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This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.
The esophagus is a hollow tubular organ whose wall comprises a mucosa or epithelium (with sparse lamina propria), submucosa (containing mucus-secreting glands and papillae), muscle (the proximal esophagus has striated muscle and the distal esophagus has smooth muscle) and adventitia. The epithelium is squamous and stratified, similar to the skin, oropharynx, trachea and anogenital tract. The stratified squamous esophageal epithelium protects against mechanical and chemical insults, and, in this context, has a barrier function. Infectious organisms, such as viruses (cytomegalovirus virus, Epstein-Barr virus, herpes simplex virus, human papillomavirus and human immunodeficiency virus), bacteria and fungi (Candida albicans) may cause esophagitis. Acid reflux from the stomach and bile reflux from gastroduodenal contents may cause erosive esophagitis, often in the distal esophagus. It is known that acid and bile reflux can cause either an incomplete intestinal metaplasia at the esophagogastric junction or Barrett’s esophagus, which is a precursor to esophageal adenocarcinoma. The other major subtype of esophageal malignancy is esophageal squamous cell carcinoma (ESCC).
The esophageal epithelium has several compartments: (i) a proliferative basal cell compartment in which cells reside on the basement membrane; (ii) a differentiating suprabasal cell compartment; and (iii) a superficial squamous cell compartment. Basal cells migrate in an outward direction to the lumen, undergoing a transition from proliferation to terminal differentiation. Eventually, cells desquamate at the luminal surface. This process is renewed on a continuous basis1. There are distinct keratins expressed in stratified squamous epithelia2. As with the skin3,4, the esophageal basal cells express cytokeratins 5 and 14 (CK5, CK14)5. Whereas the skin suprabasal and superficial layers express CK1 and CK10 (ref. 4), the esophageal suprabasal layer express CK4 (ref. 6) and CK13 (ref. 7). The superficial layer of the esophagus expresses involucrin and filaggrin8,9. The mouse esophageal epithelium has a keratinized layer on its surface; the human esophageal epithelium lacks this feature.
Human esophageal epithelial cells can be cultured in 2D, and may or may not use a 3T3 fibroblast layer as a support10,11. Apart from the human esophagus, there have been reports of cultivating esophageal epithelial cells from different species, such as rabbit12. In human esophageal epithelial cells, SV40 T-antigen was introduced to induce immortalization13. We have successfully isolated primary human esophageal cells (EPCs) that undergo replicative senescence after 42–45 population doublings. Retroviral transduction with human telomerase reverse transcriptase (hTERT) overcomes replicative senescence and these cells are immortalized14. EPC-hTERT cells can be placed into 3D OTC with recapitulation of the normal stratified squamous epithelium14. The organotypic 3D culture has been used to provide insights into mechanisms underlying normal esophageal epithelial biology and esophageal cancer biology14–18.
There are intrinsic advantages in evaluating EPCs in 3D culture versus 2D culture. The 3D OTC system for esophageal epithelial cells is unique. Its advantages relate to the normal polarization and differentiation patterns of cells, the expression of genes involved in the adherens junctions and tight junctions and the gene signatures that resemble in vivo tissues. Esophageal epithelial cells immortalized with hTERT can constitute a complete stratified squamous epithelium after exposure to a liquid-air interface14. Perturbations in the esophageal epithelium can be observed with epidermal growth factor receptor (EGFR) overexpression15, resulting in epithelial hyperplasia. The expression of inducible AKT in esophageal epithelial cells results in an expansion of the proliferating basal cells and impaired (delayed) differentiation16. Epithelial cells may be transformed by the introduction of a combination of oncogenes and/or inactivated tumor suppressor genes (e.g., EGFR, cyclin D1 and mutant p53), and the resulting transformed epithelial cells invade into the underlying matrix, thereby providing a platform to investigate properties of tumor cells and the cross talk between invading tumor cells and activated stromal fibroblasts17–19. Genes that mediate tumor invasion can be identified using laser capture microdissected cells from OTC17,20. Gene expression can be modified using retrovirally or lentivirally mediated shRNA in esophageal epithelial cells and fibroblasts16,19,21. In addition, gene expression can be modulated in the 3D context using inducible systems22. Cell signaling pathways in the reconstituted epithelia can be interrogated by pharmacological inhibitors, although such agents may influence both epithelial and fibroblast functions15,22. In addition, the self-renewal capacity of mouse esophageal stem cells can be demonstrated using OTC23.
