This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.
Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.
Esophageal squamous cell carcinoma (ESCC) is the deadliest of human squamous cell carcinomas, owing to its late diagnosis, therapy resistance, and metastasis1,2. ESCC arises from the stratified squamous epithelium, which lines the luminal surface of the esophagus. The squamous epithelium is comprised of proliferative basal cells and differentiated cells within the suprabasal cell layer. Under physiologic conditions, basal cells express markers such as p63, Sox2, and cytokeratin K5 and K14, while differentiated cells express K4, K13, and IVL. Basal cells themselves are heterogeneous and include putative stem cells defined by markers such as K153 and CD734. In homeostasis, basal cells undergo post-mitotic terminal differentiation within the suprabasal cell layer, whereas differentiated cells migrate and desquamate into the lumen to complete epithelial renewal. Reminiscent of their cells of origin, ESCC displays squamous cell differentiation to varying degrees. ESCC is often accompanied by multifocal histologic precursor lesions, known as intraepithelial neoplasia (IEN) or dysplasia, comprising atypical basaloid cells. In addition to epithelial changes, ESCC displays tissue remodeling within the subepithelial compartment, where the activation of cancer-associated fibroblasts (CAFs) and the recruitment of immune/inflammatory cells take place to foster the tumor-promoting microenvironment.
The pathogenesis of ESCC involves genetic changes and exposure to environmental risk factors. Key genetic lesions include the inactivation of the tumor suppressor genes TP53 and CDKN2A (p16INK4A) and the activation of the CCND1 (cyclin D1) and EGFR oncogenes, which culminate in impaired cell cycle checkpoint function, aberrant proliferation, and survival under genotoxic stress related to exposure to environmental carcinogens. Indeed, genetic changes interact closely with behavioral and environmental risk factors, most commonly tobacco and alcohol use. Tobacco smoke contains human carcinogens such as acetaldehyde, which is also the major metabolite of alcohol. Acetaldehyde induces DNA adducts and interstrand DNA crosslinks, leading to DNA damage and the accumulation of DNA mutations and chromosomal instability. Given excessive mitogenic stimuli and aberrant proliferation from oncogene activation, the malignant transformation of esophageal epithelial cells is facilitated by mechanisms to cope with genotoxic stress, including the activation of antioxidants, autophagy, and epithelial-mesenchymal transition (EMT). Interestingly, these cytoprotective functions are often activated in ESCC cancer stem cells (CSCs) that are characterized by high CD44 (CD44H) expression and have the capabilities of tumor initiation, invasion, metastasis, and therapy resistance5,6,7.
ESCC has been modeled in cell culture and in rodent models8,9. In the last three decades, robust genetically engineered mouse models of ESCC have been developed. These include CCND1 and EGFR transgenic mice10,11 and p53 and p120Ctn knockout mice12,13. However, single genetic changes do not typically result in rapid-onset ESCC. This challenge has been overcome with the use of esophageal carcinogens that recapitulate well the human genetic lesions in ESCC14. For example, 4-nitroquinoline-1-oxide (4NQO) accelerates ESCC development in CCND1 transgenic mice15. In recent years, putative esophageal epithelial stem cells, progenitor cells, and their respective fates have been investigated in cell lineage-traceable mouse models3,4. Furthermore, these cell lineage-traceable mice have been utilized to explore the cells of origin of ESCC and how such cells give rise to CD44H CSCs via conventional histology and omics-based molecular characterization7.
One emerging area related to these mouse models is the novel application of cell culture techniques to analyze live ESCC and precursor cells in a three-dimensional (3D) organoid system in which the architecture of the original tissues is recapitulated ex vivo7,8,9. These 3D organoids are rapidly grown from a single-cell suspension isolated from murine tissues, including primary and metastatic tumors (e.g., lymph node, lung, and liver lesions). The cells are embedded in basement membrane extract (BME) and fed with a well-defined serum-free cell culture medium. The 3D organoids grow within 7-10 days, and the resulting spherical structures are amenable for subculture, cryopreservation, and assays for analyzing a variety of cellular properties and functions, including CSC markers, EMT, autophagy, proliferation, differentiation, and apoptotic cell death.
