A new tunable 3D alveolospheres model from human alveolar epithelial type 2 cells (AEC2) with reduced heterogeneity for studying cigarette smoke extract exposure

Marina Guecamburu, Arthur Pavot, Amélie Legrix, Caroline Jeannière, Yaniss Belaroussi, Matthieu Thumerel, Emma Samaniego, Hugues Begueret, Guillaume Maucort, Fanny Decoeur, Jean-William Dupuy, Anne-Aurélie Raymond, Pauline Esteves, Leo Grassion, Gael Dournes, Patrick Berger, Elise Maurat, Katharina Raasch, Eloïse Latouille, Vincent Studer, Isabelle Dupin, Pauline Henrot, Maéva Zysman
Respiratory Research. 2026-03-17; 27(1):
DOI: 10.1186/s12931-026-03628-z

1. Respir Res. 2026 Mar 17;27(1):179. doi: 10.1186/s12931-026-03628-z.

A new tunable 3D alveolospheres model from human alveolar epithelial type 2
cells (AEC2) with reduced heterogeneity for studying cigarette smoke extract
exposure.

Guecamburu M(1)(2)(3), Pavot A(1)(2)(3), Legrix A(2), Jeannière C(4)(5),
Belaroussi Y(1)(2)(3), Thumerel M(1)(2)(3), Samaniego E(1)(2)(3), Begueret
H(1)(2)(3), Maucort G(6)(7), Decoeur F(6), Dupuy JW(8), Raymond AA(8), Esteves
P(1)(2), Grassion L(1)(2)(3), Dournes G(1)(2)(3), Berger P(1)(2)(3), Maurat
E(1)(2), Raasch K(1)(2), Latouille E(1)(2), Studer V(4)(5), Dupin I(1)(2),
Henrot P(1)(2)(3), Zysman M(9)(10)(11)(12).

Author information:
(1)Univ-Bordeaux, Centre de Recherche Cardio-thoracique de Bordeaux, U1045,
CIC1401, Pessac, France.
(2)INSERM, Centre de Recherche Cardio-thoracique de Bordeaux, U1045, CIC1401,
Pessac, France.
(3)Service de pneumologie, Service d’exploration fonctionnelle respiratoire,
Service de réanimation, Service de chirurgie thoracique, CHU de Bordeaux,
Service d’anatomopathologie, France.
(4)Interdisciplinary Institute for Neuroscience, Centre National de la Recherche
Scientifique, Bordeaux, France.
(5)Interdisciplinary Institute for Neuroscience, University of Bordeaux,
Bordeaux, France.
(6)Université de Bordeaux, CNRS, INSERM, Bordeaux Imaging Center (BIC), US4,
Bordeaux, 3420, 33000, UAR, France.
(7)France-BioImaging Core – UAR2057 CNRS UM, Bordeaux, France.
(8)Univ. Bordeaux, CNRS, INSERM, TBM-Core, US5, UAR 3427, OncoProt, Bordeaux,
F-33000, France.
(9)Univ-Bordeaux, Centre de Recherche Cardio-thoracique de Bordeaux, U1045,
CIC1401, Pessac, France. .
(10)INSERM, Centre de Recherche Cardio-thoracique de Bordeaux, U1045, CIC1401,
Pessac, France. .
(11)Service de pneumologie, Service d’exploration fonctionnelle respiratoire,
Service de réanimation, Service de chirurgie thoracique, CHU de Bordeaux,
Service d’anatomopathologie, France. .
(12)Service des Maladies Respiratoires, CHU Bordeaux, Pessac, 33604, France.
.

RATIONALE: Three-dimensional (3D) organoid models, such as alveolospheres, are
unique tools for investigating the mechanisms underlying emphysema. However,
high inter-organoid heterogeneity hampers consistent results in emphysema
research and drug testing.
OBJECTIVES: To develop a tunable 3D alveolosphere derived from human primary
type II alveolar epithelial cells (AEC2) for modeling alterations linked to
cigarette smoke exposure.
METHODS: AEC2 (HTII-280+) were isolated from 52 lung samples from both COPD and
non-COPD patients, then cultured in 3D, comparing Matrigel to preformed
photopolymerized hydrogel microwells of adjustable size and stiffness.
Topological and phenotypic characterization were performed on days (D)1, 7, and
14. Lamellar bodies (LBs) were quantified using artificial intelligence (AI)
analysis of transmission electron microscopy (TEM) serial block-face images.
Chronic exposure to 1% or 5% cigarette smoke extract (CSE) was performed for 5
consecutive days.
RESULTS: Compared to Matrigel-based spheroid cultures, alveolospheres generated
in microwells display reduced heterogeneity in size. Such alveolospheres were
maintained in culture for 14 days and exhibited central lumen formation from D7
to D14. Across different hydrogel stiffness, a stiffness of 5 kPa was found to
best support long-term organoid maintenance. The presence of tight junctions
(TEM, ZO-1 immunostaining) suggested an auto-organization. AEC1 markers (P2XR4,
PDPN) increased from D1 to D14 while AEC2 markers (ABCA3, SFTPA, SFTPC)
persisted over time, in qPCR. TEM indicated surfactant synthesis, and AI-driven
LB quantification revealed a decrease in LB-containing cells over time. CSE
exposure resulted in cell death, architectural disorganization, oxidative
stress, and inflammation. Similarly, alveolospheres derived from COPD patients
showed increased expression of inflammatory and cell death markers.
CONCLUSION: This standardized and adjustable 3D alveolosphere model, derived
from human primary AEC2, successfully reproduces key native alveolar features.
Exposure to CSE provides a relevant platform for studying responses to cigarette
smoke exposure.
SUPPLEMENTARY INFORMATION: The online version contains supplementary material
available at 10.1186/s12931-026-03628-z.

DOI: 10.1186/s12931-026-03628-z
PMCID: PMC13107669
PMID: 41845362

Conflict of interest statement: Declarations. Ethics approval and consent to
participate: Surplus lung tissue obtained after surgery was used for research
within the context of patient care, under a no-objection system for the coded
anonymous use of residual diagnostic or therapeutic tissue. The
non-interventional research protocol received approval from the Bordeaux
University Hospital’s TUBE agreement, version 1 (CHU BX 2020/54, 14/01/2021).
Consent for publication: Not Applicable. Competing interests: The authors
declare no competing interests.