The authors are grateful to Dr. Mathew Mazhavancheril, Director and Head of the Pushpagiri Research Centre in Thiruvalla, for his support in documenting the procedures at the Research Centre.

The protocol outlined herein conforms to the guidelines of the institutional human research ethics committee (IRB, Pushpagiri Research Center, Kerala). The use of extracted teeth was conducted following ethical standards to ensure the integrity, dignity, and rights of the participants. The participants selected for this study were healthy individuals under 30 years of age who required tooth extraction for orthodontic treatment. Those with extensive dental caries or severe periodontitis were excluded from the study. Decid...

The protocol outlines the isolation, culture, and characterization of dental pulp stem cells (DPSCs) from human deciduous and permanent teeth. It includes a description of the storage and proliferation of these cells, as well as the assessment of their in vitro differentiation potential into osteoblasts, adipocytes, and chondrocytes35.
Chen et al.36&#160;demonstrated that Dental Pulp Stem Cells (DPSCs) could be ob...

Stem cell research has flourished in biomedical science due to its promising applications in regenerative medicine and tissue engineering. Dental pulp stem cells (DPSCs), derived from the pulp tissue of both human deciduous and permanent teeth, have attracted significant interest as a source of stem cells due to their ready availability and multipotent capacity1,2. These cells have the potential to differentiate into various cell types, including adipocytes, osteoblasts, and chondrocytes, as confirmed by numerous studies3.
Over the past few decades, research and therapeutic applications of stem cells have surged. The expansive potential of stem cells calls for diversifying the sources from which they are obtained. Several factors influence the efficiency, viability, and stemness of chosen cells. Despite the existence of various known stem cell reservoirs, such as bone marrow and adipose tissues, the invasive procedures, site morbidity, and ethical concerns linked to these sources often limit their exploration4,5. Among the various stem cell sources, dental stem cells have gained attention due to their easy accessibility, high plasticity, and diverse potential applications. Human dental pulp stem cells, in particular, have been extensively researched for their therapeutic prospects6. Teeth, commonly discarded as medical waste, hold a wealth of mesenchymal stem cells7. Safeguarding this valuable stem cell pool requires collective efforts from patients, dentists, and doctors to ensure that these resources are not wasted, making each dental pulp stem cell available for future regenerative requirements.
Dental pulp-derived stem cells, such as human adult dental pulp stem cells (DPSCs) and stem cells from exfoliated human deciduous teeth (SHED), are located in the perivascular niche of the dental pulp. These cells are believed to originate from cranial neural crest cells and exhibit early markers for both mesenchymal stem cells (MSCs) and neuroectodermal stem cells. DPSCs and SHEDs have demonstrated multipotency and the ability to regenerate diverse tissue types8.
Potential sources of dental stem cells encompass healthy deciduous and permanent teeth. Stem cells constitute only about 1% of the total cell population in the pulp, highlighting the importance of effective isolation and expansion techniques9. Consequently, the extraction and expansion of these stem cells are pivotal steps in DPSC isolation10. Extracted or exfoliated teeth need to be stored in a nutrient-rich transport medium, such as phosphate-buffered saline (PBS) or Hanks-buffered saline solution (HBSS).
Obtaining dental pulp can be achieved through various methods, contingent on the tooth type7,11. For deciduous teeth with resorbed roots, extraction can be performed via the root apex. Similarly, sterile barbed broaches can be used to obtain pulp from permanent teeth with an immature open apex. In cases of permanent teeth with fully developed roots, accessing the pulp chamber involves separating the dental crown from the root. This is accomplished by cutting the tooth using a diamond disc at the cementoenamel junction. This incision exposes the pulp chamber, enabling retrieval of the pulp tissue12,13,14.
Dental pulp stem cells (DPSCs) can be isolated through enzymatic digestion (ED) or outgrowth from tissue explants (OG), also known as spontaneous growth. The ED method employs enzymes, primarily collagenase I and dispase, to break down the tissue into single-cell suspensions15,16. The OG method, simpler and quicker, entails chopping the pulp fragments and directly placing them into a culture plate, allowing cells to grow from the tissue explants17. Researchers have utilized and compared both techniques to assess cell proliferation rates, preservation of isolated stem cell properties, differentiation, and surface marker expression18. Establishing and standardizing protocols for acquiring DPSCs with high efficiency and stemness can pave the way for effective and safe therapies19. This protocol encompasses extracting DPSCs using enzymatic digestion, lab processing, preservation, and cell differentiation with colorimetric staining for adipogenesis, osteogenesis, and chondrogenesis.
The protocol outlined in this article presents a step-by-step procedure, beginning with the initial isolation of dental pulp from the tooth, followed by culture and maintenance of DPSCs in the laboratory, and concluding with their characterization using specific stem cell markers (Figure 1). The techniques for inducing these stem cells into different cell lineages, highlighting their multipotency, are also described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-isobuty-l-methyl-xanthine&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>AS001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>RM471</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S staining solution</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>GRM894</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase -Staining kit</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha-MEM, or Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha-MEM, or Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>012-04802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-glycerophosphate&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>G9422-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet-Laminar flow hood</td>
