This study was supported by the Jane and Aatos Erkko Foundation.

This protocol involves the use of animals and all the procedures were approved by Ethical Committees on the Use and Care of Animals and the Animal Facility at the University of Helsinki.
1. Preparation of the organ culture dish
<ol>
	<li>Perform all procedures in a laminar flow hood. Clean work surfaces with 70% ethanol and use autoclaved glass instruments and solutions. Sterilize scissors and other metal equipment in a glass-bead sterilizer.</li>
	<li>Prepare filters normally stored in 70% ethanol by washing them three times in 1x PBS to remove ethanol (Figure 1). Cut filters into rectangular pieces (3 x 3-5 x 5 mm). 
	NOTE: Washed filter pieces can be stored for several days in 1x PBS at 4 &#176;C.</li>
	<li>Prepare the culture medium (1:1 DMEM:F12 supplemented with 1% [v/v] 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, 10% [v/v] FBS, 150 &#181;g/mL ascorbic acid, and 0.2% [v/v] penicillin [10,000 I.U./mL] and streptomycin [10,000 &#181;g/mL]). Store at 4 &#176;C.</li>
	<li>Place the 30 mm metal grids (with 1-2 mm diameter holes that enable tissue imaging) in a 35 mm Petri dish. Add sufficient media to reach the grid surface without producing air bubbles. Pre-warm the prepared culture dish at 37 &#176;C until the tissue is isolated and ready for culture (in a standard incubator with 5% CO2 and 90%-95% humidity).</li>
</ol>
2. Incisor dissection and isolation of the proximal end
<ol>
	<li>Sacrifice the animals following an approved animal care protocol.</li>
	<li>Decapitate the mouse and dissect the mandible. To do so, first remove the skin to expose the mandible and cut through the masseter muscles to separate it from the maxilla and the rest of the head.</li>
	<li>Once the mandible is isolated, remove the tongue and as much soft tissue as possible.</li>
	<li>Collect all the mandibles and keep them in a Petri dish containing PBS on ice, as this enhances the viability of the tissues.</li>
	<li>Transfer one mandible to a glass Petri dish and use disposable 20/26 G hypodermic needles to dissect the incisor under a stereomicroscope. Split the mandible at the midline, cutting through the symphysis. Clean the soft and muscle tissue away from the bone surface for better visualization. 
	&#8203;NOTE: A glass Petri dish is essential, as this will not blunt the needles.</li>
	<li>Mandibles obtained from animals younger than 10 days are softer and more fragile, therefore, use disposable 20/26 G hypodermic needles to open each half of the mandible longitudinally to expose the incisor tooth. For mice older than 10 days, use tweezers to grip the mandible and break the bone to expose the tooth.</li>
	<li>Gently detach the incisor from the surrounding bone and cut off the proximal end, which contains the cervical loop.</li>
	<li>Cut the proximal end and remove the mineralized enamel and dentin matrix.</li>
	<li>Keep the collected proximal ends in Dulbecco&#39;s PBS on ice until ready for culture.</li>
</ol>
3. Culture
<ol>
	<li>Carefully place a filter rectangle on the top of a grid in a pre-warmed culture dish.</li>
	<li>Use a stereomicroscope to properly orient the tissue pieces.</li>
	<li>Place in a standard incubator at 37 &#176;C with 5% CO2 and 90%-95% humidity.</li>
	<li>Change the medium every other day and replace with fresh medium, carefully avoiding formation of air bubbles. Monitor tissue growth and photograph daily using a camera attached to the stereomicroscope.</li>
</ol>
4. Adding soluble factors to the culture
<ol>
	<li>Supplement the culture medium with soluble factors and molecules of interest to study their effect on regulation of SCs. 
	&#8203;NOTE: The administration protocol for any molecule (growth factors, signaling molecules, blocking antibodies, pharmacological compounds such as inhibitors or activators, vectors, etc.) depends on its half-life and solubility. These parameters also determine the appropriate control to be used.</li>
</ol>
5. Molecular and histological analyses
<ol>
	<li>Remove the culture medium.</li>
	<li>Carefully add ice-cold methanol to the tissues to prevent detachment from the filters.</li>
	<li>Leave methanol for 5 min.</li>
	<li>Transfer filters carrying tissue explants to sampling tubes.</li>
	<li>Fix the explants in 4% paraformaldehyde in PBS for 10-24 h at 4 &#176;C.</li>
	<li>Proceed with established protocols for histological processing (paraffin, frozen, etc.) or immunostainings.</li>
</ol>
6. Culture of tissue slices
NOTE: There are several variations to this protocol, which are all equally successful and are a matter of personal choice, depending on the speed and the skill of the user. These refer to the buffer used to collect and maintain tissue viability. For this purpose, Krebs buffer, PBS supplemented with 2% glucose and antibiotics, or PBS can be used. If Krebs buffer is used, it should be made 1 day in advance and kept at 4 &#176;C.
<ol>
	<li>Before the experiment, prepare 4%-5% low-melting point agarose by dissolving 2-2.5 g of low-melting point agarose in 50 mL of boiling 2% glucose/PBS, after which the solution should be placed in a 45 &#176;C water bath.</li>
	<li>Set up the vibratome, wash with 70% ethanol, and fill with ice-cold 2% glucose/PBS supplemented with antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin).</li>
	<li>Dissect the proximal ends of the incisor and collect them in the ice-cold 2% glucose/PBS supplemented with antibiotics.</li>
	<li>Place one proximal end of the incisor in the mold containing 4%-5% low-melting point agarose. Under a stereomicroscope, orient the piece in the desired direction and leave on ice for the agarose to harden.</li>
	<li>Trim the hardened agarose block and place it in the vibratome. Cut thick slices (150-300 &#181;m).</li>
	<li>Collect the slices in a Petri dish containing ice-cold 2% glucose/PBS supplemented with antibiotics.</li>
	<li>Use a spatula to transfer the thick slices on a filter rectangle placed on the pre-warmed grid prepared as in section 1.3.</li>
	<li>Incubate the thick slices in the incubator and proceed with imaging (Figure 2).</li>
</ol>

