# A more natural follicle culture system: Detailed steps of In Vitro 3D follicle culture with alginate gel

## TL;DR

This paper describes a detailed method for culturing ovarian follicles in 3D using alginate gel to create a more natural in vitro environment.

## Contribution

The paper provides a refined, step-by-step procedure for 3D follicle culture emphasizing follicle integrity and alginate gel hardness.

## Key findings

- Only follicles surrounded by theca cells grow healthily to the preovulatory stage.
- Alginate gel hardness is crucial for successful 3D follicle culture.
- Detailed steps improve follicular microenvironment protection and bioavailability.

## Abstract

Follicle culture is a process of dividing follicle unit structures from ovaries for continued culture in vitro in an incubator, which simulates the in vivo environment. Alginate gel is the most stable and most convenient 3D material currently used in follicle culture. We performed in vitro follicle culture following the standard operating procedure recommended by the Follicle Handbook and we have summarized our experience and skills in details. Through several experiments, we found only follicles tightly surrounded by theca cells can grow healthily until the preovulatory stage. In addition, the hardness of alginate gel is crucial for constructing the 3D culture system, and selecting appropriate tools can reduce damage to the alginate gel and shorten the time follicles are exposed to room temperature. Our detailed operation improves bioavailability and provides a more natural environment for the entire process of follicular growth.•Alginate gel is still the most suitable 3D material used for in vitro follicle culture.•Follicle integrity and the hardness of alginate gel are the keys for in vitro culture.•Detailed operation steps better protect the follicular microenvironment and improve bioavailability.

Alginate gel is still the most suitable 3D material used for in vitro follicle culture.

Follicle integrity and the hardness of alginate gel are the keys for in vitro culture.

Detailed operation steps better protect the follicular microenvironment and improve bioavailability.

Image, graphical abstract