Establishment and Characterization of Cell Lines from Primary Culture of Hemangioblastoma Stromal Cells
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.280643
Source of Support: None, Conflict of Interest: None
Keywords: Cell line, hemangioblastoma, primary cell culture, stromal cellKey Messages: Primary cell line of HB stromal cells was well-established with details on methods. Both von Hippel–Lindau disease-related and sporadic HBs might originate from hemangioblasts. Lysosomal autophagy without signs of apoptosis, as a novel phenomenon, was observed in late stage.
Central nervous system hemangioblastomas (HBs) are highly vascular tumors accounting for about 2% of all intracranial tumors and 7%–12% of tumors in posterior fossa.,, They can occur sporadically or as a manifestation of von Hippel–Lindau disease (VHL),, both of which share the same pathological features characterized by neoplastic stromal cells embedded in a dense network of vascular channels. Although HBs are benign tumors, and the surgical outcome of this disease is generally favorable, part of cases are associated with significant morbidity and mortality because of the compression of adjacent structures and multiplicity in VHL., Therefore, it would be necessary to explore the mechanisms of HBs so as to investigate the salvage therapy after failure of surgery and radiotherapy. Accordingly, a well-established cell line and suitable animal model of HBs are expected; however, establishment of HBs' cell culture is difficult because of the benign nature and slow proliferation rate of the stromal cells. Till now, only few studies reported their experience on cell culture of this tumor with limited details on the characteristics of the cultured cells, which largely impeded the basic research and further investigations of this disease.
In this study, we aimed to explore a stable way to establish the primary cell lines of HB stromal cells. Moreover, we identified the biological, morphological, and molecular features of these cells in a broader attempt to supply the theoretical foundation for the establishment of immortalized HB cell lines.
Original tumor specimen
This study was approved by the Ethics Committee of West China Hospital of Sichuan University. The specimens were obtained from 13 patients with intracranial HBs (maximal diameter of the tumor nodule ≥1 cm) who underwent microsurgical resection between January 2015 and January 2016. There were nine males and four females with age ranging from 21 to 55 years. Among them, two patients suffered from solid HBs and 11 from cystic ones. All the specimens were confirmed the diagnosis of HBs by neuropathologists and four patients were diagnosed as VHL.
Central cores of tumors from dissected fresh samples without necrosis were collected, put into Dulbecco's Modified Eagle Medium (DMEM; Gibco), supplemented with 20% fetal bovine serum (FBS; Gibco) and glucose (4500 mg/mL), stored at 4°C in a container covered with ice cubes, and immediately sent to the laboratory for cell culture. The time span should be strictly controlled within 1 h from the beginning of cutting off the blood supply of these tumors.
Samples were rinsed three times in cold phosphate-buffered saline (PBS), cut into 1-mm 3 pieces, digested with Type II collagenase (1 mg/mL; Gibco) at 37°C, and shaken for 1.5 hours. After neutralization of the collagenase with 20% FBS, the solution was filtrated through a 70-μm nylon net, and both the filtrated solution and the digested tissues were centrifuged together at 1500 rpm for 3 min. Then, the supernatant liquid was discarded and the depositional cells were subsequently resuspended in DMEM, supplemented with 20% FBS, hydroxyethyl piperazine ethyl sulfonic acid (HEPES; 5958 mg/mL; Gibco), penicillin (100 U/mL; Hyclone), streptomycin (100 μg/mL; Hyclone), l-glutamine (584 mg/mL), and recombinant human stem cell factor (100 ng/mL; R and D systems). The cultures were incubated at 37°C in a humidified 5% CO2 incubator for approximately 60 h. The cell pellets were paced in the Petri dish at a distance of 1 cm and adhered at 37°C for 5 minutes. Then, the cultures were sequentially incubated at 37°C in a humidified 5% CO2 incubator with the medium and replaced every 72 h. The place of cell pellets should be maintained without movement, and frequent exchange of the culture medium should be avoided. When the cell growth extended more than 80% of the Petri dish, they were detached with 0.25% trypsin and reseeded into fresh proliferative medium. In avoiding the cell injury and waste, multi-step trypsin should be used.
Cryopreservation and resuscitation
The whole process followed the principle of slow frozen and fast thawed in management of cells. Cells with favorable growth status were selected for cryopreservation and further resuscitation. They were digested through multi-step trypsin and centrifuged after culture medium was replaced the previous day. Then, the supernatant liquid was discarded and the depositional cells were resuspended in 1.5 mL of the medium (10% dimethyl sulfoxide and 20% FBS) and transferred into cryopreservation tubes after gentle shaking. Cells were stored at 4°C for 10 min and −20°C for 30 min until the mixture was frozen. Then frozen cells were placed directly into a −80°C refrigerator overnight and deposited in −196°C liquid nitrogen for long-term cryopreservation.
