We gratefully acknowledge Dr. Leila Shakeri &amp; Dr. Aref Dournaei for their critical discus and Mr. Mohammad Reza Khadem Sharif for his technical supports.

1. Prepare the Enzyme Solution and Proliferation Medium (PM)
<ol>
 <li>Make Collagenase Type I Solution: Weigh out collagenase type I (12 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 &mu;m syringe filter. Then place it 15 ml tube and keep it at -20 &deg;C until needed.</li>
 <li>Make dispase Solution: Weigh out dispase (16 mg/ml) and dissolve in 1 ml PBS and filter using a 0.2 &mu;m syringe filter. Then place it 15 ml tube and keep it at 4 &deg;C until needed. </li>
 <li>Make enzyme solution: Add 1 ml collagenase type I solutions (12 mg/ml) and 1 ml dispase solutions (16 mg/ml) into the 2 ml sterile PBS containing 100 mg/ml penicillin, 100 mg/ml streptomycin. Total concentration of dispase and Collagenase I in final volume should be 4 mg/ml and 3 mg/ml, receptively. Then, aliquot this volume in to four 15 ml tube, each containing 1 ml enzyme solution. Each tube could be used for one pulp digestion. </li>
 <li>Make washing solution: Add 100 mg/ml penicillin, 100 mg/ml streptomycin into PBS. </li>
 <li>Make basic media: Alpha modification of Eagle's medium (&alpha;-MEM) supplemented with 10% FBS &amp; 100 units/ml penicillin, 100 mg/ml streptomycin.</li>
 <li>Make the Proliferation Media (PM): Alpha modification of Eagle's medium (&alpha;-MEM) supplemented with 10% FBS, 100 &mu;M L-ascorbic acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B. </li>
 <li>Make Odontogenic media: Alpha modification of Eagle's medium (&alpha;-MEM) supplemented with 10% FBS, 100 &mu;M L-ascorbic acid 2-phosphate, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.01 &mu;M Dexamethasone, 5 mM &beta;-Glycerol phosphate, 1.8 mM Monopotassium phosphate. </li>
</ol>
2. Prepare Human Dental Pulp Tissue for Dental Pulp Stem Cell Isolation
<ol>
 <li>Normal human third molars were collected from adults (21-29 years of age) at the Dental Clinic of the Imam Ali under approved Institutional Review Board (IRB). </li>
 <li>Teeth were placed into the basic medium (&alpha;-MEM supplemented with 10% FBS) and were transferred into laboratory at 4 &deg;C. </li>
 <li>Under the sterile condition, working within a biohazard laminar flow hood, set-up was done one 100 mm Petri dishes for each tooth to be processed. Tooth surfaces were cleaned by 70% ethanol. </li>
 <li>While working in one of the Petri-dishes, tooth was kept by dental forceps and a scalpel blade was used to clean off debris and periodontal ligament on the roots. </li>
 <li>Cemento-enamel junction was slowly cut by using sterilized dental disc to reveal the pulp chamber. It should be considered that cutting process must be performed slowly to reduce overheating of the dental tissue. </li>
 <li>Pulp tissue was gently separated from the crown and root. </li>
 <li>Pulp tissue was cut into small pieces with the aid of a scalpel blade. </li>
</ol>
3. Human Dental Pulp Isolation
<ol>
 <li>Pulp tissues underwent either enzymatic dissociation of pulp tissue or direct cell outgrowth from pulp tissue explants for isolation of hDPSCs. </li>
 <li>Enzymatic dissociation of pulp tissue: 
 
