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 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.
Teeth have essential roles in food mastication, speech, and psychological well-being (self-image). The human tooth consists of highly mineralized tissues of varying density and hardness1. Dental enamel, the main component of the tooth crown, is the highest mineralized tissue in the human body. During enamel formation (amelogenesis), when teeth develop, dental epithelial stem cells (DESCs) differentiate into enamel-forming cells (ameloblasts). Once formed, the enamel is rarely repaired or renewed due to the apoptotic loss of the ameloblasts at the onset of tooth eruption1. Restoration of damaged enamel tissue, as caused by trauma or bacterial disease, is currently accomplished using synthetic materials; however, these are troubled with important shortcomings such as microleakage, inferior osseointegration and anchorage, finite life span, and lack of fully functional repair2. Hence, a robust and reliable culture of human DESCs with the capacity to generate ameloblasts and the potential to produce mineralized tissue would be a major step forward in the dental regenerative field.
Knowledge on human DESC phenotype and biological function are scarce3,4,5. Interestingly, DESCs of human teeth have been proposed to exist in the Epithelial Cell Rests of Malassez (ERM), cell clusters present within the dental follicle (DF), which surrounds unerupted teeth, and remains present in the periodontal ligament around the root once the tooth erupts1. ERM cells co-cultured with dental pulp have been found to differentiate into ameloblast-like cells and generate enamel-like tissue6. However, profound studies of the specific role of ERM cells in enamel (re-) generation have been limited due to the lack of reliable study models7. Current ERM in vitro culture systems are hampered by limited life span and quick loss of phenotype in the 2D conditions standardly used8,9,10,11,12. Hence, a tractable in vitro system to faithfully expand, study, and differentiate human DESCs is strongly needed.
During the last decade, a powerful technique to grow epithelial stem cells in vitro has been successfully applied to several types of (human) epithelial tissues to study their biology as well as disease13,14,15,16. This technology enables the tissue epithelial stem cells to self-develop into 3D cell constructions (i.e., organoids) when seeded into an extracellular matrix (ECM)-mimicking scaffold (typically, Matrigel) and cultured in a defined medium replicating the tissue's stem cell niche signaling and/or embryogenesis. Typical growth factors needed for organoid development include epidermal growth factor (EGF) and wingless-type MMTV integration site (WNT) activators14,15,16. The resultant organoids are characterized by lasting fidelity in mimicking the tissue's original epithelial stem cells, as well as high expandability while retaining their phenotype and functional properties, thereby overcoming the often-limited primary human tissue availability as acquired from the clinic. To establish organoids, isolation of the epithelial stem cells from the heterogeneous tissue (i.e., comprising other cell types such as mesenchymal cells) prior to culturing is not required as mesenchymal cells do not attach to, or thrive in, the ECM, eventually resulting in purely epithelial organoids13,16,17,18,19. This promising and versatile technology has led to the development of manifold organoid models from various human epithelial tissues. However, human tooth-derived organoids, valuable for deep study of tooth development, regeneration and disease, were not established yet20,21. We recently succeeded in developing such a new organoid model starting from DF tissue from third molars (wisdom teeth) extracted from adolescent patients19.
Here, we describe the protocol to develop epithelial organoid cultures from the adult human tooth (i.e., from the DF of third molars) (Figure 1A). The resultant organoids express ERM-associated stemness markers while being long-term expandable. Intriguingly, opposite to most other organoid models, the typically needed EGF is redundant for robust organoid development and growth. Interestingly, the stemness organoids show ameloblast differentiation properties, thereby mimicking ERM/DESC features and processes occurring in vivo. The new and unique organoid model described here allows exploring DESC biology, plasticity, and differentiation capacity and opens the door for taking the first steps toward tooth-regenerative approaches.
All methods described here have been approved by the Ethics Committee Research UZ/KU Leuven (13/0104U). Extracted third molars (wisdom teeth) were obtained after patients' informed consent.
1. Preparations
2. Dental follicle dissociation
3. Establishment of tooth organoid culture (Figure 1A and Figure 2A)
4. Amplification and passaging of tooth organoid culture (Figure 1B and Figure 2B)
5. Cryopreservation of tooth organoids
6. Thawing of cryopreserved tooth organoids
7. Fixation and paraffin-embedding of tooth organoids
NOTE: This procedure (including sections 8 and 9) can also be applied to the primary DF tissue.
