αmem glutamax

hBMSCs from 3 healthy male donors under 30 years old were purchased from RoosterBio Inc (USA) at passage 2 and expanded in RoosterNourish™ medium following the manufacturer's protocol. Prior to cell seeding, the scaffolds were sterilised in 70% ethanol solution for 30 ​min and rinsed 3 times with 1x phosphate buffered saline (PBS). hBMSCs at passages 4 were harvested from culture flasks using trypsin-ethylenediaminetetraacetic acid. Cells were washed with PBS and centrifuged at 800 ​g for 5 ​min. The cell pellet was resuspended in basal medium [consisting of α-minimal essential medium (αMEM GlutaMax™, ThermoFisher Scientific, UK) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (PS)]. The cell suspension was seeded onto the scaffolds at a density of 450,000 ​cells per scaffold. To ensure complete penetration of the cells into the scaffold, a syringe vacuum-assisted method was used [53 (link)]. Briefly, the scaffolds were placed in a 5 ​ml syringe and the cell suspension was aspired. The entrance of the syringe was closed by using a luer lock cap and the plunger was pulled back to create vacuum, held for 10 ​s and positioned back to the initial position for 10 more sec. These steps were repeated 3 times before placing the scaffolds in the well plate and in the incubator. Medium was refreshed every 3 days for the duration of all experiments.

Ovaries were isolated from reproductively young (9.5 weeks) and reproductively old (14-16 months) CB6F1 female mice primed with 5 IU PMSG and placed into L15/PVP/SP media. Ovaries were cut into six equal pieces which yielded 24 pieces for each age group. Each piece was encapsulated within a 0.5% alginate droplet and placed into 50 mM CaCl₂ for 2 minutes to cross-link the alginate, which created a bead around the organoids. Encapsulated organoids were then placed into maintenance media and cultured for 0, 2, 4, 6, or 8 days (only day 0 and day 8 data shown). Maintenance media (MM) consisted of 30 μL α-MEM + GlutaMAX (ThermoFisher Scientific), 150 μL 0.5% Pen-Strep (Life Technologies), 300 μL 1% fetal bovine serum (FBS) (ThermoFisher Scientific), and 30 μL 0.1% insulin/transferrin/selenium (ITS) (ThermoFisher Scientific). Organoids were removed from MM on their respective days of culture and placed in alginate lyase solution (final concentration of 10 IU) for 30-35 minutes. Tissue pieces were then fixed in Modified Davidson’s solution or Ethanol-Formalin-Glacial acetic acid (EFG; 70%, 10%, 5% v/v, respectively) fixative at room temperature for 1 hour and then at 4°C overnight. The next day, they were washed 3 times with 70% ethanol, processed and embedded, and IHC was performed on sections with Troma-1 as described above.

Primary hBM-MSC were isolated from bone marrow aspirates of 4 donors (3 male, age 24–33; 1 female, age 60; all Caucasian), expanded in α-MEM/GlutaMAX (ThermoFisher Scientific, Waltham, MA, USA) containing 15% FCS (Corning, NY, USA) and 100 U/mL penicillin/100 µg/mL streptomycin (Pen/Strep; ThermoFisher Scientific) and used in passage 3 and 4 for the experiments. hBM-MSC was characterized according to the criteria of the International Society for Cellular Therapy (ISCT) [44 (link)]. The use of hBM-MSC was approved by the institutional review board (ethics committee) of the Technische Universität Dresden. Human umbilical vein endothelial cells (HUVEC) were purchased from Promocell (Heidelberg, Germany), expanded in Endothelial Cell Growth Medium (Promocell) and used in passage 4 for the experiments.
A human mesenchymal stem cell line expressing human telomerase reverse transcriptase (hTERT-MSC) [45 (link)], kindly provided by Matthias Schieker (Laboratory of Experimental Surgery and Regenerative Medicine, University Hospital Munich (LMU), Munich, Germany), was used for the HCM production to obtain reproducible HCM batches without the variations caused by using primary cells of individual donors.

The CB-derived hMSC used in this work were previously obtained and fully characterized as described in detail in recent publications^{13 (link),14 (link),67 (link)}. The definition of LL- and SL-CBMSC was applied retrospectively based on lifespan and growth properties as previously described (Barilani, 2015). Stored cell samples were used in the present study accordingly to that definition. Culture medium consisted of αMEM-GlutaMAX (Invitrogen, Carlsdad, CA, USA) supplemented with 20% fetal bovine serum (FBS; Invitrogen) and medium changes were performed twice a week. Cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. At 80% confluence, the cells were harvested using 25% TrypLE Select 1× (Invitrogen) and were washed with PBS (Invitrogen) and cultured at a concentration of 4 × 10³ cells/cm². The authors state that this study was performed according to the amended Declaration of Helsinki. In addition, written informed consent has been obtained from all the cord blood donors involved in the study and use of human tissue and cells was approved by the Ethical Committee of our institute “Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico”.