These methods can be broadly applied to 3D organoid cultures established from any stratified squamous epithelial tissue, such as the head and neck mucosa (oral cavity, tongue, pharynx, and larynx) and even the forestomach. The head and neck mucosa are contiguous with the esophagus, and the two tissues share similar tissue organization, function, and susceptibility to disease. Both head and neck squamous cell carcinoma (HNSCC) and ESCC share genetic lesions and lifestyle-related environmental risk factors such as tobacco and alcohol exposure. Underscoring this similarity, mice treated with the tobacco smoke mimetic 4NQO readily develop both HNSCC and ESCC. Given the ease with which the protocols described below can be applied to modeling HNSCC, we include specific instructions for establishing 3D organoid cultures from these lesions.
Herein, we provide detailed protocols for generating murine esophageal 3D organoids (MEOs) representing normal, preneoplastic, and ESCC lesions that develop in mice treated with 4NQO. Various mouse strains can be utilized, including common laboratory strains such as C57BL/6 and cell lineage-traceable and other genetically engineered derivatives. We emphasize the key steps, including the isolation of normal or diseased murine esophageal epithelium, the preparation of single-cell suspensions, the cultivation and monitoring of the growing 3D organoids, subculture, cryopreservation, and the processing for subsequent analyses, including morphology and other applications.
The murine experiments were planned and performed in accordance with regulations and under animal protocol #AABB1502, reviewed and approved by Columbia University's Institutional Animal Care and Use Committee. The mice were housed at a proper animal care facility that ensures the humane treatment of mice and provides appropriate veterinary care for the mice and laboratory safety training for the laboratory personnel.
1. Treatment of mice with 4NQO to induce esophageal IEN and ESCC lesions (time consideration: up to 28 weeks)
NOTE: To generate MEOs representing neoplastic esophageal lesions, the mice are subjected to 4NQO-mediated chemical carcinogenesis as previously described by Tang et al.14. Normal/non-neoplastic MEOs are generated from untreated mice.
2. Establishment of murine esophageal organoid (MEO) culture
NOTE: This protocol can also be used to establish a murine tongue organoid culture with the addition of a step in which the tongue tissue is minced before trypsinization. See the note in step 2.2.3.
3. Preparation of organoids for paraffin embedding (time consideration: <1 h [plus 1.5 h for reagent preparation])
This protocol describes the process of generating murine esophageal organoids (MEOs) from normal esophageal tissue or ESCC tumor tissue from 4NQO-treated mice according to a specific treatment regimen consisting of 16 weeks of 4NQO administered in drinking water, followed by a 10 week to 12 week observation period (Figure 1). The mice are then euthanized for the dissection of the tongue or esophageal tissue (Figure 2 and Figure 3). We describe a method for the isolation of the epithelial layer from the intact esophagus (Figure 4) to be utilized for subsequent single-cell isolation. Esophagus-derived epithelial cells are initially plated at 5,000 cells per well in 50 µL droplets containing cells in a basement membrane matrix and well-characterized serum-free cell culture medium (Figure 5) and allowed to form organoids over the course of 7-10 days on average. Murine esophageal, tongue, and forestomach organoids can be further characterized by morphological analysis by phase-contrast/brightfield imaging and histopathology (Figure 6). The self-renewal capacity of organoids can be assessed by determining the OFR upon subculture (Figure 7). The OFR is largely influenced by the content of the proliferative basaloid cells, as revealed by the growth kinetics analysis coupled with their morphology (Figure 8). Finally, these organoids, including normal structures from 4NQO-untreated mice, grow continuously and can be maintained for a long time (Figure 9).
Figure 1: The 4NQO treatment regimen. The mice are treated with 4NQO in drinking water for 16 weeks, followed by a 10 week to 12 week observation period. Please click here to view a larger version of this figure.
Figure 2: Exposing the thoracic esophagus. The trachea (blue lines) is peeled away from the esophagus (white lines). Please click here to view a larger version of this figure.
Figure 3: Esophagus (white lines) connected to the stomach (yellow lines) at the squamous columnar junction (blue line). Abbreviations: FS = forestomach, DS = distal stomach, L = liver. Please click here to view a larger version of this figure.