8. Microtome sectioning and staining of tooth organoids (Figure 2B and Figure 3A-C)
9. RNA Extraction and RT-qPCR of tooth organoids (Figure 2B and Figure 3D)
Tooth organoid development
We provide a detailed protocol to establish organoid cultures from human DF tissue acquired following wisdom tooth extraction (Figure 1A). Isolated DF is enzymatically and mechanically dissociated. The obtained cells are cultured within BMM in media that were empirically defined for optimal organoid development and growth (tooth organoid medium; TOM)19.
The organoids typically develop within 2 weeks after DF cell seeding (P0; Figure 2A). The organoids are long-term expandable (up to 11 passages so far) (Figure 2B, shown at P4). Seeding around 20,000 cells per BMM droplet (at both P0 and further passages) yields an optimal density of organoids (Figure 2C), whereas seeding higher cell numbers leads to suboptimal organoid outgrowth (i.e., smaller organoids at too high density) as there is insufficient space to grow (Figure 2D). The eventually optimized culture conditions allow the development of organoids from DF samples at 100% efficiency19.
Tooth organoid characterization and validation
The developed organoids show a dense appearance and contain cells displaying a high nucleo-cytoplasmic ratio, as similarly observed in ERM cells7 (Figure 3A). Moreover, and in further analogy, the organoids express the ERM marker cytokeratin 14 (CK14)22, thereby confirming their epithelial origin (Figure 3B), as well as other proposed ERM markers (such as P63, CD44 and ITGα612,22,23 (Figure 3B). Moreover, organoids express SOX2, a well-known DESC marker in mice and also present in the epithelium of developing human teeth (Figure 3B)1. Interestingly, amelogenin (AMELX), the main component of the enamel matrix, also found expressed in the organoids, is also detected in the ERM24 (Figure 3C). Expression of still other ERM/stemness markers is described in our recent study19 and can be used to further certify the obtained organoids. Furthermore, the organoids retain their ERM/stemness phenotype during passaging, among others shown by stable expression of ERM/stem cell markers (Figure 3D). Finally, the tooth-derived organoids show differentiation capacity to ameloblast(-like) cells, which can also be applied to validate the obtained organoid cultures, showing expression of mature ameloblast markers such as odontogenic-ameloblast associated protein (ODAM) and amelotin (AMTN) after transfer to differentiation medium (see19).
Figure 1: Schematic workflow of tooth organoid development, characterization, and applications. (A) Tooth organoid development from dental follicle (DF) tissue isolated from unerupted third molars. (B) Amplification, characterization, and application potential of tooth organoids. d, day; P, passage. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 2: Tooth organoid development. (A) Organoid development from DF tissue. Representative brightfield images shown at different days (d) after seeding (P0; P, passage; 2.5x). (B) Brightfield images showing robust passageability of a tooth organoid line (2.5x). (C) Brightfield images showing a tooth organoid line immediately at passaging (d0; left; 2.5x) seeded at a density of 20,000 cells per well, and the resultant confluent organoid culture ready to be passaged (d14; right; 2.5x). (D) Brightfield images showing a tooth organoid line, which had been seeded at a density of >20,000 cells, leading to smaller organoids at too high density at d14 (2.5x). Scale bars: 200 µm. Please click here to view a larger version of this figure.
Figure 3: Tooth organoid characterization. (A) Brightfield images of DF-derived organoid cultures at different magnifications showing dense structures developed at 14 days in TOM (P4; 5-20x)). Hematoxylin and eosin staining of an organoid (P1, day 11). Box is enlarged. Arrows indicate cells with a high nucleo-cytoplasmic ratio. (B) Immunofluorescent staining for epithelial/ERM/stemness markers in TOM-grown organoids (20x). (C) Immunofluorescent staining for amelogenin (AMELX) in TOM-grown organoid (20x). DAPI (blue) was used to label nuclei. (D) Gene expression levels (relative to GAPDH) of ERM/stemness markers in P1 and P5 TOM-grown organoids at day 14 of culture (mean ± SEM; n = 3 biological replicates). Scale bars: 50 µm, unless indicated otherwise. Please click here to view a larger version of this figure.
Dental follicle (DF) collection medium | |
Name | Concentration |
Minimum essential medium eagle (αMEM) | |
Fetal bovine serum (FBS) | 10% |
Amphotericin B | 0.5% |
Penicillin-streptomycin (Pen/Strep) | 1% |
Table 1: Dental follicle (DF) collection medium. The table lists the constituents of the DF collection medium.