Cryopreservation tubes containing tumor cells were taken out from liquid nitrogen and rapidly placed into a 37°C water bath for resuscitation. When the cells had thawed, they were resuspended with culture medium and centrifuged at 1500 rpm for 5 min. The supernatant liquid was discarded and the depositional cells were managed again following the above-mentioned steps. Then, the cells were incubated at 37°C in a humidified 5% CO2 incubator for further cultures.
Morphological and cell growth features
Morphologic cellular changes in the cultured cells in each generation were monitored and photographed under an inverted light microscope (Leica). Cells of Passage 3 were studied to estimate the doubling time. A single suspension of 1 × 104 cells was produced in 24-well plates by trypsin and stained by Trypan Blue. Cells were counted every 24 h in triplicate for up to 7 days. The growth curve was plotted and the doubling time of the cell population was estimated.
MTT method was used to detect cell viability. Exponentially growing cells (Passage 3) were inoculated on 96-well plates at a density of 3000 cells per well and maintained at 37°C in a 5% CO2 incubator for 7 days. About 20 μL of MTT solution (5 mg/mL; Sigma) was added into each well and cells were incubated for 4 h. The supernatant was discarded, and each well was supplemented with 150 μL of dimethyl sulfoxide (Sigma) and gently shaken for 10 min to dissolve the crystalline substrate. Cell viability as a function of cell growth and contact inhibition was measured every 24 h from 1 to 7 days after plating using Microplate Spectrophotometer (Multiskan MK3; Thermo Labsystems) by measuring the absorbance value at 570 nm. The cell viability curve was plotted with time as the abscissa and the mean absorbance value of each well as the ordinate.
Immunocytochemical staining and transmission electron microscopy
The single suspended cells (Passage 4) at a density of 1 × 104/mL were inoculated on 24-well plates at 37°C in a 5% CO2 incubator. Then, cultured cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After that, 0.5% Tritonx 100–PBS (Sigma) was used to penetrate the cell membranes for 1 h, and the cells were blocked by 1% BSA (Sigma) for 30 min. After each step, cells were washed by PBS for three times. Then, the cells were treated with primary antibodies separately including anti-inhibin-α (1:200; Abcam), anti-brachyury (1:100; Abcam), anti-CD133 (1:200; Santa Cruz), anti-CD34 (1:200; Abcam), anti-CD31 (1:200; Abcam), anti-glial fibrillary acidic protein (anti-GFAP; 1:200; Santa Cruz), anti-NeuN (1:200; Abcam), anti-CD45 (1:200; Abcam), and anti-Olig2 (1:200; Abcam) at 4°C overnight. After washing with PBS for three times, the cells were incubated with corresponding secondary antibody (1:500; Invitrogen) in dark for 2 h. After washing with PBS for three times, DAPI was added into the plate in dark for 8 min, and washing with PBS for three times was performed again. Finally, the cells were analyzed and photographed under a fluorescence microscope (Leica DM4000 B).
Exponentially growing cells were selected for transmission electron microscopic observation. Cultured cells at fourth, seventh, and ninth generation were digested by trypsin and centrifuged at 2000 rpm for 10 min. The supernatant liquid was discarded and the depositional cells were fixed with 0.5% glutaraldehyde at 4°C for 10 min. After centrifugation again at 13,000 rpm for 15 min, the supernatant liquid was discarded and the depositional cells were fixed with 3% glutaraldehyde at 4°C for 10 min. Then, the cells were gradually dehydrated in a series of acetone (80%, 90%, and 100%) solutions for 30 min each and embedded in Epox812 resin. Finally, thin sections were cut and double-stained with uranyl acetate–lead citrate. Transmission electron microscope (H-600IV; Hitachi, Japan) at 8000× magnification was performed to observe the cultured cells.
Student's t-test was used to compare continuous variables and Pearson's Chi-square test was used to compare categorical variables. All statistical analyses were processed by SPSS software (version 19.0), and P < 0.05 was considered statistically significant.
The continuous primary culture of HBs stromal cells was performed in 13 cases, in which 11 cases were successfully cultured and the primary cell lines were established with a success rate of 84.6%. Two specimens from deeply located solid HBs failed to culture, whereas cystic tumors with superficial feeding arteries are the favorable samples [Figure 1]. The cryopreserved cells could be resuscitated successfully and showed favorable growth status.
Morphological characterization and cell growth
After cells were cultured in vitro, it could be observed that the cultured stromal cells grew rapidly from the cell pellet on the third day. Then, these cells showed vigorous growth status and presented as polygons or trigons with significant heterogeneity during the former five generations. Several cell protrusions were observed in this period, which represent stronger growth ability than previously reported. However, from the sixth to ninth generation, despite the stromal cell presentation as polygons with significant heterogeneity, the time span of subculture extended gradually. Until 9th to 10th generation, the cell growth ceased, and the cultured cells died in about 1–2 weeks [Figure 2]. The growth curve is plotted in [Figure 3] and the estimated doubling time was 77.2 ± 5.89 h. Cell viability measured through MTT method is shown in [Figure 3].