 <ol>
 <li>Small pieces of pulp tissues were transferred into Enzyme solution for 1 hr at 37 &deg;C. Vortex every 30 min to help breaking up tissue.</li>
 <li>Afterwards, large cell aggregates were removed and Single-cell suspensions were obtained by passing cells through a 70-&mu;M cell strainer.</li>
 <li>Single-cell suspensions were centrifuged at 1,200 rpm for 5 min at room temperature.</li>
 <li>Supernatants were carefully pipetted off and pellet was re-suspended in 1 ml proliferation medium (PM). (Note: FBS in proliferation medium terminate enzymatic dissociation.) </li>
 <li>Single-cell suspensions of dental pulp was seeded into 25 cm2 culture flask with PM and then incubated at 37 &deg;C in 5% CO2. </li>
 <li>The culture medium was changed every three days until the cell confluency was achieved. </li>
 </ol>
 </li>
 <li>Direct cell outgrowth from pulp tissue explants:
 <ol>
 <li>Pulp tissue was minced into 1-2-mm fragments, and each piece was placed in 25-cm2 culture flasks with PM and then incubated at 37 &deg;C in 5% CO2. It should be considered that total volume of the PM for outgrowth of cell must support the attachment of pulp pieces for further cell outgrowth (2-3 ml per flask). </li>
 <li>Medium was changed after outgrowth was observed. </li>
 <li>The outgrown cells at confluence were sub-cultured at ratio of 1:4 (passage 1). </li>
 </ol>
 </li>
</ol>
4. Immunophenotyping
<ol>
 <li>250,000 cells/tube of both types of cells in passage 3 were incubated by PE or FITC-conjugated anti-human antibodies with their isotypes for 30 min at 4 &deg;C in dark place. (Note: the concentration of antibodies was applied according to the manufacture protocol.) </li>
 <li>After the incubation time, 100 &mu;l PBS or FACS dilution solution was added to each tube and centrifuged at 1,200 rpm for 5 min in dark place. </li>
 <li>Supernatants were removed &amp; pellets were re-suspended in 100 &mu;l PBS or FACS dilution in dark place. </li>
 <li>Targeted surface markers (CD markers) were evaluated by BD FACSCalibur. </li>
</ol>
5. Induction of Differentiation of DPSCs into Mineralized Cells &amp; Quantified Alizarin Red S Assay 
<ol>
 <li>DPSCs that were obtained by either enzymatic dissociation of pulp tissue (known as DPSC-ED) or direct cell outgrowth from pulp tissue explants (DPSC-OG) were sub-cultured for 3 passages. </li>
 <li>At Passage 3, cells were seed into a 6 well plate at 5,000 cell/well. </li>
 <li>At confluence of 60%, odontogenic medium were replaced by conventional PM. Three wells were remained as a negative control by adding growth medium. </li>
 <li>Medium change every 3 days until day 21. </li>
 <li>At day 21, Cells were washed with PBS and were fixed by 1 ml/well 10% paraformaldehyde for 15 min at room temperature. </li>
 <li>Then paraformaldehyde was pipetted off, carefully and cells were washed 3 times (5-10 min for each time) with distilled H2O. (Note: Avoid disturbing the monolayer.)</li>
 <li>After washing, 1 ml/well Alizarin red S were added for 20 min at room temperature. </li>
 <li>Cells were washed by deionized H2O four times (5 min for each time with gentle rocking) to remove any additional Alizarin red.</li>
 <li>1 ml/well H2O were added to prevent the cells from drying.</li>
 <li>The plates were assumed to visual inspection and/or image acquisition.</li>
 <li>For Alizarin Red S quantification assay, H2O were replaced by 1 ml 10% acetic acid to each well of 6-well plate &amp; incubate for 30 min at room temperature with shaking. </li>
 <li>Then, cell monolayer/well were scrape &amp; both acetic acid &amp; cells were transferred into the separate 1.5 ml micro-centrifuge tubes. (Note: each well should be transfer into a separate tube i.e. three test wells &amp; three control wells for each ED &amp; OG groups.)</li>
 <li>Tubes were vortexed vigorously for 30 sec. </li>
 <li>Heat to 85 &deg;C for 10 min. (Note: to avoid evaporation, tubes were sealed with parafilm.)</li>
 <li>After heating, tubes were transferred into ice for 5 min. (Note: Tubes should not be opened until become cooled.)</li>
 <li> The slurries were centrifuged at 20,000 x g for 15 min. While centrifuging, Alizarin Red standards were made up.</li>
 </ol>
 Alizarin Red S standard were prepared by first diluting the dilution buffer &amp; used for making 2-fold serial dilutions based on the table. 
 <table border="1">
 <tbody>
 <tr>
 <td colspan="7">High Range concentration of dye </td>
 <td colspan="7">Low Range concentration of dye </td>
 </tr>
 <tr>
 <td>2 mM</td>
 <td>1 mM</td>
 <td>500 &mu;M</td>
 <td>250 &mu;M</td>
 <td>125 &mu;M</td>
 <td>62.5 &mu;M</td>
 <td>31.3 &mu;M</td>
 <td>15.6 &mu;M</td>
 <td>7.8 &mu;M</td>
 <td>3.9 &mu;M</td>
 <td>1.9 &mu;M</td>
 <td>0.9 &mu;M</td>
 <td>0.4 &mu;M</td>
 <td>Blank</td>
 </tr>
 </tbody>
 </table>
 <ol start="16">
 <li>After the centrifugation, 1 ml of supernatant/tube were transferred into new separate 1.5 ml micro-centrifuge tubes.</li>
 <li>The pH was neutralized with ~375 &mu;l 10% ammonium hydroxide per tube. (Note: pH rage should be 4.1-4.5.)</li>
 <li>150 &mu;l of standard &amp; samples (includes separate tests &amp; controls) were added into an opaque-walled, transparent bottom 96-well plate.</li>
 <li>The absorbance was read at 405 nm and OD values from samples were compared with standard plot for further analysis.</li>
</ol>

