The authors would like to acknowledge the National Institute of Biological Sciences, Beijing for the assistance. This work was supported by Beijing Municipal Natural Science Foundation (No. Z17110200040000).

All procedures used in this protocol were carried out in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Biological Sciences, Beijing. 8 week-old CD-1 male mice were housed in a specific pathogen free (SPF) facility until the experiments were conducted. Surgeries were performed using completely anesthetized mice (i.e., without any toe pinch responses). After surgery, mice were kept in a warm, humid room with sufficient food and fresh water. Mice were sacrificed using an overdose of anesthetic delivered by intraperitoneal injection.
1. Mouse PNX Surgery
<ol>
	<li>Fully anesthetize the mice with sodium phenobarbital (120 mg / kg body weight) and buprenorphine (0.1 mg / kg body weight) via intraperitoneal (I.P.) injection. Perform surgery when mice no longer react to toe pinching.</li>
	<li>Remove hair on the left thorax of the mice with chemical depilatory treatment (~3 x 3 cm2 area).</li>
	<li>Secure each mouse on an intubation platform with its ventral side facing the operator (Figure 1A).</li>
	<li>Pull out the mouse tongue and illuminate the vocal cords with a small animal laryngoscope containing a notch for guiding catheters18 (Figure 1A).</li>
	<li>Distinguish the vocal cords by observing the movements of the vocal cords during breathing. Gently insert a 20 G intravenous intubation cannula into the trachea at an anterior angle19.</li>
	<li>Place mice in a right lateral recumbent position and connect the cannula to a mechanical ventilator (e.g., pressure controlled; see the Table of Materials). Check the insertion of the cannula into the trachea by observing the breathing movements of the mouse chest (Figure 1B).</li>
	<li>Set the inspiratory pressure of the ventilator to 12 cm H2O and set the respiratory rate to 120 breaths per min (Figure 1B).</li>
	<li>Decontaminate the skin in the surgical area with betadine and 70% ethanol.</li>
	<li>Make a 2 - 3 cm posterolateral thoracotomy incision in the space at the 5th intercostal space, cut through skin and muscles with Noyes Spring Scissors (cutting edge: 14 mm; tip diameter: 0.275 mm) (Figure 2B, C). Surgical instruments used for thoracotomy procedure are sterilized prior to use.</li>
	<li>Make a 1.5 cm incision at the 5th intercostal space to expose the left lung (Figure 2D, E). During the operation, use a high temperature cauterizer to stop bleeding.</li>
	<li>Lift one-third of the left lung lobe from the chest with blunt tip forceps (Figure 2F), and then use a cotton swab to pull out the entire left lung (Figure 2G).</li>
	<li>Identify the pulmonary artery and bronchi of the left lung lobe (Figure 2G).</li>
	<li>Tightly ligate the bronchi and vessels at the hilum with a silk surgical suture and cut out the left lung lobe at 3 - 4 mm from the ligation (Figure 2H, I). 
	NOTE: Be careful not to cut off the suture knots on the left hilum, which can cause pneumothorax (i.e., air or gas in the cavity of the thorax).</li>
	<li>Close the chest wall with 1 suture, and then stitch the muscle layer and the skin layer sequentially, using 5 - 6 interrupted sutures. Leave a 3 - 4 mm gap between each suture (Figure 2M, 2N). 
	NOTE: Keep the surgical suture needle away from the heart; inadvertent cardiac puncture will result in immediate death.</li>
	<li>Disinfect the surgical area with povidone-iodine.</li>
	<li>After the surgical operation, place the mouse on a 38 &#176;C thermal pad and connect the mouse to the ventilator until spontaneous breathing movements commence (Figure 2O).</li>
</ol>
2. Prosthesis Implantation
<ol>
	<li>Perform steps 1.1 - 1.13 of the PNX procedure (that is, up to the point when the left lung lobe of the mouse is removed).</li>
	<li>Clamp the center of the silicone prosthesis (customer made, 12 mm in length, 3 mm in thickness, 7 mm in width, 0.2 g, ellipsoid-shape) using blunt forceps (Figure 2J). Sterilize silicone prosthesis prior to insertion.</li>
	<li>Hold the rib with forceps with one hand to expose the thoracic cavity, and then insert the prosthesis into the left empty thoracic cavity with another hand. 
	NOTE: The insertion angle is approximately 45 degrees between the frontal plane of the prosthesis and the thoracic surface (Figure 2K, L). Be very gentle when inserting the prosthesis. Excessive force will result in pleural rupture.</li>
	<li>Adjust the orientation of the prosthesis with blunt forceps to ensure that the prosthesis occupies the left empty thoracic cavity.</li>
	<li>Perform steps 1.14 - 1.16 of the mouse PNX procedure.</li>
</ol>
3. Measurement of ILV
<ol>
	<li>Prepare a custom device (&#34;inflation tube&#34;) that consists of a plunger removed from a disposable serological pipette (10 mL), a 40 cm long flexible tube with a needle adapter, a flow rate control valve, and an 18 G needle. After assembly, secure the pipette on a board with tape (Figure 3A). The distance between the top of the pipette and the experimental bench must be at least 30 cm.</li>
	<li>Prepare fresh 4% paraformaldehyde (PFA) fixation solution by dissolving 20 g PFA in 500 mL pre-heated 1x phosphate buffered saline (PBS) in a 55 &#176;C water bath, shaking manually once every 10 min until the solution is clear. After cooling to room temperature, filter the solution with a 0.45 &#181;m filter. 
	CAUTION: Wear appropriate personal protective equipment (PPE) when handling PFA.</li>
