From June 2022 to December 2022, 10 primary human meningiomas were collected after surgery at the Department of Neurosurgery at Saarland University (Homburg, Germany). Patients undergoing elective surgery for suspected or previously diagnosed meningioma were eligible for inclusion. Written informed consent was obtained from each patient as per the protocol approved by the Ethical Committee of the Medical Association of Saarland (approval no. 02/20; Saarbrücken, Germany). A total of 10 tumor samples from 9 patients (3 male patients; 6 female patients) were included in the present study. The mean age of patients at the time of surgery was 75.2±9.4 years (range, 62–86 years). All patients had a primary tumor and patients with recurrence were not included in the present study. All tumors were classified according to the actual 2021 World Health Organization (WHO) classification of tumors of the nervous system (17) by a neuropathologist.

From each meningioma, fragments of the tumor were placed by one individual in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 1% penicillin/streptomycin directly after preparation in the surgical field by a neurosurgeon in the operating theatre at room temperature (22–28°C) before being transferred to the neurosurgical laboratory with standard conditions as previously described (1). A segment of the tissue sample was prepared for culture on the same day as the surgery took place, whilst another was stored overnight in DMEM with 1% penicillin/streptomycin in a refrigerator (4–6°C). The remainder of the tissue was cryopreserved in cryotubes in a freezer at −80°C. For cell culture preparation, the tumor sample was minced using a scalpel and small scissors. Cells were suspended in DMEM containing 10% fetal calf serum (Thermo Fisher Scientific, Inc.), 1% non-essential amino acids and 1% penicillin/streptomycin, before subsequently being distributed into two different 25-cm² cell culture flasks for the next steps. The primary cultures were incubated at 37°C with 5% CO₂ in air and the medium was changed twice per week.

Primary cell cultures at passage 0 (P0) were established on the day of surgery and 1 day postoperatively, and these were classified as culture groups A and B, respectively. These cells from group A and B were stored overnight in DMEM with 1% penicillin/streptomycin in a refrigerator (4–6°C). Cultures from group A and B were split into two passage 1 (P1) cultures. The termination of the cell culture occurred when the cell culture occupied the flask space completely or at least 10 days had passed. One P1 culture was used for FISH, while the other culture was frozen in liquid nitrogen after a cell count. The latter was then thawed after 6–7 months and cultivated again at passage 2 (P2).

The second culture flask from the first passage was prepared for freezing in liquid nitrogen as aforementioned. The medium in the appropriate culture flasks was first transferred to a sterile round tube. The cell culture in each flask was then incubated with 2 ml 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) at 37°C in the incubator for ≥2 mins. Proteolysis was halted by adding 2 ml of DMEM. The cell suspension was subsequently transferred to a centrifuge tube and centrifuged at 37.02 × g for 5 min at room temperature with a brake. The supernatant was removed and discarded, and the cell pellet was resuspended in 1 ml of fresh medium.

For cell counting, 10 µl 0.4% trypan blue solution was pipetted into a 1.5-ml tube. Cell suspension (10 µl) was added and both liquids were mixed well. From this mixture, 10 µl was placed onto a cell counting slide. The LUNA-II™ cell counter (Aligned Genetics, Inc.) was used to analyze the sample, and provided the total cell count, along with the number of live and dead cells, and the viability ratio.

