This work was supported by the National&#160;Natural Science Foundation of China (82370281, 81870222), the Natural Science&#160;Foundation of Guangdong Province (2022A1515012103), and the Science, the&#160;Technology Development Special Fund Competitive Allocation Project of Zhanjiang City&#160;(2021A05086), and the Startup Foundation&#160;from the Second Affiliated Hospital of&#160;Guangdong Medical University (23H03).

The samples were obtained from mice, with ethical approval granted by the Ethics Committee of Guangdong Medical University. Detailed descriptions of materials, equipment, and software used in this protocol are provided in the Table of Materials. The details of the preparation prior to extraction are illustrated in Figure 1, while the process of Spleen-Exo extraction and enrichment is described in Figure 2.
1. Preparation
<ol>
	<li>Pre-cool high-speed and ultra-high-speed centrifuges to 4 &#176;C and set the temperature of a desktop shaker to 37 &#176;C.</li>
	<li>Prepare cell culture dishes (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />100 mm), 50 mL sterile centrifuge tubes, ice, and sterile cell strainer (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />70 &#181;m) as shown in Figure 1A, B.</li>
	<li>Sterilize scissors and forceps using 75% alcohol spray, followed by high-temperature sterilization. These tools are shown in Figure 1C.</li>
	<li>Place the spleen tissue in a sterile <img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />100 mm Petri dish on ice, and wash off the surface blood with 1x iced sterile phosphate-buffered saline (PBS). Dry the spleen tissues with sterile gauze and weigh them after drying. 
	NOTE: If the tissue is frozen, thaw it in a dish for 15 min on ice before proceeding.</li>
	<li>Moisturize the cell strainer (<img alt="Equation 1" src="/files/ftp_upload/67234/67234eq01.jpg" style="margin: auto;" />70 &#181;m) by dropping 1 mL of sterile PBS into the strainer using a sterile transfer pipet.</li>
	<li>Prepare the digestive buffer (0.1% type I collagenase) using 1x PBS with a vortex mixer. 
	NOTE: Use 10 mL of digestive buffer for every 100 mg of spleen tissue. The collagenase concentration was chosen in this study based on the product instructions. For other tissue types, researchers may need to adjust these parameters based on tissue density and composition. Preliminary experiments with varying concentrations and digestion times are recommended to determine the optimal conditions for each tissue type.</li>
</ol>
2. Tissue digestion
<ol>
	<li>Chop the tissue into uniform small pieces with scissors in a culture dish on ice (Figure 3A) and transfer to a 50 mL centrifuge tube with a sterile transfer pipet.</li>
	<li>Add digestion buffer to the tube according to the spleen tissue weight as indicated above (Figure 3B), then shake the tube with an angle of 45&#176; on a horizontal shaker at 37 &#176;C. Monitor the digestion progress and stop the digestion when tissue chunks have dispersed, and most have lost their shape. Digestion time should vary between tissues. Under the experimental conditions in this study, the digestion time is approximately 30 min.</li>
	<li>Transfer the digested mixture to a cell strainer (&#1060;70 &#181;m) at room temperature (RT) to remove fibrous and larger tissue debris. 
	NOTE: Complete filtration immediately after digestion.</li>
	<li>Collect the filtrate in a new 50 mL centrifuge tube.</li>
</ol>
3. Exosome isolation by differential centrifugation
<ol>
	<li>Centrifuge filtrates at 500 x g for 10 min at 4 &#176;C. A pellet containing residual cells, cell debris, and nuclei can be seen after centrifugation, as shown in Figure 3C.</li>
	<li>Transfer the supernatant to a new tube and centrifuge at 3000 x g for 20 min at 4 &#176;C. A pellet containing apoptotic bodies and microsomes can be seen after centrifugation, as shown in Figure 3D.</li>
