We acknowledge the funding support to AK from the Department of Health Research (DHR), ICMR, Govt. of India (DHR-NRI Grant # R.12015/01/2022-HR). SR has received funding from ICMR, Govt. of India (Grant # 2020-7593/SCR-BMS) and PS has received fellowship from CSIR, Govt. of India. We are also thankful to Ms. Sandhya Tokhi and Ms. Bhupinder Kaur for assistance in flow cytometry, and central sophisticated instrumentation core (CSIC) and PGIMER, Chandigarh for providing infrastructural support.

Establishing the Primary Culture of Dental Pulp Stem Cells

<ol>
	<li>Bhattacharyya, S., Kumar, A., Lal Khanduja, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+voyage+of+stem+cell+toward+terminal+differentiation:+a+brief+overview.">The voyage of stem cell toward terminal differentiation: a brief overview.</a> Acta Biochimica et Biophysica Sinica. 44 (6), 463-475 (2012).</li><li>Prentice, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+stem+cells.">Adult stem cells.</a> Circulation Research. 124 (6), 837-839 (2019).</li><li>Raik, S., Kumar, A., Bhattacharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+cell-free+therapeutic+approach:+Role+of+stem+cell+&amp;#34;soup-ernatant&amp;#34.">Insights into cell-free therapeutic approach: Role of stem cell &amp;#34;soup-ernatant&amp;#34.</a> Biotechnology and Applied Biochemistry. 65 (2), 104-118 (2018).</li><li>Rodriguez-Fuentes, D. E., 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=Mesenchymal+stem+cells+current+clinical+applications:+a+systematic+review.">Mesenchymal stem cells current clinical applications: a systematic review.</a> Archives of Medical Research. 52 (1), 93-101 (2021).</li><li>Yamazaki, K., Kawabori, M., Seki, T., Houkin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+trials+of+stem+cell+treatment+for+spinal+cord+injury.">Clinical trials of stem cell treatment for spinal cord injury.</a> International Journal of Molecular Sciences. 21 (11), 3994 (2020).</li><li>Kumar, 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=Molecular+spectrum+of+secretome+regulates+the+relative+hepatogenic+potential+of+mesenchymal+stem+cells+from+bone+marrow+and+dental+tissue.">Molecular spectrum of secretome regulates the relative hepatogenic potential of mesenchymal stem cells from bone marrow and dental tissue.</a> Scientific Reports. 7 (1), 15015 (2017).</li><li>Kumar, A., Kumar, V., Rattan, V., Jha, V., Bhattacharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+cues+modulate+the+neurogenic+potential+of+bone+marrow+and+dental+stem+cells.">Secretome cues modulate the neurogenic potential of bone marrow and dental stem cells.</a> Molecular Neurobiology. 54 (6), 4672-4682 (2017).</li><li>Kumar, A., Kumar, V., Rattan, V., Jha, V., Bhattacharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretome+proteins+regulate+comparative+osteogenic+and+adipogenic+potential+in+bone+marrow+and+dental+stem+cells.">Secretome proteins regulate comparative osteogenic and adipogenic potential in bone marrow and dental stem cells.</a> Biochimie. 155, 129-139 (2018).</li><li>Kumar, A., Bhattacharyya, S., Rattan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+uncontrolled+freezing+on+biological+characteristics+of+human+dental+pulp+stem+cells.">Effect of uncontrolled freezing on biological characteristics of human dental pulp stem cells.</a> Cell Tissue Bank. 16 (4), 513-522 (2015).</li><li>Raik, S., 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=Assessment+of+post-thaw+quality+of+dental+mesenchymal+stromal+cells+after+long-term+cryopreservation+by+uncontrolled+freezing.">Assessment of post-thaw quality of dental mesenchymal stromal cells after long-term cryopreservation by uncontrolled freezing.</a> Applied Biochemistry and Biotechnology. 191 (2), 728-743 (2020).</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> Proceedings of the National Academy of Sciences. 97 (25), 13625-13630 (2000).</li><li>Tsutsui, T. W. <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:+advances+to+applications.">Dental pulp stem cells: advances to applications.