Name: isolation of cd146+ resident lung mesenchymal stromal cells from rat lungs
Uploaded: 2016-06-17T15:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs at JoVE.com

JJPC is supported by a Canadian Institutes of Health Research (CIHR) postdoctoral fellowship. MAM receives a merit scholarship from the German National Academic Foundation - Studienstiftung des Deutschen Volkes and is supported by a grant from the EFCNI (European Foundation for the Care of the Newborn Infant). BT is supported by CIHR, the Canadian Lung Association, the Stem Cell Network and the Children's Hospital of Eastern Ontario Research Institute.

All procedures were approved by the Animal Care Committee of the University of Ottawa (animal ethics protocol OHRI-1696). Animal care was performed in accordance with institutional guidelines.
1. Isolation of Lung Mesenchymal Stromal Cells
<ol>
	<li>Prepare the enzyme mix in a 50 ml tube: weigh in 30 U Neutral Protease, 2,500 U Collagenase I and 500 U DNAse I. These amounts suffice for the lungs of an adult mouse or rat pup. Prepare on day of isolation and store at 4 &#176;C until use.</li>
	<li>Sacrifice rat pups at day 13 by an intra-peritoneal injection of pentobarbital sodium (0.2 ml, 65 mg/ml). Use the toe pinch reflex to establish unconsciousness. Death of the animal is ensured by opening the chest, as outlined below.</li>
	<li>Sanitize the skin by spraying the animal with 70% ethanol and carefully open the rib cage using surgical scissors, starting at the diaphragm and cutting towards the rostral side, being very careful not to damage the lungs. Spread the ribcage open using hemostatic clamps, or alternatively cut away the ribcage to provide access to the thorax.</li>
	<li>Exsanguinate the animal by removing the heart. To remove the heart, grasp the thymus and heart with small forceps and cut these away with surgical scissors. Immediately afterwards, absorb the blood with a gauze until no more blood comes out of the severed aorta and pulmonary artery.</li>
	<li>Remove the lungs from the thorax as follows: hold the trachea with small forceps, then sever the trachea with surgical scissors on the rostral side. While gently pulling the lung package out of the thorax, cut away any connective tissue on the dorsal side along the ribcage to free the lungs.</li>
	<li>Sever the lungs from the aorta and esophagus by cutting along the diaphragm with surgical scissors. Now that the lungs are completely free from the thorax remove any remaining blood gently with a gauze.</li>
	<li>Remove the trachea and bronchi with surgical scissors and carefully transfer the lung lobes to a 50 ml tube containing cold 35 ml 30% Citrate-Phosphate-Dextrose Adenine (CPDA-1) anticoagulant (26.30 g trisodium citrate dihydrate, 3.27 g ascorbic acid monohydrate, 2.22 g monosodium dihydrogen phosphate, 31.80 g D-glucose, 0.275 g adenine in 1 L purified H2O; sterile filter the solution using a 0.22 &#181;m membrane filter before use) in phosphate buffered saline (PBS) to remove blood and debris.</li>
	<li>After a 5 min rinse in 30% CPDA-1/PBS, transfer the lung to a new 50 ml tube containing 35 ml sterile phosphate buffered saline (PBS) (RT), invert gently to remove citrate. Transfer to a tube containing 35 ml Dulbecco&#39;s PBS + Sodium-pyruvate + Glucose (DPBS++) (RT). Each rinsing step should take approximately 5 min. 
	Note: DPBS++ can be ordered commercially or made up as follows21: calcium chloride dihydrate 132.4 mg, magnesium chloride hexahydrate 100 mg, potassium chloride (anhydrous) 200 mg, potassium phosphate monobasic (anhydrous) 200 mg, sodium chloride (anhydrous) 8,000 mg, sodium phosphate dibasic (anhydrous) 1,144.5 mg, D-glucose 1000 mg, sodium pyruvate 36 mg in 1 L purified H2O, pH 7.3. Sterile filter the solution using a 0.22 &#181;m membrane filter before use.</li>
	<li>Dissolve the enzyme mix by adding 10 ml DPBS++ (pre-warmed to 37 &#176;C) and inverting gently until dissolved.</li>
	<li>Transfer the lungs to a new 50 ml tube and chop the lung on the wall of the tube using a scalpel until finely minced. Add the enzyme mix to the tissue (10 ml enzyme mix/ per adult mouse/rat pup lung), close the tube tightly and incubate at 38 &#176;C with gentle agitation (either in a thermal mixer, a shaking water bath, or a water bath with regular manual agitation). The duration depends on the type of tissue: fetal tissue 60 min, juvenile/ adult tissue 90-120 min, fibrotic adult tissue 120-150 min.</li>
