title: Cryopreservation and recovery of human endometrial epithelial cells with high viability, purity, and functional fidelity.
abstract: OBJECTIVE
  To develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo.
  
  
  METHODS
  In vitro study using human endometrial cells.
  
  
  METHODS
  University research laboratory.
  
  
  METHODS
  Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures.
  
  
  METHODS
  Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte Serum-Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium.
  
  
  METHODS
  Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity.
  
  
  RESULTS
  Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN.
  
  
  CONCLUSIONS
  We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Chen
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Jacquelyn R
authors.initials: JR
authors.lastname: Hoffman
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Ripla
authors.initials: R
authors.lastname: Arora
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Lila A
authors.initials: LA
authors.lastname: Perrone
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Gonzalez-Gomez
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Kim Chi
authors.initials: KC
authors.lastname: Vo
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Laird
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Irwin
authors.affiliation: Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California. Electronic address: Linda.Giudice@ucsf.edu.
authors.forename: Linda C
authors.initials: LC
authors.lastname: Giudice
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D016764:Cell Polarity; D002469:Cell Separation / Q000379:methods*; D048430:Cell Shape; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D004717:Endometrium / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D010539:Permeability; D010641:Phenotype; D019108:Tight Junctions / Q000378:metabolism
chemical_list: D015415:Biomarkers
keywords: Cryopreservation; endometrial epithelial cells; endometrial epithelium; endometrium; freezing medium