A special freezing medium was used for freezing of the cells. This was prepared from 10 ml dimethyl sulfoxide (DMSO) and 90 ml DMEM, aliquoted into 5-ml portions in round tubes and stored at −20°C. DMSO protects cells from mechanical damage due to ice crystals during freezing. The added medium was centrifuged at 37.02 × g for 5 min at room temperature with a brake. The supernatant was pipetted out and discarded. The pellets were then resuspended in 1 ml freezing medium and transferred to cryotubes, which were placed inside a freezing box. The freezing container filled with isopropanol allowed for controlled cooling of the cells in the cryotubes, achieving a cooling rate of approximately −1°C per min. The container was frozen overnight at −80°C. The following day, the cryotubes were removed and transferred to liquid nitrogen at −196°C, where the cells were stored for subsequent analysis. After 6–7 months, the corresponding cryotubes were thawed and the cells were recultivated at P2. The cultures from P2 were then treated in the same manner as the primary cultures (as described for P1) except for the stage involving the splitting of the initial cultures into two separate cultures. Medium changes and termination of the cultures occurred as soon as a dense cell lawn was observed under the light microscope (Olympus CKX; Olympus Corporation) in daily observations.

In addition to cell culture, one tumor fragment from each sample at P1 and P2 was used for the preparation of dabbed slides. To achieve this, a fragment of the tumor was dabbed onto object slides coated with silane for 5 sec at room temperature, and was fixed with DeLauney fixative, comprising 1:1 acetone/ethanol with 0.05% trichloroacetic acid, at room temperature for 5 min. Samples were stored at −20°C.

Primary tumor cells were dispersed with 0.05% trypsin-EDTA and subsequently suspended by centrifugation at 30.85 × g for 8 min at room temperature. The supernatant was discarded, and the pellet was treated with 0.075 M KCl solution at room temperature for 5 min and resuspended. The cell suspension was centrifuged once more at 30.85 × g for 8 min at room temperature. The supernatant was once again removed and the cells were fixed with methanol/acetic acid (3:1) for 1 h at room temperature. Subsequently, the cell suspension was dropped onto the object dry slides. Additionally, dabbed slides were used to compare both methods as partially described previously (1).

The slides were treated with RNase A (Fisher Scientific; Thermo Fisher Scientific, Inc.) for 30 min at 37°C and placed three times in 2X saline sodium citrate (SSC) for 5 min at room temperature. Cells were digested in 100 ml 0.01 M hydrogen chloride with 10 mg pepsin (SERVA Electrophoresis GmbH) at 0.7 mA for 1 min and 45 sec at 37°C. Slides were dipped in 1X PBS for 5 min at room temperature, 4% paraformaldehyde/1X PBS for 10 min at room temperature for fixation and 1X PBS for 5 min at room temperature. Samples were subsequently dehydrated in 70, 80 and 95% ethanol and air-dried. Dual-probe hybridization was carried out using locus-specific probes for band 36 on the short arm of chromosome 1 (1p36; D6021-100-OG; MetaSystems) and band 11 on the long arm of chromosome 22 (22q11; D5117-100-OG; MetaSystems).

The probes were then pipetted onto the slides and denatured for 2 min at 75°C. Afterwards, samples were incubated overnight at 37°C in a humidified chamber. Stringency washes were performed in 0.4X SSC for 2 min at 72°C and 2X SSC/0.05% Tween-20 for 30 sec at room temperature. Following this, slides were counterstained with DAPI antifade (Vectashield; Vector Laboratories, Inc.; Maravai LifeSciences).

In total, ≥200 non-overlapping nuclei per sample were evaluated according to the Hopman criteria (18) using an Olympus BX43 fluorescence microscope (Olympus Corporation). Cut-offs for alterations were determined by comparison with human lymphocytes as control samples at 10% for deletions of 1p36 and 22q11.

PCR-based microsatellite analysis was performed on meningioma probes directly from the operating theatre (group A) in a manner similar to the protocols previously described for oligodendrogliomas, using different probes (18). For the PCR reaction 1.3 µl DNA, 1.0 µl primer mix (both forward and revers primer at a concentration of 20 µM; Eurofins Genomics), 10.2 µl water and 12.5 µl HotStarTaq Master Mix Kit (Qiagen GmbH) were mixed (25.0 µl total reaction volume). The PCR reaction was performed according to the protocol developed by Hartmann et al (19).