	<li>Stop the digestion by chelating the bivalent cations with 200 &#181;l ethylenediaminetetraacetic acid (EDTA, 0.15 M, pH 7.4. Sterile filter the solution using a 0.22 &#181;m membrane filter before use).</li>
	<li>Add 20 ml 5% fetal bovine serum (FBS)/PBS and pass the entire suspension through 100 &#181;m cell strainer into a new 50 ml tube. Rinse the tube and cell strainer with 10 ml 5% FBS/PBS, bringing the total volume to 40 ml.</li>
	<li>Centrifuge for 5 min at 500 x g, RT.</li>
	<li>Remove the supernatant (pellet should be clearly visible), resuspend in 40 ml 5% FBS/PBS and repeat centrifugation step 1.13.</li>
	<li>Resuspend in 4 ml 5% FBS/PBS (RT) and layer carefully over 3 ml density gradient media (1.073 g/cm3) in a 15 ml tube14. To obtain a good interphase, it is crucial that the layering of the single cell suspension occurs at a &#62;45&#176; angle at very low speed.</li>
	<li>Centrifuge 20 min at 900 x g, medium (5/10) acceleration, no deceleration, 19 &#176;C.</li>
	<li>Collect the cells present in the interphase and resuspend them in 10 ml sterile PBS.</li>
	<li>Centrifuge for 5 min at 500 x g, 21 &#176;C.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 10 ml pre-warmed completed L-MSC culture medium (minimum essential media Eagle, alpha modification (&#945;MEM), supplemented with 2 mmol L-Glutamine per 500 ml &#945;MEM, 20% vol/vol FBS and 1% vol/vol Penicillin/ Streptomycin/Amphotericin B.</li>
	<li>Repeat centrifugation step 1.18. Subsequently, remove supernatant and resuspend in 10 ml pre-warmed fresh culture medium.</li>
	<li>Count cells using a hemocytometer or automated cell counter and plate at a high density, since the majority of cells at this stage are not plastic adherent. The exact plating density depends on the animal species and age, e.g., the cells of an adult mouse lung are seeded at 105 cells/ cm2, whereas cells from a rat pup lung are sufficiently dense at a seeding density of 1-2 x 104 cells/ cm2. Use 0.2 ml L-MSC culture medium/cm2.</li>
	<li>Culture L-MSCs in a humidified 5% O2, 5% CO2 atmosphere at 37 &#176;C.</li>
	<li>Change medium 24 hr after the initial seeding, and subsequently every 3 days after rinsing once with PBS. Cells should be cultured until 80-90% confluence, which takes 3-5 days on average depending on the animal of origin. Higher confluence will promote differentiation into fibroblasts.</li>
</ol>
2. Selection of CD146+ Lung Mesenchymal Stromal Cells
<ol>
	<li>Coating of Magnetic Beads with Secondary Antibody
	<ol>
		<li>Coat the magnetic beads with biotinylated secondary antibody, at a ratio of 10 &#181;g secondary antibody/ 100 &#181;l magnetic beads in a round bottom sterile 2 ml cryovial in sterile conditions. Use 10 &#181;l secondary antibody per expected 0.5 x 106 cells, and use a minimum of 100 &#181;l magnetic beads and 10 &#181;g antibody. 
		Note: The ratio of antibodies used per volume of beads may differ between manufacturers, please consult the manufacturer&#39;s instructions for the beads of choice. For this protocol, please refer to table 1 for the beads and antibodies that were used in the optimization of this protocol.</li>
		<li>Incubate in a rotating sample mixer for 30 min at 30 rotations/min (rpm), at RT.</li>
		<li>Resuspend the beads in 1.5 ml sterile 0.1 % bovine serum albumin (BSA)/PBS and transfer to a 3 ml round bottom tube (flow cytometry tube). Rinse the cryovial with an additional 1.5 ml 0.1 % BSA/PBS and transfer to the 3 ml tube.</li>
		<li>Place the tube on an appropriate magnet and wait 2 min until all beads are attached to the rear wall of the tube and the solution remains clear. Remove runoff and repeat wash with 3 ml 0.1 % BSA/PBS two times.</li>
		<li>Resuspend the beads in 3 ml 0.1 % BSA/PBS and store at 4 &#176;C, protected from light. Use within 5 days.</li>
	</ol>
	</li>
	<li>Labelling CD146+ L-MSCs
	<ol>
		<li>Harvest the mesenchymal cells after rinsing with PBS using a gentle non-trypsin solution. 
