VIA Extractor™ tissue disaggregator for AT2 organoid prep in IPF research

Abstract - AT2 organoids generation from lung tissue using automated technologies

In this study, we will be optimizing the manual workflow of human type 2 alveolar epithelial (AT2) organoids:

• AT2 cells are successfully isolated from healthy human tissue (lung) using the Omics bundle - VIA Extractor™ tissue disaggregator.
• Evaluation and establishment of AT2 organoids are optimized with the Beckman Coulter CytoFLEX SRT cell sorter.

Introduction

In this article, we’ll explore research to elucidate the pathophysiology of chronic interstitial pneumonia, particularly an intractable disease called idiopathic pulmonary fibrosis (IPF), and develop therapeutic drugs based on these findings (1).

Recent advances in developmental biology have brought attention to 3D cultures such as cerebral organoids, which model organogenesis using tissue-derived stem or progenitor cells. While our work focus is lung disease, these insights extend across organ systems, guiding us in establishing organoid models from specific tissue sources, including lung epithelium.

In IPF, the spotlight has been on the aberrant basaloid cells in the pathological exacerbation mechanism. Studies show that human type 2 alveolar epithelium (AT2) transdifferentiates into aberrant basaloid cells, though the details of this process await further clarity. Additionally, inflammation stimulation using AT2 organoids has been found to increase the KRT17 gene expression, a marker of aberrant basaloid cells, prompting further research into culture methods.

To advance studies in this domain, efficiently establishing and cultivating specific tissue, such as human type 2 alveolar epithelial (AT2) organoids, is crucial. At Osaka University, we’ve been using a manual method for isolating AT2 cells from healthy human tissue (lung) to establish AT2 organoids in our workflow. However, the introduction to the VIA Extractor™ tissue disaggregator allows researchers, including those new to this field, to work more efficiently with tissue-derived samples and achieve higher cell viability. Therefore, we will evaluate the efficiency of Cytiva’s VIA Extractor™ tissue disaggregator and Beckman-Coulter CytoFLEX SRT cell sorter with our organoids to compare the performance against the manual method.

Materials and methods

Samples of normal human lung tissue were dissociated into single cells with the VIA Extractor™ tissue disaggregator after 12 hours from surgery, stained with EpCAM and HTII-280, and cells were isolated by CytoFLEX SRT in areas positive for EpCAM and HTII-280 markers (Figures 1-3). By 3D culture (tissue-like structures) using Matrigel, the establishment efficiency of AT2 organoids was evaluated.

Fig 1. Workflow for establishing AT2 organoids using the VIA Extractor™ tissue disaggregator and the CytoFLEX SRT Cell Sorter.

Fig 2. Distribution of tissue sample to use in VIA Extractor™ tissue disaggregator (about 300 mg each).

• Dissociation starts at 37℃ and with a pedal speed of 200 rpm
• Visually check the cell dissociation every 10 minutes and after 40 minutes when the entire cell suspension is collected
• Centrifugation after placing samples in a cell strainer
• Counting of cells is performed using Countess

1. Insert the tissue sample in the individual compartment of the omics pouch with the omics applicator/syringe
2. Place the omics pouch on the Omics clamp and secure the clamp
3. Heat seal the opening of the omics pouch
4. Use the syringe to add 5 mL of collagenase/dispase
5. Use the same syringe to remove the excess air
6. Assemble and tighten the omics pouch to the omics clamp

Fig 3. Process of tissue dissociation using the VIA Extractor™ tissue disaggregator (above).

Results

This time, we achieved a survival rate of 90% using the VIA Extractor™tissue disaggregator with normal lung tissue weighing 300 mg, which yielded 10⁶ cells (see Table 1). As a result, an AT2 organoid with the same efficiency as the manual method (Figures 4-6) was established.

Table 1: Viability and cell count results with VIA Extractor™ tissue disaggregator

Fig 4. Brightfield image of AT2 organoids cultured under Matrigel for 12 days after cells were isolated by CytoFLEX SRT.

Fig 5. Immunostaining of organoids cultured for 12 days after cell separation with CytoFLEX SRT (left), HE staining, (middle) HTll-280, (right) SFTPC are shown above.

Fig 6. Number of organoid colonies formed in the manual method (left) and when using the VIA Extractor™ tissue disaggregator (right).

Conclusion

Isolating enough AT2 cells for organoid culture using manual methods is challenging, especially for those unfamiliar with the process, as it requires skilled technique.

The most significant advantage of using the VIA Extractor™ tissue disaggregator in this study was that it provided an easy, efficient way to establish organoids within a single day, even before the enzymes and temperature conditions were fully optimized.

Compared to the manual method, the VIA Extractor™ tissue disaggregator enabled faster, high-quality single-cell isolation from specific tissue, reducing overall experimental time and improving workflow efficiency.

With the ability to customize settings based on the hardness, tissue size, and enzyme type, the VIA Extractor™ tissue disaggregator delivers consistent results regardless of user experience or tissue condition, whether normal or diseased. Importantly, it maintained the Expression of the critical surface marker HTII-280 essential for organoid establishment; the VIA Extractor™ tissue disaggregator preserved this marker’s brightness, confirming its gentle dissociation process.

This reliability benefits not only researchers working with lung organoids or expanding into other tissue-derived models and basic research like ours, but also for pharmaceutical companies seeking stable, reproducible data. An automated system that consistently produces high-quality single cells is a valuable technology to accelerate progress across diverse research fields.

In combination with CytoFLEX SRT, which supports this offering with a simple start-up like an analyzer, and intuitive flow sorting protocols that minimize cell damage and boost organoid establishment efficiency, this system aids researchers across both developmental biology and translational research. Beyond its essential features like plate sorting, its compact size and quiet operation make it ideal for busy labs. From fibrosis studies to modelling organogenesis in miniaturized, 3D systems, the technology helps accelerate progress.

Streamline AT2 organoid prep

Discover how VIA Extractor™ tissue disaggregator enables fast, reproducible lung tissue dissociation for fibrosis and organoid research.