NTERA-2/cl.D1 cells (NT2) were obtained from the CNDR, University of Pennsylvania School of Medicine. Undifferentiated NT2 cells were routinely cultivated in standard tissue culture flasks (Nunc) and maintained in OptiMEM medium (Invitrogen) supplemented with 5% (v/v) of fetal bovine serum (FBS, Hyclone) and 100 U/mL of penicillin- streptomycin (P/S, Invitrogen), according to method described at Brito et al..
Stirred suspension culture
Undifferentiated NT2 cell expansion in spinner vessels
Undifferentiated NT2 cells (passage 60-62) were cultured as 3D-aggregates in 125-mL spinner vessels (Wheaton) equipped with a ball impeller and maintained at 37°C and 5% CO2 for up to 7 days. The agitation rate was increased during cultivation in order to avoid aggregate clumping and to control aggregate size (day 0 to 2 - 60 rpm, day 2 to 3 - 70 rpm, day 3 to 4 - 80 rpm, day 4 upwards - 90 rpm). Two independent experiments were performed for each expansion strategy.
Inoculum Concentration Experiments
Cells were cultured in a batch operation mode in Dulbecco's Modified Eagle's Medium- High Glucose (DMEM-HG, 25 mM glucose) (Invitrogen) supplemented with 10% (v/v) FBS and 100 U/mL of P/S (complete DMEM-HG). The cell inoculum concentrations evaluated were: 0.4 × 105, 1 × 105 and 4 × 105 cell/mL; for an easier reading the nomenclature used was SP-0.4B, SP-1B and SP-4B, respectively. In SP-0.4B and SP-1B, cells were cultured in 75 mL of medium at 50 rpm during the first 4-8 h, to promote cell aggregation.
Culture Operation Mode Experiments
Glucose fed-batch and medium exchange culture operation modes were performed using an inoculum cell density of 4 × 105 cell/mL; the nomenclature used for these experiments were SP-4FB (SP- spinner, FB- fed-batch) and SP-4ME (SP- spinner, ME- media exchange), respectively. In SP-4FB, the culture medium was DMEM-Base (Sigma) supplemented with 10% (v/v) FBS, 4 mM of glutamine (Invitrogen), 100 U/mL P/S and 1.4 mM of glucose (Merck). During culture time, glucose concentration was monitored twice a day and maintained at lower levels (<1.4 mM); refeeds were performed accordingly to the consumption rates (calculated from 2 consecutive samples). SP-4ME was cultured in similar conditions to those described for SP-4B, except that medium was partially exchanged daily from the day 3 onwards as follows: fifty percent of culture media was collected in sterile conditions and centrifuged at 200 × g for 5 min; the supernatant was discarded and the recovered cell aggregates gently resuspended in an equivalent volume of pre-warmed complete DMEM-HG.
For all spinner cultures, sampling (2.5 mL) was performed 4 h after inoculation and daily from then on. Cell aggregates were monitored under an inverted microscope (Leica DM IRB). Cell concentration, metabolite concentration and lactate dehydrogenase activity were analyzed as described below.
NT2 culture in a fully controlled bioreactor
To ensure fully controlled cell culture environment, a stirred tank bioreactor  equipped with ball impeller and pH and dissolved oxygen (pO2) measuring probes (Mettler-Toledo) was used for the expansion and differentiation of NT2 cells. The pH was kept at 7.2 by injection of CO2 and addition of base (NaOH, 0.2 M). The pO2 was maintained at 25% via surface aeration. The temperature was kept at 37°C by water recirculation in the vessel jacket controlled by a thermocirculator module. Data acquisition and process control were performed using MFCS/Win Supervisory Control and Data Acquisition (SCADA) software (Sartorius-Stedim, Germany).
NT2 cell expansion
The SP-4ME experiment was reproduced in the bioreactor system, using undifferentiated NT2 cells with 60-62 passages in static conditions. Moreover, cells used for the inoculum (day 0) and at days 3 and 6 of cultivation in the bioreactor, were characterized using immunofluorescence tools and the neuronal differentiation potential evaluated (see below).
