Co-culture Model Using Two Types of Adherent Cell Lines

Summary

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Abstract

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Introduction

Despite extensive research on human pregnancy, the molecular mechanisms at the maternal-fetal interface during implantation and early pregnancy remain poorly understood¹. The human endometrium is mainly composed of two cell types: endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Implantation progresses through three stages: apposition, attachment, and invasion, which lead to the development of a competent embryo and receptive endometrium². Considering the ethical constraints of in vivo studies on human subjects, as well as the difficulties in simulating the human co....

Protocol

NOTE: All reagents used in this protocol can be found in the Table of Materials. Unless otherwise specified, all media were pre-equilibrated to 37 °C before use.

1. 3D printing of the scaffold and model construction

NOTE: The steps here were performed according to the manual book of the commercial 3D-metal printing machine. The steps are briefly described below (Supplementary File 1).

1. Print bed.......

Discussion

A simplified and cost-effective protocol is described for the indirect co-culture of endometrial stromal and epithelial cells. This method utilizes a homemade scaffold for cell slides, which comprises an upper support ring tailored for attachment to standard 12-well cell culture plates, complemented by a basal cell slide holder featuring four L-shaped rod-like structures. This setup facilitates the separation of endometrial stromal and epithelial cells while allowing for their interaction through the exchange of signalin.......

Materials

Name	Company	Catalog Number	Comments
10x Hanks′ Balanced Salt solution	Solarbio	H1046	1/10
12-well Clear TC-treated Plates	Corning	3513	-
25 cm² Cell Culture Flask	Corning	430639	-
Aluminum	Markforged	6061-T6	-
DMEM/F12	Sigma-aldrich	D2906	-
Dulbecco’s Modified Eagle Medium	Gibco	C11995500BT
FBS	Gibco	10099141C	1/10
Fetal bovine serum	Gibco	10099141C
ITS Premix	Biocoat	354350	1/100
Matrigel Matrix	Corning	354248	ECM
Metal X	Markforged	M F-PR-5000	-
Penicillin-Streptomycin	Gibco	15140122	1/100
Round Coverslip	Biosharp	BS-18-RC	-
TrypLE Select (10X)	Gibco	A1217701	dissociation enzyme