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Biol. 238, 120-132 (2001).</li><li>Gandia, C., 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=Human+dental+pulp+stem+cells+improve+left+ventricular+function,+induce+angiogenesis,+and+reduce+infarct+size+in+rats+with+acute+myocardial+infarction.">Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction.</a> Stem Cells. 26, 638-645 (2008).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proc. Natl. Acad. Sci. U. S. A.. 97, 13625-13630 (2000).</li><li>Graziano, A., d'Aquino, R., Laino, G., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+pulp+stem+cells:+a+promising+tool+for+bone+regeneration.">Dental pulp stem cells: a promising tool for bone regeneration.</a> Stem Cell Rev. 4, 21-26 (2008).</li><li>Kerkis, I., 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=Early+transplantation+of+human+immature+dental+pulp+stem+cells+from+baby+teeth+to+golden+retriever+muscular+dystrophy+(GRMD)+dogs:+Local+or+systemic.">Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic.</a> J. Transl. Med. 6, 35 (2008).</li><li>Onyekwelu, O., Seppala, M., Zoupa, M., Cobourne, M. 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Surg. 19, 204-210 (2008).</li><li>Tirino, V., 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=Methods+for+the+identification,+characterization+and+banking+of+human+DPSCs:+current+strategies+and+perspectives.">Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives.</a> Stem Cell Rev. 7, 608-615 (2011).</li><li>Tirino, V., Paino, F., De Rosa, A., Papaccio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification,+isolation,+characterization,+and+banking+of+human+dental+pulp+stem+cells.">Identification, isolation, characterization, and banking of human dental pulp stem cells.</a> Methods Mol. Biol. 879, 443-463 (2012).</li><li>Eslaminejad, M. 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No conflicts of interest declared.

This protocol describes the process of isolation and characterization of hDPSCs from dental pulp using two methods, enzymatic dissociation and direct outgrowth of stem cells from pulp tissue explants. In addition, in vitro differentiation of these cells into odontoblasts, was assessed by quantitative Alizarin Red S assay &amp; QPCR.
Existing protocols for isolating pulp tissue from human tooth had been used various instruments such as pliers (bone forceps)9, extirpation ...

Stem cells are clonogenic cells which possess two remarkable features, known as multi-potency and self-renewal15. Among all stem cells with different replicative potencies, dental stem cells as the postnatal stem cells have drawn attention in recent years because of their accessibility, plasticity, and high proliferative ability in comparison with other adult stem cells16. Characteristically, similar to the mesenchymal stem cells, dental pulp stem cells are adherent clonogenic cells which have multiple differentiation capacity into mesenchyme and/or non-mesenchyme lineages, both in vitro and in vivo.1-8,17,18 DPSCs are identified by their negative expression of hematopoietic antigens (e.g., CD45, CD34, CD14), and positive expression of CD90, CD29, CD73, CD105, CD44 and STRO-1.19,20
Easy obtained potential with minimum pain &amp; morbidity make human DPSCs as a valuable source of MSCs compared to the ordinary sources, such as bone marrow mesenchymal stem cells21. In general, DPSCs have been isolated by either outgrowth method, i.e., migration of stem cells from pulp tissue explants (DPSC-OG)22-24, and/or by enzymatic digestion (DPSC-ED)4,6,25. Previous studies have shown that the isolation method and culture conditions can induce different populations or lineages under in vitro passages26,27. In the case of permanent teeth (pDPSCs), Huang et al revealed that enzymatic digested pDPSCs have higher proliferation potential compared to the outgrown DPSCs.26 Moreover, in the case of deciduous teeth (dDPSCs), it was demonstrated that STRO-1 &amp; CD34 markers expressed more in dDPSC-ED in comparison with dDPSC-OG. In addition, dDPSC-ED displayed higher mineralization rate in defined osteo/odonto medium.27 Therefore, due to the outstanding potential of DPSCs in regenerative medicine, more studies will be required for better understanding of possible various populations which are derived from different isolation methods.
Here, it was attempt to introduce easy way of pulp extraction, by using one-step dental diamond disk to facilitate the process of pulp extraction. Moreover, after the isolation of human pulp-derived stem cells by applying ED or OG methods, general properties &amp; differentiation capacity between two groups were also investigated.