Tooth organoid medium (TOM) | |
Name | Concentration |
Serum-free defined medium (SFDM) | See Table 3 for composition |
A83-01 | 0.5 µM |
B27 (without vitamin A) | 2% |
Cholera Toxin | 100 ng/mL |
FGF2 (= basic FGF) | 20 ng/mL |
FGF8 | 200 ng/mL |
FGF10 | 100 ng/mL |
L-Glutamine | 2 mM |
IGF-1 | 100 ng/mL |
N2 | 1% |
N-acetyl L-cysteine | 1.25 mM |
Nicotinamide | 10 mM |
Noggin | 100 ng/mL |
RSPO1 | 200 ng/mL |
SB202190 (p38i) | 10 µM |
SHH | 100 ng/mL |
WNT3a | 200 ng/mL |
Table 2: Tooth organoid medium (TOM). The table lists the constituents and their respective concentrations required to prepare the tooth organoid medium.
Serum-free defined medium (SFDM) (pH 7.3) | |
Name | Concentration |
Sterile H2O | |
DMEM 1:1 F12 without Fe | 16.8 g/L |
Transferrin | 5 mg/L |
Insulin from bovine pancreas | 5 mg/L |
Penicillin G sodium salt | 35 mg/L |
Streptomycin sulfate salt | 50 mg/L |
Ethanol absolute, ≥99.8% (EtOH) | 600 µL/L |
Catalase from bovine liver | 50 µL/L |
Sodium Hydrogen Carbonate (NaHCO3) | 1 g/L |
Albumin Bovine (cell culture grade) | 5 g/L |
Table 3: Serum-free defined medium (SFDM) (pH 7.3). The table lists the composition of the serum-free defined medium.
Dissociation medium | |
Name | Concentration |
Phosphate buffered saline (PBS) | |
Collagenase IV | 3 mg/mL |
Dispase II | 4 mg/mL |
Table 4: Dissociation medium. List of constituents and their required concentrations for preparing the dissociation medium.
Medium A (pH 7.3) | |
Name | Concentration |
Sterile H2O | |
DMEM powder high glucose | 13.38 g/L |
HEPES | 5.958 g/L |
Sodium-pyruvate (C3H3NaO3) | 110 mg/L |
Penicillin G sodium salt | 35 mg/L |
Streptomycin sulfate salt | 50 mg/L |
Sodium Chloride (NaCl) | 0.5 g/L |
Sodium Hydrogen Carbonate (NaHCO3) | 1 g/L |
Albumin Bovine (cell culture grade) | 3 g/L |
Dnase* | 0.2 mg/mL |
*add when mentioned |
Table 5: Medium A (pH 7.3). The table lists the concentration of the constituents used to prepare Medium A.
Primary Antibodies | ||
Name | Host | Concentration |
AMELX | mouse | 1:100 |
CD44 | mouse | 1:200 |
CK14 | mouse | 1:200 |
ITGA6 | rabbit | 1:200 |
P63 | rabbit | 1:1000 |
SOX2 | rabbit | 1:2000 |
Secundary Antibodies | ||
Name | Host | Concentration |
mouse IgG (Alexa 555) | donkey | 1:1000 |
rabbit IgG (Alexa 488) | donkey | 1:1000 |
Table 6: List of antibodies and their dilutions. The table lists the antibodies and their respective dilutions used in this study.
Primers | ||
Gene | Forward primer | Reverse primer |
GAPDH | GGTATCGTGGAAGGACTCATGAC | ATGCCAGTGAGCTTCCCGTTCAG |
P63 | CAACGCAGTAGACACCATTTCC | CCCAAAACCTTCTCGCTTGTT |
ITGA6 | GGCGGTGTTATGTCCTGAGTC | AATCGCCCATCACAAAAGCTC |
SOX2 | GCTGGGACATGTGAAGTCTG | CCCTGTGGTTACCTCTTCCT |
PITX2 | CAGCGGACTCACTTTACCAG | ATTCTTGAACCAAACCCGGAC |
Table 7: List of primers. The table lists the primers of GAPDH, P63, ITGA6, SOX2, and PITX2 used in this study.
This protocol describes the efficient and reproducible generation of organoids starting from the human tooth. To our knowledge, this is the first methodology for establishing current-concept (epithelial) organoids starting from human dental tissue. The organoids are long-term expandable and display a tooth epithelial stemness phenotype, duplicating DESCs previously reported in the ERM compartment of the DF7. Moreover, the organoids replicate functional DESC/ERM characteristics, including the unfolding of an ameloblast differentiation process7,25,26. Findings are robust since comparable results were found with independent patient organoid lines19.