			<td>Labconco Corporation,MO 64132-2696,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD90, CD105, CD73, CD34, CD45, and HLA-DR</td>
			<td>BioLegend, Inc.CA 92121,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m )</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP025</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>REMI Elektrotechnik Limited (REMI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>1101 | 1102</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp. NJ 08701, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp. NJ 08701, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Growth Medium</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>AT006</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Conical tubes (15 or 50 )</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td>546021P/546041P</td>
			<td>15 mL and 50 mL</td>
		</tr>
		<tr>
			<td>Cryo freezing container</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>15-350-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryolabels</td>
			<td>Label India:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial storage boxes&#160;</td>
			<td>Cryostore Storage Boxes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials (1.8 mL)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>PW1282</td>
			<td>Self standing</td>
		</tr>
		<tr>
			<td>Culture flask (25 cm&#178;)</td>
			<td>Corning Inc.NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCG4/TCG6</td>
			<td>T25/T75</td>
		</tr>
		<tr>
			<td>Culture Plates</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP129/TCP008</td>
			<td>60 mm/100 mm</td>
		</tr>
		<tr>
			<td>Dental Diamond Discs</td>
			<td>Komet SC 29730, USA</td>
			<td></td>
			<td>Komet&#160;</td>
		</tr>
		<tr>
			<td>Dental Spoon Excavator</td>
			<td>Brasseler,GA 31419,USA</td>
			<td>5023591U0</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethosone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethosone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TC185</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche Diagnostics,Mannheim,Germany.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche Diagnostics GmbH, Mannheim,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>MB106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol -70%</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>Extraction forceps&#160;</td>
			<td>Dentsply Sirona, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>RM9954</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin-coated tissue culture plate</td>
			<td>Corning Inc.Corning, NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences,CA 95131,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry buffer</td>
			<td>BD Biosciences,CA 95131,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slip 22 x 22 mm</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Lonza Group Ltd,4002 Basel, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed dental handpiece&#160;</td>
			<td>NSK Ltd,Tokyo 8216, Japan</td>
			<td></td>
			<td>Ti-Max Z series</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX, or 3-isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Pfizer Inc. NY 10017,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>I5523</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>I5523</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS) premix</td>
			<td>Corning Incorporated,MA 01876,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus Corp.,Tokyo 163-0914,Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (60% )</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>MB063</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine mixed with epinephrine</td>
			<td>DENTSPLY,NC 28277,USA</td>
			<td></td>
			<td>Citanest</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Air Liquide,75007 Paris,France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen storage tank</td>
			<td>Cryo Scientific Systems Pvt. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf AG,22339 Hamburg,Germany</td>
			<td>30020</td>
			<td>Accupipet-2-20 &#181;L</td>
		</tr>
		<tr>
			<td>Mini tissue grinder</td>
			<td>Bio-Rad Lab, Inc. CA 94547,USA</td>
			<td></td>
			<td>ReadyPrep mini grinders</td>
		</tr>
		<tr>
			<td>Minus 80 freezer</td>
			<td>Blue Star Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld Superior,arktheidenfeld,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oil red O stain</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>1024190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Osteogenic Differentiation Medium (ODM)&#160;</td>
			<td>STEMCELL Technologies Inc.Vancouver, BC, V5Z 1B3,Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL119</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution (PBS) without Ca++ and Mg++</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TS1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific,MA, USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td>P3813-1PAK</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Proline&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade Size 15&#160;</td>
			<td>Swann-Morton Ltd, Sheffield, S6 2BJ,UK</td>
			<td>BDF-6955C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>AS102</td>
			<td>4% w/v solution</td>
		</tr>
		<tr>
			<td>Sterile centrifuge tubes</td>
			<td>Tarsons Products Pvt. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile container -20 mL</td>
			<td>3M Center, MN 55144-1000,USA</td>
			<td></td>
			<td>3 M</td>
		</tr>
		<tr>
			<td>Sterile phosphate-buffered saline (PBS)</td>
			<td>Sigma Aldrich, USA</td>
			<td>P3813-1PAK</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Sterile pipettes (2, 5, and 10 mL )</td>
			<td>Eppendorf AG,22339 Hamburg,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes and tips</td>
			<td>Eppendorf India Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade Handle</td>
			<td>Becton, Dickinson and Co.,NJ,USA</td>
			<td>371030</td>
			<td>BP Handle 3</td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>BSW-01D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture, and characterization of dental pulp stem cells from human deciduous and permanent teeth