<table border="1">
<tbody>
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue 			or Lot. number</td>
 <td>Comments 			(optional)</td>
 </tr>
 <tr>
 <td>&alpha;-MEM</td>
 <td>GIBCO</td>
 <td>11900-073</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase 			type I</td>
 <td>Sigma-Aldrich</td>
 <td>C0130-100MG</td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>GIBCO</td>
 <td>17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin/streptomycin</td>
 <td>GIBCO</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Amphotericin 			B</td>
 <td>GIBCO</td>
 <td>15290-018</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal 			Bovine serum defined (FBS)</td>
 <td>HyClone</td>
 <td>SH30070.03</td>
 <td></td>
 </tr>
 <tr>
 <td>L-ascorbic 			acid 2-phosphate</td>
 <td>Sigma</td>
 <td>A8960-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>GIBCO</td>
 <td>25030-024</td>
 <td></td>
 </tr>
 <tr>
 <td>Dexamethasone</td>
 <td>Sigma</td>
 <td>D4902</td>
 <td></td>
 </tr>
 <tr>
 <td>&beta;-Glycerol 			phosphate disodium salt hydrate, BioUltra</td>
 <td>Sigma</td>
 <td>G9422-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium 			phosphate monobasic</td>
 <td>Sigma-Aldrich</td>
 <td>P5655</td>
 <td></td>
 </tr>
 <tr>
 <td>Osteogenesis 			Assay Kit</td>
 <td>Millipore</td>
 <td>PS01802031</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse 			IgG2b-PE isotype control</td>
 <td>BD 			pharmingen</td>
 <td>50808088029</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC 			mouse IgG2b isotype control</td>
 <td>BD 			pharmingen</td>
 <td>559532</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC 			mouse IgG1 &#x03BA; isotype</td>
 <td>BD 			pharmingen</td>
 <td>11471471</td>
 <td></td>
 </tr>
 <tr>
 <td>FITC/PE 			mouse anti-human CD34/CD45</td>
 <td>BD 			pharmingen</td>
 <td>341071</td>
 <td></td>
 </tr>
 <tr>
 <td>PE 			anti-human CD146</td>
 <td>BD 			pharmingen</td>
 <td>550315</td>
 <td></td>
 </tr>
 <tr>
 <td>Monoclonal 			mouse anti-human CD90/FITC</td>
 <td>Daka</td>
 <td>00034418</td>
 <td></td>
 </tr>
 <tr>
 <td>PE 			mouse anti-human CD73</td>
 <td>BD 			pharmingen</td>
 <td>550257</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-h 			CD105/Endoglin PE</td>
 <td>BD 			pharmingen</td>
 <td>FAB10971P</td>
 <td></td>
 </tr>
 <tr>
 <td>PE 			mouse anti-human CD11b/Mac1</td>
 <td>BD 			pharmingen</td>
 <td>5553888</td>
 <td></td>
 </tr>
 <tr>
 <td>CD44 			PE mouse anti human</td>
 <td>BD 			pharmingen</td>
 <td>555479</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate 			buffer Solution (PBS)</td>
 <td>GIBCO</td>
 <td>003000</td>
 <td></td>
 </tr>
 <tr>
 <td>70-&mu;m 			cell strainer</td>
 <td>Falcon</td>
 <td>352360</td>
 <td></td>
 </tr>
 <tr>
 <td>0.2 			&mu;m syringe filter</td>
 <td>Millex-GV</td>
 <td>SLGV033RB</td>
 <td></td>
 </tr>
 <tr>
 <td>25 			cm2 			culture flask</td>
 <td>Sigma-Aldrich</td>
 <td>Z707481</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>BD 			FACSCalibur</td>
 <td>BD</td>
 <td>342975</td>
 <td></td>
 </tr>
 <tr>
 <td>multiskan 			microplate spectrophotometer </td>
 <td>Thermo scientific</td>
 <td>51119200</td>
 <td></td>
 </tr>
 <tr>
 <td>Fleurcense 			Microscope</td>
 <td>Olympus</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Flowing 			Software version 2.3.1</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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

<ol>
 <li>DPSCs that were obtained by enzymatic dissociation (DPSC-ED) are shown here at day 10, 15,18 (Figure 1). The colonies initiated to form on almost 3 to 5 days after the isolation. </li>
 <li>Outgrown DPSCs (DPSC-OG) are shown in Figure 2 on day 5, 10, 13 &amp; 18. Fibroblast-like cells began to migrate from pulp tissue into the flask by parallel ordering by almost 5 days after seeding. </li>
 <li>DPSCs at passage 3 using both methods are shown in Figure 3. Both types of DPSCs ...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

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

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-characterization-comparative-differentiation-human-dental

Research

JoVE Journal

Medicine

26.3K vistas.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. El método se describe el aislamiento y caracterización de células humanas de la Pulpa Dental madre (hDPSCs) mediante el uso de cualquiera de los dos Disociación enzimática de la pulpa (DPSC-ED) O Consecuencia directa de las células madre a partir de explantes de tejido pulpar (DPSC-OG). A continuación, seguido por In vitro Diferenciación comparativo de ambos tipos de hDPSCs en odontoblastos.

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

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 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 ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

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, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...