NT2 neuronal differentiation
Undifferentiated NT2 cells with up to 62 passages in static conditions were expanded in the bioreactor, in complete DMEM-HG, using an inoculum concentration of 4 × 105 cell/mL. Differentiation was initiated in the middle of the exponential phase (day 3), following the differentiation protocol developed by Serra et al . Briefly, neuronal differentiation was induced by addition of retinoic acid (RA, Sigma) to the culture media, at a final concentration of 10 μM. A 50% media exchange was performed 3 times a week on a regular basis for up to 24 days. Two bioreactor independent experiments were performed.
Samples were collected from the bioreactor at 3 time points: day 9, 16 and 23 (corresponding to 1, 2 and 3 weeks of differentiation process). Cell concentration and neurosphere size were determined and culture was characterized using immunofluorescence microscopy. Neurospheres harvested at the referred time points were transferred to coverslips or culture flasks (5 × 104 cell/cm2) coated with poly-D-lysine (PDL, Sigma) and Matrigel (MG, Becton-Dickinson) and cultured for up to 7 days in mitosis inhibitor (MI) medium: DMEM-HG supplemented with 5% FBS, 100 U/mL of P/S, 1 μM cytosine arabinosine (Sigma), 10 μM fluorodeoxyuridine (Sigma) and 10 μM uridine (Sigma). Neurons were selectively trypsinized [22, 23] using a 0.015% Trypsin-EDTA solution (prepared from Trypsin-EDTA 1X, liquid 0.05% Trypsin, Invitrogen), counted and transferred to coverslips coated with PDL and MG for characterization by immunocytochemistry. Neuronal differentiation efficiency was defined as the ratio between the number of neurons obtained after 7 days of culture in MI medium and the total amount of cells harvested at the 3 different harvesting times.
Cell concentration determination
Cell aggregates were dissociated by a 2 min incubation with Trypsin-EDTA (0.05%) at 37°C followed by cell resuspension in complete DMEM-HG. Cell density was assessed using a Fuchs-Rosenthal haemocytometer (Brand, Wertheim, Germany) and cell viability estimated by the standard trypan blue exclusion test.
Aggregate size in each culture sample was determined using a micrometer coupled to an inverted microscope (Leica, DM IRB). Two perpendicular diameters of a minimum of 15 aggregates were measured and the average diameter was calculated. Aggregates less than 20 μm in diameter (generally cell doublets or triplets) were not considered for calculations as they represent a small percentage of the total cell number in culture.
Lactate dehydrogenase activity
Lactate dehydrogenase (LDH) activity from the culture supernatant was determined as an indirect way of assessing cell death. LDH activity was determined by following spectrophotometrically (at 340 nm) the rate of oxidation of NADH to NAD+ coupled with the reduction of pyruvate to lactate. The specific rate of LDH release (qLDH, U.day-1.cell-1) was calculated for every time interval using the following equation: qLDH = ΔLDH/(Δt ΔXV), where ΔLDH (U) is the change in LDH activity over the time period Δt (day) and ΔXv (cell) is the average of total cells during the same time period. The cumulative value qLDHcum was estimated by qLDHcum i+1 = qLDH i + qLDH i+1. The fold increase of the specific LDH release rates achieved throughout 6 days of cultivation were determined by calculating the ratio between the values of qLDHcum obtained at day 6 and day 0. These values indirectly represent the fold increase in cell lysis obtained within 6 days of culture.
Glucose (GLC), lactate (LAC) and glutamine (GLN) concentrations in the culture medium were analyzed using an YSI 7100MBS (YSI Incorporated, USA). Ammonia was quantified enzymatically using a commercially available UV test (Roche, Germany).
The specific metabolic rates (qMet., mol.day-1.cell-1) were calculated using the equation: qMet. = ΔMet/(Δt ΔXv), where ΔMet (mol) is the variation in metabolite concentration during the time period Δt (day) and ΔXv (cell) the average of adherent cells during the same time period.
Apparent growth rate and fold increase in cell expansion
Apparent growth rates and fold increase parameters were calculated for all expansion cultures. Apparent growth rates (μ, day-1) were calculated using a first order kinetic model for cell expansion: dX/dt = μX, where t (day) is the culture time and × (cell) is the value of viable cells for a specific t. The μ values were estimated applying the model to the slope of the curves during the exponential phase. The fold increase in cell expansion (FI) was defined as the ratio XMAX/X0, where XMAX is the peak cell density (cell/mL) and X0 is the inoculation cell density (cell/mL).
