This work was supported by the Natural Science Foundation of Zhejiang Province (grant no. LY19H060001) and the Zhejiang Traditional Chinese Medicine Science and Technology Plan Project (no. 2022ZB093).

All experiments in this study were conducted in accordance with the animal experiment guidelines of the Zhejiang Chinese Medical University Laboratory Animal Research Center (Approval No: IACUC-20181029-11).
1. Cell extraction
<ol>
	<li>Put the Sprague-Dawley rats (SD rats, 1-10 days old, male or female) in the euthanasia cages filled with CO2 at a balanced rate of 30%-70% container volume/minute. After the rats lose consciousness (20-60 min), euthanize the rat by cervical dislocation to ensure a painless death.</li>
	<li>Immerse the rats in 75% alcohol for 10 min for disinfection.</li>
	<li>Carefully remove all the limbs of the rat with scissors and forceps, aspirate with PBS using a pipette and flush the blood adhering to the limbs.</li>
	<li>Add 5 mL of penicillin/streptomycin solution into 500 mL of DMEM and mix well. Take a 50 mL sterile centrifuge tube, add 5 mL of FBS and 45 mL of DMEM medium, and mix thoroughly to obtain a 10% FBS DMEM containing 1% penicillin/streptomycin solution. Add 2 mL of the above culture medium into a 5 mL tube.</li>
	<li>Transfer the limb bones into the 5 mL tube. Use scissors to cut the limb bones in the tube into small pieces (1-3 mm) and mix the homogenate to re-suspend the bone marrow cells into the culture medium. Stand for 5 min until the tissue fragments settled to the bottom of the tube.</li>
	<li>Add 10&#160;mL of 10% FBS DMEM into a 100 mm culture dish, and then transfer the supernatant into the culture dish. When aspirating the supernatant, avoid transferring the tissue debris into the culture dish.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for approximately 24 h. After this incubation time, the majority of mesenchymal stem cells will adhere to the culture dish wall and grow slowly, while most BMMs will still be suspended in the culture medium.</li>
	<li>Transfer the cell suspension in the 100 mm culture dish to a new 25 cm2 flask and continue to cultivate the cells at 37 &#176;C and 5% CO2 for an additional 24 h. BMMs will adhere to the flask wall at 24-48 h of culture. Remove the old medium carefully and replace with fresh medium after BMMs have adhered to the flask wall.</li>
	<li>Sub-culture when the cells in the flask reached 80%-90% confluency. 
	&#8203;NOTE: All the cells were cultured at 37 &#176;C and 5% CO2. During culture, the cell morphology was gradually unified. Cells were large, irregularly shaped, radially growing and attached to the flask in a form of disc, where multiple nuclei could be observed.</li>
</ol>
2. FACS staining of the cell
<ol>
	<li>Trypsin digest using 1-2 mL of commercial trypsin at 37 &#176;C and 5% CO2 for 5 min and count secondary adherent cells to ensure a final cell count of 100,000/sample (count cells with Neubauer hemocytometer).</li>
	<li>Divide the cells into three groups (500 &#181;L of PBS contains 100,000 cells): (1) the blank control group containing unstained cells; (2) the isotype control group; and (3) the experimental group (CD11b/c staining).</li>
	<li>Incubate primary antibodies (no antibody for the blank control group; anti-CD11 isotype control for the isotype control group, 0.6 &#181;L of antibody/sample, 1 &#181;g/sample; anti-CD11b/c for the experimental group, 1 &#181;L of antibody/sample, 1 &#181;g/sample) on ice for 30 min.</li>