The steps involved in the development of OTC are outlined in Figure 1. The process involves the casting of an acellular collagen matrix on the bottom of an insert, followed by the casting of a layer of esophageal fibroblasts mixed with collagen type I and Matrigel. Matrigel is not required for the formation of stratified squamous epithelium, but it facilitates invasion of transformed epithelial cells or ESCC cells. These two layers serve as a substitute for the esophageal ‘mesenchyme’ and are cultured initially for up to 7 d, thereby allowing for fibroblast-mediated constriction of the collagen matrix. Sources of epithelial cells for OTC are the following: (i) primary mouse esophageal keratinocytes, (ii) primary mouse esophageal stem cells using fluorescence-activated cell sorting (FACS), (iii) primary human esophageal keratinocytes (EPCs), (iv) immortalized human esophageal keratinocytes (EPC-hTERT cells) and (v) esophageal cancer cell lines. On day 5, the epithelial cells are seeded on the surface of the constricted matrix. The medium of the OTC is changed every 2 d and the epithelium is exposed to air to create a liquid-air interface, thereby promoting epithelial stratification and differentiation. Finally, on day 15, the resulting OTC may be processed for histology, and subsequent immunohistochemistry or immunofluorescence. In addition, the epithelium may be peeled away from the matrix and processed for RNA or protein isolation. Specific cell populations (e.g., epithelial cells, regions of fibroblasts in the matrix) may be isolated using laser capture microdissection (LCM) for RNA isolation and subsequent in vitro RNA amplification and microarray analysis or quantitative reverse-transcription PCR (Fig. 2). The conditioned medium from OTC has been used for the detection of proteins by western blot analysis, ELISA or proteomics.
Use to the KSFM 500 ml of KSFM without CaCl2. Add 30 μl of 300 mM CaCl2 (final concentration 0.018 mM). Add EGF to a final concentration of 1 ng ml −1, 25 mg of BPE (the whole content of the vial supplied) and 5 ml of penicillin-streptomycin. For primary culture, add gentamicin sulfate (5 μg ml −1) and nystatin (100 U ml −1) for the first 2 weeks. Store at 4 °C for up to 1 month. ▲ CRITICAL Warm medium to 37 °C before use for primary culture to avoid cell stress.
Use 500 ml of KSFM with 0.09 mM CaCl2. Add EGF (final concentration 1 ng ml −1), 25 mg of BPE and 5 ml of penicillin-streptomycin as in m-KSFM. For primary culture, add gentamicin sulfate (5 μg ml −1) and nystatin (100 U ml −1) for the first 2 weeks. Store at 4 °C for up to 1 month. ▲ CRITICAL Warm medium to 37 °C before use.
Use 500 ml of DMEM supplemented with 50 ml of 100% FBS and 5 ml penicillin-streptomycin. Store at 4 °C for up to 1 month.
Dissolve 250 mg of soybean trypsin inhibitor in 1 liter of Dulbecco’s PBS. Filter-sterilize with a filter unit (1,000-ml capacity) and dispense into 50-ml tubes and store at 4 °C for up to 6 months. ▲ CRITICAL Trypsin inhibition is essential before human and mouse esophageal keratinocytes are seeded in KSFM-base medium as it contains a low level of protein (i.e., EGF and BPE).