For the investigation of the short arm of chromosome 1 (1p), the probes D1S 1608, D1S 1161 and D1S 1184 (created by Qiagen GmbH for the Institute of Neuropathology, Saarland University, Homburg, Germany), and a probe for the gene locus of AT-rich interaction domain 1A were used. For the long arm of chromosome 22 (22q), the following probes were used: D22S 445, D22S 684, D22S 268 and D22S 258 (created by Qiagen GmbH for the Institute of Neuropathology, Saarland University). PCR products were visualized on high-resolution Spreadex^® EL 800 Wide Mini gels (AL-Labortechnik & Diagnostik GmbH) using an Origins electrophoresis system (AL-Labortechnik & Diagnostik GmbH) and SYBR™-Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific, Inc.). Gel electrophoresis was conducted for 90 min at 120 V in TRIS-acetate-EDTA-buffer (cat. no. 42548.01; TAE buffer, SERVA Electrophoresis GmbH) at 54°C. Subsequently, gels were washed once with distilled water, stained with staining solution for 30 min at room temperature [final buffer concentration, 0.8X TAE (cat. no. 42548.01; SERVA Electrophoresis GmbH), 0.8X Destaining Solution (cat. no. 3037.01; SERVA Electrophoresis GmbH), 2X SYBR™ Gold] and washed again with distilled water. The results were documented using the ‘EOS Utility’ program (Canon, Inc.). Blood samples for LOH analysis were obtained during clinical routine as a standard procedure for neuropathological diagnostics and were stored at room temperature in EDTA tubes for further analysis within 12–48 h.

The graphing and analysis of data were performed using SPSS (version 27.0; IBM Corp.) and Microsoft Excel (Microsoft 365; Microsoft Corporation). The variables were initially tested for normal distribution using the Shapiro-Wilk test. Normality was assumed if the P-value exceeded the significance level. Fisher's exact test was also used for calulations. Since some variables were not normally distributed, the P-values were calculated using the Mann-Whitney U test. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Primary cultures and native tissue samples from 10 meningiomas were established, derived from 3 male and 6 female patients. Notably, 1 patient, designated patient 7, had two tumor probes from different localizations (T9175 and T9177). These two different tumors were surgically treated using two separate surgical approaches and were treated as two separate tumor pathologies in the clinical and scientific setting. All tumors showed soft tumor tissue and could be cut for further preparation of tumor samples. The mean age of patients at the time of surgery was 75.2±9.4 years. Histopathological analysis revealed 8 meningiomas classified as WHO grade 1 and 2 meningiomas classified as WHO grade 2. The tumors were located at the sphenoid wing (n=3), convexity (n=2), parasagittal (n=3), posterior fossa (n=1) and anterior skull base (n=1). Further details are provided in Table I.

A total of 8 out of 10 meningioma samples showed signs of proliferation in both cultures tested from the day of surgery and from 1 day after surgery. In one case, only the sample in group A P0 showed proliferation, whereas in another case, no proliferation was observed in the group A P0 cell culture flask and in the group B P0 cell culture flask. Overall, 17 out of 20 primary cultures proliferated, whilst 3 did not show proliferation after 10 days of observation (Table II). The proliferation rate was calculated as 85% for P0.

There was sufficient cell proliferation in approach A to transfer the P0 cultures into two P1 culture flasks, with 9 successful cultures among a total of 10 cultures (90%) in both. The splitting process led to faster cell proliferation, as seen in daily microscopic observations of the cell cultures, ensuring that the P1 flasks contained enough cells to use one culture for FISH and freeze the other in liquid nitrogen. Representative images of cell culture are shown in Fig. 1.

For approach B, widespread proliferation of meningioma cells was observed in 8 out of 10 cultures (80%) at P0, allowing for further processing. Cultures initiated on the first postoperative day exhibited lower proliferation rates compared with those established on the day of surgery. However, this difference was not statistically significant (P>0.35) (Fig. 2; Table II).

