Dpbs cts

Human bone marrow-derived MSCs from two donors (35 (link), 36 (link)) at passage 4 were thawed, and 1,200 cells/cm² were plated on 10 cm plates (Nunclon™ Delta Surface, Thermo Fisher Scientific) in 10 ml α-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 20 mM HEPES (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco). The cells were incubated at 37°C, 5% CO₂ for 5 days, and the medium was renewed after 24 h. The cells were washed with 5 ml warm endotoxin-free phosphate-buffered saline (PBS) with or without (w/o) Ca²⁺/Mg²⁺ (Gibco DPBS CTS™) and detached with 1.5 ml TrypLE™ Express (Gibco). The detachment process was stopped with 5 ml warm 10% FBS (Sigma-Aldrich) in RPMI Medium 1640, GlutaMAX™ Supplement (Gibco), and the cells were centrifuged at 500 g for 5 min. The pellet was suspended with 10% FBS in RPMI Medium 1640, GlutaMAX™ Supplement, and cells were counted with NucleoCounter^® NC-100™ (ChemoMetec).

Cryopreservation and thawing of cortex samples were performed according to established protocols with modifications [32 (link)–37 ]. In brief, tissue samples were incubated in L-15 Leibovitz’s medium (Gibco Life technologies, NY, U.S.A.) supplemented with 11% human serum albumin [HSA] (Irvine Scientific, Santa Ana, USA), 10% dimethyl sulfoxide [DMSO] (CryoSure DMSO, WAK Chemie, Steinbach, Germany) and equilibrated for 35 min prior slow freezing procedure. Cortex samples were transferred to 1.8 ml vials (Nunc, Thermo Fisher Scientific, Denmark) and cryopreserved with a cooling rate of − 2 °C per min. After seeding, samples were cooled at a rate of − 0.3 °C per min to − 40 °C and at − 10 °C per min to − 140 °C. For thawing, tissue samples were brought to room temperature for 40 s and kept in a water bath for 130 s at 37.2 °C. Transfer of tissue was conducted in 15-min steps to 3 thawing solutions of decreasing sucrose concentrations based on Dulbecco’s phosphate buffered saline [DPBS] (DPBS CTS, Gibco Life technologies, NY, U.S.A.) supplemented with 11% HSA (Irvine Scientific, Santa Ana, USA) prior 2 washing intervals.

calcein AM (Merck, Darmstadt, Germany) was dissolved with DMSO (Cryosure DMSO, WAK Chemie, Steinbach, Germany. Follicular viability was determined 24 h after tissue preparation and thawing/rapid warming. Prealiquoted calcein (Merck, Darmstadt, Germany) was dissolved with prewarmed DPBS CTS (Gibco Life technologies, NY, U.S.A.) and transferred to prealiquoted collagenase type 1A (Merck, Darmstadt, Germany) to obtain a viability working solution of 2 µmol/l calcein AM and 1 mg/ml collagenase type 1A. 2 × 2 mm biopsy punches were added to 500 µl of viability working solution and incubated for 90 min at 37.2 °C, protected from light. The solution was resuspended cautiously after 60 and 70 min to enable evenly bottoming of follicles prior fluorescence measurements at room temperature. Number of vital follicles was determined with fluorescence microscopy (Nikon, Ti2, Düsseldorf) as indicated in Fig. 6.Fig. 6

Viability staining of ovarian cortex samples after digestion with collagenase. Viable follicles can be identified by a layer of granulosa cells enclosing the oocyte emitting green fluorescence (495 nm) and by their size difference to stroma cells (A, B). Picture with tenfold (A) magnification displaying viable follicles. Zoomed in section of A with 4 viable follicles (B) at 20-fold magnification. Scalebar 100 µm