To assess the neuronal differentiation potential along the expansion assays, 2.3 × 106 cells were collected from the suspension cultures and plated in a T75 flask (Nunc) (Figure 1). NT2 cells were differentiated into post-mitotic neurons according to Pleasure et al . Briefly, cells were cultured for 5 weeks in complete DMEM-HG supplemented with 10 μM RA. Cells were splitted at 1:4.5 ratio and cultured in MI medium for 12 days. After this period, neurons were selectively trypsinized, as described above, counted and transferred to coverslips coated with PDL and MG for characterization by immunocytochemistry. Neuronal differentiation efficiency was defined as the ratio between the number of neurons obtained after culture in MI medium and the total amount of cells harvested after RA treatments.
In expansion cultures, cell aggregates were collected at day 3 and 6, dissociated using Trypsin-EDTA (0.05%) at 37°C followed by cell resuspension in complete DMEM-HG, and transferred to glass coverslips. Three days after plating, cultures were characterized. In differentiation assays neurospheres were harvested from the bioreactor cultures at day 9, 16 and 23, and processed for cryosection or transferred to coverslips coated with PDL and MG (see Figure 1).
Cells in coverslips were washed in PBS with 0.5 mM MgCl2 and fixed in 4% (w/v) paraformaldehyde solution in PBS with 4% (w/v) sucrose, for 20 min. For cryosection, neurospheres were washed in PBS, transferred to a tissue protecting compound (Tissue Teck, OCT™ Compound) and frozen at -80°C. Ten μm sections, obtained using a cryostat (Leica), were rehydrated with PBS and fixed in methanol, at -20°C, for 10 min. After fixation, the same procedure was followed for cryosections and coverslips.
For staining intracellular epitopes, cells were permeabilized with 0.1% (w/v) Triton X-100 (TX-100) in PBS, for 15 min. After 1 h in blocking solution (0.2% (w/v) fish skin gelatin in PBS), cells were incubated with primary antibody for 2 h. The coverslips were washed 3 times with PBS and overlaid with secondary antibody for 1 h. Primary and secondary antibodies were diluted in 0.125% (w/v) fish skin gelatin in PBS with 0.1% (w/v) TX-100. Samples were mounted in ProLong mounting medium (Molecular Probes), supplemented with DAPI for nucleus staining. For surface epitopes staining, cells were not permeabilized with TX-100. Samples were visualized using a fluorescence microscope (Leica DMRB).
Primary antibodies used were: mouse anti-tumor related antigen-1-60 (Tra-1-60) (Santa Cruz Biotechnology), mouse anti-stage specific embryonic antigen-4 (SSEA-4) (Santa Cruz Biotechnology), mouse anti-Oct-4 (Santa Cruz Biotechnology), mouse anti-nestin (Chemicon), mouse anti-type III β-tubulin (βIII-Tub) (Chemicon), mouse anti-microtubule associated protein 2A and 2B (MAP2) (Chemicon). The secondary antibodies were goat anti-mouse IgM-AlexaFluor488, goat anti-mouse IgG-AlexaFluor 594, goat anti-mouse IgG-AlexaFluor 488 and rabbit anti-mouse IgG-AlexaFluor 594 (Invitrogen).
Cells used for the inoculum (day 0) and from day 3 of the bioreactor expansion culture were dissociated into single cells and analyzed by flow cytometry (Figure 1). Samples were fixed in CytofixCytoperm reagent (BD Pharmigen) for 10 min, blocked with 1% BSA in PBS at 4°C for 30 min and, in the case of intracellular antigens, permeabilized with 1% TX-100 for 10 min. Primary antibodies were mouse anti-Tra-1-60 and anti-Oct-4. Secondary antibodies were anti-mouse IgM-AlexaFluor488 and anti-mouse IgG-AlexaFluor488. Ten thousand events were registered per sample with a CyFlow® space (Partec) instrument, using the appropriate scatter gates to avoid cellular debris and aggregates. A cell was considered to be positively stained if the measured fluorescence intensity exceeded the signal obtained by cells incubated with an isotype control antibody (Santa Cruz Biotechnology).
For each spinner and bioreactor assays, two independent experiments were performed. The results were expressed as the mean ± standard deviation. The statistical test used, One-way ANOVA, was performed in SPSS 13.0 for Windows for a level of confidence of 95% (a = 0.05) followed by the Scheffé multiple comparison test.