Generation and Differentiation of Human Olfactory Epithelial Organoids from Adult Stem Cells

Summary

This protocol outlines an established approach for isolating olfactory progenitor cells from adult human superior turbinate tissue to generate olfactory epithelial organoids. It also details a chemical induction strategy to drive the differentiation of these organoids into mature, olfactory sensory neurons, thereby enabling the modeling of human olfactory neurogenesis.

Abstract

The human olfactory epithelium possesses remarkable neurogenic capacity, sustained by a resident population of basal stem cells; however, establishing a dynamic in vitro model that faithfully recapitulates this regenerative process has been proven challenging. Here, we present an established protocol for generating olfactory organoids from progenitor cells isolated from adult human superior turbinate tissue. These organoids maintain stemness, as evidenced by SOX2 expression, and display a spatially organized differentiation pattern, characterized by uniform expression of the immature neuronal marker GAP43 and peripheral localization of the mature olfactory sensory neuron marker OMP. Subsequent targeted differentiation with the Notch inhibitor LY411575, the Wnt activator CHIR-99021, and retinoic acid generated marker-positive olfactory sensory neuron–like cells that co-expressed OMP and the neuronal marker Tuj1 (class III β-tubulin). This human-derived model circumvents the limitations of murine systems and provides a robust in vitro platform for studying olfactory neurogenesis, COVID-19–associated anosmia, and other olfactory-related neurodegenerative disorders.

Introduction

The human olfactory system supports continuous neurogenesis,¹ a process primarily driven by basal stem cells in the olfactory epithelium, specifically, horizontal basal cells (HBCs) and globose basal cells (GBCs).^2,3 GBCs constitutively differentiate into olfactory sensory neurons (OSNs), whereas HBCs remain quiescent until activated by injury.^4,5 Olfactory perception begins when odorant molecules bind to receptors on the cilia of OSNs, triggering a signal transduction cascade that relays information to the olfactory bulb and cortex.⁶ The development and maintenance of this sensory function are governed by the olfactory epithelium.

Olfactory dysfunction caused by physical, chemical, or biological insults often resolves spontaneously.⁷ Nevertheless, it affects 1.5% to 25% of the global population⁸ and can result from diverse etiologies, including trauma,⁹ infections,¹⁰ and neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease¹¹; in these cases, spontaneous recovery is frequently incomplete or absent. Current therapeutic approaches, such as olfactory training and corticosteroid administration, show limited efficacy.^12,13 This therapeutic gap stems from an incomplete understanding of the mechanisms governing olfactory neuroepithelium survival and regeneration following injury, a knowledge gap further exacerbated by the inadequacy of existing in vitro models for studying human olfactory biology.

In recent years, the development of diverse human organoid models has created new technological platforms for studying human diseases.¹⁴ Organoids derived from adult stem cells (AdSCs), also referred to as tissue-resident stem cells, have been successfully generated for a range of endoderm-derived tissues, including the stomach, liver, pancreas, lungs, and bladder.^{15,16,17,18,19} In contrast, establishing human olfactory epithelial organoids from the ectoderm-derived olfactory placode remains technically challenging. A major practical challenge is the difficulty of consistently obtaining viable olfactory epithelial tissue from the human superior turbinate. The location and extent of the olfactory region vary considerably among individuals and undergo pronounced age-related degeneration. Inadvertent collection of respiratory rather than olfactory epithelium compromises culture outcomes. To address this, we refined the 2021 protocol by Ren et al., who successfully cultured cell clusters from human olfactory mucosa,²⁰ to reliably generate human olfactory epithelial organoids. Our approach generates human olfactory epithelial organoids with a three-dimensional, hierarchically organized architecture and supports their differentiation into olfactory sensory neurons. This model provides a valuable experimental platform for investigating the mechanisms that determine the fate of olfactory epithelial stem cells and for screening potential therapeutic agents for olfactory disorders.