Figure 4: Separating the stomach and esophagus and isolating the epithelium. Upper: The stomach is separated from the esophagus. The red line indicates where the esophagus should be detached during dissection. Middle: The epithelium (white line) is peeled away from the muscle layer. Lower: The esophagus bearing tumors following the treatment schedule described in Figure 1. Please click here to view a larger version of this figure.
Figure 5: Plating the BME droplet containing single cells using a wide bore tip. The organoids begin to form around days 4-7 and are ready to be passaged after 7-10 days on average. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 6: Characterizing the organoids by morphological analysis. (A) Representative images of normal and ESCC MEOs. Upper left: Brightfield image of a normal MEO. Lower left: H&E staining of a normal MEO. Upper right: Brightfield image of an ESCC MEO from a 4NQO-treated mouse. Lower right: H&E staining of an ESCC MEO from a 4NQO-treated mouse displaying nuclear atypia and abrupt keratinization, the latter being reminiscent of a keratin pearl, representing a well-differentiated compartment within an SCC tumor. (B) Representative images of normal mouse tongue and forestomach organoids (MTO and MFO, respectively). Upper left: Brightfield image of a normal MTO. Lower left: H&E staining of a normal MTO. Upper right: Brightfield image of a normal MFO. Lower right: H&E staining of a normal MFO. All scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 7: Determining organoid renewal capacity. (A) Experimental design to determine the organoid renewal capacity by subculture. The growing primary organoids (P0) are dissociated at various time points and made into single-cell suspensions. These cells are passaged to determine the OFR at day 7 in the subculture (P1). (B) Representative OFR data from passaged MEOs generated from 4NQO-untreated mice. Note that the OFR decreases as a function of time, reflecting decreased proliferative basaloid cells in the matured organoids, which contain more cells that have undergone post-mitotic terminal differentiation (see Figure 8). *** p < 0.001 (n = 6) OFR at day 7, day 11, and day 21 versus OFR at day 4. Please click here to view a larger version of this figure.
Figure 8: Growth kinetics of organoids as revealed by their morphology at various time points. Representative bright-field and H&E staining images of a normal MEO from 4NQO-untreated mice are shown. Note that the keratinization of the inner core of organoids becomes prominent by day 10. The proliferative basaloid cells remain in the outermost cell layer even at the day 14 and day 21 time points. For immunohistochemistry/immunofluorescence, accumulated keratin may cause non-specific staining, as is the case for original squamous epithelial tissues, warranting a careful optimization of the staining conditions, controls (e.g., secondary antibody only), and data interpretation. All scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 9: A population doubling curve of a representative normal MTO generated from 4NQO-untreated mice. These organoids were previously generated, cryopreserved, and thawed to demonstrate their steady growth over multiple passages and long-term culture. Please click here to view a larger version of this figure.
There are several critical steps and considerations for the generation and analysis of MEOs in the protocols described here. To ensure reproducibility and rigor in MEO experiments, biological and technical replicates are both important. For biological replicates, two to three independent mice bearing ESCC are generally sufficient per experimental condition. However, the appropriate number of biological replicates may vary depending on the parameters to be tested in individual studies. For example, it is currently unknown how early and how frequently neoplastic organoids may be detected from 4NQO-treated mice without macroscopically visible lesions or histologic IEN and ESCC lesions. Although 4NQO induces esophageal lesions in various mouse strains14, MEOs have been generated from C57BL/6 mice only. The development and progression of ESCC are accelerated in mice with genetically engineered genetic lesions such as Trp53 loss and cyclin D1 overexpression7,14,15. In such mice, neoplastic MEOs may emerge more frequently and earlier than in wild-type C57BL/6 mice during or after 4NQO treatment. If necessary, pilot studies should be performed to estimate the appropriate sample size of mice in planned experiments. With multi-well cell culture plates, technical replicates (n = 3-6) are easily created. However, careful pipetting and practice are warranted to minimize well-to-well variability as the cells are dispensed in a viscous medium containing BME.