</a> Stem Cells and Cloning. 13, 33-42 (2020).</li><li>Huang, G. T. -. J., Gronthos, S., 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=Mesenchymal+stem+cells+derived+from+dental+tissues+vs.+those+from+other+sources:+their+biology+and+role+in+regenerative+medicine.">Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine.</a> Journal of Dental Research. 88 (9), 792-806 (2009).</li><li>La Noce, M., 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=Dental+pulp+stem+cells:+state+of+the+art+and+suggestions+for+a+true+translation+of+research+into+therapy.">Dental pulp stem cells: state of the art and suggestions for a true translation of research into therapy.</a> Journal of Dentistry. 42 (7), 761-768 (2014).</li><li>Xiao, L., Tsutsui, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+dental+pulp+cells-derived+spheroids+in+serum-free+medium:+stem+cells+in+the+core.">Characterization of human dental pulp cells-derived spheroids in serum-free medium: stem cells in the core.</a> Journal of Cellular Biochemistry. 114 (11), 2624-2636 (2013).</li><li>Kumar, A., Yun, H., Funderburgh, M. L., Du, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+therapy+for+the+cornea.">Regenerative therapy for the cornea.</a> Progress in Retinal and Eye Research. 87, 101011 (2022).</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 (3), 638-645 (2008).</li><li>Yong, 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=Comparison+of+the+angiogenic+ability+between+SHED+and+DPSC+in+a+mice+model+with+critical+limb+ischemic.">Comparison of the angiogenic ability between SHED and DPSC in a mice model with critical limb ischemic.</a> Tissue Engineering and Regenerative Medicine. 19 (4), 861-870 (2022).</li><li>Zhang, X. M., 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=Therapeutic+potential+of+dental+pulp+stem+cell+transplantation+in+a+rat+model+of+Alzheimer's+disease.">Therapeutic potential of dental pulp stem cell transplantation in a rat model of Alzheimer's disease.</a> Neural Regeneration Research. 16 (5), 893-898 (2021).</li><li>Kabir, R., 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=Imperative+role+of+dental+pulp+stem+cells+in+regenerative+therapies:+a+systematic+review.">Imperative role of dental pulp stem cells in regenerative therapies: a systematic review.</a> Nigerian Journal of Surgery. 20 (1), 1-8 (2014).</li><li>Kumar, 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=Dental+pulp+stem+cell+secretome+ameliorates+d-galactose+induced+accelerated+aging+in+rat+model.">Dental pulp stem cell secretome ameliorates d-galactose induced accelerated aging in rat model.</a> Cell Biochemistry and Function. 40 (5), 535-545 (2022).</li><li>Hirata, M., 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=Multifaceted+therapeutic+benefits+of+factors+derived+from+dental+pulp+stem+cells+for+mouse+liver+fibrosis.">Multifaceted therapeutic benefits of factors derived from dental pulp stem cells for mouse liver fibrosis.</a> Stem Cells Translational Medicine. 5 (10), 1416-1424 (2016).</li><li>Fujii, 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=regeneration+by+human+dental+pulp+stem+cells+using+a+helioxanthin+derivative+and+cell-sheet+technology.">regeneration by human dental pulp stem cells using a helioxanthin derivative and cell-sheet technology.</a> Stem Cell Research &amp; Therapy. 9 (1), 24 (2018).</li><li>Ferrua, C. P., 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=How+has+dental+pulp+stem+cells+isolation+been+conducted?+A+scoping+review.">How has dental pulp stem cells isolation been conducted? A scoping review.</a> Brazilian Oral Research. 31, e87 (2017).</li><li>Pilbauerova, N., Soukup, T., Suchankova Kleplova, T., Suchanek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+isolation,+amplification+and+characterization+of+dental+pulp+stem+cells.">Enzymatic isolation, amplification and characterization of dental pulp stem cells.</a> Folia Biologica. 