Immunocytochemical findings and transmission electron microscopy observation
Inhibin-α, as the diagnostic marker of HBs stromal cells, was expressed in all the cultured cells, located in nucleus and cell membrane. Whereas GFAP (marker of glial cells), CD31 (marker of endothelial cells), NeuN (marker of neuronal lineage), CD45 (marker of microglial cells), and Olig2 (marker of oligodentrocyte) were all negative in all the cultured cells. Moreover, brachyury, CD133, and CD34 were also positive in all these cells; brachyury and CD133 were concentrated in nucleus with limited expression in cytoplasm, whereas CD34 was mainly expressed in cytoplasm [Figure 4].
As seen from transmission electron microscopy (TEM), it can be confirmed that the cultured cells were the stromal cells because of the typical structure of intracellular lipid droplets. After the fourth generation, the stromal cells were in vigorous growth status presenting as large size cells and typical lipid droplets in cytoplasm, while no endothelial cells and pericytes were observed. After sixth generation, the intracellular lipid droplets shrunk gradually with subculture time, and until the 9th to 10th generation, the autophagy of the lysosome was commonly observed, whereas no signs of apoptosis were observed [Figure 5].
Central nervous system HBs are benign tumors, and most cases are curable after surgical resection; however, for those lesions deeply located in the brainstem and VHL patients with multiple HBs, effective therapeutic strategy is still lacking.,,, Thus far, cytological origin and pathogenesis of HBs remain unclear and controversial. Moreover, based on the data from case reports and some clinical trials, management of HBs with antiangiogenic drugs seemed disappointed, including ineffectiveness in reducing the tumor size and increasing the risk of recurrence after withdrawal of treatment.,,, Therefore, for further investigations, a well-established cell line of HBs is required.
Establishment of primary cultures of hemangioblastoma cells
Currently, studies on cell culture of central nervous system HBs are rare, let alone the establishment of cell lines, largely because of the benign nature of this tumor, which leads to difficulty in maintaining rapid growth when cultured. In 1975, Spence and Rubinstein first reported the tissue culture of a HB from cerebellar vermis of a 21-year-old man on gelatin sponge foam matrices and Millipore filter platforms. The cultures were maintained for up to 48 days with stable structures, and superficial invasion of matrix was observed. However, stromal cells, as the neoplastic component of HBs, were not separately cultured. In 1996, Hatva et al. cultured the tumor tissue in RPMI medium supplemented with 10% FBS, penicillin, and l-glutamine. The cultured cells only passaged three to four times and grew for several weeks. Moreover, they depicted the morphological features of stromal cells as small, round, and uniform cells in light microscopy, which was inconsistent with our study. Albinana et al. cultured the HB cells through similar methods; however, the details on successful rate and growth kinetics were lacking. Therefore, a well-established primary cell line of HBs is still lacking. In this study, continuous cell culture of HB stromal cells was performed in 13 cases, in which 11 cases were successfully subcultured with a success rate of 84.6%. According to our experience, four key points may contribute to the successful establishment of primary cell lines of HB stromal cells. (1) Appropriate preparation of tumor specimen. It was mentioned by some authors that solid HBs should be selected for further cell culture, because it could provide more tumor cells. However, in this study, two specimens from solid HBs failed to culture. We considered that solid HBs were commonly deeply located lesions with severe adhesion to the surrounding structures and complex blood supply; samples from surgical resection were associated with long-time ischemia and severe cauterization, which were difficult to be cultured. Therefore, rather than solid HBs with large size, we consider that cystic HBs with superficial feeding arteries are the favorable samples. (2) Short duration of devascularization. HBs are highly vascular tumors, which require en bloc resection. En bloc resection of HBs is time-consuming, while the tolerance to deficiency of blood supply of these tumors is poor. Therefore, the time span between initiation of cutting off the blood supply and tissue digestion should be strictly controlled within 1 h. (3) Ideal digestion time. Long time digestion can reduce cell activity, whereas if the digestion time is too short, it will lead to insufficient cells available for cell culture. We used filtrated cell solution and digested tissues for cell culture, which not only increased the utilization rate but also reduced cell impairment caused by long time digestion. (4) Nutritious medium. After comparing among different mediums in the preliminary experiment, we found that stromal cells were easier to grow in DMEM with high glucose than those in RPMI 1640 and DMEM with low glucose. Some authors cultured stromal cells in serum-free medium which were available to investigate tumor-initiating cells of HBs and their evolving process; however, the success rate of these cells in less nutritious medium needs further examination. In addition, Ding et al. successfully cultured stromal cells of HBs in DMEM supplemented with 20% FBS and other components including heparin, HEPES, penicillin, streptomycin, and l-glutamine. After the culture medium was changed three to four times every 3–5 days, it was observed that the stromal cells grew rapidly presenting as polygons or trigons, whereas the endothelium showed apoptosis gradually. Eventually, they failed to establish the cell line of HBs, probably because the proliferation of stromal cells, similar to stem cells, was based on the microenvironment in vivo, which was difficult to simulate in vitro. According to their study, we compared culturing stromal cells in medium with or without stem-cell factor through the MTT method in our preliminary experiment, which showed that the cultured cells in medium with stem cell factor had stronger cell viability than those without it. Nevertheless, other studies used even more nutritious components which could not only promote cell proliferation but also lead to differentiation of stromal cells. Park et al. performed cell culture by single-cell suspensions of tumors in DMEM supplemented with 30% FBS and other components including BSA, β-mercaptoethanol, l-glutamine, penicillin, streptomycin, transferrin, selenium, insulin, ferrous sulfate, ferric nitrate, dexamethasone, stem-cell factor, and erythropoietin. Although the cultured cells could grow as rapidly as ours and were generated for at least 90 days, plenty of nourishing components could lead to differentiation of the stromal cells, and the establishment of primary cell lines was not mentioned.
