This work was supported by the National Natural Science Foundation of China (81772133, 81902444), the Guangdong Natural Science Fund (2020A1515010269, 2020A1515011367), the Guangzhou Citizen Health Science and Technology Research Project (201803010034, 201903010072), and the Military Medical Innovation Project (17CXZ008).

All animal care and procedures were in accordance with China Nanfang Hospital policies for heath and well-being (application No: NFYY-2019-73).
1. Mouse preparation
<ol>
	<li>Anesthetize the mouse.
	<ol>
		<li>Prepare sodium pentobarbital (50 mg/kg) in a syringe.</li>
		<li>Grab the mouse (8-week-old male C57BL/6) with the left hand so that its belly is facing up and its head is lower than its tail. Disinfect the abdominal skin with 75% alcohol.</li>
		<li>Holding the syringe in the right hand, pierce the abdomen white line with the needle slightly to the right side of the skin by 3-5 mm. Ensure that the syringe needle and the skin are at a 45&#176; angle, inject the pentobarbital slowly into the abdominal cavity.&#160;&#160;After shaving, disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution.</li>
		<li>Check whether the anesthesia was successful by the lack of a righting reflex.</li>
	</ol>
	</li>
	<li>Inject Rhodamine B isothiocyanate&#8211;dextran into the caudal vein.
	<ol>
		<li>Prepare 100 &#181;L of 10 mg/mL Rhodamine B isothiocyanate&#8211;dextran in a syringe.</li>
		<li>Wipe the mouse&#8217;s tail with 75% alcohol.</li>
		<li>Hold the tail with the left hand, hold the syringe in the right hand with the needle parallel to the vein, and inject the 100 &#181;L dye.</li>
		<li>Stop the bleeding with a cotton swab after the injection.</li>
	</ol>
	</li>
	<li>Soak the mouse&#8217;s abdominal fur with gauze wetted with sterile water, and shave the mouse&#8217;s abdomen using a razor, making strokes in the direction of the fur.After shaving, disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution.</li>
	<li>After wiping a heating pad with 75% alcohol, open the heating pad, and place the mouse on its back in the heating pad to maintain its body temperature at 37 &#176;C.</li>
</ol>
2. Fixing the mouse liver with the body organ imaging frame
NOTE: The commercial organ imaging frame has not been released yet.
<ol>
	<li>Place a clean suction cup in a fixed position.</li>
	<li>Install an organ imaging fixture (Supplementary Figure 1) and wipe the heating pad and suction cup with 75% alcohol.</li>
	<li>Connect the suction cup (5 mm diameter) to the vacuum pump hose and turn on the vacuum pump.</li>
	<li>On a sterile table sterilized with 75% alcohol, disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution, then disinfects around the mouse&#8217;s incision site 3 times alternately with 75% alcohol and Chlorhexidine solution, then use surgical scissors to cut 2 cm of skin off the lower sternal border of the mouse and expose the liver.</li>
	<li>Place the mouse together with the heating pad on the holder base.</li>
	<li>Adjust the organ imaging fixture so that the suction cup holds the liver. Then use a negative pressure of 30-35 kPa for suction so that the liver attaches to the suction cup (Supplementary Figure 2).</li>
</ol>
3. Adjusting the multiphoton laser scanning microscope
<ol>
	<li>Turn on the multiphoton microscope and select the 60x/1.00 W objective.</li>
	<li>Fix the frame and mouse under the objective lens.</li>
	<li>Add a drop of normal saline that can cover the entire lens, which is the center of the suction cup, and adjust the objective lens to just touch the normal saline.</li>
	<li>Double-click on the computer icon to turn on the laser software and turn on the switch. Then press and hold the power button and shutter for 3 s.</li>
	<li>Set the wavelength to 800 nm.</li>
	<li>Start the microscope operating software using the computer.</li>
	<li>For the Acquisition Setting, set Laser 800.</li>
</ol>
4. Observation using multiphoton laser scanning microscope
<ol>
	<li>For Image Acquisition Control, click the Fluorescent switch.</li>
	<li>Ensure that the room and equipment lights are switched off. To reduce noise levels, use the microscope in a darkened environment.</li>
	<li>Open the light path shutter on the microscope.</li>
	<li>Rotate the fluorescence filter to the fourth gear and pull the two levers of the optical switch.</li>
	<li>Looking through the eyepiece, use the coarse and fine focusing quasispirals to adjust the focal length, and use the X/Y axes to adjust the field of view to find the target area.</li>
</ol>
5. Multiphoton laser scanning micrography
<ol>
	<li>Turn off the fluorescent switch on the software, switch the fluorescent filter to the second gear (second gear is RXD1), and push the two levers of the optical switch.</li>
	<li>Click on Focus &#215;2 to preview the target area and adjust the acquisition setting and image acquisition control parameters (image resolution: 1024).</li>
	<li>Press Control + C and adjust the high voltage, gain, and offset (to reduce noise levels).</li>
	<li>Click Stop to stop preview, click the XY button to scan in two dimensions, and save after completion (Figure 1).</li>
	<li>Select a region, click on Depth, and then Preview to select the end set and start set (see the complete required part of the blood vessel) in the Microscope setting. Scan 3D images and save after completion (Figure 2).</li>
	<li>Select a region, click on Time, and adjust other acquisition setting and image acquisition control parameters (Step Size: 1 &#956;m; Slices: 10; Time Scan Number: 40). Scan different slices and save after completion to obtain moving images (Video 1). 
	NOTE: Have someone care for the mice during the scan and add anesthesia accordingly.</li>
	<li>Sacrifice the mouse by neck dissection.</li>
</ol>

