Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

Summary

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Abstract

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

Introduction

The FRT has the dual function of protecting against pathogens while allowing implantation and pregnancy¹. To accomplish this, the FRT is compartmentalized, with each anatomical region displaying unique histological, immunological, and functional characteristics¹.

DCs present at mucosal surfaces make contact with microbes in the lung, the gut, and the genital tract, and provide immune surveillance for potential pathogens². DCs have the unique ability to prime naïve T-cells and trigger adaptive immune responses³. DCs in the FRT are also specia....

Protocol

Studies involving human subjects were conducted according to the principles expressed in the Declaration of Helsinki. Studies were approved by Dartmouth College Institutional Review Board and the Committee for the Protection of Human Subjects (CPHS). Written informed consent was obtained before surgery from HIV-negative women undergoing hysterectomies at Dartmouth-Hitchcock Medical Center (Lebanon, NH). Trained pathologists selected tissue samples from the EM, CX, and ECX, free of pathological lesions and distant from the sites of pathology. Blood samples were obtained from volunteer healthy donors recruited at Dartmouth Hitchcock Medical Center. Blood donors were ano....

Results

Following tissue digestion, the release of epithelial sheets and glands can be observed, as shown in Figure 1A; this is a positive control that indicates the enzymatic digestion was successful. The number of total viable cells and DCs recovered per gram of tissue are also shown in Figure 1B and Figure 1C, respectively. Immune cells represent between 5-30% of the stromal cells prior to density centrif.......

Discussion

Mucosal DCs are a rare cell population strongly influenced by the tissue environment, which changes their phenotype and function once they enter the tissues³. While blood derived DCs are a very useful model, they do not fully represent the diversity of DC populations found in tissues. Therefore, to understand the unique characteristics of mucosal DCs, isolation of primary cells from tissues is necessary. Isolation of DCs from different anatomical sites in the FRT offers the opportunity to study DC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hank's Balanced Salt Solution (HBSS)	Hyclone	SH30015.03
Penicillin-streptomycin	Hyclone	SV30010
HEPES (1M)	Hyclone	15630-080
Collagenase IV	Sigma	C5138
Deoxyribonuclease I	Worthington Biochemical	LS002140
D-glucose	Sigma Aldritch	50-99-7
0.22 um Stericup 500 mL  filter	Millipore	SCGPU05RE
100mm x 15mm polystyrene petri dish	Fisherbrand	FB0875712
150mm x 15mm polystyrene petri dish	Fisherbrand	FB0875714
150mm x 25mm polystyrene dish	Corning	430599	Treated cell culture dish
Isotemp Incubator	Fisher Scientific	FICO3500TABB	5.0% CO2
American Rotator V	American DADE	R4140
250 um nylon mesh	Sefar	03-250/50
20 um nylon mesh	Sefar	03-20/14
Beckman GS-6R Centrifuge	Beckman	358702
X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L	Lonza	04-418Q
Human AB Serum	Valley Biomedical	HP1022
Histopaque-1077	Sigma Aldritch	10771
Phosphate Buffer Solution (PBS)	National Diagnostics	CL-253	pH 7.4
Dead Cell Removal Kit	Miltenyi Biotec	130-090-101
Pre-separation filter (30um)	Miltenyi Biotec	130-041-407
LS column	Miltenyi Biotec	130-042-410
Quadro MACS Separator	Miltenyi Biotec	130-090-976
MACS multi-stand	Miltenyi Biotec	130-042-303
EDTA	USB	15694
CD1a Microbeads, human	Miltenyi Biotec	130-051-001
CD14 Microbeads, human	Miltenyi Biotec	130-050-201
eFluor 670 cell proliferation dye	eBiosciences	65-0840-85
96 well round bottom plate	Falcon	9/8/2866
Zombie yellow viability dye	Biolegend	423104
CD3 APC/Cy7, anti-human	Tonbo Biosciences	25-0038-T100
CD4 PE, anti-human	eBiosciences	12-0048-42
CD8a FITC, anti-human	Tonbo Biosciences	35-0086-T100
Gallios Flow Cytometer	Beckman Coulter Life Sciences	B43618	10 color, VBR
MACSquant Analyzer 10	Miltenyi Biotec	130-096-343	8 color, VBR