		Note: Volumes depend on the size of culture flask (0.2 ml/cm2 medium or PBS).
		<ol>
			<li>Remove culture medium and rinse cells three times with sterile PBS.</li>
			<li>Add the appropriate volume of gentle non-trypsin solution (see Table 1). Incubate at 37 &#176;C in the incubator until the cells have detached, approximately 10 min&#160;when using the solution in Table 1.</li>
			<li>Inactivate the enzyme by adding L-MSC culture medium, and centrifuge for 5 min at 500 x g.</li>
		</ol>
		</li>
		<li>Remove supernatant, resuspend cells in 0.1 % BSA/PBS and count the cells with a hemocytometer or automated cell counter. 
		Note: The volume of 0.1% BSA/PBS depends on the size of culture flask: aim for 5 or 10 ml 0.1% BSA/PBS.</li>
		<li>Repeat step 2.2.2 and resuspend the dedicated amount of cells in 3 ml 0.1 % BSA/PBS (blocks unspecific binding sites and keeps the cells alive). Keep cells on ice.</li>
		<li>Prepare a 3 ml round bottom tube with the primary anti-CD146 antibody, and keep on ice. 
		Note: The concentration of primary antibody that is added to the dedicated amount of cells depends on the antibody that is used. For a suggested anti-rat CD146 antibody see Table 1.</li>
		<li>Add the total volume of cells from step 2.2.3 to the tube with the primary antibody. Close tightly and incubate 30 min&#160;on a rotating sample mixer at 15 rpm, 4 &#176;C.</li>
		<li>Centrifuge cells for 5 min at 500 x g, 4 &#176;C, remove supernatant and resuspend cell pellet in 3 ml 0.1 % BSA/PBS. Repeat centrifuge step.</li>
		<li>Resuspend the cells in the 3 ml solution containing secondary antibody coated beads that was prepared according to the instructions in section 2.1 and incubate 30 min on a rotating sample mixer at 15 rpm, 4 &#176;C.</li>
	</ol>
	</li>
	<li>Selecting CD146+ L-MSCs
	<ol>
		<li>Place the tube containing the labelled CD146+ L-MSCs on the magnet. The positive cells will be drawn to the back of the tube. Without moving the tube or touching the beads, harvest the supernatant with the negative cells slowly with a sterile Pasteur pipette (these can be either collected or discarded based on the experimental design).</li>
		<li>Remove the tube from the magnet and resuspend the cells in 3 ml 10 % BSA/PBS. Repeat the selection procedure described in step 2.3.1. four more times (&#931; 5 selection steps).</li>
		<li>Resuspend the CD146+ L-MSCs in pre-warmed L-MSC culture medium and return to the magnet.</li>
		<li>Remove supernatant, resuspend in L-MSC culture medium and plate in an appropriately sized culture flask (0.2 ml/cm2). As a rule of thumb, plate cells in the same size culture flask from which the cells were lifted prior to bead-selection. The number of CD146+ L-MSCs, and therefore the plating density, will depend on the age, species and strain of the source animal. Always aim for a plating density of ~5,000 cells/cm2. 
		Note: The beads will not disturb the cell growth and will gradually disappear as the cells proliferate.</li>
	</ol>
	</li>
</ol>
3. Freezing Cells
<ol>
	<li>Use the following freezing media: 60% pentastarch solution (10% pentastarch in 0.9% NaCl), 20% FBS, 12.5% CPDA-1, 2.5% NaCl (0.9%), 5% dimethyl sulfoxide (DMSO)22. 
	Note: If not all ingredients are available, a solution containing 5% DMSO, 30% FBS and 65% &#945;MEM is a good alternative.</li>
	<li>Resuspend cells at 0.5 x 106 cells/ml, aliquot in cryovials of 1 ml each and freeze O/N at -80 &#176;C using a freezing container.</li>
	<li>Transfer to a liquid nitrogen storage tank the next day.</li>
</ol>
4. Thawing Cells
<ol>
	<li>Thaw a vial of cells in a 37 &#176;C water bath for 5 min.</li>
	<li>Resuspend cells in 10 ml pre-warmed culture medium and centrifuge for 5 min at 500 x g, 21 &#176;C.</li>
	<li>Remove supernatant and resuspend the cell pellet in 10 ml culture medium. Seed cells in a vented cap cell culture treated flasks at 5,000 cells/cm2 in 0.2 ml/cm2 media (e.g., 15 ml for a 75 cm2 flask).</li>
	<li>Culture cells in 5% O2, 5% CO2 until 80-90% confluent for harvesting and/or experimental use. Over-confluency will induce differentiation. When seeding at 5,000 cells/cm2, L-MSCs will reach 80-90% confluency within 3-4 days of culture.</li>
</ol>

