Del objetivo específico nº 1: análisis de los modelos de planeación

This experiment was performed on one of the tumour cell lines of human origin available from the stock (Ludwig Institute for Cancer Research), which was derived from a meningioma. The stability of this meningioma cell line (IN1067) allowed for observations upon parameters with possible variability in time. Techniques and protocols that were going to be used for the preparation and analysis of all the samples for all the experiments described in this thesis were used in this reproducibility test.

Meningioma cells were immunocytochemically labelled with specific antibodies at all stages in culture (protocols described in Section 2.3). The following antibodies were used for cell characterization at all passages: A2B5, aGalC, aGFAP [7,71,72], aFN and aVim [84,85]. The purpose of immunocytochemical characterization of the cultures of meningioma cells was threefold:

1). to assess cell subpopulations present in the tumour lines examined {i.e. the homogeneity of the cell populations present in the culture flasks at each passage);

2). to detect possible differences in the cell composition of the culture, and antigen expression by cells, that would appear due to passaging procedures;

One vial containing 10^ cells at the third passage in culture {i.e. subcultured 3 times, P3) of the human meningioma cell line was thawed out from the stock of cell lines preserved in liquid nitrogen. The amount of cells was divided in three equal parts that were plated onto three 25 cm^ flasks, as described in the protocol 2.1.3. When cells reached confluence, each T25 was passaged into one T175 (P4) following the protocol described in 2.1.6. Cells from each of these 3xT175 were further passaged at confluence (P5) onto five T175 flasks (3x(5xT175)). The first three cell extracts were then prepared at passage five (P5) when cells reached confluence. Each of the extracts was prepared from 4xT175 (times 3, equal 12 flasks), while the remaining parallel 3xT175 flask were further passaged, each onto 5xT175 flasks (Figure 3.1).

The passaging and harvesting steps were repeated as above, by continuously subculturing the cells and obtaining cell extracts until P8, each sample representing a fresh culture from the previous passage. Three cell extracts (replicate samples) were obtained at each passage (P5, P6, P7 and P8), and a total of 12 cell samples were obtained. All cell cultures were passaged or extracted when 95-100% confluent. After harvesting, some cells from the final 4xT175 flasks (P8) were plated onto coverslips in order to obtain an assessment of the immunocytochemical labelling of cells at P9.

Cells were harvested by trypsinization (Section 2.1.6) 24 h after a final medium change. PCA extracts were obtained following the method described in Section 2.4. Samples were prepared for NMR analysis as described in Section 2.6. One dimensional and in some cases (at P7) two-dimensional (2D-PFG-C0SY) ^H-NMR spectra from processed cell extracts were acquired (protocols given in Section 2.7).

Quantitative and qualitative assessments of metabolites in cell extracts were obtained from the single pulse ‘H-NMR spectra. HPLC analyses of amino acids, NAA and NAAG were also performed on diluted NMR samples (Section 2.8). Metabolite amounts were referenced to the amount of protein in the sample (Section 2.9), as nmol/mg pr. Statistical analysis was performed on both sets of data, from HPLC determinations, and from NMR spectra. Analysis of variance (oneway ANOVA, Section 2.10) was carried out on individual amounts of metabolites at each passage, against the passage number.

P3 1 vial of cells (10^ cells) o 3xT25 harvested and extracted harvested and extracted P4 PS T25* — ► T175*— ► 5xT175 P4 PS T25* — ► T 1 7 5 * —► 5xT175 P4 PS T25* —► T175*— ► 5xT175 4xT175 lxT175* P6 harvested and extracted 5xT175 4xT175 lxT175* P7

/

Legend: * - cells were p la ted onto coverslips during passage, ju s t before plating onto flasks; Pn - passage number n; Tn - culture fla sk o f n cm^ volume.

Figure 3.1. Schematic representation of the experiment assessing the reproducibility of cell culture techniques, harvesting and extraction procedures in cell preparations within a passage number, and across passages.

Single-pulse *H-NMR spectra were acquired from solutions (20 mM concentration) of putrescine and spermidine. Characteristic HPLC profiles of these polyamines were also obtained by running the solutions on the HPLC column used for the identification and quantification of amino acids.

3.3. RESULTS

3.3.1. Immunocytochemical characterization of the cells at all passages in culture

All preparations of IN1067 meningioma cell line contained cells with a flat, bipolar or tripolar morphology at the passages studied (P5-P9). The cells had the following general immunocytochemical characteristics: A2B5', GalC', GFAF. A very low positive immunocytochemical labelling of cells with A2B5 was obtained at P5, but this can only be interpreted as an experimental artifact, as no other positive labelling with A2B5 was obtained at passages either before P5 {i.e. P4), or after (P6, P7, P8, P9). The strong background coloration present in the coverslips labelled with A2B5 at that passage could be a possible explanation for such a result.

Positive immunocytochemical labelling was obtained for FN and Vim (Table 3.1). The amount of FN expressed by cells in culture was very similar in cell preparations at the same passage. The proportion of FN"^ cells in culture increased with the passage number, in a consistent manner in parallel cell preparations. Vim was strongly expressed by the meningioma cells irrespective of the passage number (100% Vim"^ positive cells). The types and amounts of antigen expression were consistent within a passage number. With the exception of FN, the same consistency was maintained at all the passages examined.

3.2. Identification and quantification of metabolites in NMR spectra, and