65 (3), 124-133 (2019).</li><li>Tatullo, M., Marrelli, M., Shakesheff, K. M., White, L. J. <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:+function,+isolation+and+applications+in+regenerative+medicine.">Dental pulp stem cells: function, isolation and applications in regenerative medicine.</a> Journal of Tissue Engineering and Regenerative Medicine. 9 (11), 1205-1216 (2015).</li><li>Hilkens, P., 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=Effect+of+isolation+methodology+on+stem+cell+properties+and+multilineage+differentiation+potential+of+human+dental+pulp+stem+cells.">Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells.</a> Cell and Tissue Research. 353 (1), 65-78 (2013).</li><li>Hendijani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture:+An+advantageous+method+for+isolation+of+mesenchymal+stem+cells+from+human+tissues.">Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.</a> Cell Proliferation. 50 (2), e12334 (2017).</li><li>Aryal Ac, S., Islam, M. S., Samsudin, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+effect+of+a+time+delay+on+the+characteristics+and+survival+of+dental+pulp+stem+cells+from+extracted+teeth.">Investigation of the effect of a time delay on the characteristics and survival of dental pulp stem cells from extracted teeth.</a> Archives of Oral Biology. 119, 104896 (2020).</li><li>Langenbach, F., Handschel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dexamethasone,+ascorbic+acid+and+&#946;-glycerophosphate+on+the+osteogenic+differentiation+of+stem+cells+in+vitro.">Effects of dexamethasone, ascorbic acid and &#946;-glycerophosphate on the osteogenic differentiation of stem cells in vitro.</a> Stem Cell Research &amp; Therapy. 4, 117 (2013).</li><li>Jaiswal, N., Haynesworth, S. E., Caplan, A. I., Bruder, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+purified,+culture-expanded+human+mesenchymal+stem+cells+in+vitro.">Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro.</a> Journal of Cellular Biochemistry. 64 (2), 295-312 (1997).</li><li>Bernar, A., Gebetsberger, J. V., Bauer, M., Streif, W., Schirmer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+alizarin+red+S+assay+by+enhancing+mineralization+of+osteoblasts.">Optimization of the alizarin red S assay by enhancing mineralization of osteoblasts.</a> International Journal of Molecular Sciences. 24 (1), 723 (2022).</li><li>Coe, C. L., Lubach, G. R., Schneider, M. L., Dierschke, D. J., Ershler, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+rearing+conditions+alter+immune+responses+in+the+developing+infant+primate.">Early rearing conditions alter immune responses in the developing infant primate.</a> Pediatrics. 90, 505-509 (1992).</li><li>Bakopoulou, 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=Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo/odontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth.">Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.</a> Calcified Tissue International. 88 (2), 130-141 (2011).</li><li>Arthur, A., Rychkov, G., Shi, S., Koblar, S. A., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+dental+pulp+stem+cells+differentiate+toward+functionally+active+neurons+under+appropriate+environmental+cues.">Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues.</a> Stem Cells. 26 (7), 1787-1795 (2008).</li><li>Kim, B. 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=Osteoblastic/cementoblastic+and+neural+differentiation+of+dental+stem+cells+and+their+applications+to+tissue+engineering+and+regenerative+medicine.">Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine.</a> Tissue Engineering. Part B, Reviews. 18 (3), 235-244 (2012).</li><li>Ge, 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=Distal+C+terminus+of+CaV1.2+channels+plays+a+crucial+role+in+the+neural+differentiation+of+dental+pulp+stem+cells.">Distal C terminus of CaV1.2 channels plays a crucial role in the neural differentiation of dental pulp stem cells.</a> PLoS One. 8 (11), e81332 (2013).</li></ol>