The authors have nothing to disclose.

The isolation and culture of primary L-MSCs presents an opportunity to better understand their function and their interaction with other cell populations at a cellular level, and their role in lung development, health and disease. This is especially important as the lack of a specific single marker of these cells makes it nearly impossible to study these cells in situ. As with all primary cell populations, one should keep in mind that these cells are more likely to change their character the longer they are kept...

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. During development, including in the lung, the mesenchyme is an important source of developmental cues, and resident MSCs are a likely candidate to be at the center of this. Moreover, evidence is emerging that resident MSCs are perturbed in adult diseases, including cancer1,2 and fibrosis3. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells4. Stem cell antigen-1 (Sca-1) was identified in mice as a marker for a variety of tissue stem cells, and can be used for the isolation of L-MSCs5, but has unfortunately no known orthologs in other species6. Researchers have reported a variety of different isolation methods for the isolation of L-MSCs from either lung tissue or fluid. These vary from fluorescence activated cell sorting (FACS) based methods selecting for CD31-/CD45-/CD90+ cells7, CD31-/CD45-/epithelial cell adhesion molecule (EpCAM)-/Sca-1+ cells8, multidrug resistance transporter ATP binding cassette G (ABCG2) positive cells9 or Hoechst 33342 dye efflux10, to plastic adherence11,12 and migration out of minced tissue13.
The advantages of the herein presented method are several fold. By using a gentle enzymatic digestion and density gradient14, one obtains all the cells of the density range that include MSCs but exclude epithelial or endothelial cells. The subsequent plastic adherence step ensures that only the mesenchymal cells adhere and stay in culture, eliminating leukocytes. Most importantly however, the CD146+ selection step allows for the elimination of fibroblasts, as these cells do not express CD146. Expression of the cell adhesion molecule CD146 is positively correlated with multipotency, and is therefore a good marker to weed out fibroblasts from a mesenchymal cell population15-19. This is an advantage over using CD90 as a selection marker, as it is not only expressed in MSCs but also in lipofibroblasts5,20. In this protocol we have explicitly chosen a magnetic bead selection, as it is gentler on the cells, and the entire procedure can be done in sterile conditions. Another important advantage of this isolation method as opposed to the outgrowth method, is that it is relatively fast, 6-10 days as opposed to a month or more for the outgrowth method. Three to five days after the initial isolation the mesenchymal population is ready for CD146+ selection; after another three to five days the CD146+ cells are ready to be used for experiments or can be frozen for later use. The decreased time of culture improves the quality of the cells as MSCs transdifferentiate towards fibroblasts in prolonged ex vivo culture19. Lastly, because of the nature of the protocol, it is possible to apply this method to other species by simply choosing appropriate antibodies, or even to other organ systems by adjusting the choice of digestion enzymes and incubation time.
A detailed protocol of this isolation method is given below, and a schematic overview of the isolation and subsequent selection of the CD146+ subpopulation is provided in Figure 1A and 1B respectively. Additionally, details are included for passaging, freezing and thawing these cells.
