# Culture Strategy Determines the Differentiation Status of Sweat Gland Cells

## TL;DR

This paper shows that how sweat gland cells are cultured in the lab affects their ability to maintain their specialized function, which is important for creating realistic skin substitutes.

## Contribution

The study introduces an optimized isolation method and compares 2D and 3D culture strategies to preserve sweat gland cell identity.

## Key findings

- 2D culture leads to loss of sweat gland-specific markers like AQP5 and α-SMA.
- 3D spheroids preserve SGC markers but hinder growth and structure.
- SGCs cultured in 2D cannot regain glandular features in 3D conditions.

## Abstract

Reliable methods for the isolation and culture of human eccrine sweat gland cells (SGCs) are essential for studying glandular biology and developing tissue-engineered skin substitutes (TESs) that restore full skin function. However, maintaining the glandular phenotype of SGCs in vitro remains a major challenge. In this study, we present an optimized isolation protocol combining enzymatic digestion with mechanical separation to improve SGC yield and purity, while also enabling keratinocyte isolation from a single human skin biopsy. We then evaluated two culture strategies, 2D monolayers and 3D spheroids, to determine their impact on SGC identity and proliferation. While 2D culture supported cell expansion, SGCs and keratinocytes exhibited highly similar marker expression profiles, with the absence of functional SGC markers (AQP5, α-SMA) reflecting a shift toward less differentiated phenotypes. In contrast, SGCs cultured in 3D spheroids preserved the expression of SGC-specific markers (AQP5, K18, α-SMA), distinguishing them from keratinocytes; however, their growth and structural organization were suboptimal under these 3D conditions. Moreover, SGCs expanded in 2D did not regain their glandular features when reintroduced into 3D culture, suggesting potential limitations in phenotype recovery. These results highlight the need for improved culture systems that maintain SGC identity while supporting expansion. Advancing such methods is a critical step toward integrating functional sweat glands into TESs and achieving complete skin regeneration for clinical applications.