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.

The successful execution of the outlined protocol yielded dental pulp stem cells (DPSCs) capable of multilineage differentiation, demonstrating their multipotency.
Viability assays 
The viability of the DPSCs was assessed using a Trypan Blue exclusion assay at various time points. The results show consistently high viability (greater than 95%) throughout the culture period, demonstrating the robustness of our isolation and culture protocol.
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Read this Scientific Article Protocol about Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth at JoVE.com

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

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816 Views.  Hamad Medical Corporation. This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.

Video: Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the...

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

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...

primary culture of dental pulp stem cells

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...

Establishing the Primary Culture of Dental Pulp Stem Cells

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Primary Culture of Dental Pulp Stem Cells

establishment of primary culture of dental pulp stem cells (dpscs) from human dental tissue

Establishment of Primary Culture of Dental Pulp Stem Cells (DPSCs) from Human Dental Tissue

single-cell sorting of immunophenotyped mesenchymal stem cells from human exfoliated deciduous teeth

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based...

Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth

isolation, characterization and microrna-based genetic modification of human dental follicle stem cells

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell...

isolation, propagation, and prion protein expression during neuronal differentiation of human dental pulp stem cells

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Here we present a protocol for human Dental Pulp Stem Cells isolation and propagation in order to evaluate the prion protein expression during neuronal differentiation...

rapid isolation of single cells from mouse and human teeth

Rapid Isolation of Single Cells from Mouse and Human Teeth

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human...

isolation and culture of dental epithelial stem cells from the adult mouse incisor

Isolation and Culture of Dental Epithelial Stem Cells from the Adult 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...

in vitro induction of human dental pulp stem cells toward pancreatic lineages

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

isolation of epithelial cells from human dental follicle

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

isolation and culture of adult epithelial stem cells from human skin

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell...

mesenchymal stem cell isolation from pulp tissue and co-culture with cancer cells to study their interactions

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell...

establishing a co-culture of dental pulp cells and trigeminal neurons for cross-communication

This video showcases a technique for establishing a co-culture of trigeminal neurons and primary dental pulp cells using a transwell filter. In this physically separated condition, mesenchymal cells obtained from dental pulp tissue secrete paracrine signaling molecules that traverse through the pores, promoting the differentiation and migration of neurons towards the mesenchymal cells establishing a co-culture...

Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

isolation, culture, and characterization of primary schwann cells, keratinocytes, and fibroblasts from human foreskin

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

isolation and culture of neural crest stem cells from human hair follicles

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair...

isolation and culture of bone marrow mesenchymal stem cells from the human mandible

Isolation and Culture of Bone Marrow Mesenchymal Stem Cells from the Human Mandible

The present protocol describes an efficient procedure for isolating and culturing of human mandibular bone marrow-derived mesenchymal stem cells using the whole bone marrow adherence method. The cultured cells were identified by cell proliferation assays, flow cytometry, and multilineage differentiation...

isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this...

isolation and characterization of human umbilical cord-derived mesenchymal stem cells from preterm and term infants

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of...

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...