A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Summary

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Abstract

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

Introduction

Unlike standard 2D cultures, 3D cell culture surrogate models allow for the study of epithelial cell behavior in a physiologically relevant context, one resembling a tissue. 3D cultures of the mammary gland have helped elucidate many aspects of mammary gland development and neoplasia. However, most of the 3D culture models currently available are unsuitable to study hormone action because the human epithelial cell lines used for the task lack hormone receptor expression ^6,7,9.

Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action ¹². This model uses the commercia....

Protocol

1. Preparation of Reagents

1. Dissolve the synthetic progestagen promegestone (R5020) and 17-β-estradiol (E2) in ethanol to make 10^-3 M stock solutions. Dissolve prolactin in distilled deionized water to make a 1 mg/ml stock solution. Dissolve the antiestrogen ICI 182,780 in DMSO to make a 10^-2M stock solution. Store these solutions at -20 ° C for up to 6 months.
2. Charcoal dextran (CD) stripped serum and CDFBS medium:
   1. Heat-inactivate fetal bovine serum (FBS) in a 57 °C water bath for 30 min.
      Note: Use only acid-washed glassware for the remainder of this protocol.
   2. Calculate the amount....

Results

Figure 1 summarizes the procedure for preparing the hormone-sensitive 3D cultures. Epithelial structures are observed in whole mounts of gels cultured for 2 weeks in the presence of E2 alone and in combination with other hormones. Only single cells or groups of 2-3 cells are present when no hormones are added to the culture medium (CDFBS medium) (Figure 2). This condition serves as a negative control.

Cells in 3D culture form structures that vary in shape, siz.......

Discussion

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels ¹². One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures ¹.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12-well Tissue Culture Plates (Falcon)	Fisher Scientific	08-772-29
15 ml polystyrene conical Tubes	Fisher Scientific	14-959-49D
Activated Charcoal	Sigma	C-5510
Carmine Alum	Sigma	C1022-100G
Collagenase, Type 3	Worthington	S0C11784
Confocal Microscope	Zeiss	LSM510	Equiped with HeNe 633nm laser
Dextran T-70	Abersham/Pharmacia	17-0280-01
DMEM/F-12, HEPES, no phenol red	Life Technologies	11339-021	Phenol red-free media for hormone use
DMEM, low glucose, pyruvate, no glutamine, no phenol red	Life Technologies	11054-020	Phenol red-free media for hormone use
17-β-Estradiol	EMD Millipore	3301	Dissolved in Ethanol
Ethanol	Koptec	V1001
Fetal Bovine Serum	Hyclone	SH30070.03	For use with hormones, must be Charcoal Dextran stripped
Filters (115 ml)	Nalgene	380-0080, 245-0045, 120-0020	0.88, 0.45, 0.20 micron, respectively
Formalin, 10%	Fisher Scientific	SF93-20
L-Glutamine (200 mM)	Life Technologies	25030-081
ICI 182,780 (fulvestrant)	Sigma Aldrich	I4409-25MG	Dissolved in DMSO
Microtome	Leica	RM2155
Tissue embedding media	McCormick Scientific	39502004
Penicillin	Sigma	7794-10MU	Dissolved in 10 ml of distilled deionized water
Permount	Fisher Scientific	SP15-500
Phosphate Buffered Saline pH 7.4	Sigma Aldrich	P3813-10PAK
Prolactin	Sigma Aldrich	L4021-50UG	Dissolved in distilled deionized water
Promegestone	Perkin Elmer	NLP004005MG	Dissolved in Ethanol
Rat-Tail Collagen	Corning	354236	Lots may contain varying concentrations, note accordingly
Scalpel	Miltex	4311
Semi-enclosed Benchtop Tissue Processor	Leica	TP1020
Sodium Hydroxide	Sigma Aldrich	S5881	Prepare 1N NaOH stock
StaticMaster Anti-static brush	Amstat	C3500
Stripette Serological Pipettes	Corning	4101
T-25 flasks	Corning	430168
Tissue Cassettes	Fisher Scientific	15-200-403E
Wheaton Vials, Glass, 20 ml	Fisher Scientific	03-341-25D
Xylene	VWR	95057-822