When executing this tooth organoid protocol, several critical points need to be taken into account. First, the addition of Rho-associated kinase (ROCK) inhibitor Y-27632 at initial seeding and immediately after each passaging is essential to prevent the single cells from undergoing anoikis27. In addition, Amphotericin B is required in all media refreshments during P0 to avoid (oral) fungal outgrowth. Second, it is advised to immediately process the freshly isolated DF tissues for the optimal organoid formation and growth efficiency, rather than to start from cryopreserved tissue, which results in lower efficiency. Third, when thawing a cryopreserved organoid line for culturing, perform steps as rapidly as possible and avoid too long thawing time as well as too long intervals between the steps as time prolongation decreases cell survival. Fourth, it should be noted that the number of organoids at early passage (P0-P3) may remain limited, also because only limited numbers of ERM (stem) cells may be present in the specific isolated DF tissue samples. Hence, the organoid cultures at early passage should be handled with care and consideration. Therefore, it is recommended to (i) avoid rapid expansion of the organoid culture (i.e., only start to split at 1:3 or more starting from P3-4, and before at 1:0.5 or 1:1); (ii) use the appropriate splitting method (low passage – higher passage) as described. Within this context, it is advised to collect unerupted wisdom teeth of young adolescent patients (15 to 19 years old) since ERM cells decrease in number with tooth development and age28. Fifth, remaining undispersed hard tissue fragments from the DF tissue in the cell suspension (even after filtering) cause the BMM drop to be less stable and more likely to dislodge during culture. A higher percentage of BMM (such as 80%) is recommended if multiple undispersed hard tissue fragments are observed in the dissociated DF cell suspension. Sixth, it is strongly advised to passage the organoids between day 10 and day 14 of culture since longer culture will negatively affect organoid expandability because of less optimal dissociation. If for one or the other reason, organoids are cultured longer than 14 days, TryplE Express quantity and incubation time for organoid dissociation could be extended for efficient dissociation, although 15 min of enzymatic exposure should not be surpassed. Within the same context, the culture medium must be refreshed every 2-3 days to prevent nutrient and growth factor exhaustion. In case organoids do not expand properly, regardless of the critical points mentioned above, one should focus on keeping all tools (BMM, ice-cold SFDM for pre-coating tip, microcentrifuge tubes) used during passaging on ice. In addition, it is crucial to correctly apply the distinct passaging methods (low passage and higher passage method) for efficient passaging of the organoids.
Before, other groups have reported in vitro growth of primary human DESC/ERM tissue8,9,10,11,12,21. However, cultures were mainly 2D (monolayers) and not 3D, such as this organoid model, moreover only showing short-term growth and phenotype retention. Alternatively, often (spontaneously) immortalized cells were used, which, however, are not physiological and show only limited resemblance to the tissue or cells of origin. Moreover, these cell lines were derived from embryonic tissue and/or from animals. Furthermore, ameloblast differentiation is either not described or only limitedly documented. Thus, the organoid model presented here offers several advantages, being (i) faithful recapitulation of the tissue/cells of origin, (ii) long-term expandable, (iii) cultured in 3D more closely representing the in vivo configuration, (iv) of human origin and postnatal age, and (v) capable of differentiating into mature dental cells (ameloblast cell type) (see19).
Thus, we generated a valuable research tool, not reported before, holding several interesting applications (Figure 1B). The organoids can be applied to study human DESC/ERM stemness and plasticity. It provides the opportunity to gain further insight into the biology of the still enigmatic ERM cell population by means of immunofluorescent, gene expression, and (single-cell) transcriptomic analyses. In addition, organoids are particularly suited for human disease modeling to decipher pathogenetic mechanisms, identify (new) therapeutic targets and generate drug discovery and screening tools29. More specifically, this model can be applied to odontogenic cysts (for which no reliable research model is available), which can be compared to healthy tooth-derived organoids. In addition, this tooth organoid approach may be harnessed to model and study tooth diseases ranging from the impact of bacteria to genetic mutations associated with tooth anomalies (such as mutations in P63, which could be introduced using cutting-edge gene-editing methods such as CRISPR-Cas)30, eventually leading to potential, and novel, therapeutic targets, and treatments. Other applications of the tooth organoid protocol may include biobanking (currently already available for dental pulp, such as the Future Health Biobank)31 to collect organoid lines from manifold persons and diseases (e.g., for basic and translational research such as drug screening). Furthermore, several reports on composite organoid models containing not only epithelial but also other cell types of the tissue of origin have recently been published32,33. As tooth composition is rather complex, accommodating mesenchymal, immune, and endothelial cells, applying this epithelial organoid model in combination with these cell types to more in detail represent their in vivo counterpart is an appealing perspective. Also, this system allows to explore amelogenesis in the human tooth, at present only poorly understood, but certainly relying on epithelial-mesenchymal interactions. Deciphering ameloblast development is expected to represent an important leap forward in the dental scientific and clinical world since the production of enamel, the quintessential component of our teeth, is a highly chased goal in dental tissue repair. Moreover, the organoid modeling detailed in this study may signify the start toward the formation of mineralized tissues in vitro and pave the way toward developing a bioengineered tooth (or at least parts) for replacement therapy.