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The authors declare no conflicts of interest.

In vitro organ culture has been used extensively to study inductive potential and epithelial-mesenchymal interactions that govern organ growth and morphogenesis. The Thesleff laboratory has demonstrated how to adapt the Sax&#233;n modification of the Trowell-type organ culture and use it to study tooth development14. The reproducible conditions and advancements in fluorescent reporters have made this a useful method for monitoring tooth morphogenesis and the distinct cell populations within. This paper described how to apply this protocol to study epithelial stem cells and the microenvironment in which they reside.
A technique for isolating the proximal end of the incisor that contains the SC niche from postnatal pups and from older animals was described here. The success of this method depends on the isolation time (which directly impacts tissue viability) and on the ability to isolate an undamaged proximal end with an intact labial cervical loop. This is particularly challenging in older animals with mineralized bone. The protocol shows how to use controlled mechanical force to break the mineralized bone and isolate fully intact and viable soft tissue in a significantly shorter time when compared to other published protocols25. Once isolated, the tissue can be cultured in vitro, either as a whole or as a thick slice. The disadvantage of the thick tissue slicing method is that only a portion of the cervical loop is present in each slice; thus, obtaining a reproducibly identical portion of the cervical loop in different sections is not possible. Organ culture provides an easy and reproducible method to study the molecular and cellular mechanisms that regulate SCs and their niche over time. However, there are limitations to the total culture period, mainly due to the progressive loss of mesenchyme after the initial 3-4 days of culture.
The isolated tissue can also be further processed to obtain a single cell suspension enriched in incisor epithelial SCs26. Enzymatic separation of the cervical loop from the rest of the proximal end and the use of specific fluorescent reporters enables more detailed mRNA and protein analyses (such as qPCR, single-cell RNA sequencing) and a greater insight into specific cell populations within the niche.
In vitro culture provides a suitable platform for screening various genetic and pharmacological manipulations for their regulatory effect on tooth epithelial SCs and their niches. It should be noted that the success of the pharmacological treatments depends on the properties of the tested molecules, such as half-life, solubility, metabolic stability, dosage, and cell toxicity. In combination with a fluorescent reporter, the regulatory effect can be assigned to a specific cell population within a tissue, thus providing a powerful tool to uncover the molecular and cellular mechanisms that regulate tooth epithelial SCs and their niche.
With the use of specific fluorescent reporter mice, it is possible to identify and follow specific cell populations within live tissue over time. Recently, it has been demonstrated that in vitro organ culture can be used for live imaging of tooth development16. In addition, cell behavior can be imaged over a short period of time using in vitro culture of thick tissue slices7. As previously indicated, only a portion of the SC niche can be imaged and the imaging duration is brief. Ideally, the entire SC niche should be imaged. However, 4D time-lapse imaging of this structure is challenging, mainly due to the thickness of the niche and the limitations of currently available imaging methods. Nevertheless, continuous advances in imaging technology promise an exciting future in unravelling cellular behaviors that constitute the incisor SC niche.

