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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

Published: April 5th, 2021

DOI:

10.3791/62300

1Department of Cell and Developmental Biology and the Rocky Mountain Taste and Smell Center, University of Colorado Anschutz Medical Campus
* These authors contributed equally

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

In rodents, lingual taste buds are housed in fungiform papillae distributed anteriorly, bilateral foliate papillae posteriorly, as well as a single circumvallate papilla (CVP) at the posterodorsal midline of the tongue1. Each taste bud is composed of 50-100 short-lived, rapidly renewing taste receptor cells (TRCs), which include type I glial-like support cells, type II cells that detect sweet, bitter, and umami, and type III cells that detect sour2,3,4. In the mouse CVP, LGR5+ stem cells along the basal lamina produce all TRC types as well a....

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All the animal procedures were performed in an AAALAC-accredited facility in compliance with the Guide for the Care and Use of Laboratory Animals, Animal Welfare Act, and Public Health Service Policy, and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus. Lgr5EGFP-IRES-CreERT2 mice used in this protocol are from The Jackson Laboratory, Stock No. 008875.

NOTE: The following steps should be completed before b.......

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Mice have one CVP, located posteriorly on the tongue, from which LGR5+ stem cells can be isolated (Figure 1A, black box). Injection of an enzyme solution under and around the CVP (Figure 1B) results in slight swelling of the epithelium and digestion of the connective tissue. Sufficient digestion is achieved following a 33 min incubation, which allows easy separation of the CVP epithelium from the underlying tissue. When attempting to .......

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Reported here is an efficient and readily repeatable method for culturing, maintaining, and processing lingual organoids derived from adult mouse taste stem cells. It was found that using three CVPs from 8 to 20-week-old Lgr5-EGFP mice is sufficient to obtain ~10,000 GFP+ cells for experimental use, resulting in 50 wells plated at a density of 200 cells per well in 48-well plates. Removal of CVP trench epithelia is optimized by injecting the lingual epithelium with freshly made Dispase II and type-I C.......

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The authors would like to thank Dr. Peter Dempsey and Monica Brown (University of Colorado Anschutz Medical Campus Organoid and Tissue Modeling Shared Resource) for providing WNR conditioned media and valuable discussions. We also thank the University of Colorado Cancer Center Cell Technologies and Flow Cytometry Shared Resources, especially Dmitry Baturin, for cell sorting expertise. This work was funded by: NIH/NIDCD R01 DC012383, DC012383-S1, DC012383-S2, and NIH/NCI R21 CA236480 to LAB, and R21DC016131 and R21DC016131-02S1 to DG, and F32 DC015958 to EJG.