One of the limitations of the organoid model is that it solely represents the epithelial component of tissue. However, as described in detail above, this shortcoming could be solved by the addition of other cell/tissue types, such as the dental mesenchyme19. Another aspect that may be recognized as a limitation is the origin of the BMM used here (Matrigel). This BMM is derived from a sarcoma (Engelbrecht-Holm-Swarm) of a mouse and therefore must be replaced before translating the organoid approach to the clinic. Recently, several efforts have been made to replace Matrigel with synthetic hydrogels34,35. However, more research is needed to successfully grow organoids in such non-natural gels. Although the organoid technology provides an interesting approach for future dental regenerative therapy – for instance, the development of a bioengineered tooth – ethical questions should be raised regarding the privacy of cell donors as well as the commercialization of human organoids and tissues derived thereof. So far, no conclusions regarding organoid commercialization for regenerative purposes have been reached36. Dental pulp biobanks have been on the rise31, as well as organoid biobanks from several, mainly cancerous tissues, for drug screening purposes. Given that organoids cannot be categorized as cells, gametes, tissues, or organs (which all are regulated by law), there is an urgent need for depicting its juridical status for its use in clinical, scientific, or commercial settings. Even though the organoids have shown to deposit mineralized tissue when subcutaneously transplanted in vivo19, further studies are required to analyze their potential to deposit enamel similar to that of a natural human tooth.
Altogether, the new organoid model developed presents a promising, valuable tool to study human tooth (stem cell) biology and amelogenesis, both at present only poorly explored, with future perspectives toward tooth disease modeling and regenerative therapies.
The authors have nothing to disclose.
We are grateful to all staff members of the Oral and Maxillofacial Surgery (MKA) of UZ Leuven, as well as the patients, for their invaluable help in collecting freshly extracted third molars. We would also like to thank Dr. Reinhilde Jacobs and Dr. Elisabeth Tijskens for their help with the sample collection. This work was supported by grants from KU Leuven (BOF) and FWO-Flanders (G061819N). L.H. is an FWO Ph.D. Fellow (1S84718N).
1.5 mL Microcentrifuge tube | Eppendorf | 30120.086 | |
15 mL Centrifuge tube | Corning | 430052 | |
2-Mercaptoethanol | Sigma-Aldrich | M-6250 | |
48-well flat bottom plates | Corning | 3548 | |
50 mL Centrifuge tube | Corning | 430290 | |
A83-01 | Sigma-Aldrich | SML0788 | |
Agarose | Lonza | 50004 | |
Albumin Bovine (cell culture grade) | Serva | 47330.03 | |
AMELX antibody | Santa Cruz | sc-365284 | |
Amphotericin B | Gibco | 15200018 | |
B27 (without vitamin A) | Gibco | 12587-010 | |
Cassette | VWR | 7202191 | |
Catalase from bovine liver | Sigma-Aldrich | C100 | |
CD44 antibody | Abcam | ab34485 | |
Cell strainer, 40 µm | Falcon | 352340 | |
Cholera Toxin | Sigma-Aldrich | C8052 | |
Citric acid | Sigma-Aldrich | C0759 | |
CK14 antibody | Thermo Fisher Scientific | MA5-11599 | |
Collagenase IV | Gibco | 17104-019 | |
Cover glass | VWR | 6310146 | |
Cryobox | Thermo Scientific | 5100-0001 | |
Cryovial | Thermo Fisher Scientific | 375353 | |
Dimethylsulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
