Generation of Mosaic Mammary Organoids by Differential Trypsinization

Summary

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Abstract

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

Introduction

The mammary gland (MG) is a tree-like, tubular epithelial structure embedded within an adipocyte rich stroma. The bilayered ductal epithelium comprises an outer, basal layer of contractile, myoepithelial cells (MyoECs) and an inner layer of luminal, secretory epithelial cells (LECs), encircling a central lumen¹. During lactation when the outer MyoECs contract to squeeze milk from the inner alveolar LECs, the MG undergoes numerous changes that are under the control of growth factors (e.g., EGF and FGF) and hormones (e.g. progesterone, insulin, and prolactin). These changes cause the differentiation of specialized structures, alveoli, which synth....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, Santa Cruz.

1. Day 1: Mammary gland digestion

1. Prepare to harvest the MGs from mature female mice 10-14 weeks of age.
   1. Perform the harvesting on an open bench under aseptic conditions.
   2. Sterilize all surgical supplies, cork boards, and pins by autoclaving and soaking in 70% alcohol for 20 min prior to surgery.
   3. Anesthetize animals with sodium pentobarbital (2X anesthetic dose of 0.06 mg/g body weight) delivered via intraperitoneal injection with a 0.5 mL insulin s....

Results

The protocol presented here describes a method for investigating specific lineage contributions of mammary epithelial cells by making use of mosaic organoids. To obtain primary murine cells for organoids, the mammary gland epithelium must first be isolated from the surrounding adipocyte rich stroma (Figure 1). This process is described briefly here and is also described in a previously published study¹⁸. To obtain enough cells, it is r.......

Discussion

Here, a method is presented detailing how researchers can generate 3D organoid cultures using primary MG cells. The difference between this and other protocols is that we detail a method to separate the two, distinct MG cell compartments: the outer basal MyoECs and inner LECs. Our method employs a two-step trypsin-EDTA (0.05%) treatment that we call differential trypsinization¹⁹. This procedure allows researchers to isolate MyoECs and LECs without using sophisticated flow cytometry and thus can be.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 ml High-Clarity Polypropylene Conical Tube (BD Falcon)	Fisher Scientific	352096
24 well ultra-low attachment plate (Corning)	Fisher Scientific	CLS3473-24EA
35 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon)	Fisher Scientific	353001
50 ml High-Clarity polypropylene conical tube (BD Falcon)	Fisher Scientific	352098
60 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon)	Fisher Scientific	353004
70 µM nylon cell strainer (Corning)	Fisher Scientific	08-771-2
Antibiotic-Antimycotic (100X)	Thermo Fisher Scientific	15240062	Pen/Strep also works
B27 supplement without vitamin A (50x)	Thermo Fisher Scientific	12587010
B6 ACTb-EGFP mice	The Jackson Laboratory	003291
BD Insulin syringe 0.5 mL	Thermo Fisher Scientific	14-826-79
Class 2 Dispase (Roche)	Millipore Sigma	4942078001
Class 3 Collagenase	Worthington Biochemical	LS004206
Corning Cell Recovery solution	Fisher Scientific	354253	Follow the guidelines for use – Extraction of Three-Dimensional Structures from Corning Matrigel Matrix
Corning Costar Ultra-Low Attachment 6-well	Fisher Scientific	CLS3471
Dexamethasone	Millipore Sigma	D4902-25MG
DMEM/F12, no phenol red	Thermo Fisher Scientific	11039-021
DNase (Deoxyribonuclease I)	Worthington Biochemical	LS002007
Donkey anti-Goat 647	Thermo Fisher Scientific	A21447	Use at 1:500, Lot: 1608641, stock 2 mg/mL, RRID:AB_2535864
Donkey anti-Mouse 647	Jackson ImmunoResearch	715-606-150	Use at 1:1000, Lot: 140554, stock 1.4 mg/mL
Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)	Thermo Fisher Scientific	11330-057
Dulbecco's phosphate-buffered saline (DPBS)	Thermo Fisher Scientific	14190-250	Without Mg2+/Ca2+
EGF	Fisher Scientific	AF-100-15-100ug
Fetal Bovine Serum	VWR	97068–085	100% US Origin, premium grade, Lot: 059B18
Fluoromount-G (Southern Biotech)	Fisher Scientific	0100-01	Referred to as mounting media in text
Gentamicin	Thermo Fisher Scientific	15710064
Glycine	Fisher Scientific	BP381-5
Goat anti-WAP	Santa Cruz Biotech	SC-14832	Use at 1:250, Lot: J1011, stock 200 µg/mL, RRID:AB_677601
Hoechst 33342	AnaSpec	AS-83218	Use 1:2000, stock is 20mM
Insulin	Millipore Sigma	I6634-100mg
KCl	Fisher Scientific	P217-500
KH2PO4	Fisher Scientific	P285-500
KRT14–CreERtam	The Jackson Laboratory	5107
Matrigel Growth Factor Reduced (GFR); Phenol Red-Free; 10 mL	Fisher Scientific	CB-40230C	Lot: 8204010, stock concentration 8.9 mg/mL
MillexGV Filter Unit 0.22 µm	Millipore Sigma	SLGV033RS
Millicell EZ SLIDE 8-well glass, sterile	Millipore Sigma	PEZGS0816	These chamber slides are great for gasket removal but other brands can work well (e.g. Lab Tek II).
Mouse anti-SMA	Millipore Sigma	A2547	Use at 1:500, Lot: 128M4881V, stock 5.2 mg/mL, RRID:AB_476701
N-2 Supplement (100x)	Thermo Fisher Scientific	17502048
NaCl	Fisher Scientific	S671-3
NaH2PO4	Fisher Scientific	S468-500
Nrg1	R&D	5898-NR-050
Ovine Pituitary Prolactin	National Hormone and Peptide Program		Purchased from Dr. Parlow at Harbor-UCLA Research and Education Institute
Paraformaldahyde	Millipore Sigma	PX0055-3
Pentobarbital	Millipore Sigma	P3761
R26R-EYFP	The Jackson Laboratory	6148
Rho inhibitor Y-27632	Tocris	1254
R-spondin	Peprotech	120-38
Sodium Hydroxide	Fisher Scientific	S318-500
Sterile Filtered Donkey Serum	Equitech-Bio Inc.	SD30-0500
Sterile Filtered Donkey Serum	Equitech-Bio Inc.	SD30-0500
Triton X-100	Millipore Sigma	x100-500ML	Laboratory grade
Trypsin EDTA 0.05%	Thermo Fisher Scientific	25300-062