Murine Dermal Fibroblast Isolation by FACS

Summary

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Abstract

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin^-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Introduction

Fibroblasts are frequently defined morphologically as spindle-shaped cells that adhere to plastic substrates. Fibroblasts are the principle cell type responsible for synthesizing and remodeling the extracellular matrix in embryonic and adult organs¹. Fibroblasts are thus critical to mammalian development and contribute substantially to the extracellular milieu that influences the behavior of neighboring cell types present in each tissue and organ.

Fibroblasts are also the principal cell type behind a diverse set of medical conditions that cause enormous clinical burden. Pathologic fibroblast activity impairs normal tissue functio....

Protocol

This protocol follows methods approved by the Stanford University Administrative Panel on Laboratory Animal Care.

1. Digestion of Murine Dermis

1. Euthanize mice by cervical dislocation after anesthesia with an intraperitoneal injection of ketamine 100 mg/kg + xylazine 20 mg/kg + acepromazine 3 mg/kg.
   Note: Various ages and backgrounds can be used.
2. Shave and depilate the dorsal skin. Approximately 100,000 cells can be isolated from a piece of dorsal skin 60 mm x 100 mm.
3. Submerge the mouse in 70% ethanol and place on a clean, sterile surface to dry.
4. Immediately harvest dorsal mouse skin using ste....

Results

The validity of this approach (Figure 1) has been verified in a number of ways, which can be examined in detail in our recent publication²⁷. These include immunocytochemistry of sorted cells and mass cell and single cell transcriptional analysis of freshly sorted cells. Sorting fibroblasts directly rather than relying on culture more accurately captures their in vivo phenotype. Using a lineage negative depletion approach (Figure 2A) rather than a positive selection approach avoids pre-sel.......

Discussion

The protocol described in this manuscript offers a means to isolate fibroblasts by FACS-based sorting, in comparison to existing methods, which either select for a subpopulation or require time in cell culture before subsequent analyses. The time required from harvesting of the skin to sorting of fibroblasts is approximately 6 hr; however, the number of mice used in the harvest will influence this estimate.

Several points in the protocol require particular care. The first is limiting adipocyte.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Surgical Forceps	Kent Scientific	INS650916
Micro-scissors	Kent Scientific	INS600127
Povidone Iodine Prep Solution	Dynarex	1415
Nair (depilatory cream)	Church and Dwight Co.	22600267058
Collagenase IV	Gibco	17104-019
Elastase	Abcam	ab95133
DMEM	Life Technologies	A14430-01
Fetal Bovine Serum	Gibco	16000-044
Ammonium-Chloride-Potassium (ACK) lysing buffer	Gibco	A10492-01
40 μm filters	Fisher Scientific	08-771-1
70 μm filters	Fisher Scientific	08-771-2
100 μm filters	Fisher Scientific	08-771-19
CD31	BioLegend	102421
CD45	BioLegend	103125
Tie2	BioLegend	124005
Ter-119	BioLegend	116233
EpCAM (CD326)	eBioscience	48-5791
DAPI	Invitrogen	D3571
propidium iodide (PI viability stain)	BioLegend	421301