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 (eEC) retaining molecular and functional characteristics of endometrial epithelium in vivo. Design This is an in vitro study using human endometrial cells. Setting University research laboratory. Patients Endometrial biopsies were obtained from premenopausal women undergoing benign gynecological procedures. Interventions Primary eEC were cryopreserved in 1% fetal bovine serum (FBS)/10% dimethyl sulfoxide (DMSO) 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. Main Outcome Measures Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity. Results eEC recovered after cryopreservation (n=5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared to eSF, recovered eEC displayed increased (P<0.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<0.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eEC secrete levels of cytokines and growth factors comparable to freshly cultured eEC. Recovered eEC can formed 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. Conclusion We have developed a protocol for cryopreservation of eEC in which recovered cells after thawing demonstrate morphological, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.

Alterations in Epithelial Cell Polarity During Endometrial Receptivity: A Systematic Review

“…In terms of utilizing newer technologies and methods, the use of three-dimensional cell culture models incorporating primary epithelial and stromal cells from fertile women would increase the translatability of results to the normal endometrium. Emerging studies of cryopreservation and recovery of human endometrium has expanded capacity to use primary epithelial cells for the assessment of epithelial cell polarity and could be employed in future studies (98). To progress the studies toward translation into the clinic, standardized analysis of marker PMT signatures and determinations of the functional significance of the PMT makers using appropriate models will facilitate translation.…”

In vitro evidence that platelet-rich plasma stimulates cellular processes involved in endometrial regeneration

“…All cell culture experiments were conducted with the use of second to fourth cell passages. Primary cells are archived in our Tissue Bank, and we have extensively studied their stability and response to steroid hormones after thawing, plating, and multiple passages in vitro [37]. Once cultured, these cells retain their viability and steroid hormone response uniformly over passages 1-4 [38].…”

Infection of human uterine fibroblasts by Zika virus in vitro: implications for viral transmission in women

“…Cryopreserved UF were revived as recommended by the American Type Culture Collection (ATCC, Manassas, VA, USA) and the cells cultured to confluency using methods reported previously. 8 Individual flasks of UF were then infected with either VR-84 or VR-1843 (ATCC). The inoculum was prepared in 1:100 and 1:1000 dilutions for each strain in low serum UF medium.…”