A method for the dispersal and characterization of leukocytes from the human female reproductive tract.
OBJECTIVE
The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina.


METHODS
This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250-microm mesh screen to facilitate cell dispersion.


RESULTS
The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T-cell marker CD3, monocyte/macrophage marker CD14 and B-cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight.


CONCLUSIONS
These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.
White
H D
HD
Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756-0001, USA. hillary.d.white@dartmouth.edu
Prabhala
R H
RH
Humphrey
S L
SL
Crassi
K M
KM
Richardson
J M
JM
Wira
C R
CR
D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002469:Cell Separation; D005260:Female; D005836:Genitalia, Female / Q000166:cytology* / Q000276:immunology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D007962:Leukocytes / Q000276:immunology*
D015704:CD4 Antigens; D016827:CD8 Antigens; D017493:Leukocyte Common Antigens