# An Optimized Protocol for the Generation of Alveolospheres from Wild-Type Mice

## TL;DR

This paper presents an improved method to generate alveolospheres from wild-type mice, eliminating the need for transgenic models and ensuring purity.

## Contribution

The novel contribution is a refined protocol that enables pure alveolosphere generation without transgenic mice.

## Key findings

- The optimized protocol eliminates the need for transgenic mice to isolate alveolar epithelial cells.
- The method produces pure alveolospheres without contamination from bronchiolar or bronchioalveolar organoids.
- This approach enhances the standardization of alveolosphere generation for lung research.

## Abstract

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.