The authors declare no financial or non-financial conflict of interest.

Stem cells have pinned the hopes of curing numerous diseases, owing to their plasticity, robustness, immunomodulatory properties, paracrine mechanisms, and homing efficiencies. Dental pulp tissue is considered the most potent and valuable source of stem cells, with eminent plasticity and a regenerative capability. Here, we demonstrate the isolation of DPSCs, utilizing the widely adopted explant culture method, in which the cells migrate from pieces of pulp tissue or explants to grow into a homogenous cell culture that mo...

Adult stem cells have transpired into a powerful therapeutic tool for cell-directed treatments and therapies due to their plasticity, paracrine mechanisms, and immunomodulatory properties1,2,3. The encouraging data from stem cell-based preclinical studies have inspired researchers to work for the bench to-bedside translation. The type of stem cells used for stem cell therapy plays a significant role in successful outcomes. In preclinical and clinical studies, the most widely reported source for mesenchymal stem cells (MSCs) remain bone marrow4,5. However, major drawbacks to&#160;using bone marrow-derived stem cells (BMSCs) include their rare population, highly invasive procedures for isolation, and their limited ability to expand. Therefore, alternative sources of MSCs are being explored. In this regard, dental tissues, with their ease of accessibility, enormous plasticity, high regenerative potential, and high proliferative ability, have now been deemed as a rich and potential alternative source of stem cells6,7,8,9,10.
Dental pulp stem cells (DPSCs) were the first type of dental stem cells to be isolated and characterized by Gronthos in 200011. DPSCs have grabbed the attention for tissue engineering applications because of their high proliferation rate, significant differentiation potential, ease of accessibility with effortless culturing, and, most importantly, their ability to be obtained from a discarded tooth without any ethical concern12.&#160;The limitations posed by other stem cell sources, such as BMSCs and adipose-derived stem cells (ADSCs), in their isolation and inadequate self-renewal capacities are circumvented by DPSCs13. Human DPSCs can be obtained from human primary teeth, permanent teeth, wisdom teeth, exfoliated deciduous teeth (SHEDs), and apical papillae. Moreover, DPSCs can also be isolated from supernumerary teeth, which are generally discarded14. DPSCs express neural crest-associated markers and have the potential to differentiate into neuronal cells both in vitro and in vivo15. In addition to their neurogenic potential, DPSCs can differentiate into other cell lineages, such as osteogenic, chondrogenic, adipogenic, hepatic, and myogenic, when given specific differentiation conditions13. Thus, these multipotent cells hold great potential for cell-based therapy and can be employed for the regeneration of various tissues. Studies have also reported the potential role of DPSCs in the reconstruction of the cornea16, repair of myocardial infarction17, and their potential therapeutic role in diseases like limb ischemia18, Alzheimer&#39;s 19, Parkinson&#39;s 20, and aging21. Therefore, dental tissue-derived stem cells can be used not only for dental regeneration, but also for the repair and regeneration of non-dental organs like eyes16, hearts17, livers22, bones23 etc.
There are two particular methods for the isolation of an MSC population from pulp tissue -&#160;enzymatic digestion and explant culture24,25. Successful establishment of primary cultures without any significant difference in the quantity and properties of DPSCs have been reported by both these methods26. In this study, we have focused on the isolation of DPSCs by the explant method27, since this method generates DPSCs without contamination of hematopoietic and endothelial cells, as compared to enzymatic digestion which can result in fibroblast contamination28.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>Costar</td>
			<td>3516</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>EZstain chondrocyte staining kit, HiMedia</td>
			<td>CCK029</td>
			<td></td>
		</tr>
		<tr>
			<td>alizarin red S stain</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-008</td>
			<td>Osteogenic stain</td>
		</tr>
		<tr>
			<td>Antibiotic cocktail</td>
			<td>Himedia</td>
			<td>A002-5X50ML</td>
			<td>To prevent culture contamination</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Himedia</td>
			<td>TC094-25G</td>
			<td>Chondrogenic induction</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>bFGF ( basic Fibroblast Growth Factor)</td>
			<td>Gibco</td>
			<td>PHG0024</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>CD 105</td>
			<td>BD-Pharmingen</td>
			<td>560839</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 35</td>
