Name: multiphoton microscopic observation of vessels in mouse liver tissue
Uploaded: 2021-05-17T12:30:00
Description: Watch this Scientific Journal Video about Multiphoton Microscopic Observation of Vessels in Mouse Liver Tissue at JoVE.com

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 ha...

<ol>
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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...

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 surr...

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 ...

This method can help us to visualize the movements of blood vessels within the mouse liver along evolution of the dynamics of the liver in multiple dimensions. Using this method, we can visualize the dynamics of blood vessels within the liver in three dimensions. We have demonstrated the use vascular dye, but you can perform the vessel staining with your substance of interest before microscopic visualization.After confirming a lack of righting reflex in an anesthetized eight week old male C 57 black six mouse. Wipe the mouse's tail with 75%alcohol and use a one microliter syringe equipped with a 30 gauge needle to inject 10 milligrams per milliliter of freshly prepared Rhodamine B Isothiocyanate-Dextran in 100 microliters of Rhodamine B Isothiocyanate-Dextran into the caudal tail vein. When all of the solution has been delivered use a cotton swab to apply pressure to the puncture site and use gauze wet with sterile water to soak the abdominal fur.Use a razor to shave the abdomen, making strokes in the direction of the fur, and place the mouse onto a 37 degrees Celsius, alcohol disinfected heating pad. To fix the mouse liver with a body organ imaging frame, First place a clean five millimeter diameter suction cup in a fixed position and wipe the heating pad and suction cup with 75%alcohol. Connect the suction cup to the vacuum pump hose and turn on the pump.On a 75%alcohol sterilized table use surgical scissors to cut two centimeters of skin from the lower sternal border of the mouse and expose the liver. Place the mouse and the heating pad onto the holder base of the body frame and adjust the organ imaging fixture so that the suction cup can hold the liver. Then use a negative pressure of 30 to 35 kilopascals for suction so that the liver attaches to the cup.To set up the Multiphoton Laser Scanning Microscope turn on the Microscope and select the 60 X objective. Fix the frame and the mouse under the objective and add a drop of normal saline to the cup large enough to cover the lens. Adjust the objective so the lens just touches the normal saline and turn on the laser software.Turn on the laser and press and hold the power button and shutter for three seconds. Then start the microscope operating software and set the laser to 800 nanometers. To set the image acquisition control click the fluorescent switch and turn off the room and equipment lights.Open the light path shutter on the microscope and rotate the fluorescence filter to the fourth gear. Pull the two levers of the optical switch and looking through the eye piece, use the course and fine focusing quasi spirals to adjust the focal length. Then use the X Y axes to adjust the field of view to locate the target area.For imaging, turn off the fluorescent switch in the software and switch the fluorescent filter to the second gear. Push the two levers of the optical switch and click focus times two to preview the target area adjust the acquisition settings and the image acquisition control parameters and press control plus C to adjust the high voltage gain and offset. Click stop to stop the preview, click the X Y button to scan in two dimensions and click save.Select a region and click depth and preview to select the end and start sets in the microscope settings. Scan 3D images and save. After scanning, select a region click time and adjust the other acquisition settings and image acquisition controlled parameters.Then scan through the different slices and save to obtain the resulting image movie. Here visualization of the distribution of blood vessels within the liver using multi photon microscopy as demonstrated, can be observed. This blood vessel is divided into a plurality of branches emanating from a trunk and distributed into the surrounding space.The outer circumference of the blood vessel can be observed in red while the inner cavity is dark, as observed in the video non red objects moving within the vessels could be cells. The darkness of the tissue observed in this video is likely due to the liver not having been fixed well over time. It is important to remember that how scar-less liver is fixed, affects the stability of the image.This methodology can also be applied to the position of other structures within the liver for the direct observation of the distribution or changes in specific labor structures of interest.

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

Research

JoVE Journal

Bioengineering

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 ...

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. ...

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 ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...