<ol>
	<li>Wu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+microscopy+in+cardiovascular+research.">Multi-photon microscopy in cardiovascular research.</a> Methods. 130, 79-89 (2017).</li><li>Zhou, M., Ling, W., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrahepatic+mass-forming+cholangiocarcinoma+growing+in+a+giant+hepatic+hemangioma:+A+case+report.">Intrahepatic mass-forming cholangiocarcinoma growing in a giant hepatic hemangioma: A case report.</a> Medicine (Baltimore). 98 (27), 16410 (2019).</li><li>Werkmeister, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+for+blood+vessel+imaging:+new+non-invasive+tools+(Spectral+SHG,+FLIM).">Multiphoton microscopy for blood vessel imaging: new non-invasive tools (Spectral SHG, FLIM).</a> Clinical Hemorheology and Microcirculation. 37 (1-2), 77 (2007).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+optical+microangiography+provide+accurate+imaging+of+capillary+vessels?:+validation+using+multiphoton+microscopy.">Does optical microangiography provide accurate imaging of capillary vessels?: validation using multiphoton microscopy.</a> Journal of Biomedical Optics. 19 (10), 1-5 (2014).</li><li>Upputuri, P. K., Sivasubramanian, K., Mark, C. S., Pramanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+vascular+imaging+techniques+in+tissue+engineering+and+regenerative+medicine.">Recent developments in vascular imaging techniques in tissue engineering and regenerative medicine.</a> Biomed Research International. 2015, 783983 (2015).</li><li>Ustione, A., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+introduction+to+multiphoton+microscopy.">A simple introduction to multiphoton microscopy.</a> Journal of Microscopy. 243 (3), 221-226 (2011).</li><li>Centonze, V. E., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+excitation+provides+optical+sections+from+deeper+within+scattering+specimens+than+confocal+imaging.">Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.</a> Biophys Journal. 75 (4), 2015-2024 (1998).</li><li>Vogt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatic+multiphoton+imaging+of+the+whole+brain.">Chromatic multiphoton imaging of the whole brain.</a> Nature Methods. 16 (6), 459 (2019).</li><li>Bacskai, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+amyloid-&#946;+deposits+in+brains+of+living+mice+permits+direct+observation+of+clearance+of+plaques+with+immunotherapy.">Imaging of amyloid-&#946; deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy.</a> Nature Medicine. 7 (3), 369-372 (2001).</li><li>Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+dendritic+spines+in+the+developing+rat+barrel+cortex+in+vivo.">Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo.</a> Nature. 404 (6780), 876-881 (2000).</li><li>Svoboda, K., Denk, W., Kleinfeld, D., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dendritic+calcium+dynamics+in+neocortical+pyramidal+neurons.">In vivo dendritic calcium dynamics in neocortical pyramidal neurons.</a> Nature. 385 (6612), 161-165 (1997).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Deep-Brain+Structural+and+Hemodynamic+Multiphoton+Microscopy+Enabled+by+Quantum+Dots.">In vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.</a> Nano Letters. , (2019).</li><li>Sandoval, R. M., Molitoris, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+multiphoton+microscopy+as+a+tool+for+studying+renal+physiology+and+pathophysiology.">Intravital multiphoton microscopy as a tool for studying renal physiology and pathophysiology.</a> Methods. 128, 20-32 (2017).</li><li>Shear, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peer+Reviewed:+Multiphoton-Excited+Fluorescence+in+Bioanalytical+Chemistry.">Peer Reviewed: Multiphoton-Excited Fluorescence in Bioanalytical Chemistry.</a> Analytical Chemistry. 71 (17), 598-605 (1999).</li><li>Heymann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> Journal of Visualized Experiments. (97), (2015).</li><li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+first+fully+human+anti-TEM1+scFv+in+models+of+solid+tumor+imaging+and+immunotoxin-based+therapy.">Characterization of the first fully human anti-TEM1 scFv in models of solid tumor imaging and immunotoxin-based therapy.</a> Cancer Immunology &amp; Immunotherapy. 66 (3), 367-378 (2017).</li><li>Williams, R. M., Zipfel, W. R., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiphoton+microscopy+in+biological+research.">Multiphoton microscopy in biological research.</a> Current Opinion in Chemical Biology. 5 (5), 603-608 (2001).</li></ol>

The authors have nothing to disclose.