	<li>Centrifuge at 300-350 x g for 5 min, discard the supernatant, and resuspend the cells with 500 &#181;L of PBS. Repeat the above procedure once to ensure that the unbound primary antibodies are washed away.</li>
	<li>Incubate the corresponding secondary antibody (no antibody for the blank control group; goat anti-rabbit IgG for the isotype control and the experimental groups, 0.25 &#181;L antibody/sample, 1:2,000) on ice in the dark for 30 min.</li>
	<li>Centrifuge at 300-350 x g for 5 min, discard the supernatant, and resuspend the cells with 500 &#181;L of PBS. Repeat the above procedure once to ensure that the unbound second antibodies are washed away.</li>
	<li>Detect the CD11b/c positive cells by flow cytometry (10,000 cells/sample) and analyze the data by software (e.g., FlowJo 7.5). 
	&#8203;NOTE: To obtain the final percentage of CD11b/c+ cells, the following formula was used: Percentage of CD11b / c + cells in the experimental group - the percentage of CD11 + cells in the isotype control group.</li>
</ol>
3. Wright-Giemsa staining
<ol>
	<li>Seed the adherent secondary cells that have been passaged three times onto 35 mm2 cell climbing sheets (1 &#215; 106 cells/well) and culture at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Discard the culture medium and wash thrice with PBS.</li>
	<li>Add Wright-Giemsa dye solution (0.5 mL-0.8 mL) to the cell climbing sheet for 1 min.</li>
	<li>Mix the dye with distilled water (0.5 mL-0.8 mL) with a cotton swab, and stand for 10 min.</li>
	<li>Wash the dye solution with distilled water, and then dry for 1-3 min. Observe under a microscope.</li>
</ol>
4. TRAP staining
<ol>
	<li>Seed the adherent secondary cells that have been passaged three times onto 35 mm2 cell climbing sheets (1 &#215; 106 cells/well) and culture at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Replace the old medium with the 10% FBS DMEM or osteoclast induction medium supplemented with 50 ng/mL receptor activator of nuclear factor-&#954;B ligand (RANKL) and 30 ng/mL of macrophage colony-stimulating factor (M-CSF), and culture at 37 &#176;C and 5% CO2 for an additional 7 days.</li>
	<li>Stain the cells with the TRAP staining kit according to manufacturer&#39;s protocol and observe under a microscope. 
	&#8203;NOTE: TRAP-positive cells were defined as osteoclasts, which were purple under a light microscope. The number of TRAP-positive cells was measured using ImageJ software.</li>
</ol>
5. Western blot
<ol>
	<li>Seed the adherent secondary cells that have been passaged three times on 35 mm2 cell climbing sheets (1 &#215; 106 cells/well) and culture for 24 h.</li>
	<li>Replace the old medium with fresh 10% FBS DMEM or osteoclast induction medium (50 ng/mL of RANKL and 30 ng/mL of M-CSF) and culture for an additional 7 days at 37 &#176;C and 5% CO2.</li>
	<li>Extract total cellular proteins with RIPA buffer, separate by 10% SDS-PAGE, and transfer to polyvinylidene fluoride membranes18,19.</li>
	<li>Block with 5% skimmilk powder (25 mL) for 2 h and wash three times, for 10 min each time, with TBS-Tween 20 (TBST).</li>
	<li>Incubate primary antibodies at 4 &#176;C overnight (anti-&#946;-actin, anti-TRAP, and anti-cathepsin K; all primary antibodies were diluted 1:1,000, 10 mL diluted antibody/band), and wash thrice with TBST.</li>
	<li>Incubate the secondary antibody (goat anti-rabbit IgG, 1:2,000 dilution, 10 mL diluted antibody/band) at room temperature for 2 h, and wash thrice with TBST. Visualize the protein bands using a developing solution. 
	NOTE: The expression levels of the above proteins were normalized to &#946;-actin.</li>
</ol>