<img alt="Figure 1" src="/files/ftp_upload/53782/53782fig1.jpg" /> 
Figure 1.&#160;Schematic overview of the isolation of pulmonary mesenchymal cells (A) and subsequent CD146+ cell selection (B). min = minutes; EDTA = Ethylenediaminetetraacetic acid; 2nd Ab = secondary antibody; &#945;-CD146 Ab = primary anti-CD146 antibody; -ve cells = CD146 negative cells; +ve cells = CD146 positive cells. <a href="https://www.jove.com/files/ftp_upload/53782/53782fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Neutral Protease</td><td>Worthington Biochemical Corporation</td><td>LS02104</td><td>Prepare on day of isolation and store at 4 &amp;deg;C until use</td></tr><tr><td>Collagenase I</td><td>Worthington Biochemical Corporation</td><td>LS004196</td><td>Prepare on day of isolation and store at 4 &amp;deg;C until use</td></tr><tr><td>DNAse I</td><td>Sigma-Aldrich</td><td>D5025</td><td>Prepare on day of isolation and store at 4 &amp;deg;C until use</td></tr><tr><td>Pentobarbital sodium (Euthanyl)</td><td>Bimeda-MTC Animal Health Inc, Dublin, Ireland</td><td>-</td><td>Use 0.2 ml for rat pups between 20-30 g; larger animals may need more.</td></tr><tr><td>Dulbecco&amp;rsquo;s PBS + Sodium-pyruvate + Glucose</td><td>Life Technologies</td><td>14287-072</td><td>Very crucial to use this type of D-PBS, as the calcium and magnesium that are present in this D-PBS facilitate facilitate the enzymatic digestion</td></tr><tr><td>Ficoll-Paque PREMIUM (1.073 g/cm&lt;sup&gt;3&lt;/sup&gt;)</td><td>GE Healthcare</td><td>17-5446-52</td><td>Density gradient media. To obtain a good interphase, it is crucial that the layering of the single cell suspension occurs at a &gt;45&amp;deg; angle at very low speed, and that the subsequent centrifugation is done at 19 &amp;deg;C.</td></tr><tr><td>&amp;alpha;MEM</td><td>Sigma-Aldrich</td><td>M8042</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>200 mM L-Glutamine</td><td>Life Technologies</td><td>25030-164</td><td></td></tr><tr><td>100x Antibiotic-Antimycotic</td><td>Life Technologies</td><td>15240-062</td><td>Penicillin/Streptomycin/Fungizone</td></tr><tr><td>M-280 Dynabeads</td><td>Life Technologies</td><td>11205D</td><td>Magnetic beads</td></tr><tr><td>biotinylated polyclonal rabbit-anti-mouse IgG</td><td>Dako, Agilent Technologies</td><td>E0464</td><td>Biotinylated secondary antibody that matches with the anti-rat CD146 antibody described below</td></tr><tr><td>DynaMag5-magnet</td><td>Life Technologies</td><td>12303D</td><td>Magnet that is recommended for use with Dynabeads. NOTE: magnet should be chosen based on the manufacturer&amp;rsquo;s instructions of the magnet beads of choice.</td></tr><tr><td>TrypLE express&amp;nbsp;</td><td>Life Technologies</td><td>12605-028</td><td>Gentle non-trypsin alternative, use 1 ml of TrypLE express/25 cm&lt;sup&gt;2&lt;/sup&gt; surface area. After detachment, 10 ml L-MSC culture medium is sufficient to inactivate 3 ml TrypLE</td></tr><tr><td>anti-rat CD146 antibody</td><td>Lifespan Biosciences Inc.</td><td>C35841</td><td>12.5 &amp;mu;l per 0.5 x 10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Pentaspan (pentastarch solution)</td><td>Bristol-Myers Squibb Canada</td><td>-</td><td>Can be obtained through local blood donation services or hematology departments. Alternatively, one could use the PSI 20% Pentastarch solution (Preservation Solutions, PST001) diluted 1:1 with 0.9% NaCl.</td></tr><tr><td>Mr. Frosty freezing container</td><td>ThermoFisher Scientific</td><td>5100-0001</td><td>Improves viability when freezing L-MSCs overnight at -80 &amp;deg;C</td></tr></tbody></table>