Dispase II | Sigma-Aldrich | D4693 | |
DMEM 1:1 F12 without Fe | Invitrogen | 074-90715A | |
DMEM powder high glucose | Gibco | 52100039 | |
Dnase | Sigma-Aldrich | D5025-15KU | |
Donkey serum | Sigma-Aldrich | D9663 – 10ML | |
Embedding workstation, 220 to 240 Vac | Thermo Fisher Scientific | 12587976 | |
Ethanol absolute, ≥99.8% (EtOH) | Fisher Chemical | E/0650DF/15 | |
Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
FGF10 | Peprotech | 100-26 | |
FGF2 (= basic FGF) | R&D Systems | 234-FSE-025 | |
FGF8 | Peprotech | AF-100-25 | |
GenElute Mammaliam Total RNA Miniprep Kit | Sigma-Aldrich | RTN350-1KT | Includes 1% β-mercaptoethanol dissolved in lysis buffer |
Glass Pasteur pipette | Niko Mechanisms | 170-40050 | |
Glycine | VWR | 101194M | |
HEPES | Sigma-Aldrich | H4034 | |
IGF-1 | PeproTech | 100-11 | |
InSolution Y-27632 (ROCK inhibitor, RI) | Sigma-Aldrich | 688001 | |
Insulin from bovine pancreas | Sigma-Aldrich | I6634 | |
ITGA6 antibody | Sigma-Aldrich | HPA012696 | |
L-Glutamine | Gibco | 25030024 | |
Matrigel (growth factor-reduced; phenol red-free) | Corning | 15505739 | |
Microscope slide | Thermo Fisher Scientific | J1800AMNZ | |
Millex-GV Syringe Filter Unit, 0.22 μm | Millipore | SLGV033R | |
Minimum essential medium eagle (αMEM) | Sigma-Aldrich | M4526 | |
mouse IgG (Alexa 555) secondary antibody | Thermo Fisher Scientific | A-31570 | |
N2 | Gibco | 17502-048 | |
N-acetyl L-cysteine | Sigma-Aldrich | A7250 | |
Nicotinamide | Sigma-Aldrich | N0636 | |
Noggin | PeproTech | 120-10C | |
P63 antibody | Abcam | ab124762 | |
Pap Pen | Sigma-Aldrich | Z377821-1EA | Marking pen |
Paraformaldehyde (PFA), 16% | Merck | 8.18715 | |
Penicillin G sodium salt | Sigma-Aldrich | P3032 | |
Penicillin-streptomycin (Pen/Strep) | Gibco | 15140-122 | |
Petri dish | Corning | 353002 | |
Phosphate buffered saline (PBS) | Gibco | 10010-015 | |
Pipette (P20, P200, P1000) | Eppendorf or others | 2231300006 | |
Plastic transfer pipette (3.5 mL) | Sarstedt | 86.1171.001 | |
Rabbit IgG (Alexa 488) secondary antibody | Thermo Fisher Scientific | A21206 | |
RSPO1 | PeproTech | 120-38 | |
SB202190 (p38i) | Biotechne (Tocris) | 1264 | |
Scalpel (surgical blade) | Swann-Morton | 207 | |
SHH | R&D Systems | 464-SH-200 | |
Silicone molds (Heating block) | VWR | 720-1918 | |
Sodium Chloride (NaCl) | BDH | 102415K | |
Sodium Hydrogen Carbonate (NaHCO3) | Merck | 106329 | |
Sodium-pyruvate (C3H3NaO3) | Sigma-Aldrich | P-5280 | |
SOX2 antibody | Abcam | ab92494 | |
StepOnePlus | Thermo Fisher Scientific | Real-Time PCR System | |
Stericup-GP, 0.22 µm | Millipore | SCGPU02RE | |
Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 μm | Millipore | SCGP00525 | |
Sterile 1000 μL pipette tips with filter | Greiner | 740288 | |
Sterile 20 μL pipette tips with filter | Greiner | 774288 | |
Sterile 200 μL pipette tips with and without filter | Greiner | 739288 | |
Sterile H2O | Fresenius | B230531 | |
Streptomycin sulfate salt | Sigma-Aldrich | S6501 | |
Superscript III first-strand synthesis supermix | Invitrogen | 11752-050 | Reverse transcription kit |
Tissue processor | Thermo Scientific | 12505356 | |
Transferrin | Serva | 36760.01 | |
Triton X-100 | Sigma | T8787-50ML | |
TrypLE express | Gibco | 12605-010 | |
Vectashield mounting medium+DAPI | Labconsult NV | H-1200 | Antifade mounting medium with DAPI |
WNT3a | Biotechne (Tocris) | 5036-WN-500 | |
Xylenes, 99%, for biochemistry and histology | VWR | 2,89,75,325 |