	<li>Sacrifice mice with an overdose injection of anesthetic (0.8% phenobarbital sodium, 1,000 U/mL heparin).</li>
	<li>Secure each mouse on a polystyrene dissection plate and spray it with 70% alcohol.</li>
	<li>Carefully open the mouse chest and cut out the sternum using scissors to thoroughly expose the lung lobes.</li>
	<li>Remove excessive tissue using scissors to expose the trachea. Make sure to separate the trachea from the esophagus.</li>
	<li>Cut the abdominal aorta and insert a 25-gauge needle into the right ventricle of heart; connect the needle to a 20 mL syringe prior to this insertion. Slowly push 1x PBS into the heart to remove blood cells until lungs turn white. Typically, 5 - 10 mL PBS is required to clear the pulmonary blood vessels.</li>
	<li>Fill the custom-constructed inflation tube with 4% fresh PFA and remove all the bubbles from the inflation tube.</li>
	<li>Insert the 18-gauge needle of the inflation tube into the trachea and clip the trachea with vessel clips to avoid fluid leakage.</li>
	<li>Inflate lungs with 4% PFA at a constant transpulmonary pressure of 25 cm/H2O2,20. Incubate the lungs at room temperature for 2 h to achieve fully expanded lungs. This &#34;pre-fix&#34; step is critical for preserving lung morphology.
	<ol>
		<li>By monitoring the inflation tube, record the value of the initial 4% PFA volume and record the final volume. The internal lung volume equals the initial 4% PFA volume minus the final 4% PFA volume.</li>
	</ol>
	</li>
	<li>Ligate the trachea and using scissors, gently dissect out the lungs (keeping the lungs intact) from surrounding connective tissues. Be very gentle to avoid damaging the lungs.</li>
	<li>Incubate the lungs in a 50-mL conical tube filled with 4% PFA for 12 h at 4 &#176;C with gentle shaking on an shaker (50 rpm). Proceed to tissue processing and staining (see section 4).</li>
</ol>
4. Tissue Embedding, Sectioning, and Hematoxylin &#38; Eosin (H&#38;E) Staining
<ol>
	<li>After fixation, use Noyes Spring Scissors to trim the heart and excessive connective tissues off the lungs. Gently separate the individual lung lobes by cutting off the bronchus that connects the lung lobes to the trachea.</li>
	<li>Extensively wash the lung lobes 3 - 4 times in 50 mL 1x PBS (30 min/wash) on an orbital shaker (50 rpm).</li>
	<li>Following the final wash, cryoprotect the lung lobes by immersing them in a 30% sucrose solution (in 1x PBS) at 4 &#176;C until the tissue sinks to the bottom of the 50-mL conical tubes (approximately 12 h).</li>
	<li>Prior to embedding and cryosectioning the tissues, remove the lung lobe samples from the tubes with forceps, retain the accessory lobes for the histological analysis, dab the remaining sucrose solution from the surface of the accessory lobe samples, and then thoroughly immerse the sample into a Petri dish containing optimal cutting temperature (O.C.T) compound for approximately 30 min.</li>
	<li>Freeze the O.C.T-embedded accessory lobe samples in liquid nitrogen using cryomolds. Position the largest surface area of the lobe parallel to the bottom of the mold.</li>
	<li>Prepare a total of three 10-&#181;m-thick sections for each sample during cryosectioning for histological analysis. Discard the first 1 mm of tissue, collect one 10-&#181;m-thick section, discard 0.5 mm of tissue, collect another section, discard 0.5 mm of tissue, and collect the third (final) section.</li>
	<li>Air dry the sections for 1 h before performing H&#38;E staining.</li>
	<li>Perform H&#38;E staining
	<ol>
		<li>Wash the sections in 3 - 4 changes of tap water and then stain the sections in fresh hematoxylin for 2 min; rinse the section under running tap water; immerse the section two times in a 1% HCl-70% ethanol solution to remove excess hematoxylin.</li>
		<li>Stain the section in fresh eosin for 3 min; dehydrate the sections with two successive 30 s washes in 95% ethanol and two 30 s washes with 100% ethanol; clear the sections in xylene for 30 s, repeat the clearing step once in fresh xylene; mount the slides with mounting medium using glass coverslips.</li>
	</ol>
	</li>
</ol>
5. Quantification of MLI
<ol>
	<li>Acquire digital images of the H&#38;E stained accessory lobe sections (20X magnification) using a bright-field microscope.</li>
	<li>To quantify the MLI, select a total of 15 non-overlapping views (1,000 &#181;m x 1,000 &#181;m) randomly from the suitable areas (without arteries and veins, major airways, and alveolar ducts) of 3 sections.</li>
	<li>Place a grid with 10 evenly-distributed vertical lines and 10 equally-distributed horizontal lines of defined length (1,000 &#181;m) on the chosen areas of view using a ruler tool; each line is thus spaced 100 &#181;m apart (Figure 4B).</li>
	<li>Define the value of one intercept as the linear length between two adjacent alveolar epithelia. Measure the values of all intercepts along each 1,000 &#181;m length line.</li>
	<li>For each grid, quantify the values of all intercepts among the 10 horizontal 1,000 &#181;m length lines and the 10 vertical 1,000 &#181;m length lines. 
	NOTE: MLI is the average value of the intercept lengths from a total of 15 grids analyzed from among the 3 sections prepared for each of the accessory lobes.</li>
</ol>
6. Calculation of ISA
<ol>
	<li>Calculate the ISA using Equation 1 (see the Introduction). Refer to section 3 for the measurement of ILV and refer to section 5 for the quantification of MLI.</li>
</ol>