Grow cells at 37 °C in a 5% CO2 incubator. If required, perform retrovirus/lentivirus-mediated stable gene transduction in human and mouse esophageal fibroblasts, keratinocytes (i.e., EPC and EPC-hTERT derivatives) and esophageal cancer cell lines with the vectors listed in Table 1 and used for 3D OTC.
Add penicillin-streptomycin (1% (vol/vol)), gentamicin sulfate (5 μg ml −1) and nystatin (100 U ml −1) to 1 liter of HBSS and store the mixture at 4 °C for up to 6 months.
Dispase I is shipped as a vial containing ~2 mg (≥20 U per vial) lyophilized enzyme; dissolve in HBSS to 10 mg ml −1. Sterilize through a 0.2-μm filter membrane, aliquot and store at −20 °C for up to 1 year. Dilute further in HBSS to 1 U ml − 1 as a working solution.
Reconstitute (≥125 U mg− 1) collagenase in HBSS to 1 mg ml− 1 (≥125 U ml− 1). Sterilize through a 0.2-μm filter membrane, aliquot and store at − 20 °C for up to 1 year.
Store 10× EMEM and 7.5% (wt/vol) NaHCO3 at room temperature (25 °C) for up to 2 years. Avoid repeating freezing and thawing cycles more than four times. Prepare aliquots of the reagent and store at − 20 °C after filter sterilization through a 0.2-μm membrane. When reconstituted in a 100-ml scale, the authors’ laboratory makes 20–50 small aliquots in 1.7-ml tubes (~1,400 μl per tube). The rest is stored at − 20 °C as a large aliquot (12–30 ml per tube) in 15-ml/50-ml tubes. Thaw aliquots of FBS, newborn calf serum, L-glutamine, hydrocortisone, ITES, O-phosphorylethanolamine, adenine, progesterone and triiodothyronine in a water bath at 37 °C. Transfer them onto ice immediately. Matrigel is stored at − 20 °C. However, it should be thawed at 4 °C overnight; Matrigel thawed at 37 °C solidifies rapidly and can no longer be used. ▲ CRITICAL All OTC reagents should be chilled on ice before use.
Prepare aliquots of ~40 ml and store them at 4 °C for up to 6 months. Chill on ice before use. Approximately 35 ml of collagen will be needed to prepare two six-well Transwell carriers (12 wells).
Dissolve 0.0269 g (molecular weight (MW) = 362.46) in 2.5 ml of absolute ethanol. Dilute with 97.5 ml of DMEM to obtain a 74.2 μM stock solution. Filter (0.2 μm), label aliquots ‘H’ and store at − 20 °C for up to 1 year.
Dissolve 0.705 g (MW = 141.06) in 100 ml of DMEM to obtain a 5 mM stock solution. Filter (0.2 μm), label aliquots ‘O’ and store at − 20 °C for up to 1 year.
Dissolve 1.55 g (MW = 171.59) in 100 ml of double-distilled H2O to obtain a 90 mM stock solution. Filter (0.2 μm), label aliquots ‘A’ and store at − 20 °C for up to 1 year.
Dissolve 1 mg (MW = 314.46) in 1 ml of absolute ethanol. Add 14.7 ml of double-distilled H2O. Dilute 1 ml of this in 100 ml of DMEM (final concentration of 2 μM) as a stock solution. Filter (0.2 μm), label aliquots ‘P’ and store at − 20 °C for up to 1 year.
Dissolve 1 mg (MW = 672.96) in 1 ml of 1 N NaOH. Add 19 ml of DMEM. Further dilute 4 μl of this solution in 31 ml of DMEM to obtain a 10 nM stock solution. Filter (0.2 μm), label aliquots ‘T’ and store at − 20 °C for up to 1 year.