A near 100% success rate may be expected for MEO establishment in the protocols described. However, successful MEO culture depends on the viability of the initial isolated esophageal epithelial cells, which is largely related to the quality of the starting material. When the mice cannot be immediately dissected upon euthanasia, the carcasses may be kept at 4 °C overnight to isolate the esophagi the following day. However, the carcasses should not be frozen, as frozen tissue will not yield viable cells. The dissociated esophageal epithelial cells may be cryopreserved as a single-cell suspension in a freezing medium (90% FBS and 10% DMSO) and stored in liquid nitrogen to generate organoids at a later date. The esophagi (epithelial sheets) may be cryopreserved in a similar fashion, albeit less optimally. While MEOs can be initiated with cells purified by fluorescence-activated cell sorting (FACS) and other methods to purify unique subsets of esophageal epithelial cells, the cell viability may be diminished after purification. It should be noted that cell viability assays such as the trypan blue exclusion test can distinguish live cells from dead cells but not dying cells, thus underestimating the cell viability prior to organoid culture initiation. After establishment, MEOs from both 4NQO-treated and untreated mice can be passaged indefinitely (>20 passages), with >80% viability expected at each passage in the medium specified. It is noteworthy that there are organoid medium components whose requirements remains unclear. Zheng et al. recently demonstrated that a supplement comprising Wnt3A, RSpondin-1, and Noggin is required to grow murine esophageal organoids16. However, we do not utilize Wnt3A in this protocol as organoids from the tongue, esophagus, and forestomach grow and can be passaged multiple times (>10 passages) without it7,3,17. It should be noted that the requirements of these factors have not been tested individually.
Epithelial sheets are utilized to generate MEOs from the normal esophagus or the esophageal mucosa without macroscopically visible lesions such as tumors. The use of epithelial sheets permits the enrichment of epithelial cells as a starting material. However, the isolation of the epithelial sheets from the tumor-bearing esophageal mucosa is difficult due to ESCC invasion into the subepithelial compartments. The whole esophagus can be used to initiate MEO culture, although the presence of non-epithelial cell populations may decrease the organoid formation rate (OFR) in the initial culture. The OFR is expected to rise in the subsequent passages, as the current MEO culture conditions allow epithelial cells to grow within the BME. Of note, the protocols described here can also be applied to other organs, especially the oral tongue and the forestomach, both of which are susceptible to 4NQO-induced squamous cell carcinogenesis. Of note, we have generated mouse tongue organoids (MTOs) and mouse forestomach organoids (MFOs) from the whole tissues without isolating the epithelial cells. If necessary, these organoids can be made from a subpopulation of epithelial cells purified by fluorescence-activated cell sorting (e.g., cancer stem cells characterized by high CD44 expression, CD73+ putative esophageal stem cells).
Additionally, non-epithelial cells, fibroblasts in particular, may migrate out of the BME to grow in a monolayer on the plastic surface, thereby allowing the concurrent establishment of murine esophageal fibroblasts, including cancer-associated fibroblasts. The selectivity for epithelial cell growth in the 3D BME may limit the co-culturing of MEOs with immune cells and other cell types under the current conditions described here. Optimization for co-culture experiments is underway. Additionally, the tumor invasive front may be better recapitulated in the epithelial-stromal interface modeled by alternative 3D culture methods such as organotypic 3D culture8,18. Moreover, MEOs may not be a suitable platform with which to measure the epithelial barrier function, which can be better evaluated by utilizing air-liquid interface culture to assess the transepithelial electrical resistance19,20,21.
The foremost advantage of MEOs, and indeed MTOs and MFOs, is the rapid growth of single cell-derived structures that recapitulate the original epithelial structures, both benign and malignant. The doubling time (DT) of these organoids is typically estimated to be <20 h. The growth kinetics of normal MTOs from 4NQO-untreated mice illustrate both the capability for long-term culture of normal organoids, as well as the expected population doubling (PD) level and DT (Figure 9). In this example, roughly 10 population doublings occurred during each passage, with a calculated DT of 18.5 h and 18.3 h, on average. After the initial primary culture, an OFR of 15%-25% is typically expected, regardless of seeding density, and MEOs may emerge as early as day 4 (Figure 8), though they may be smaller in size. Under phase-contrast microscopy, organoids from the normal esophageal mucosa display spherical structures with smooth surfaces. As a function of time, normal organoids display a concentric structure as the inner cell mass undergoes post-mitotic terminal differentiation, generating a differentiation gradient that mimics the stratified squamous epithelium. Neoplastic organoids show irregular surfaces, reflecting the high proliferative activity of the basaloid cells, which may expand in an outward fashion.The ability of MEOs to display a proliferation-differentiation gradient can be utilized to study how proliferative esophageal basal keratinocytes may propagate and undergo post-mitotic terminal differentiation. By subculturing dissociated MEO cells, one may evaluate epithelial cell self-renewal (Figure 7), as well as regeneration or transformation under genotoxic stress induced by 4NQO. Similar studies may also be carried out utilizing the MTO model in the context of oral cancer, broadening the application of the described 4NQO mouse organoid model to head and neck SCC. While epithelial renewal may imply stem cells, one potential weakness of the MEO system is that it may not detect quiescent or rarely dividing stem cells, as MEO formation depends upon cell proliferation. A recent single-cell analysis study on MEOs provided substantial insight into esophageal epithelial cell heterogeneity22.
