Nucleocounter nc 200

Cell viability was determined using an NC-200™ NucleoCounter^® (Chemometec). Prior to their suspension in the bio-ink for printing, neuroblastoma cells were stained with CellTracker™ Red CMTPX dye (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol with prolonged incubation times, then washed in PBS and counter-stained with CellTox™ Green dye (Promega, Madison, WI, USA) according to the manufacturer’s protocol. The stained 3D tumor models were analyzed using a 2-photon microscope (LaVision Biotec GmbH, Bielefeld, Germany). A region of interest comprising 2.5 x 2.5 x 0.3mm from 5 models was imaged two and eleven days after printing. Imaris Software 7.6.5 (Oxford Instruments, Oxford, UK) was used to count red cells (total cell number) and green cell cores (dead cells) were counted. Live/dead cell ratios in percent were calculated using the simple formula:

Proliferating forebrain and midbrain NSCs (passage 21–27) were analysed using a handheld automated cell counter (Scepter 2.0; Millipore) and 40 µm sensors (Millipore). After trypzination and resuspension, 100 µl cell suspension from each cell line was measured with the Scepter automated cell counting system. Datasets on size distribution estimated as the number of counts in each dedicated size span were processed with the Scepter software 2.0 (Millipore).
Proliferating forebrain and midbrain NSCs were plated onto PLL coated 24-well or 6-well plates (Nunc) at a density of 10,000 cells/cm² and grown in HNSC100 medium with 20 ng/ml EGF and 20 ng/ml bFGF for 4 days before fixation or analysis using the NucleoCounter NC200 (Chemometec, Allerød, DK). Cells were grown at 36°C at either high (20%) or low (3%) oxygen tension. Cells grown in 24-well plates were fixed and immunostained as described later. The NucleoCounter was used to obtain total cell numbers and cell viability. In brief, medium was removed from 6-well plates, adherent cells washed in D-PBS followed by addition of 500 µl/well Trypsin-EDTA for 3-5 min at 36°C. Detached cells were transferred to individual Eppendorf tubes containing 1 ml HNSC100 medium and immediately analyzed by the NucleoCounter according to the manufacturer's instructions.

CD3⁺ cells were purified from healthy donors’ Leukopaks received from Hemacare (Northridge, CA, USA). Upon reception, the leukapheresis material was analyzed using the Sysmex pocH-100i Automated Hematology Analyzer (Sysmex, Milton Keynes, UK), diluted with CliniMACS PBS/EDTA buffer (Miltenyi, Bisley, UK), supplemented with 0.5% human AB serum (Seralab, Hayward Heath, UK) and centrifuged at 300 × g for 15 min at room temperature to remove platelets. The CD3⁺ fraction was purified from the plasma on the CliniMACS Plus instrument (Miltenyi) using the CliniMACS CD4 and CD8 reagents (Miltenyi) anti-CD4 and anti-CD8 monoclonal antibodies conjugated to superparamagnetic iron dextran particles. The viability of the final CD3⁺ product was assessed using the Vi-CELL XR (Beckman Coulter, High Wycombe, UK) and Nucleocounter NC-200 (ChemoMetec, Allerod, Denmark). The number of CD45⁺ cells, CD3⁺ cells, CD4⁺ cells, and CD8⁺ cells was evaluated before and after the cell separation on the CliniMACS by flow cytometry on the Fortessa (BD Biosciences, San Jose, CA, USA). The acceptance criteria for the CD3⁺ cell purification were set as follows: (1) viability over 95% and (2) number of CD3⁺ cells over 95%. Following purification, a working cell bank of CD3⁺ cells was cryopreserved in CryoStor CS10 (Sigma, Gillingham, UK) until use.

Peripheral blood was collected into sodium heparin tubes (9‐mL Heparin S‐Monovette; Starstedt AG & Co KG) and PBMCs were isolated with the use of a Lymphoprep™ (Sigma‐Aldrich) density gradient after centrifugation at 2100 rpm, for 30 min, at room temperature (25°C). After centrifugation, the mononuclear cell layer was collected, washed twice and counted by using an automated cell counter (NucleoCounter NC‐200; ChemoMetec).

Cell number and viability were evaluated by the NucleoCounter NC-200 Automated Cell Counter (ChemoMetec, Denmark). Results were verified by a manual count following a Trypan blue staining (Biological Industries, Israel).