isolation of cd146+ resident lung mesenchymal stromal cells from rat lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Two of the most reliable physical characteristics of MSCs, density and plastic adherence, are used in the first part of this protocol to obtain the mesenchymal cell fraction of the lung that contains L-MSCs. Although the density gradient interphase will include monocytes and macrophages in addition to lung mesenchymal cells, the plastic adherence followed by 3-5 days of culture ensures that only the lung mesenchymal cells remain. Indeed this cell population expresses the classic MSC surfa...

Watch this Scientific Journal Video about Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs at JoVE.com

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

isolation-cd146-resident-lung-mesenchymal-stromal-cells-from-rat

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8.3K Views.  Ottawa Hospital Research Institute. This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Video: Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Immunology and Infection

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To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

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MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

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Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Isolation of Immune and Non&mdash;Immune Cells from Mouse Lungs for Flow Cytometry

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

Niche-Based Isolation of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

Medicine

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Author Spotlight: Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Isolation of Lung Retinoid-Containing Cells by Cell Sorting

Retinoids (vitamin A and its metabolites) are an essential lipid component of the alveolar microenvironment, and cell-type specific retinoid metabolism is required to maintain the functional health of the developing and adult lungs. Lung cells utilize specific pathways, allowing for the efficient uptake of circulating retinoids from the blood as retinol (ROH), followed by intracellular stepwise conversion of ROH into the transcriptionally active retinoid species, all-trans-retinoic acid (ATRA). ATRA-mediated (or retinoid-mediated) signaling is crucial for regulating lung alveolarization, surfactant production, angiogenesis, permeability, and immunity. Importantly, specific lung cells, including fibroblasts, can accumulate retinoids in the form of retinyl esters (RE), which can be stored or further mobilized as ROH for transfer to the neighboring cells when needed. Lung retinoid-containing cells can be isolated and collected from the single-cell suspension of digested lungs by making use of retinoid autofluorescence (the emission at 455 nm upon excitation at 350 nm) and by employing fluorescence-activated cell sorting (FACS). Additional cell-specific in vivo labeling of lung cells with red fluorescent protein allows isolating and collecting specific retinoid-containing lung cell populations. The collected cells can be directly analyzed or cultured for further analyses of cell morphology, gene expression, and responsiveness to pharmacological manipulations. This technique of isolation and application is important for animal model studies of lung health and lung injury to gain deeper insight into cellular aspects of retinoid metabolism in the lungs and lipid-mediated cellular communications.

The protocol described below is a simple and effective way to isolate retinoid-containing cells from highly heterogeneous lung cell populations by making use of specific retinoid autofluorescence and by employing fluorescent-activated cell sorting.

Retinoids (vitamin A and its metabolites) are an essential lipid component of the alveolar microenvironment, and cell-type specific retinoid metabolism is ...

Isolation of CD146⁺ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

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