Malignant transformation can be assessed by the careful morphological assessment of individual MEO structures and the nuclear atypia of cells comprising MEOs. The molecular profiling of MEOs by whole-exome sequencing and RNA sequencing may reveal the distinct nature of preneoplastic and ESCC lesions. However, the ultimate test for malignant transformation may require the transplantation of MEOs into either immunodeficient or immunocompetent mice to document the tumorigenicity of the ESCC cells. ESCC MEOs transplanted in syngeneic immunocompetent mice may also provide an excellent platform to investigate the tumor immune microenvironment.
The applications of MEOs are broad. Besides morphology, biochemistry, and multi-omics approaches, flow cytometry can be utilized not only to analyze cellular processes such as DNA synthesis, apoptosis, autophagy, and mitochondrial respiration but also cell surface markers that define unique subsets of cells within MEOs corresponding with genetic and pharmacological modifications ex vivo7,23. Furthermore, organoids may be used for co-culture experiments to evaluate the interactions between the epithelial cells and the stroma, the immune cells, or even the microbiome24. Finally, MEOs can be generated from mice carrying cell lineage-traceable genetic modifications, such as fluorescent reporter protein expressed under a cell type-specific gene regulatory promoter (e.g., Sox2, cytokeratins Krt5, and Krt15)3,4,7,25.
In summary, MEOs are an invaluable tool for the study of ESCC. The protocols described here aim to show that MEOs represent a versatile model that is relatively easy to generate, maintain, and characterize and has the ability to recapitulate the features of ESCC and esophageal neoplasia.
The authors have nothing to disclose.
We thank the Shared Resources (Flow Cytometry, Molecular Pathology, and Confocal & Specialized Microscopy) at the Herbert Irving Comprehensive Cancer Center at Columbia University for technical support. We thank Drs. Alan Diehl, Adam J. Bass, and Kwok-Kin Wong (NCI P01 Mechanisms of Esophageal Carcinogenesis) and members of the Rustgi and Nakagawa laboratories for helpful discussions. This study was supported by the following NIH Grants: P01CA098101 (H.N. and A.K.R.), R01DK114436 (H.N.), R01AA026297 (H.N.), L30CA264714 (S.F.), DE031112-01 (F.M.H.), KL2TR001874 (F.M.H.),3R01CA255298-01S1 (J.G.), 2L30DK126621-02
(J.G.) R01CA266978 (C.L.), R01DK132251 (C.L.), R01DE031873 (C.L.), P30DK132710 (C.M. and H.N.), and P30CA013696 (A.K.R.). H.N. and C.L. are recipients of the Columbia University Herbert Irving Comprehensive Cancer Center Multi-PI Pilot Award. H.N. is a recipient of the Fanconi Anemia Research Fund Award. F.M.H. is the recipient of The Mark Foundation for Cancer Research Award (20-60-51-MOME) and an American Association for Cancer Research Award. J.G. is the recipient of the American Gastroenterological Association (AGA) award.
