P-325 Comparing slow-freezing and vitrification endometrial biopsy cryopreservation protocols for tissue cryodamage and functional organoid formation
Authors: Jiang Y , Saare M , Wróbel M , Rodriguez-Wallberg K  A , Reyes Palomares A , Kask K , Kalinina A , Apostolov A , Minejeva A , Kiisholts K , Pathare A , Laudański P , Peters M , Salumets A 
Published: July 2024

Abstract

Study question
Are slow-freezing and vitrification suitable methods for cryopreserving endometrial biopsies intended for downstream research applications and organoid formation?

Summary answer
Both slow-frozen and vitrified endometrial biopsies resulted in successful organoid formation thus can be considered similar to fresh endometrial biopsies in preserving the cellular viability.

What is known already
Passive slow freezing (PSF) protocols have demonstrated efficacy for cryopreserving endometrial biopsies with subsequently viable isolated epithelial and stromal cells. Additionally, single-cell transcriptomic analyses and successful generation of endometrial organoids suggest a little impact of cryopreservation. By avoiding ice crystal formation, vitrification (VT) could potentially prevent mechanical injury and improve tissue integrity compared to slow freezing.

Study design, size, duration
Experimental study using endometrial biopsies collected using a Pipelle from eight healthy volunteers. Samples were retrieved at three cycle phases: proliferative (n = 2), mid-secretory (7 days post-LH surge, LH + 7, n = 2) and late-secretory phase (LH + 11, n = 4). Each biopsy was divided into fresh, slow-freezing and vitrification sub-samples for further comparison of outcomes according to microscopic histological analysis, transmission electron microscopy (TEM), gene expression profiling evaluation and generation of endometrial epithelial organoids (from 2 slow-frozen and 2 vitrified samples).

Participants/materials, setting, methods
PSF was performed using Mr Frosty with samples embedded in 1x DMEM, 30% FBS and 7.5% DMSO at -80ºC. Vitrification was performed using 40% ethylene glycol (EG); 30% Ficoll; 0.5 M Sucrose and 10 mg/ml HSA, and preserved in liquid nitrogen. Tissue morphology was evaluated by traditional histology, TEM and gene expression profiling. The viability of endometrial cells and the ability to form epithelial organoids were evaluated before and after two different freezing protocols.

Main results and the role of chance
Our analysis showed that both PSF and vitrification techniques are appropriate for cryopreservation and storage of endometrial biopsies aimed at organoid generation, as no significant differences were found between fresh and cryopreserved tissue samples.
However, ultrastructural changes were observed by TEM in PSF samples indicating an impact on mitochondria of epithelial and stromal cells with increased swelling. Changes were also observed in epithelial cells in vitrified samples, with decreased nuclear granularity, clarity, and open karyoplasm. On TEM analyses changes in the chromatin status were more evident in vitrified samples compared to fresh and PSF samples.
PSF treated samples showed similarly high quality of epithelial and stromal nuclear staining when compared to the fresh samples in haematoxylin and eosin-stained slides, while VT of samples resulted in a significant decrease of both epithelial and stromal nuclear clarity.
However, despite these changes observed in the tissue morphology, all three groups displayed similar transcriptional profiles and endometrial organoids were generated from both slow-frozen and vitrified samples, indicating very little or no impact of the freezing method used for post-thaw tissue functionality.

Limitations, reasons for caution
Although our study demonstrated relatively mild damages in both freezing protocols, we cannot exclude the absence of additional changes not evaluated in this study, such as those concerning the epigenetic constitution of the cells. Therefore, more extended research with larger sample size is needed for further validation of the results.

Wider implications of the findings
Our findings indicate that both slow-freezing and vitrification methods applied to endometrial biopsies are effective in maintaining tissue morphology and functionality. Further optimization of cryopreservation methods of endometrium could contribute to future experimental and clinical use of cryopreserved endometrial tissue.

Trial registration number
not applicable