To summarize explicitly, the primary objective of this method is to establish a standardized workflow for generating and differentiating human olfactory epithelial organoids from adult progenitor cells. The rationale for this approach is to address the critical gap created by the absence of physiologically relevant in vitro models of the human olfactory system. Compared with traditional two-dimensional (2D) monolayer cultures or murine models, this three-dimensional (3D), human-derived system offers the distinct advantage of faithfully recapitulating the complex spatial organization and human-specific genetic context of the olfactory mucosa. For researchers to consider this protocol, it is essential to recognize that precise isolation of viable olfactory epithelium from the human superior turbinate, while avoiding contamination with respiratory epithelium, is exceptionally challenging and highly influenced by patient age and individual anatomical variation. Consequently, this protocol is particularly well-suited for investigators who have consistent access to human clinical specimens and seek to model human olfactory neurogenesis, virus-induced anosmia, or olfactory deficits associated with neurodegenerative disorders.

Protocol

This study was conducted with approval from the Institutional Review Board (IRB) of West China Hospital of Sichuan University (Approval No. 2024 (31)). Written informed consent was obtained from all participating patients prior to tissue collection and data publication. Superior turbinate tissue specimens were obtained from patients undergoing endoscopic skull base surgery or ethmoid sinus surgery for cerebrospinal fluid rhinorrhea.

1. Tissue sample processing and transport

1. Immediately after collection, place the tissue samples in a prechilled, sterile transport medium, composed of DMEM/F12/HEPES basal medium supplemented with 10% fetal bovine serum and 5% penicillin–streptomycin solution, and transport them to the laboratory at 4 °C.
   ​NOTE: The serum helps maintain cell viability, while the antibiotic mixture prevents potential microbial contamination during transit. Keep transportation time under 2 h to preserve cell viability.

2. Tissue preprocessing (performed on ice)

1. Transfer the sample to a prechilled, sterile culture dish, remove the transport medium, and wash the tissue with a modified PBS buffer containing 5% penicillin-–streptomycin solution. Perform the wash as follows: add 5 mL of prechilled wash solution to the dish, gently tap the tissue 5–7x, then completely aspirate the liquid. Repeat this wash cycle 3x to remove blood cells and tissue debris.
2. Using sterile microscissors, mince the tissue into fragments of approximately 1–2 mm³.
   NOTE: During this step, maintain tissue hydration, secure it with blunt-tipped forceps, and minimize trimming time to preserve cell viability.

3. Enzymatic digestion

1. Transfer the mechanically dissociated tissue into a 15 mL centrifuge tube and add 3 mL of prewarmed (37 °C) compound digestion buffer (containing 0.125% trypsin and 10 U/mL DNase I), maintaining a 3:1 enzyme-to-tissue volume ratio. Incubate the tube in a 37 °C water bath with gentle agitation for 20 min. Inspect the sample every 5 min to monitor digestion progress and prevent tissue clumping.
2. Stop the digestion by adding an equal volume of termination solution, DMEM/F12/HEPES basal medium supplemented with 10% fetal bovine serum. Gently pipette the mixture up and down 10x to ensure complete mixing and effective contact between the enzymes and the inhibitory components.
   ​NOTE: The α1-antitrypsin present in the serum efficiently neutralizes trypsin activity.

4. Preparation of single-cell suspension

1. Place a 40 µm cell strainer on top of a 50 mL centrifuge tube. Using a trimmed 1 mL pipette tip, transfer the digested tissue and liquid from the 15 mL tube onto the strainer. Gently swirl the material on the filter with the pipette tip to facilitate passage, then rinse the strainer with an additional 1–2 mL of termination solution. Once all liquid has passed through the strainer, transfer the filtrate from the 50 mL tube into a new 15 mL centrifuge tube.
2. Centrifuge the suspension at 300 × g for 5 min at 4 °C. Observe the distinct cell pellet at the bottom of the tube and carefully aspirate the supernatant, leaving approximately 100 µL to avoid disturbing or losing the cell pellet.

