Cryo s freezing tubes

For cryopreservation experiments, Bioblock-derived hPS cells generated by above described dissociation procedure were used. E8 + RI-suspended cells were counted and the respective amount of cells was transferred into a 15 mL conical tube (Greiner AG) for subsequent centrifugation at 300×g and 4 °C for 3 min. Collected cells were re-suspended in a respective amount of cryopreservation medium consisting of 90% E8, 10% dimethyl sulfoxide (DMSO; Merck), 0.1% Pluronic F-68 (Thermo Fisher Scientific) and 10 µM RI (Tocris Bioscience) to achieve the desired cell concentration followed by aliquoting of 1 mL into CRYO.S freezing tubes (Greiner AG). Cryopreservation was performed with a Planer Kryo 10 Series III controlled-rate freezer (Planer Limited) with a defined protocol (Additional file 1; Table S4) followed by long-term storage at −150 °C. Cells have been cryopreserved for at least 3 weeks before further cultivation. For direct suspension culture inoculation in bioreactors, cells were thawed in a water bath at 37 °C for ≈3 min, diluted in cooled 10 mL of E8 + RI and collected by centrifugation at 300 × g and 4 °C for 3 min for re-suspension in 20 mL E8 + RI followed by cell counting. Bioreactors were inoculated as described above at the established density of 5 × 10⁵ cells/mL.

Blood samples for analysis were collected within a median of 1 day (range 0–109 days) before surgery. In five patients, time from blood sampling to resection was >30 days (33, 36, 45, 46, 109 days). These patients are further described in the Supplement. All samples were centrifuged at 2300 g at 4°C for 10 min, and serum was then aliquoted in Greiner tubes (Cryo.s™ Freezing Tubes, 2 ml, GR‐121280, Greiner Bio‐One GmbH). The serum was subsequently stored at −80°C. Upon collection, samples were thawed at room temperature, mixed using a vortex mixer, and centrifuged at 1600 g for 10 min. Then, 250 μl was aliquoted to tubes (2.0 ml Graduated w/o Ribs Screw Tubes, Natural from SSIbio, CA, USA), labeled with an individual number, and stored at −80°C until analysis at BioXpedia.

The blood for biochemical analysis was drawn at the time of diagnosis and before surgery or the commencement of chemotherapy. The samples were processed according to the BIOPAC protocol. All blood samples were centrifuged at 2300× g at 4 °C for 10 min, and serum was aliquoted in Greiner tubes (Cryo.s™ Freezing Tubes, 2 mL, GR-121280, Greiner Bio-One GmbH, Baden-Württemberg, Germany). The serum was subsequently stored at −80 °C.