane voltage hyperpolarizer eNpHR3 (excitation: 590 nm). These three functional proteins can be activated by spectrum-orthogonal laser lines in a crosstalk-free manner, enabling all-optical two-way signaling communications (readout and control) of membrane electrophysiology. Using an integrated opto-electronics system and a manual patch-clamp, the authors have validated the all-optical control and readout of the membrane voltage (Vm) in single human cancer cells and multicellular tumor spheroids. The all-optical electrophysiology interrogation opens the possibility for detailed explorations of previously inaccessible bioelectricity in cancer cells, which may help advance tumor biology from a new axis.

The overall goal of this method is to enable all-optical multi-functional imaging and analysis of living cells. An all-in-one imaging program that enables the automatic coordination of multi-functional optoelectronic devices will reduce labor-intensive and error-prone manual operations and is essential for researchers to conduct long-term live-cell imaging1,2,3,4. However, most existing public programs in the biomedical research community either only apply to limited optoelectronic devices or require additional hardware for the coordination of different equipment5,6,7,8,9. Recently, an open-source and software-based program has been developed, titled &#34;Automatic Multi-functional Integration Program (AMFIP),&#34; enabling multi-channel and time-lapse imaging. Based on Java language and the Application Programming Interface (API) of &#181;Manager11,12, AMFIP was developed as a plugin in &#181;Manager that executes customized Java scripts to accomplish software-based communications of multiple optoelectronic hardware and software platforms, including but not limited to those from Nikon. The establishment of AMFIP opens the possibility for programmable and multi-functional interrogation of cell behaviors. An integrated experimental and computational system is developed in this paper and combines AMFIP with digital imaging analysis and cell traction force microscopy. The system enables the elucidation of the distinct YAP mechanobiology in CRISPR/Cas9-engineered human normal B2B (Figure 1) and lung cancer PC9 (Figure 2) cell lines. The system provides the scientific community a comprehensive solution that avoids the demand of purchasing additional controlling devices that may not be available for and/or compatible with every imaging system.
The protocols presented in this paper introduce how to (1) apply AMFIP to conduct automatic long-term imaging for both CRISPR/Cas9-engineered cell lines that express mNEonGreen2-tagged YAP; and (2) combine Fiji ImageJ, MATLAB, and Origin for the quantitative analysis of YAP nuclear/cytoplasm (N/C) ratio based on their fluorescent intensity (Figure 3 and Figure 4), cellular displacement field (Figure 1C and Figure 2C), and cellular traction field (Figure 1D and Figure 2D). The results suggest that (1) during the first 10 h of cell spreading on the substrates that have physiologically relevant mechanical stiffness13,14,15,16,17,18, the YAP N/C ratio of single B2B cells shows more noticeable time-dependent variation and fluctuation compared to that of single PC9 cells (Figure 5 and Figure 6); and (2) PC9 cancer cells generate noticeable traction in their peri-nuclear regions (Figure 7). The integrated system and methodologies described in this protocol transcend the specific types of cells and optogenetic molecules. Researchers can apply the protocols to customize their specific live-cell interrogation experiments and elucidate multifaceted signaling dynamics in the context of cell physiology and pathology.

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			<td>Fiji ImageJ</td>
			<td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation</td>
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			<td>Gibco</td>
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			<td>Gibco</td>
			<td>10010023</td>
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			<td>New England Biolabs</td>
			<td>&#160;F-553S</td>
			<td>high-fidelity DNA polymerase</td>
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			<td>&#956;Manager</td>
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all-optical mechanobiology interrogation of yes-associated protein in human cancer and normal cells using a multi-functional system

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

Watch this Scientific Journal Video about All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System at JoVE.com

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...

all-optical-mechanobiology-interrogation-yes-associated-protein-human

Research

JoVE Journal

Bioengineering

2.9K Views.  University of Florida. This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP). 

Video: All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches2-4. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM5. Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis6.
Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates7 requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines8. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency9,10.
Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D11. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC)12 and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

A 3D culture system for hematopoiesis is described using human cord blood and leukemic bone marrow cells. The method is based on the use of a porous synthetic polyurethane scaffold coated with extracellular matrix proteins. This scaffold is adaptable to accommodate a wide range of cells.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens

A detailed understanding of the complexity and relative variability within the human cardiac venous system is crucial for the development of cardiac devices that require access to these vessels. For example, cardiac venous anatomy is known to be one of the key limitations for the proper delivery of cardiac resynchronization therapy (CRT)1 Therefore, the development of a database of anatomical parameters for human cardiac venous systems can aid in the design of CRT delivery devices to overcome such a limitation. In this research project, the anatomical parameters were obtained from 3D reconstructions of the venous system using contrast-computed tomography (CT) imaging and modeling software (Materialise, Leuven, Belgium). The following parameters were assessed for each vein: arc length, tortuousity, branching angle, distance to the coronary sinus ostium, and vessel diameter.
	CRT is a potential treatment for patients with electromechanical dyssynchrony. Approximately 10-20% of heart failure patients may benefit from CRT2. Electromechanical dyssynchrony implies that parts of the myocardium activate and contract earlier or later than the normal conduction pathway of the heart. In CRT, dyssynchronous areas of the myocardium are treated with electrical stimulation. CRT pacing typically involves pacing leads that stimulate the right atrium (RA), right ventricle (RV), and left ventricle (LV) to produce more resynchronized rhythms. The LV lead is typically implanted within a cardiac vein, with the aim to overlay it within the site of latest myocardial activation. 
	We believe that the models obtained and the analyses thereof will promote the anatomical education for patients, students, clinicians, and medical device designers. The methodologies employed here can also be utilized to study other anatomical features of our human heart specimens, such as the coronary arteries. To further encourage the educational value of this research, we have shared the venous models on our free access website: <a href="http://www.vhlab.umn.edu/atlas" target="_blank">www.vhlab.umn.edu/atlas</a>.

The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

A detailed  understanding of the complexity and relative variability within the human  cardiac venous system is crucial for the development of cardiac ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

3D Bioprinting for Rapid Vascular Network Fabrication Using Vessels-on-a-Plate

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

Author Spotlight: Automated Bioprinting for High-Throughput Vascular Model Fabrication

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...