	<li>Transfer the supernatant to a new tube and centrifuge at 10,000 x g for 30 min at 4 &#176;C. Pellet containing microvesicles and plasma membrane can be seen after centrifugation, as shown in Figure 3E.</li>
	<li>Ultracentrifuge the resulting supernatant at 120,000 x g for 2 h at 4 &#176;C. A pellet can be seen at the bottom of the tube after centrifugation, as shown in Figure 3F.</li>
	<li>Discard the supernatant, wash the pellet with PBS, and ultracentrifuge again at 120,000 x g for 2 h at 4 &#176;C to obtain exosomes. A pellet can be seen at the bottom of the tube after centrifugation, as shown in Figure 3G.</li>
	<li>Dissolve the isolated exosomes with sterile PBS (200 &#181;L per spleen) and store the isolated exosomes in a new 2 mL centrifuge tube at -80 &#176;C.</li>
</ol>
4.&#160;Exosome characterization by transmission electron microscopy (TEM)
NOTE: TEM was used to determine whether the extracted Exo displayed vesicle characteristics. The Exo samples identified by electron microscopy must be fresh samples or stored briefly at 4 &#176;C for a short time.
<ol>
	<li>Fix exosomes (2-6 &#181;g/&#181;L) with 2% electron microscopy (EM)-grade paraformaldehyde at RT for 2 h.</li>
	<li>Place 10 &#181;L of exosome solution on a copper mesh, incubate at RT for 10 min, rinse with sterile distilled water, and blot excess liquid with absorbent paper.</li>
	<li>Stain the copper mesh with 2% uranyl acetate for 1 min, blot excess fluid with filter paper, and dry under an incandescent lamp for 2 min.</li>
	<li>Observe the prepared sample under a transmission electron microscope at 80 kV.</li>
</ol>
5. Size distribution and particle concentration measurement of exosomes
<ol>
	<li>Load the standard polystyrene nanoparticles (250 nm) to the nano-flow cytometer.</li>
	<li>Measure the protein concentration of Exo samples using a BCA protein assay kit. Dilute Exo sample with PBS according to BCA Protein Assay results (dilute the exosomes to 1-10 ng/&#181;L), then load the Exo samples to the nano-flow to measure the side scatter intensity (SSI).</li>
	<li>Calculate Exo concentration according to the ratio of SSI to particle concentration in the standard polystyrene nanoparticles. 
	NOTE: For size measurement, a standard curve is generated by loading standard silica nanoparticles with gradient sizes (68 nm, 91 nm, 113 nm, 155 nm) to the nano-flow cytometer. The loading of the Exo sample follows this. The size distribution of Exo was calculated according to the standard cure.</li>
</ol>
6. Lysis and immunoblot confirmation of exosome proteins
<ol>
	<li>Add 100 &#181;L of radio immunoprecipitation assay (RIPA) lysis buffer to the exosome pellet for protein analysis.</li>
	<li>Vortex vigorously, cover with parafilm, rock for 20 min at RT, then vortex again.</li>
	<li>Centrifuge briefly at 1000 x g for 30-60 s to collect the sample, transfer to a new 1.5 mL microcentrifuge tube, and store at -20 &#176;C to -80 &#176;C until analysis.</li>
	<li>For immunoblot, mix samples with 5x loading buffer to achieve a 1x concentration and prepare for gel electrophoresis.</li>
	<li>If whole-tissue homogenate controls are desired, use a strong lysis buffer with a protease inhibitor to lyse the tissue, as detailed above. Then, centrifuge the samples at 10,000 x g for 10 min and discard the remaining extracellular matrix, whole cells, or intact cellular debris.</li>
	<li>Boil the sample buffer containing exosomes or tissue homogenate at 95 &#176;C for 5-10 min. Load equal protein per lane into a 10% SDS-PAGE gel. Load equal protein of tissue homogenate as control.</li>
	<li>Proceed with gel electrophoresis under a voltage of 80 V for approximately 2 h using the conventional electrophoresis buffer. Perform western blot analysis to confirm Exo protein expression in the purified lysates and compare relative Exo abundance in fractions.</li>
</ol>