Observing a specific living tissue is an effective means of understanding the changes, localization, and biological effects of the material inside the tissue17. In this experiment, the important steps are fixing the liver with an organ imaging fixture, which can solve the problem of motion artifacts due to breathing and heartbeats, and the use of a multiphoton microscope for observation. Using this method, the internal tissues of the liver in vivo are observed through a multiphoton microscope, and the blood vessels are fluorescently labeled to clearly observe their position and three-dimensional structure. This method can be used to observe specific effects by fluorescently labeling a substance and dynamically following the substance in the living tissue to provide further visualizations.
Multiphoton microscopy has evolved from being a novel technique to become an indispensable tool for collecting information from cellular events in an organized tissue environment. However, the organ must be fixed in place to produce a stable image so that the method can be effective in observing certain tissues and organs. Movement artifacts from breathing and the heartbeat can prevent the liver from being directly imaged, so a body organ fixture is used, which uses the principle of vacuum adsorption to secure the organ and prevent image shake. The vacuum chuck can also fix a variety of body organs. However, there is a risk of tissue rupture if the appropriate negative pressure is not used or the imaging time extends beyond 2 h.
Although the resolution of multiphoton microscopy is slightly less than that of confocal microscopy, the depth of penetration is increased to 9-10 &#181;m, enabling the provision of images for deeper tissue observation, cellular events, and material effects.

Blood vessels can provide nutrients for various organ tissues of the human body, and exchange substances. At the same time, many cytokines, hormones, drugs, and cells also function through vascular transport to specific locations. Observing vascular changes in liver tissue can help in understanding the distribution of blood flow in liver tissue and the transport of substances, and assist in the analysis of certain vascular-related diseases1,2.
There are many ways to observe the blood vessels of the liver in mice. Among them, optical microscopy has many limitations in observing opaque vascular tissue3. Multiphoton microscopy can be used to image the blood vessels of living livers with noninvasive high resolution4. Not only can three-dimensional images of blood vessels be obtained, but the technique can also be used to help organize the tissue to observe biological effects therein; furthermore, the whole tissue can be imaged rather than only the microvessels as in computed tomography and magnetic resonance imaging5.
Multiphoton microscopy can be used to effectively detect scattered fluorescent signals in deep living tissue, with less phototoxicity6. Therefore, the activity of living tissue can be ensured, and the amount of damage can be reduced. Multiphoton microscopy has better penetrating power than confocal microscopy, allowing deeper layers to be observed7, providing unique 3D imaging. Multiphoton microscopy is now often used in imaging cranial nerves8 and has been extended to the study of neuronal dynamics in live mice9,10,11.
In this experiment, after fluorescent labeling of mouse blood vessels, the liver is fixed in a frame, and the dynamics of blood vessels in living liver tissue can be seen using multiphoton microscopy. This experiment demonstrates how to mark specific substances, use multiphoton microscopy to help observe a location within the tissue, observe cellular events in the intercellular tissue, make photochemical measurements12,13,14, and observe the material dynamics inside the living tissue15. For example, tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors, marking single-chain variable fragment (scFv) 78 against TEM1, and then multiphoton microscopy can be used for mouse hemangioma location and evaluation of tumors16.

<table>
	<tbody>
		<tr>
			<td>1 mL syringe x 2</td>
			<td>Hunan Pinan Medical Devices Technology</td>
			<td>YA0551</td>
			<td></td>
		</tr>
		<tr>
			<td>5 W heating pad</td>
			<td>BiolinkOptics Technology</td>
			<td>BL336</td>
			<td></td>
		</tr>
		<tr>
			<td>75% absolute ethanol</td>
			<td>Guangdong Guanghua Sci-Tech</td>
			<td>1.17113.023</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent cotton ball</td>
			<td>Healthy Sanitation Kingdom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse surgical instrument</td>
			<td>RWD Life Science</td>
			<td>SP0001-G</td>
			<td>Including scissors and tweezers</td>
		</tr>
		<tr>
			<td>Multiphoton microscopy</td>
			<td>Olympus</td>
			<td>FV1200MPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Organ imaging fixture</td>
			<td>BiolinkOptics Technology</td>
			<td>BL336</td>
			<td>Including suction cup, hose, negative pressure pump and bracket</td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate&#8211;Dextran</td>
			<td>Sigma</td>
			<td>R9379</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaving machine</td>
			<td>Lei Wa</td>
			<td>RE-3201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Sigma</td>
			<td>P3761-25G</td>
			<td></td>
		</tr>
	</tbody>
</table>