			<td>Biolegend</td>
			<td>343604</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 45</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 73</td>
			<td>Biolegend</td>
			<td>344016</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 90</td>
			<td>Biolegend</td>
			<td>328107</td>
			<td>Characterization</td>
		</tr>
		<tr>
			<td>cetyl pyridinium chloride (CPC)</td>
			<td>Sigma-Aldrich</td>
			<td>1104006</td>
			<td>For Alizarin Red extraction</td>
		</tr>
		<tr>
			<td>Dexamethasone 21-phosphate disodium</td>
			<td>Sigma-Aldrich</td>
			<td>D1159-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Himedia</td>
			<td>TS1006-5L</td>
			<td>For washing purpose</td>
		</tr>
		<tr>
			<td>EGF (Epidermal Growth Factor)</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>For hepatic and neural&#160; induction</td>
		</tr>
		<tr>
			<td>EVOS LED microscope</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For&#160; fluorescence imaging</td>
		</tr>
		<tr>
			<td>EZ stain Chondrocyte staining kit</td>
			<td>Himedia</td>
			<td>CCK029-1KT</td>
			<td>Chondro stain Kit</td>
		</tr>
		<tr>
			<td>FACS Canto flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>For cell characterization</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For primary culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>G5 supplement</td>
			<td>Gibco</td>
			<td>17503012</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>HGF( Hepatocyte Growth Factor)</td>
			<td>Sigma-Aldrich</td>
			<td>H1404</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>Biolegend</td>
			<td>307605</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Human TGF-&#946;3</td>
			<td>Peprotech</td>
			<td>#100-36E-10U</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenous acid premix</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>ITS premix</td>
			<td>Corning</td>
			<td>354350</td>
			<td></td>
		</tr>
		<tr>
			<td>LDL Uptake Assay kit</td>
			<td>Abcam</td>
			<td>ab133127</td>
			<td>For hepatic characterization</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td>For hepatic induction</td>
		</tr>
		<tr>
			<td>MAP2 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>M4403</td>
			<td>For neural characterization</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>Neural Basal Media</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>NFM antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
			<td>For neural characterization</td>
		</tr>
		<tr>
			<td>Nikon Elipse TS100 microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>For&#160; fluorescence imaging</td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>01391-250Ml</td>
			<td>Adipogenic stain</td>
		</tr>
		<tr>
			<td>Oncostatin M</td>
			<td>R&#38;D Systems</td>
			<td>295-OM-010/CF</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Tarson</td>
			<td>460090-90MM</td>
			<td>For tissue cutting</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>15655-100G</td>
			<td>Osteogenic induction</td>
		</tr>
		<tr>
			<td>Propan-2-ol</td>
			<td>Thermo Fisher</td>
			<td>Q13827</td>
			<td>For Oil Red O extraction</td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution</td>
			<td>Sigma life sciences</td>
			<td>S8636-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td>For cell passaging</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>merck</td>
			<td>WHA1001325</td>
			<td>filter paper</td>
		</tr>
		<tr>
			<td>&#945;- Minimum Essential Media (&#945;-MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643-10X 1L</td>
			<td>Media for primary culture</td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422-50G</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of dental pulp stem cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Here, we describe how researchers can establish a pure culture of DPSCs via the explant method6,7,8,9,10 and induce them toward multiple lineages to establish the purity of culture for downstream applications.
We established a primary culture of DPSCs from the small tissue of pulp extracted from the third molar tooth of pat...

Read this Scientific Article Protocol about Author Spotlight: Advancing Tissue Regeneration and Disease Modelling with Dental Pulp Stem Cells at JoVE.com

Author Spotlight: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...

primary-culture-of-dental-pulp-stem-cells

Research

JoVE Journal

Biology

2.0K Views.  PGIMER. This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

Video: Author Spotlight: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Primary Culture of Rat Hepatic Cells

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

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

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

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...