Mouse incisors grow continuously due to life-long preservation of the stem cells (SC) that support the unceasing production of tooth components. These include epithelial SCs, which generate enamel-producing ameloblasts, and mesenchymal stem cells (MSCs), which generate dentin-producing odontoblasts, among other cells1. The epithelial SCs in the continuously growing incisors were initially identified as label-retaining cells2,3 and have since been shown to express a number of well-known stemness genes, including Sox24. These cells share common features with epithelial SCs in other organs and reside within the SC niche called the cervical loop located on the labial side of the incisor. The niche is a dynamic entity composed of cells and extracellular matrix that control SC activity5. Lineage-tracing studies have demonstrated that Sox2+ epithelial SCs can regenerate the whole epithelial compartment of the tooth and that they are crucial for successional tooth formation6,7. MSCs with dentin reparative or regenerative potential are largely recruited from outside the organ through blood vessels and nerves8,9,10,11, therefore, providing a suitable model to study recruitment, migration, and housing of the MSC population.
To study SCs in vivo is not always feasible, since many of the genetic and/or pharmacological manipulations can affect organ homeostasis and/or have lethal consequences. Therefore, organ culture provides an excellent tool to study regulation of SCs and their niches in vitro. The organ culture system that utilizes a metal grid was initially developed by Trowell12 to study organ development and has been further modified by Saxen13&#160;to study inductive signals in kidney development. Since then, this in vitro method of culturing the whole or part of the organ has been successfully applied in different fields. In the field of tooth development, this method has been widely used to study the epithelial-mesenchymal interactions that govern tooth development14&#160;and successional tooth formation15. The work of the Thesleff laboratory has demonstrated the utility of this system for temporal analysis of tooth growth and morphogenesis, for analysis of the effect of various molecules and growth factors on tooth growth, and for time-lapse live imaging of tooth development16,17. More recently, this method has been utilized to study regulation of incisor SCs and their niche18,19, which is described in detail here.

<table><tbody><tr><td>1-mL plastic syringes</td><td></td><td></td><td></td></tr><tr><td>Disposable 20/26 gauge hypodermic needles</td><td>Terumo</td><td></td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>61965-026</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Gibco</td><td>14287</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>F.S.T.</td><td>14084-08</td><td></td></tr><tr><td>F-12</td><td>Gibco</td><td>31765-027</td><td></td></tr><tr><td>FBS South American (CE)</td><td>LifeTechn.</td><td>10270106</td><td>divide in aliquotes, store at -20&amp;deg;C</td></tr><tr><td>Glass bead sterilizer, Steri 250 Seconds-Sterilizer</td><td>Simon Keller Ltd</td><td>4AJ-6285884</td><td></td></tr><tr><td>GlutaMAX-1 (200 mM L-alanyl-L-glutamine dipeptide)</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Isopore Polycarb.Filters, 0,1 um 25-mm diameter</td><td>MerckMillipore</td><td>VCTP02500</td><td>Store in 70% ethanol at room temperature.</td></tr><tr><td>L-Ascobic Acid</td><td>Sigma</td><td>A4544-25g</td><td>diluted 100 mg/ml in MilliQ, filter strerilized and divided in 20&amp;mu;l aliquotes, store at dark, -20&amp;deg;C</td></tr><tr><td>Low melting agarose</td><td>TopVision</td><td>R0801</td><td></td></tr><tr><td>Metal grids</td><td></td><td></td><td>Commercially available, or self-made from stainless-steel mesh (corrosion resistant, size of mesh 0.7 mm). Cut approximately 30 mm diameter disk and bend the edges to give 3 mm height. Use nails to make holes.</td></tr><tr><td>Micro forceps</td><td>Medicon</td><td>07.60.03</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000U/ml) sol.</td><td>Gibco</td><td>15140-148</td><td></td></tr><tr><td>Petri dishes, Soda-Lime glass</td><td>DWK Life Sciences</td><td>9170442</td><td></td></tr><tr><td>Petridish 35 mm, with vent</td><td>Duran</td><td>237554008</td><td></td></tr><tr><td>Petridish 90 mm, no vent classic</td><td>Thermo Fisher</td><td>101RT/C</td><td></td></tr><tr><td>Small scissors</td><td></td><td></td><td></td></tr></tbody></table>

