Co-culture assay
A co-culture of NG108-15 neurons with PA5 cells after expansion on microcarriers was used to assess neuron outgrowth. NG108-15 cells (ATCC HB-12317) are a hybrid rodent glioma-neuroblastoma cell line. As a positive control, a rat SCL 4.1/F7 (ECACC 93031294) Schwann cell line was used.
NG108-15 and F7 cells were grown for two passages on T-flasks coated with poly-L-lysine (PLL, Sigma, 100 µg/mL) or uncoated, respectively. When 60-80% confluency was reached, NG108-15 cells were passaged by hitting the flask and re-plated at 6000 cells/cm2, while F7 cells were passaged following the same protocol used for PA5 hOMCs and re-plated at 6000 cells/cm2. For the co-culture assay PA5 hOMCs were plated at 6000 cells/cm2 onto freshly coated 24-well plates with poly-L-lysine (PLL, Sigma, 100 µg/mL). After 24 hours, NG108-15 cells were plated on wells with PA5 hOMCs or F7 cells using a seeding density of 500 cells/well. Medium changes were performed every two days and samples fixed after 5 days. 4-OHT was not added in the medium for this assay. Immunocytochemistry was performed as previously described. Images were acquired with EVOS FL Imaging System (Thermo-Scientific) at a 100x total magnification. 15 frames were acquired per condition (5 per technical repeat) and neurite quantification was performed manually using the NeuronJ (Meijering et al., 2004) plugin in ImageJ.
Statistical methods
The Kolmogorov-Smirnov test was used for data normality and Levene’s test was used for homogeneity of variance. For pairwise-comparisons, one-way ANOVA was used with post-hoc Tukey’s or Games-Howell test. A difference of p<0.05 was considered significant. All tests were performed using OriginPro 2016 (OriginLab, USA).
Formulae
The specific growth rate, µ (days-1):
\begin{equation} \ln\left(X\right)=\ \mu t+ln(X_{0})\nonumber \\ \end{equation}
Where \(X\) is the concentration of cells, \(X_{0}\) is the concentration of cells at \(t_{0}\). \(\mu\) for the exponential phase, considered to be from day 3 to 7.
Doubling time, \(t_{d}\) (days):
\begin{equation} t_{d}=\frac{ln(2)}{\mu}\nonumber \\ \end{equation}
Fold-increase:
\begin{equation} Fold\ increase=\ \frac{X_{f}}{X_{0}}\nonumber \\ \end{equation}
Where \(C_{X(f)}\) is the cell concentration at the end of the culture and \(C_{X(0)}\) is the initial cell concentration.
Specific metabolite consumption rate, \(q_{\text{met}}\)(pmol.cell-1.day-1)
\begin{equation} q_{\text{met}}=\frac{\mu}{X_{0}}\frac{C_{met(f)}-C_{met(0)}}{e^{\text{μt}}-1}\nonumber \\ \end{equation}
Where \(C_{met(t)}\) and \(C_{met(0)}\) are the concentration of metabolite at the end and start of the exponential phase.
Lactate yield from glucose, YLac/Glc
\begin{equation} Y_{Lac/Glc}=\frac{[Lac]}{[Glc]}\nonumber \\ \end{equation}
Where \([Lac]\) is the lactate production and\([Glc]\) is the glucose consumption during the exponential phase.