Characterization of primary cultures of hemangioblastoma cells
Recent studies have identified embryonic hemangioblasts as the source of VHL-related HBs and hemangioblasts are responsible for neovascularization of HBs.,, Accordingly, we believe that a well-established cell line of HBs with clear characterization is required to treat these tumors by persuading hemangioblasts to differentiate or using their unique proteins to target toxic drugs to HBs.
In this study, the expression of inhibin-α was positive in all the cultured cells, whereas the expression of GFAP, CD31, NeuN, CD45, and Oligo2 was all negative. Inhibin-α, a member of transforming growth factor beta, is widely accepted as the pathologically diagnostic sign of HBs, which located in nucleus and cell membrane of stromal cells., Meanwhile, GFAP is considered as the marker of glial cells, CD31 as the most common marker of endothelial cells, NeuN as the marker of neuronal lineage, CD45 as the marker of microglial cells, and Olig2 as the marker of oligodendrocyte. Negative expression of GFAP, CD31, NeuN, CD45, and Olig2 in all the cultured cell lines excluded the existence of glial cells, endothelial cells, neuron, microglial cells, and oligodendrocyte, and positive expression of inhibin-α identified all the cell lines as stromal cells of HBs. Furthermore, the results from TEM also confirmed that cultured cells were stromal cells with typical structure of intracellular lipid droplets. After the sixth generation, the intracellular lipid droplets shrunk gradually with subculture time, which indicated that cell viability gradually reduced with generations. Interestingly, until the 9th to 10th generations the cultured cells were in senescent condition; however, the phenomenon of lysosomal autophagy without apoptotic cells was commonly observed, which implied that cultured cells were still in the state of stress rather than apoptosis. Therefore, these primary cell lines of stromal cells might be continued to subculture through modifying the condition of culture in late stage. In addition, all the cultured cell lines of stromal cells demonstrated positive expression of brachyury, CD133, and CD34. Brachyury is expressed only during early mesoderm development,, and CD133 as well as CD34 are hematopoietic stem-cell markers., Previous study identified their expressions in VHL-related HBs suggesting these cells might originate from embryologically arrested hemangioblasts. In this study, all the cultured cells demonstrated expression of brachyury, CD133, and CD34, which implied that both VHL-related HBs and sporadic HBs may share a similar origin.
This study focused on the details of establishment of primary cell lines of HB stromal cells, which provided the foundation for further investigation on this tumor. In this study, cultured cells were unable to continuously passage; however, the results from TEM showed that the cultured cells in the late stage still had the potential to subculture, which required further investigation on modification of the condition of culture so as to establish immortalized HB cell lines.
Appropriate preparation of tumor specimen, short duration of devascularization, ideal digestion time, and nutritious medium are critical points for well establishment of the primary cell line of HB stromal cells. Immunocytochemical findings and transmission electron microscopy confirmed that the cultured cells were HB stromal cells demonstrating positive expression of brachyury, CD133, and CD34, which implied that both VHL-related HBs and sporadic HBs might originate from embryologically arrested hemangioblasts. The phenomenon of lysosomal autophagy without apoptotic cells was commonly observed, which indicated that the cultured cells still had the potential to subculture through modifying the condition of culture in late stage. Therefore, our study provided the foundation for the establishment of HBs' infinite cell line and further investigations.
Liu and Zhang contributed equally in this study as the co- first author.
Financial support and sponsorship
The current study was supported by Youth Program of National Natural Science Foundation of China (No. 81801178).
Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]