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The authors declare that they have no competing interests.

Osteoclasts are one of the most significant cell types involved in the occurrence and development of bone diseases, as well as one of the primary objects of bone disease research20. Monocyte/macrophages can differentiate into osteoclasts. Since mononuclear macrophages (RAW264.7 cells) are too expensive to buy and are easily activated during culture, it is difficult to perform in vitro differentiation experiments using this cell line. Although several methods have been developed for extrac...

It has been reported that monocyte-macrophage lineage cells existing in the bone marrow can differentiate into blood monocytes and tissue macrophages1,2. The above cells, which can differentiate into osteoclasts to balance bone growth and development, are commonly used as a cell model to induce osteoclasts in vivo3,4. Bone marrow is a special tissue containing several different types of cells, which include but are not only limited to bone marrow mesenchymal stem cells, bone marrow monocyte-macrophage cells (BMMs), hematopoietic stem cells, endothelial cells, and immune cells. In fact, several previous studies suggested that adherent cells rushed out of the bone marrow cavity of the long bone could differentiate into osteoblasts, osteoclasts, chondrocytes, or adipocytes5,6,7,8. Although, different isolation and culture methods have been used to produce different homogeneous cell populations, there are still great challenges in isolating and culturing BMMs from a variety of different cell types.
Several methods have been developed to extract bone marrow mononuclear macrophages (BMSCs). However, the majority of these methods are complex9,10,11. For example, density gradient centrifugation requires a specialized kit and the operation is time-consuming and cumbersome. This method is suitable for the isolation of BMMs from high-volume blood samples, but not from bone marrow samples9,12,13. In addition, extracting tissue samples using collagenase digestion is a complex and time-consuming procedure; this method is not recommended for the isolation of BMMs from bone marrow samples14,15. In addition, although flow separation can result in highly purified monocyte/macrophage populations, it requires very large sample sizes and high instrument and equipment requirements10,16. Additionally, the microbead enrichment method is extremely expensive and is not feasible in a general laboratory17.
Therefore, in the current study a convenient, fast, and cheap method was proposed for the isolation of mononuclear macrophages from the bone marrow. Bone marrow cells adhered for different time points were used to isolate BMMs using a secondary adherence method. BMMs extracted with the above method could induce the formation of osteoclasts in vitro, thus providing a simple and convenient cell model for the future study of osteoporosis in vitro.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm2 cell climbing slices</td>
			<td>NEST Biotechnology</td>
			<td>80102</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-cathepsin K</td>
			<td>Abcam</td>
			<td>ab19027</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-CD11 isotype control</td>
			<td>Abcam</td>
			<td>ab172730</td>
			<td>1 &#956;g/test,1.675 mg/Ml</td>
		</tr>
		<tr>
			<td>Anti-CD11b/c</td>
			<td>Absin</td>
			<td>abs124232</td>
			<td>&#160;1&#956;g/test, 1 mg/mL</td>
		</tr>
		<tr>
			<td>Anti-TRAP</td>
			<td>Abcam</td>
			<td>ab191406</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin</td>
			<td>Beyotime</td>
			<td>&#160;AF5003</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Cell climbing slices</td>
			<td>NEST Biotechnology</td>
			<td>80102</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td>for IF, 1:2,000</td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td>for WB, 1:2,000</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Pepro tech</td>
			<td>400-28</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biosharp</td>
			<td>BL302A</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>Pepro tech</td>
			<td>400-30</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rat</td>
			<td>Shanghai SLAC Laboratory Animal Co, Ltd</td>
			<td></td>
			<td>1-10 days old</td>
		</tr>
		<tr>
			<td>SDS-PAGE gel preparation kit</td>
			<td>Solarbio</td>
			<td>P1200</td>
			<td></td>
		</tr>
		<tr>
			<td>TRAP/ALP Staining Kit</td>
			<td>Wako</td>
			<td>294-67001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Biosharp</td>
			<td>BL512A</td>
			<td></td>
		</tr>
		<tr>
			<td>Wright-Giemsa solution</td>
			<td>Keygen Biotech</td>
			<td>KGA225-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of monocyte-macrophage lineage cells from rat bones by secondary adherence method

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and development of bones are mainly regulated by osteoblasts and osteoclasts. It has been widely accepted that osteoclasts are derived from bone marrow monocyte-macrophage cells (BMMs). BMMs and other hematopoietic stem cells are located in the bone marrow cavity. Therefore, isolating single stable BMMs from different and heterogeneous cell populations is a huge challenge. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. Adherent cells cultured for 24-48 h in primary culture were collected. Flow cytometric analysis showed that approximately 37.94% of the cells were CD11b/c+ (monocyte-macrophage surface antigen). Tartrate resistant acid phosphatase (TRAP) staining and western blot analysis demonstrated that BMMs could differentiate into osteoclasts in vitro. The above findings suggested that the secondary adherence cells could be considered as a suitable cellular model for osteoclast differentiation research.

The secondary adherent cell population was stable and uniform. With the continuous cell proliferation, the majority of cells became larger, with irregular shape and grew into a radial adherent disk (Figure 2C,D). Flow cytometry showed that the percentage of cells expressing CD11b/c, a molecular marker on the surface of monocyte-macrophage lineage cells, was approximately 37.94% (Figure 2A,B). To ...

Watch this Scientific Journal Video about Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method at JoVE.com

Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method.

With a decrease of bone mineral density, bones are more likely to fracture, thus negatively affecting a patient's quality of life. The growth and ...

isolation-monocyte-macrophage-lineage-cells-from-rat-bones-secondary

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3.4K Views.  Zhejiang Chinese Medical University. Here we present a protocol for the isolation of BMMs from SD rats, called the secondary adherence method. 

Video: Isolation of Monocyte-Macrophage Lineage Cells from Rat Bones by Secondary Adherence Method

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...