Culture and Imaging of Human Nasal Epithelial Organoids

Summary

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Abstract

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

Introduction

Introduction to the technique
Ex vivo culture-based assays are an increasingly utilized tool for precision medicine and the study of disease pathophysiology. Primary human nasal epithelial (HNE) cell culture has been used in numerous studies of cystic fibrosis^{1,2,3,4,5,6,7,8,9,10,}

Protocol

HNE samples were collected at the Children's of Alabama hospital. All procedures and methods described here have been approved by the IRB University of Alabama at Birmingham (UAB IRB #151030001). To facilitate the expansion and improve the function of human nasal epithelial cells (HNEs), the present culturing methods are adapted from the well-known air-liquid interface (ALI) culture method^28,34. HNEs were initially collected by brush biopsy as previously desc.......

Discussion

This manuscript provides detailed methodologies for comprehensive live and fixed imaging of the airway epithelial organoids derived from HNE brush biopsy. It describes functional assays that can determine CFTR activity in an individual. HNEs provide a minimally invasive, primary tissue for a variety of applications. The expansion techniques offered here can be used for modeling airways disease, including organoids. Organoids can be used for precision therapeutic approaches and to monitor the stability of gene or mRNA-bas.......

Materials

Name	Company	Catalog Number	Comments
Nasal brush	Medical Packaging CYB1	CYB-1	Length: 8 inches, width approximately 7 mm
Large-Orifice Pipette Tips	ThermoFisher Scientific	02-707-141	Large bore pipette tips
Accutase	ThermoFisher Scientific	A1110501	Cell detachment solution
0.05% trypsin -EDTA	Gibco	25300-054
Trypsin inhibitor from soybean	Sigma	T6522	Working solution: 1mg/mL in 1XDPBS
Matrigel matrix	Corning	356255	Extracellular matrix (EM)
µ-Slide Angiogenesis	Ibidi	81506	15-well slide
24-Well Transwell	Corning	7200154	Culture insert
Chambered Coverglass	ThermoFisher Scientific	155409	8-well glass-bottom chamber slides
Cell-Tak Cell and Tissue Adhesive	ThermoFisher Scientific	354240	Cell adhesive
Paraformaldehyde	Electron Microscopy Sciences	50980487
Triton X-100	Alfa Aesar	A16046
BSA	ThermoFisher Scientific	BP1600-100
NucBlue	ThermoFisher Scientific	R37605	DAPI
Eclipse Ts2-FL (Inverted Routine Microscope)	Nikon		Inverted epi-fluorescence microscope or bright-field microscope
Nikon A1R-HD25	Nikon		Confocal microscope
NIS Elements- Basic Research	Nikon		manual imaging analysis software
Histogel	ThermoFisher Scientific	HG-4000-012
Disposable Base Molds	ThermoFisher Scientific	41-740
Lionheart FX	BioTek	BTLFX	Automated image system
Lionheart Cover	BioTek	BT1450009	Environmental Control Lid
Humidity Chamber	BioTek	BT1450006	Stage insert (environmental chamber)
Gas Controller for CO2 and O2	BioTek	BT1210013	Gas controller
Microplate/Slide Stage Insert	BioTek	BT1450527	Slide holder
Gen5 Imaging Prime Software	BioTek	BTGEN5IPRIM	Automated imaging analysis software
4x Phase Contrast Objective	BioTek	BT1320515
10x Phase Contrast Objective	BioTek	BT1320516
LED Cube	BioTek	BT1225007
Filter Cube (DAPI)	BioTek	BT1225100	DAPI
CFTRinh-172	Selleck Chemicals	S7139
Forskolin	Sigma	F6886
IBMX	Sigma	I5879
Expansion Media
DMEM	ThermoFisher Scientific	11965
F12 Nutrient mix	ThermoFisher Scientific	11765
Fetal Bovine Serum	ThermoFisher Scientific	16140-071
Penicillin/Streptomycin	ThermoFisher Scientific	15-140-122
Cholera Toxin	Sigma	C8052
Epidermal Growth Factor (EGF)	ThermoFisher Scientific	PHG0314
Hydrocortisone (HC)	Sigma	H0888
Insulin	Sigma	I9278
Adenine	Sigma	A2786
Y-27632	Stemgent	04-0012-02
Antibiotic Media
Ceftazidime	Alfa Aesar	J66460-03
Tobramycin	Alfa Aesar	J67340
Vancomycin	Alfa Aesar	J67251
Amphotericin B	Sigma	A2942
Differentiation Media
DMEM/F-12 (1:1)	ThermoFisher Scientific	11330-32
Ultroser-G	Pall	15950-017
Fetal Clone II	Hyclone	SH30066.03
Bovine Brain Extract	Lonza	CC-4098
Insulin	Sigma	I-9278
Hydrocortisone	Sigma	H-0888
Triiodothyronine	Sigma	T-6397
Transferrin	Sigma	T-0665
Ethanolamine	Sigma	E-0135
Epinephrine	Sigma	E-4250
O-Phosphorylethanolamine	Sigma	P-0503
Retinoic Acid	Sigma	R-2625
Primary antibodies
Human CFTR antibody	R&D Systems	MAB1660	Dilution: 100x
ZO-1 antibody	Thermo Fisher	MA3-39100-A647	Dilution: 1000x
Anti-MUC5B antibody	Sigma	HPA008246	Dilution: 100x
Anti-acetylated tubulin	Sigma	T7451	Dilution: 100x
Anti-beta IV Tubulin antibody	Abcam	Ab11315	Dilution: 100x
Secondary antibodies
Donkey anti-Mouse IgG (H+L), Alexa Fluor 488	Invitrogen	A21202	Dilution: 2000x
Donkey anti-Rabbit IgG (H+L), Alexa Fluor 594	Invitrogen	A21207	Dilution: 2000x