Refining Techniques for Murine Spleen-Derived Exosome Isolation

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R., Hoenderop, J. G. J., Rigalli, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rigorous+method+to+enrich+for+exosomes+from+brain+tissue.">A rigorous method to enrich for exosomes from brain tissue.</a> J Extracell Vesicles. 6 (1), 1348885 (2017).</li><li>Hoshino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+and+particle+biomarkers+define+multiple+human+cancers.">Extracellular vesicle and particle biomarkers define multiple human cancers.</a> Cell. 182 (4), 1044-1061.e18 (2020).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yiwei+decoction+promotes+apoptosis+of+gastric+cancer+cells+through+spleen-derived+exosomes.">Yiwei decoction promotes apoptosis of gastric cancer cells through spleen-derived exosomes.</a> Front Pharmacol. 14, 1144955 (2023).</li><li>Useckaite, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+paired+human+liver+homogenate+and+tissue-derived+extracellular+vesicles.">Proteomic profiling of paired human liver homogenate and tissue-derived extracellular vesicles.</a> Proteomics. 24 (11), e2300025 (2023).</li><li>Hurwitz, S. 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The authors declare that they have no competing interests.

Recent research has reported that spleen-derived exosomes (Spleen-Exo) play a critical role in the treatment of gastric cancer12. To further elucidate the functions of Spleen-Exo, it is necessary to establish a reproducible and optimal method for their extraction from spleen tissue. While protocols exist for isolating exosomes from brain and liver cancer13,14, methods for other tissues remain underdeveloped and require further validation. ...

Exosomes (Exo) are a subgroup of extracellular vesicles (EVs), ranging from 30 to 150 nm in size, encapsulating a diverse array of biomolecules, including proteins, nucleic acids, and lipids1. These biomolecules are derived from the parental cells and are released into the extracellular space. Exo facilitate biomolecular information exchange between cells and their surrounding microenvironment, playing roles in both prokaryotic and eukaryotic systems2. The characteristics of exosomes, such as content, size, membrane components, and cellular origin, are highly variable and influenced by the originating cell type, cellular state, and environmental conditions.
Exosomes are commonly classified into three categories based on their source: cell culture-derived, body fluid-derived, and tissue-derived (Ti-Exo). While cell culture-derived exosomes have been extensively studied due to their accessibility and consistent yield, their use is limited by potential alterations in cell characteristics after prolonged culture, which may misrepresent their biological functions3,4,5. Moreover, most cell cultures are maintained in two-dimensional environments that do not mimic the complex in vivo intercellular interactions, potentially impacting data interpretation6. Conversely, exosomes isolated from biological fluids offer a minimally invasive option to monitor disease progression in real time7. However, these samples often contain a mixture of exosomes from various origins, complicating the accurate identification of their primary source8. Given these challenges, there is an increasing interest in Ti-Exo, which reside in the extracellular interstitium and are key mediators of intercellular signaling.
The spleen plays a crucial role in immune function and maintaining internal homeostasis. Despite existing protocols for isolating Ti-Exo from organs such as the brain, liver, and tumors, practical methods for spleen-derived exosomes are limitedly reported9,10. This study aimed to establish a practical protocol for isolating exosomes from spleen tissue in mice modified from a previous report11. We detail a method involving collagenase digestion followed by ultracentrifugation, which minimizes the disruption of the cell membrane and ensures high purity and yield of spleen-derived Exo. The characteristics of isolated Exo using this established protocol were validated through electron microscopy (TEM), the nano-flow cytometer (Nano-FCM), and western blot, confirming the protocol&#39;s efficacy for further experimental research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70 &#181;m Cell Strainer</td>
			<td>Biologix Group Co., Ltd.USA</td>
			<td>15-1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti CD9 antibody</td>
			<td>Cell Signaling Technology, Inc (CST), USA</td>
			<td>983275</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti GM130 antibody</td>
			<td>Proteintech Group, Inc.China&#160;</td>
			<td>66662-1-lg</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti TSG101 antibody</td>
			<td>Abcam Plc, UK</td>
			<td>125011</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Wuxi NEST Biotechnology Co.,Ltd, China</td>
			<td>704001</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Wuxi NEST Biotechnology Co.,Ltd, China</td>
			<td>788211</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Sigma-Aldrich Corp., USA</td>
			<td>C2674</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Thermostatic Shaker</td>
			<td>Shanghai bluepard instruments Co., Ltd., China</td>
			<td>THZ-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis buffer</td>
			<td>Wuhan Servicebio Technology Co., Ltd., China&#160;</td>
			<td>G2081-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced BCA Protein Assay Kit</td>
			<td>Shanghai Beyotime Biotechnology Co., Ltd., China&#160;</td>
			<td>P0010S</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow NanoAnalyzer</td>
			<td>NanoFCM Inc., China</td>
			<td>N30E</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence/Chemiluminescence imaging system&#160;</td>
			<td>Guangzhou Biolight Biotechnology Co., Ltd., China</td>
			<td>GelView 6000Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat anti-Mouse IgG</td>
			<td>Proteintech Group, Inc.China&#160;</td>
			<td>15014</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat anti-Rabbit IgG</td>
			<td>Proteintech Group, Inc.China&#160;</td>
			<td>15015</td>
			<td></td>
		</tr>
		<tr>
			<td>microplate reader</td>
			<td>Molecular Devices, USA</td>
			<td>CMax Plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors</td>
			<td>Shanghai&#160;Medical&#160;Instruments (group)&#160;Co., Ltd.,China</td>
			<td>WA1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic tweezers</td>
			<td>Shanghai&#160;Medical&#160;Instruments (group)&#160;Co., Ltd.,China</td>
			<td>WA3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Multifuge X1R Pro centrifuge</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>75009750</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic scissors</td>
			<td>Shanghai&#160;Medical&#160;Instruments (group)&#160;Co., Ltd.,China</td>
			<td>Y00030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic tweezers</td>
			<td>Shanghai&#160;Medical&#160;Instruments (Group)&#160;Co., Ltd., China&#160;</td>
			<td>JD1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XPN-100 Ultrafiltration centrifuge</td>
			<td>Beckman Coulter, USA</td>
			<td>A94469</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS)</td>
			<td>Beijing Solarbio Science &#38; Technology Co.,Ltd., China</td>
			<td>P1003</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis buffer (strong, without inhibitors)</td>
			<td>Shanghai Beyotime Biotechnology Co., Ltd., China&#160;</td>
			<td>P0013K</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Deli&#160;Manufacturing&#160;Company, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE Sample Loading Buffer, 5x</td>
			<td>Shanghai Beyotime Biotechnology Co., Ltd., China&#160;</td>
			<td>P0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>Biologix Group Co., Ltd.USA</td>
			<td>30-0238A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Hitachi, Japan</td>
			<td>H-7650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifugation tube</td>
			<td>Beckman Coulter, USA</td>
			<td>355618</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Transfer Buffer&#160;</td>
			<td>Wuhan Servicebio Technology Co., Ltd., China&#160;</td>
			<td>G2028-1L</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes derived from mouse spleen tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Isolation and purification of spleen-derived exosomes 
To isolate exosomes from mouse spleen tissue, we employed a combination of collagenase digestion and differential ultracentrifugation (Figure 2). The initial steps involved pre-treating the spleen tissue to remove surface blood, followed by digestion with Type I collagenase. This enzymatic treatment facilitated the breakdown of extracellular matrix components, thus liberating the exosomes while preserving their inte...