5. Establishment of three-dimensional culture system

1. Without resuspending the cell pellet in culture medium, directly combine it with Matrigel using a prechilled, low-binding pipette tip to achieve a precise density of 10,000 cells/µL. Dispense 40 µL of this cell–Matrigel mixture into each well of a 24-well plate and 20 µL into each glass-bottom confocal dish. Incubate the plates at 37 °C for 30 min to allow the Matrigel to polymerize and form a three-dimensional scaffold.
2. After Matrigel solidification, gently add 500 µL of serum-free olfactory neural stem cell culture medium along the well wall to avoid disrupting the gel. Maintain cultures in a humidified incubator at 37 °C with 5% CO₂, replacing the medium every 3 days with fresh prewarmed medium.
   ​NOTE: The olfactory neural stem cell culture medium consists of DMEM/F12/HEPES supplemented with the following components: R-spondin 1 (200 ng/mL), Noggin (100 ng/mL), Wnt3a (50 ng/mL), EGF (50 ng/mL), Y-27632 (10 µM), a primary antimicrobial agent (100 µg/mL), N2 supplement (1%), B27 supplement (2%), and L-glutamine (1%).

6. Passaging and differentiation

1. Prechill the organoid recovery solution on ice for 10 min before use; aspirate the culture medium from the wells, then add 1 mL of ice-cold recovery solution to each well. Tilt the plate to a 45° angle and gently aspirate and dispense the solution 5–7x to dislodge organoids from the basement membrane matrix and transfer the resulting suspension to a 15 mL centrifuge tube. Incubate the tube on ice for 3–5 min, then centrifuge at 300 × g for 5 min at 4 °C, and carefully remove the supernatant, leaving behind approximately 100 µL of liquid to prevent loss of the pellet.
2. Add 1 mL of organoid dissociation reagent directly to the cell pellet and incubate the tube at 37 °C with gentle shaking at 220 rpm for 5 min. Immediately terminate digestion by adding an equal volume of Stop Solution and gently pipette the mixture up and down 5x to ensure homogeneity. Centrifuge at 300 × g for 5 min at 4 °C to collect the dissociated cells.
3. Resuspend the cell pellet in Matrigel at a volume of 40 µL per well for 24-well plates or 20 µL per well for glass-bottom confocal culture dishes; mix thoroughly using a prechilled, low-binding pipette tip to ensure a uniform suspension. After seeding into the appropriate plates or dishes, incubate the constructs at 37 °C for 30 min to allow Matrigel polymerization and three-dimensional scaffold formation. Then, gently add 500 µL of specialized serum-free spheroid culture medium for olfactory neural stem cells along the well walls to avoid disturbing the gel. Maintain cultures in an incubator at 37 °C with 5% CO₂ and 95% relative humidity.
4. Approximately 7 days after passaging, once the new generation of olfactory epithelial organoids has reformed into spheroids, aspirate the expansion medium from the glass-bottom confocal dishes and replace it with differentiation medium. Replace the differentiation medium with fresh, pre warmed medium every 3 days. Morphologically distinct olfactory neurons typically become apparent under microscopy after approximately 7 days of differentiation.
   NOTE: The differentiation medium consists of DMEM/F12/HEPES supplemented with R-Spondin 1 (200 ng/mL), Noggin (100 ng/mL), Wnt3a (50 ng/mL), EGF (50 ng/mL), Y-27632 (10 µM), LY411575 (5 µM), CHIR99021 (3 µM), retinoic acid (RA, 1 µM), a primary antimicrobial agent (100 µg/mL), N2 supplement (1%), B27 supplement (2%), and L-glutamine (1%).