use of trowell-type organ culture to study regulation of dental stem cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The epithelial SCs reside in a niche called the cervical loop, which is located at the proximal end of the incisor (Figure 3A). Cervical loops are morphologically distinct structures composed of inner and outer enamel epithelium that encase the stellate reticulum, a core of loosely arranged epithelial cells (Figure 3B,C). There are two cervical loops in each incisor (Figure 3A), but only the labial cervical loop contains SCs. The epithelial SCs are localized to the stellate reticulum and the adjacent enamel epithelium at the tip of the cervical loop20,21. They generate progeny characterized by Sfrp5 expression, which differentiate into highly proliferative transit-amplifying cells that produce various cells, including enamel-secreting ameloblasts2,7. Ameloblast differentiation from SCs can be recapitulated in the organ culture system described here2.
In recent years, reporter mice in which a fluorescent protein (such as GFP) is under the control of specific gene regulatory elements have become a widely used tool to identify and isolate cells from various tissues and to follow cell fate and lineage progression in vivo and in vitro22,23. The use of these animal models is beneficial for analyzing the effect of various regulatory molecules, since the intensity and the pattern of fluorescent reporter expression can be used as a readout of endogenous gene activity, or as a reporter of proliferation status (i.e., Fucci reporter mouse model).
The Sox2-GFP transgenic reporter mouse model24, in which the enhanced GFP (EGFP) expression is under the control of a 5.5 kb fragment of the upstream regulatory element of the Sox2 promoter, enables identification and visualization of Sox2-expressing incisor epithelial stem cells (Figure 4A-C). Generation of mice that carry several fluorescent reporters can be useful for identification of more than one cell population. For example, in cervical loops from Sox2-GFP;Fucci-mKO transgenic animals stem cells (GFP+, green, Figure 4D) and non-proliferative cells (Fucci-mKO+, red, Figure 4D) can be identified. In addition, Sox2-GFP expression can be used as a reporter when analyzing the effect of various molecules. Addition of the Wnt/&#946;-catenin signaling activator BIO negatively affects Sox2-expressing stem cells and consequently GFP expression (Figure 4E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62462/62462fig01.jpg" /> 
Figure 1: Preparation of the culture chamber. (A) Wash the filter in 1x PBS. (B) Cut the filter into rectangular pieces. (C) Prepare media in the hood and pipet it through the grid. Avoid air bubble formation. (D) Prewarm the pre-prepared culture dish at 37 &#176;C prior to adding tissue explants on a filter placed on the grid. (E) Cartoon representation of the culture chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62462/62462fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62462/62462fig02.jpg" /> 
Figure 2: Culturing tissue slices. The proximal end of a 2-day-postnatal (2PN) mouse incisor was dissected and sectioned by vibratome. Slices (150 &#181;m thick) were cultured and hard tissue formation (arrows) was observed after a 6-day culture. Scale 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62462/62462fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62462/62462fig03.jpg" /> 
Figure 3: Cervical loop. (A) Proximal end of the incisor-containing labial (outlined by black dashed line) and lingual (outlined by grey dashed line) cervical loops. (B) Histological section of the labial cervical loop, which represents a niche for epithelial stem cells. (C) Cartoon representation of the labial cervical loop and its components. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62462/62462fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62462/62462fig04.jpg" /> 
Figure 4: Use of fluorescent reporter mouse models in an organ culture system. (A) Brightfield, (B) fluorescent, and (C) overlay image of the proximal end of the incisor isolated from 2-day postnatal Sox2-GFP mice. (D) Sox2-GFP and Fucci-mKO expression in the proximal end of the incisor isolated from 2-day postnatal Sox2-GFP; Fucci-mKO mice. (E) Effect of BIO on the stem cells expressing Sox2-GFP and the stem cell niche (cervical loop, outlined by yellow dashed line). Scale 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62462/62462fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells at JoVE.com

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

use-trowell-type-organ-culture-to-study-regulation-dental-stem

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

2.1K Views.  University of Helsinki. De Trowell-type orgaankweek wordt gebruikt om weefselexplantaten te kweken. Hier laten we zien hoe je deze methode kunt gebruiken om de stamcellen in continu groeiende tanden te bestuderen. Met deze techniek kunnen we de stamcellen en hun gedrag in de niche bestuderen en het effect van verschillende moleculen op de overleving en het onderhoud van stamcellen screenen.Deze methode kan worden toegepast om ook de stamcellen van andere organen te bestuderen, zoals huid of borstklier. Begin ...

Video: Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

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

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

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 products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetics

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

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

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 interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Cancer Research

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

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

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

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

Medicine

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Neuroscience

Live Tracking of Dental Cells via Two-Photon Microscopy

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Author Spotlight: Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

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) ...

Bioengineering

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 ...

Biology

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.

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 ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

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