0.05% trypsin-EDTA | Thermo Fisher Scientific | 25-300-120 | |
0.25% trypsin-EDTA | Thermo Fisher Scientific | 25-200-114 | |
0.4% Trypan Blue | Thermo Fisher Scientific | T10282 | |
1 mL tuberculin syringe without needle | BD | 309659 | |
1.5 mL microcentrifuge tube | Thermo Fisher Scientific | 05-408-129 | |
100 µm cell strainer | Thermo Fisher Scientific | 22363549 | |
15 mL conical tubes | Thermo Fisher Scientific | 14-959-53A | |
200 µL wide bore micropipette tips | Thermo Fisher Scientific | 212361A | |
21 G needles | BD | 305167 | |
24 well plate | Thermo Fisher Scientific | 12-556-006 | |
4-Nitroquinoline-1-oxide (4NQO) | Tokyo Chemical Industry | NO250 | |
50 mL conical tubes | Thermo Fisher Scientific | 12-565-270 | |
6 well plate | Thermo Fisher Scientific | 12556004 | |
70 µm cell strainer | Thermo Fisher Scientific | 22363548 | |
99.9% ethylene propylene glycol | SK picglobal | ||
Advanced DMEM/F12 | Thermo Fisher Scientific | 12634028 | |
Amphotericin B | Gibco, Thermo Fisher Scientific | 15290018 | Stock concentration 250 µg/mL, final concentration 0.5 µg/mL |
Antibiotic-Antimycotic | Thermo Fisher Scientific | 15240062 | Stock concentration 100x, final concentration 1x |
B-27 supplement | Thermo Fisher Scientific | 17504044 | Stock concentration 50x, final concentration 1x |
Bacto agar | BD | 214010 | |
CO2 incubator, e.g.Heracell 150i | Thermo Fisher Scientific | 51026406 | or equivalent |
Countess II FL Automated Cell Counter | Thermo Fisher Scientific | AMQAX1000 | or equivalent |
Cryovials | Thermo Fisher Scientific | 03-337-7D | |
DietGel 76A | Clear H2O | 72-07-5022 | |
Dimethyl sulfoxide (DMSO) | MilliporeSigma | D4540 | |
Dispase | Corning | 354235 | Stock concentration 50 U/mL, final concentration 2.5–5 U/mL |
Dissecting scissors | VWR | 25870-002 | |
Dulbecco's phosphate-buffered saline (PBS) | Thermo Fisher Scientific | 14190250 | Stock concentration 1x |
Fetal bovine serum (FBS) | HyClone | SH30071.03 | |
Forceps | VWR | 82027-386 | |
Freezing container | Corning | 432002 | or equivalent |
Gelatin | Thermo Fisher Scientific | G7-500 | |
GlutaMAX | Thermo Fisher Scientific | 35050061 | Stock concentration 100x, final concentration 1x |
HEPES | Thermo Fisher Scientific | 15630080 | Stock concentration 1 M, final concentration 10 mM |
Hot plate/stirrer | Corning | PC-420D | or equivalent |
Lab Armor bead bath (or water bath) | VWR | 89409-222 | or equivalent |
Laboratory balance | Ohaus | 71142841 | or equivalent |
Matrigel basement membrane extract (BME) | Corning | 354234 | |
Microcentrifuge Minispin | Eppendorf | 22620100 | or equivalent |
Microcentrifuge tube rack | Southern Labware | 0061 | |
N-2 supplement | Thermo Fisher Scientific | 17502048 | Stock concentration 100x, final concentration 1x |
N-acetylcysteine (NAC) | Sigma-Aldrich | A9165 | Stock concentration 0.5 M, final concentration 1 mM |
Parafilm M wrap | Thermo Fisher Scientific | S37440 | |
Paraformaldehyde (PFA) | MilliporeSigma | 158127-500G | |
Pathology cassette | Thermo Fisher Scientific | 22-272416 | |
Phase-contrast microscope | Nikon | or equivalent | |
Recombinant mouse epidermal growth factor (mEGF) | Peprotech | 315-09-1mg | Stock concentration 500 ng/µL, final concentration 100 ng/mL |
RN cell-conditioned medium expressing R-Spondin1 and Noggin (RN CM) | N/A | N/A | Available through the Organoid and Cell Culture Core upon request, final concentration 2% |
Sorval ST 16R centrifuge | Thermo Fisher Scientific | 75004380 | or equivalent |
Soybean trypsin inhibitor (STI) | MilliporeSigma | T9128 | Stock concentration 250 µg/mL |
ThermoMixer C | Thermo Fisher Scientific | 14-285-562 PM | or equivalent |
Y-27632 | Selleck Chemicals | S1049 | Stock concentration 10 mM, final concentration 10 µM |