multiphoton microscopic observation of vessels in mouse liver tissue

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of the mouse liver. A mouse is injected with a dye that can stain blood vessels. To observe the mouse liver in vivo, it is exposed and fixed in a frame. Two and three-dimensional images of the blood vessels in the liver tissue are obtained using a multiphoton microscope. Images of the tissues at the selected sites are continuously acquired to observe long-term changes; the dynamic changes of blood vessels in the liver tissues are also observed. Multiphoton microscopy is a method for observing cell and cell function in deep tissue sections or organs. Multiphoton microscopy has sensitivity to tissue microstructure and enables imaging of biological tissues at high spatial resolution in vivo, providing the ability to capture the biochemical information of the organization. Multiphoton microscopy is used to observe part of the liver but fixing the liver to make the image more stable is problematic. In this experiment, a special vacuum suction cup is used to fix the liver and obtain a more stable image of the liver under the microscope. In addition, this method can be used to observe dynamic changes of specific substances in the liver by marking such substances with dyes.

The distribution of blood vessels in the liver can be seen in Figure 1, obtained using multiphoton microscopy. The blood vessel is divided into a plurality of branches emanating from a trunk and distributed to the surrounding space. The outer circumference of the blood vessel is red, the inner cavity is dark, and there are many things inside. The clearer the image, the closer to the plane of observation it is. There are also some red spots around, probably because the dye penetrates the surrounding tissue to stain other substances. In Video 1, there are non-red things that could be cells moving in the red blood vessels. At the end of the video, some of these things are darkened. This is probably because the fixed liver has not been fixed well over time. This method will allow imaging for 2 hours.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60932/60932fig01.jpg" /> 
Figure 1: The blood vessels in the liver tissue under multiphoton microscopy.&#160;The blood vessels are red. A two-dimensional image of the blood vessel with a resolution of 2048 and an HV of 512, can clearly image the red blood vessels after staining. There are also some red spots around, and the dye may also penetrate the surrounding tissue to dye other substances. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60932/60932fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60932/60932fig02.jpg" /> 
Figure 2: Three-dimensional image of living liver tissue in the same position.&#160;The thickness is 9 &#181;m, the morphology of the blood vessels is observed from different angles, and dynamic images are collected. After synthesis, the cells in the blood vessels (dark) are seen flowing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60932/60932fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Body organ imaging frame. (a) Heating pad: It maintains the mouse body temperature. (b) Bracket: It can provide a base to place the mouse and the adjustable suction cup position with knobs. (c) Suction cup: It is fixed by the bracket. The negative pressure provided by the negative pressure pump causes the liver to adsorb to the transparent lens in the center of the suction cup. (d) Flexible pipe: It connects the suction cup with the pressure pump. The pressure pump provides negative pressure to the suction cup. (e) Centrifuge tube: The centrifuge tube in the middle of the flexible pipe can hold the liquid, to prevent these liquids from being sucked into the vacuum pump. (f) Negative pressure pump: It is connected to the suction cup by the flexible pipe. The negative pressure pump can provide negative pressure to the suction cup to make the liver cling to the lens. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60932/60932supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: A close-up image of the liver attached to transparent lens. The transparent lens is in the center of the suction cup. The pump provides negative pressure to the suction cup to make it suck the liver and the liver is attached to the transparent lens. Because the suction-faced liver is a bit away from the mouse, the effect of breathing and the heartbeat on artifacts is reduced. The transparent lens can provide a stable view for the multiphoton microscope. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60932/60932supfig02.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue at JoVE.com

Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate&#8211;dextran that can stain blood vessels. After the liver is exposed and fixed, a specific part of the liver can be selected to observe the deep tissue in the living body using multiphoton microscopy.

Observing the intravascular dynamics of mouse liver tissue allows us to conduct further in-depth observations and studies on tissue-related diseases of ...

multiphoton-microscopic-observation-of-vessels-in-mouse-liver-tissue

Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.6K Views.  Southern Medical University. In this experiment, a mouse is injected in its tail vein with Rhodamine B isothiocyanate–dextran that can stain blood vessels. After the liver is exposed and fixed, a specific part of the liver can be selected to observe the deep tissue in the living body using multiphoton microscopy.

Video: Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...