7. Immunofluorescence

1. Aspirate the culture medium and gently wash the samples once with 500 µL of PBS. Remove the PBS, then add 500 µL of 4% paraformaldehyde and fix the samples at room temperature for 15 min. Aspirate and discard the fixative; then wash the fixed samples with PBS (3 x 500 µL) and store them at 4 °C for up to 48 h.
2. Aspirate the PBS and add 500 µL of permeabilization solution (0.2% Triton X-100 in PBS), Incubate for 15 min at room temperature, then aspirate and discard the solution. Wash the samples with 0.02% PBST (0.02% Triton X-100 in PBS; 3 x 500 µL).
3. Add 500 µL of blocking solution (3% BSA in PBS) and incubate for 1 h at room temperature.
4. Aspirate the blocking solution and replace it with 500 µL of primary antibody solution, prepared by diluting the appropriate primary antibody (e.g., anti-GAP43 at 1:160, anti-OMP at 1:50, or anti-SOX2 at 1:50) in blocking solution, and incubate overnight at 4 °C. The following day, aspirate the primary antibody solution and wash the samples with 0.02% PBST (3 x 500 µL).
5. Add 500 µL of secondary antibody solution, containing an Alexa Fluor 488– or Alexa Fluor 555–conjugated secondary antibody diluted 1:500 in blocking solution, and incubate at room temperature in the dark for 1 h. Aspirate the secondary antibody solution and perform additional washes with 0.02% PBST (3 x 500 µL).
6. Add 500 µL of DAPI working solution (DAPI diluted in PBS) and incubate in the dark for 10 min to stain nuclei. Aspirate the DAPI solution and wash the samples with 0.02% PBST (3 x 500 µL). Store the stained samples at 4 °C for up to 48 h prior to imaging.

8. Confocal microscopy imaging

1. Perform confocal microscopy using a Z-axis size of 1 µm for organoids with a diameter of ≤100 µm, and 0.5 µm for organoids larger than 100 µm. Acquire images in multi-channel sequential mode, scanning in the following order: DAPI, FITC, TRITC, and Cy5. Import the acquired Z-stack image sequence files into the 3D rendering software (e.g., Imaris). Utilize the 3D volume rendering function to generate a complete three-dimensional model of the organoid. Observe this reconstructed model to visualize the spatial distribution of specific cellular markers (e.g., SOX2, GAP43, and OMP) and to qualitatively confirm the hierarchical architecture and neuronal differentiation status of the olfactory epithelial organoids as the final endpoint.

Results

Isolation of olfactory epithelial tissue and culture of primary olfactory epithelial organoids
To establish a culture system for olfactory epithelial organoids, we isolated olfactory epithelium from human superior turbinate tissue and embedded it in a three-dimensional Matrigel matrix (Figure 1). Building on the defined culture conditions reported by Ren et al., we validated and fine-tuned the concentrations of key growth factors, EGF, Noggin, Wnt3a, and R-pondin 1, toge...

Discussion

Organoids represent a unique in vitro model because their self-organizing, three-dimensional architecture closely recapitulates that of native human tissues and, in some cases, can be histologically indistinguishable from actual organs.¹⁴ Establishing human organoid models is therefore critical for studying human diseases. Here, we present a protocol for generating an organoid culture system from primary human olfactory epithelial cells that reliably differentiate into mature olfactory se...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ALEXA FLUOR 488 DONKEY	Thermo	A11055	1:500
ALEXA FLUOR 555 DONKEY	Thermo	A31572	1:500
Anti-GAP43 antibody	Abcam	ab75810	1:160
Anti-OMP antibody	SANTA CRUZ	sc-365818	1:50
Anti-Sox-2 antibody	SANTA CRUZ	sc-365823	1:50
B-27 Supplement	Gibco	17504044	2%
CHIR99021	Novoprotein	HY-10182	3 μM
DMEM/F12/HEPES	Procell	PM150310	/
Glutamax (L-glutamine supplement)	Gibco	35050061	1%
LY411575	Novoprotein	HY-50752	5 μM
Matrigel (basement membrane matrix)	Corning	356231	4% (Vol/Vol)
N-2 Supplement	Gibco	17502048	1%
Organoid Dissociation Reagent	Precedo	PRS-ODR	/
Organoid Recovery Solution	Thewellbio	MS04-100	/
Primocin (primary antimicrobial agent)	Invivogen	ant-pm-05	100 μg/mL
Recombinant Human EGF	Novoprotein	C029	50 ng/mL
Recombinant Human Noggin	Novoprotein	CB89	100 ng/mL
Recombinant Human RSPO1	Novoprotein	CX83	200 ng/mL
Recombinant Human Wnt3a	Novoprotein	C18K	50 ng/mL
Retinoic acid	MCE	HY-14649	1 μM
Y27632	MCE	HY-10071	10 μM