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The authors have nothing to disclose.

In this protocol, we provide detailed descriptions about the measurement of pulmonary parameters after mouse left lung PNX and prosthesis implantation. ISA is now considered to be a key metric for the assessment of respiratory function in many pulmonary diseases and in injury-induced alveolar regeneration. However, although the pulmonary research community is in agreement about the utility of ISA as a useful metric, to date, there has been little consideration of the standardization of the measurement of ILV and MLI, the...

The fundamental function of the lung is the exchange of oxygen and carbon dioxide between blood vessels and the atmosphere. Lung diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and acute respiratory infections, result in decreased ISA2. Researchers studying lung disease have developed several quantitative methods to evaluate morphological changes in lungs, including MLI, ILV, number of gas exchange units, ISA, and lung tissue compliance2,3. Pioneering studies by Weibel et al.4 and Duguid et al.5 together established that ISA can be used as a direct measure of lung gas-exchange capacity in human lungs and can be used as a criterion to determine emphysema severity. A number of studies published in the last five years have used lung morphological parameters (e.g., ISA and MLI) to assess morphological and functional changes in the lungs of mice during development6 and during recovery from injury PNX1,7. ISA is calculated using Equation 18,9:
<img alt="Equation" src="/files/ftp_upload/56114/56114eq1.jpg" />
, where ILV is the internal lung volume and MLI is an intermediary parameter that represents the pulmonary peripheral airspace size10.
PNX, the surgical removal of one or more lung lobes, has been widely reported to induce alveolar regeneration in many species, including humans11, mice1, dogs12, rats13, and rabbits14,15. A study of mice lungs at fourteen days post-PNX showed that both the expansion of pre-existing alveoli and the de novo formation of alveoli contribute to the restoration of ISA, ILV, and the number of alveoli in the remaining lung tissues1. We and others have shown that the insertion of materials such as sponge, wax, or a custom-shaped prosthesis into the empty thoracic cavity following PNX (i.e., prosthesis implantation) impairs alveolar regeneration. It is now firmly established that mechanical force functions as one of the most important factors for initiating alveolar regeneration1,16,17. Such studies have highlighted the effectiveness of using ISA values from PNX-treated and Prosthesis-implanted lungs as a criterion to quantitatively evaluate alveolar regeneration.
Observer bias is known to significantly influence measured values for lung morphological parameters (e.g., MIL and ILV). Standardized protocols can be used to obviate this bias in determining both ILV and MLI, which are the two parameters used in the calculation of ISA. Here, we provide highly-detailed, standardized protocols for measuring these lung parameters. Importantly, the ability to accurately quantify ISA promises to improve the reliability and reproducibility of studies of lung function in injury-induced alveolar regeneration models and should facilitate mechanistic discoveries in multiple pulmonary diseases.