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

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JoVE Journal

Medicine

436 Views.  Guangdong Medical University. Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Video: Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

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 ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone splenectomy regardless of precipitating causes are prone to develop an overwhelming post-splenectomy infection and experience increased risks of deep venous thrombosis and malignancies. Recently, epidemiological studies indicated that splenectomy might be associated with the occurrence of cardiovascular diseases, suggesting that physiological functions of the spleen have not yet been fully recognized. Here, we introduce a mouse model of vascularized heterotopic spleen transplantation, which not only can be utilized to study the function and behavioral activity of splenic immune cell subsets in different biologic processes, but also can be a powerful tool to test the therapeutic potential of spleen transplantation in certain diseases. The main surgical steps of this model include donor spleen harvest, the removal of recipient native spleen, and spleen graft revascularization. Using congenic mouse strains (e.g., mice with CD45.1/CD45.2 backgrounds), we observed that after syngeneic transplantation, both donor-derived splenic lymphocytes and myeloid cells migrated out of the graft as early as post-operative day 1, concomitant with the influx of multiple types of recipient cells, thus generating a unique chimera. &#160;Despite relatively challenging techniques, this procedure can be performed with &#62;90% success rate. This model allows tracking the fate, longevity, and function of splenocytes during steady state and in a disease setting following a spleen transplantation, thereby offering a great opportunity to discover the distinct role for spleen-derived immune cells in different disease processes.

This protocol details the surgical steps of a mouse model of vascularized heterotopic spleen transplantation, a technically challenging model that can serve as a powerful tool in studying the fate and longevity of spleen cells, the mechanisms of distinct spleen cell populations in disease progression, and transplant immunity.

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Ultracentrifuge-Based Isolation of Extracellular Vesicles from Human ADSCs

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

Author Spotlight: Standardizing and Improving the Extraction and Purification of Extracellular Vesicles from Human ADSCs

This protocol describes all procedures, from culturing human adipose-derived mesenchymal stem cells (ADSCs) and collecting supernatant to extracting extracellular vesicles (EVs) using ultracentrifugation.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research ...