Cell outgrowth from tissue was observed after an average of 1.4±0.9 days. Cultures from group A showed initial cell proliferation after an average of 1.3±1.0 days, whereas cultures in group B exhibited proliferation after an average of 1.5±0.9 days (data not shown). The difference between these two groups was not statistically significant.

Splitting of cultures at P0 could be performed after 8.24±3.05 days on average. Culture group A was split after an average of 7.78±3.27 days, whilst culture group B was split after 8.75±2.91 days (Table II). This indicated that group B cultures were split nearly a day later on average; however, this difference was not statistically significant.

The average duration before termination of primary (P0 and P1) cultures for FISH was 11.94±3.15 days. Cultures from group A were terminated after an average of 11.56±2.78 days, whereas group B cultures were terminated after 12.38±3.66 days. The difference was not statistically significant (Table II).

The mean cell viability across all primary cultures was 81.74±10.17%. The viability of culture group A was 79.14±11.27%, while the viability of group B was 84.77±8.70%. No significant differences were observed between the two groups (group A and B for P1; Fig. 3).

In the secondary generation, 9 cultures from culture A and 8 from culture B were recultivated after freezing in liquid nitrogen for 6–7 months. Of the 17 thawed cultures, 17 were able to continue proliferating, resulting in a re-growth rate of 94.1% for P2. All cultures that proliferated again had established cell attachment within 24 h. Representative cell culture images are shown in Fig. 4.

When recultivating cultures that were initially established the day after surgery, 7 out of 8 cultures (87.5%) exhibited widespread proliferation on the culture surface in the secondary generation (Fig. 2).

Comparing the two generations of cell cultures, primary cultures showed an 85.0% cultivation success rate, whilst secondary cultures had a success rate of 94.1%. Out of the 37 cultures derived from vital tissue or cells, 33 exhibited widespread growth, resulting in an overall proliferative rate of 89.1% (Table II). Statistical comparison between generations was not feasible because primary cultures used tissue as the starting material, whereas secondary cultures involved pre-prepared cells.

The average time before culture termination for FISH was 7.94±1.67 days. Culture A was terminated after an average of 7.67±1.65 days, whereas culture B was terminated after 8.25±1.75 days. The difference was minor and not statistically significant.

The average viability of all secondary cultures was 82±2%. Culture A had a viability of 85±9%, while culture B exhibited an average viability of 78±3%. The difference observed was not statistically significant (P=0.47; Mann-Whitney U test) (Fig. 3). There was no evaluable cell counting possible for culture groups A and B for samples T9077 and T9092 at P1 although there were cells detected due to technical reasons. Therefore, there were only 7 instead of 9 tumors for group A at P1 and 6 instead of 8 tumors for group B at P1.

In four meningiomas, LOH analysis revealed no chromosomal aberrations (Fig. S1). A total of three tumors showed a combined loss of 1p and 22q. Additionally, an isolated 22q loss in two cases and an isolated 1p loss in one case were observed in meningiomas (Table I).

For each meningioma, a dabbed preparation was made, and for both culture approaches, drop preparations of both the P1 and P2 cell cultures were primed when cultures were established. In total, up to five preparations per meningioma were analyzed using FISH. Representative images of FISH analysis are shown in Fig. 5. The results from all preparations of a single meningioma, regardless of whether they were derived from P1, P2 or frozen tissue, were consistent. FISH analysis revealed an isolated loss of 22q (22q-) in three meningiomas, combined losses of both regions 1p and 22q in three meningiomas, and loss of 1p (1p-) in one meningioma. No chromosomal losses were detected in three tumors.

FISH and LOH analysis yielded identical results in 9 out of 10 meningiomas. In 1 case, there were discrepancies: Sample T9177 exhibited a diploid chromosome set in LOH analysis. By contrast, FISH detected a notable loss of 22q in both drop preparations at P1 and P2, in addition to in the dabbed preparation.