|50-ml tube ‘A’ acellular layer components||Transwell carriers
|× 1 (6 Inserts)||× 2 (12 Inserts)|
|10× EMEM||690 μl||1,380 μl|
|FBS||700 μl||1,400 μl|
|L-Glutamine||60 μl||120 μl|
|Sodium bicarbonate||140 μl||280 μl|
|Bovine collagen type I||5.6 ml||11.2 ml|
|50-ml tube ‘C’ cellular layer components||Transwell carriers
|× 1 (6 Inserts)||× 2 (12 Inserts)|
|10× EMEM||1.8 ml||3.6 ml|
|FBS||2 ml||4 ml|
|L-Glutamine||160 μl||320 μl|
|Sodium bicarbonate||380 μl||760 μl|
|Bovine collagen||11.4 ml||22.8 ml|
|Matrigel||3.8 ml||7.6 ml|
|6 × 105 per ml fibroblasts||1.6 ml||3.2 ml|
|Presaturation medium||Transwell carriers
|× 1 (6 Inserts)||× 2 (12 Inserts)|
|DMEM||60 ml||120 ml|
|Ham’s F12||20 ml||40 ml|
|For two Transwell carriers (12 wells; two medium changes)||EPM1 (300 ml), store at 4 °C||EPM2 (200 ml), store at 4 °C|
|DMEM||218 ml||95 ml|
|Ham’s F12||72 ml||95 ml|
|L-Glutamine||6 ml||4 ml|
|Hydrocortisone (H)||600 μl||400 μl|
|ITES||600 μl||400 μl|
|O-Phosphorylethanolamine (O)||600 μl||400 μl|
|Adenine (A)||600 μl||400 μl|
|Progesterone (P)||600 μl||—|
|Triiodothyronine (T)||600 μl||400 μl|
|NBCS||300 μl||4 ml|
|Gentamicin sulfate||300 μl||200 μl|
Troubleshooting advice can be found in Table 3.
Depending on the quality of the fibroblasts used, contraction of the matrix should be detected between days 2 and 4. The contracted matrix forms a concave surface with a depressed center and slightly rising walls that allow for seeding of keratinocytes on the surface of matrix. The matrix may contract further during the remaining cultivation. Examination of the H&E stained section should reveal a mature stratified epithelium on the surface of the matrix with embedded fibroblasts (Fig. 4). The CK14-positive basal layer contains Ki-67–positive cells (Fig. 4c), on top of which should be progressively more flattening epithelial layers and a nuclear keratinized surface, positive for CK4 and CK13 (Fig. 4e,f). Mouse cell cultures have fewer layers than human cell-derived cultures (compare Fig. 4a and Fig. 5a). Epithelial maturation is impaired by DNMAML1, a genetic pan-Notch inhibitor (Fig. 5a), which affects epithelial formation and expression of differentiation markers such as involucrin (Fig. 5b). Transformed epithelial cells and ESCC cells form dysplastic epithelia and show invasive growth into the underlying collagen matrix compartment (Fig. 2 and Supplementary Fig. 1).
We acknowledge support from the US National Institutes of Health (NIH)/National Cancer Institute (NCI) P01-CA098101, NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) P30-DK050306 and NIH/NCI U01-CA143056. The Molecular Pathology and Imaging, Molecular Biology/Gene Expression, Cell Culture, Transgenic and Chimeric Mouse Core Facilities of the P30-DK050306 NIH/NIDDK Center for Molecular Studies in Digestive and Liver Diseases at the University of Pennsylvania support this work. We also thank the Penn Flow Cytometry and Microarray Array Core Facilities for supporting this work. Additional sources of support include the American Cancer Society Research Professorship (A.K.R.), NIH/NIDDK R01DK077005 (H.N.), NIH/NCI T32-CA115299 (G.W.) and NIH T32-CA009140-37 (M.E.V.). We thank S. Naganuma for the photomicrographs given in Supplementary Figure 1.
AUTHOR CONTRIBUTIONS J.K., G.S.W., M.E.V. and M.N. contributed to the experimental results; E.S.R., M.H., H.N. and A.K.R. contributed to the experimental design; all authors contributed to the manuscript preparation and writing.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
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