<table width="881">
	<tbody>
		<tr height="21">
			<td height="21" style="width:411px;height:21px;">Low cost cautery kit</td>
			<td style="width:167px;">Fine Science Tools</td>
			<td style="width:136px;">18010-00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Noyes scissors</td>
			<td>Fine Science Tools</td>
			<td>15012-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard pattern forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel clips</td>
			<td>Fine Science Tools</td>
			<td>18374-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">I. V. Cannula-20 gauge</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>29P0601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical suture</td>
			<td>Jinhuan Medical Product Co., LTD.</td>
			<td>F602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse intubation platform</td>
			<td>Penn-Century, Inc</td>
			<td>Model MIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Laryngoscope</td>
			<td>Penn-Century, Inc</td>
			<td>Model LS-2-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO Small Animal Ventilator</td>
			<td>Kent Scientific</td>
			<td>RSP1006-05L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal pad</td>
			<td>Stuart equipment</td>
			<td>SBH130D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital sodium salt</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin Solution</td>
			<td>Sigma</td>
			<td>GHS132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin Y solution, alcoholic</td>
			<td>Sigma</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 ml Pipette</td>
			<td>Thermo Scientific</td>
			<td>170356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryosection machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Base Molds</td>
			<td>Fisher HealthCare</td>
			<td>22-363-553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 gauge needle</td>
			<td>Becton Dickinson</td>
			<td>305199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone iodine</td>
			<td>Fisher Scientific</td>
			<td>19-027132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP82011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Infusion sets for single use</td>
			<td>Weigao</td>
			<td>SFDA 2012 3661704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Gibco</td>
			<td align="right">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tapes</td>
			<td>3M Scotch</td>
			<td style="width:136px;">8915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton pad</td>
			<td>Vinda</td>
			<td>Dr.P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone prosthesis</td>
			<td>Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brightfield microscope</td>
			<td>Olympus</td>
			<td>VS120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ruler tool</td>
			<td>Adobe Photoshop</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a standardized method for measuring internal lung surface area via mouse pneumonectomy and prosthesis implantation

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), representing the gas-exchange capacity of the lung, is a critical criterion to assess respiratory function. However, observer bias can significantly influence measured values for lung morphological parameters. The protocol that we describe here minimizes variations during measurements of two morphological parameters used for ISA calculation: internal lung volume (ILV) and mean linear intercept (MLI). Using ISA as a morphometric and functional parameter to determine the outcome of alveolar regeneration in both pneumonectomy (PNX) and prosthesis implantation mouse models, we found that the increased ISA following PNX treatment was significantly blocked by implantation of a prosthesis into the thoracic cavity1. The ability to accurately quantify ISA is not only expected to improve the reliability and reproducibility of lung function studies in injured-induced alveolar regeneration models, but also to promote mechanistic discoveries of multiple pulmonary diseases.

We performed here an experiment with a PNX-treated group and a prosthesis implantation (Prosthesis-implanted) group. These groupings are the same as the groupings used in a previously-published study from our research group14.
The mouse PNX and prosthesis implantation procedures are shown in Figure 2. 8 week-old CD-1 male mice are used for the surgeries and for the quantifica...

Watch this Scientific Journal Video about A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation at JoVE.com

A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models.

Pulmonary morphology, physiology, and respiratory functions change in both physiological and pathological conditions. Internal lung surface area (ISA), ...

a-standardized-method-for-measuring-internal-lung-surface-area-via

Research

JoVE Journal

Developmental Biology

13.1K Views.  Peking University. Internal lung surface area (ISA) is a critical criterion for assessing lung morphology and physiology in lung diseases and injury-induced alveolar regeneration. We describe here a standardized method that can minimize the measurement bias for ISA in both lung pneumonectomy and prosthesis implantation mouse models. 

Video: A Standardized Method for Measuring Internal Lung Surface Area via Mouse Pneumonectomy and Prosthesis Implantation

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...