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Name Company Catalog Number Comments
Antibodies
Alexa Fluor 546 Donkey anti Goat IgG Molecular Probes A11056, RRID: AB_142628 1:2000
Alexa Fluor 546 Goat anti Rabbit IgG Molecular Probes A11010, RRID:AB_2534077 1:2000
Alexa Fluor 568 Goat anti Guinea pig IgG Invitrogen A11075, RRID:AB_2534119 1:2000
Alexa Fluor 647 Donkey anti Rabbit IgG Molecular Probes A31573, RRID:AB_2536183 1:2000
Alexa Fluor 647 Goat anti Rat IgG Molecular Probes A21247, RRID:AB_141778 1:2000
DAPI (for FACS) Thermo Fischer 62247
DAPI (for immunohistochemistry) Invitrogen D3571, RRID:AB_2307445 1:10000
Goat anti-CAR4 R&D Systems AF2414, RRID:AB_2070332 1:50
Guinea pig anti-KRT13 Acris Antibodies BP5076, RRID:AB_979608 1:250
Rabbit anti-GUSTDUCIN Santa Cruz Biotechnology Inc. sc-395, RRID:AB_673678 1:250
Rabbit anti-NTPDASE2 CHUQ mN2-36LI6, RRID:AB_2800455 1:300
Rat anti-KRT8 DSHB TROMA-IS, RRID: AB_531826 1:100
Equipment
2D rocker Benchmark Scientific Inc. BR2000
3D Rotator Lab-Line Instruments 4630
Big-Digit Timer/Stopwatch Fisher Scientific S407992
Centrifuge Eppendorf 5415D
CO2 tank Airgas CD USP50
FormaTM Series 3 Water Jackeed CO2 Incubator Thermo Scientific 4110 184 L, Polished Stainless Steel
Incucyte Sartorius Model: S3 Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus
MoFlo XDP100 Cytomation Inc Model: S13211997  Gates Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus
Orbital Shaker New Brunswick Scientific Excella E1
Real-Time PCR System Applied Biosystems 4376600
Refrigerated Centrifuge Eppendorf 5417R
Spectrophotometer Thermo Scientific ND-1000
 Stereomicroscope Zeiss Stemi SV6
Thermal Cycler Bio-Rad 580BR
Vortex Fisher Scientific 12-812
Water bath Precision 51220073
Media
A83 01 Sigma SML0788-5MG Stock concentration 10 mM, final concentration 500 nM
Advanced DMEM/F12 Gibco 12634-010
B27 Supplement Gibco 17504044 Stock concentration 50X, final concentration 1X
Gentamicin Gibco 15750-060 Stock concentration 1000X, final concentration 1X
Glutamax Gibco 35050061 Stock concentration 100X, final concentration 1X
HEPES Gibco 15630080 Stock concentration 100X, final concentration 1X
Murine EGF Peprotech 315-09-1MG Stock concentration 500 µg/mL, final concentration 50 ng/mL
Murine Noggin Peprotech 250-38 Stock concentration 50 µg/mL, final concentration 25 ng/mL
N-acetyl-L-cysteine Sigma A9165 Stock concentration 0.5 M, final concentration 1 mM
Nicotinamide Sigma N0636-100g Stock concentration 1 M, final concentration 1 mM
Pen/Strep Gibco 15140-122 Stock concentration 100X, final concentration 1X
Primocin InvivoGen ant-pm-1 Stock concentration 500X, final concentration 1X
SB202190 R&D Systems 1264 Stock concentration 10 mM, final concentration 0.4 µM
WRN Conditioned media Received from Dempsey Lab (AMC Organoid and Tissue Modeling Share Resource). Derived from L-WRN (ATCC® CRL-3276™) cells
Y27632 dihydochloride 10ug APExBIO A3008-10 Stock concentration 10 mM, final concentration 10 µM
Other
1 ml TB Syringe BD Syringe 309659
2-Mercaptoethanol, min. 98% Sigma M3148-25ML β-mercaptoethanol
2.0 mL Microcentrifuge Tubes USA Scientific 1420-2700
48-well plates Thermo Scientific 150687
5 3/4 inch Pasteur Pipets Fisherbrand 12-678-8A
Albumin from bovine serum (BSA) Sigma Life Science A9647-100G
Buffer RLT Lysis buffer QIAGEN 1015750
Cell Recovery Solution Corning 354253
Cohan-Vannas Spring Scissors Fine Science Tools 15000-02
Collagenase from Clostridium histolyticum, type I Sigma Life Science C0130-1G
Cultrex RGF BME, Type 2, Pathclear R&D Systems 3533-005-02 Matrigel
Dispase II (neutral protease, grade II) Sigma-Aldrich (Roche) 4942078001
Disposable Filters Sysmex 04-0042-2316
Dulbecco’s Phosphate Buffered Saline pH 7.4 (1X) (Ca2+ & Mg2+ free) Gibco 10010-023
Dulbecco’s Phosphate Buffered Saline with Ca2+ & Mg2+  Sigma Life Sciences D8662-500ML
Dumont #5 Forceps Fine Science Tools 11252-30
EDTA, 0.5M (pH 8.0) Promega V4231
Elastase Lyophilized Worthington Biochemical LS002292
Extra Fine Bonn Scissors Fine Science Tools 14084-08
Fetal Bovine Serum (FBS) Gibco 26140-079
Fluoromount G SouthernBiotech 0100-01
HEPES Solution Sigma Life Science H3537-100ML
HyClone Tryspin 0.25% + EDTA Thermo Scientific 25200-056
iScript cDNA Synthesis Kit Bio-Rad 1706691
Modeling Clay, Gray Sargent Art 22-4084
Needle BD Syringe 305106
Normal Donkey Serum Jackson ImmunoResearch 017-000-121
Normal Goat Serum Jackson ImmunoResearch 005-000-121
Paraformaldehyde Sigma-Aldrich 158127
PowerSYBR Green PCR Master Mix Applied Biosystems 4367659
RNeasy Micro Kit QIAGEN 74004
Safe-Lock Tubes 1.5 mL Eppendorf 022363204
Sodium Chloride Fisher Chemical 7647-14-5
Sodium Phosphate dibasic anhydrous Fisher Chemical 7558-79-4
Sodium Phosphate monobasic anhydrous Fisher Bioreagents 7558-80-7
SuperFrost Plus Microscope Slides Fisher Scientific 12-550-15
Surgical Scissors - Sharp Fine Science Tools 14002-14
Triton X-100 Sigma Life Science T8787-100ML
VWR micro cover glass VWR 48366067 22x22mm

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