A human malignant giant cell tumor of bone cell line and its use

Abstract

The present application relates to the technical field of tumor biology, and specifically discloses a human malignant giant cell tumor of bone cell line and application thereof, the human malignant giant cell tumor of bone cell line is preserved on April 9, 2025, the preservation address is China Center for Type Culture Collection, located in Wuhan University in Wuhan, Hubei Province, and the preservation number is CCTCC NO:C202539.The human malignant giant cell tumor of bone cell line of the present application is from the tumor tissue after surgical resection of a Chinese malignant giant cell tumor of bone patient, and is suitable as cell material for developing malignant giant cell tumor of bone related immunotherapy, diagnosis and drug development.The cell line of the present application contains H3F3A G34W mutation, and has high sensitivity and specificity in the diagnosis of malignant giant cell tumor of bone.This cell line can be used as a tool cell for researching target drugs with H3F3A as a target, provides a cell model for Chinese malignant giant cell tumor of bone research, and provides an experimental platform for mechanism research of malignant giant cell tumor of bone.

Description

Technical Field

[0001] This invention relates to the field of tumor biology technology, and more specifically, to a human malignant giant cell tumor of bone cell line and its applications. Background Technology

[0002] Malignant giant cell tumors of bone are extremely rare, accounting for less than 10% of all giant cell tumors of bone. Based on clinical background, malignant giant cell tumors of bone can be divided into two main types: primary and secondary. The former refers to cases where classic giant cell tumors and high-grade sarcoma components are present at initial diagnosis, while the latter refers to cases where malignant transformation or recurrence of high-grade sarcoma has occurred at the primary site after intervention such as surgery or radiotherapy. The prognosis of malignant giant cell tumors of bone is similar to that of high-grade sarcomas, with the prognosis of secondary malignant subtypes generally being worse than that of primary malignant subtypes. Currently, there is no standard treatment regimen for malignant giant cell tumors of bone; treatment often follows the model used for high-grade sarcomas, employing a comprehensive strategy of preoperative chemotherapy-surgery-postoperative chemotherapy. Especially for malignant giant cell tumors of bone with osteosarcoma phenotypes, osteosarcoma chemotherapy regimens are typically used along with extensive surgical resection. However, due to the low incidence and lack of effective comprehensive treatment methods, the recurrence rate and mortality rate are extremely high. At the molecular diagnostic level, monoclonal antibodies targeting the histone H3.3 G34W epitope exhibit high sensitivity and specificity in the diagnosis of giant cell tumor (GCTB) and malignant giant cell tumor of bone. H3F3A gene G34W mutations account for 97.8% of GCTB cases. H3F3A gene mutations can be detected by immunohistochemistry or molecular biology methods and are typically preserved in malignant cell populations. Therefore, in-depth exploration of the molecular pathogenesis of malignant giant cell tumor of bone and research into drugs targeting H3F3A may hold significant promise for the treatment of this disease. Currently, there is no widely used, standardized cell line for malignant giant cell tumor of bone, either domestically or internationally. This significantly limits the ability to study the mechanisms of malignant transformation and develop targeted therapies for these patients.

[0003] Therefore, finding methods for culturing malignant giant cell tumor of bone, establishing mature malignant giant cell tumor cell lines, and conducting corresponding drug research are urgent problems to be solved in current clinical research. This will not only help to fully reveal the biological diversity of this disease, but also provide more representative and widely applicable research resources for optimizing treatment plans and carrying out personalized treatment. Summary of the Invention

[0004] In view of this, the present invention proposes a human malignant giant cell tumor cell line and its application, aiming to solve the problem that a widely used and standardized malignant giant cell tumor cell line has not yet been formed at home and abroad.

[0005] On the one hand, the present invention proposes a human malignant giant cell tumor cell line, which was deposited on April 9, 2025, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan City, Hubei Province, with accession number CCTCC NO:C202539 and classified as human malignant giant cell tumor cell line MGCTB-CZ1.

[0006] Furthermore, the human malignant giant cell tumor cell line MGCTB-CZ1 contains the H3F3A G34W mutation and can be continuously passaged in vitro while maintaining the characteristics of malignant giant cell tumor cells.

[0007] Furthermore, the method for constructing the human malignant giant cell tumor of bone cell line MGCTB-CZ1 is as follows:

[0008] Tumor tissue samples were obtained from malignant giant cell tumors of bone after surgical resection.

[0009] After pretreatment, the tumor tissue sample was added to DMEM / F12 medium containing 5% fetal bovine serum and tissue digestion solution, and digested at 37°C for 1 hour to obtain a suspension.

[0010] The suspension was filtered sequentially through 70μm and 40μm cell sieves. The filtered suspension was centrifuged at 800 rpm for 5 minutes, and the precipitate was collected to obtain tumor cells. The tumor cells were resuspended in tumor cell culture medium and cultured at 37°C in a 5% CO2 atmosphere. The tumor cell culture medium was changed every 2-3 days during the culture.

[0011] When the cell density reaches 80%-90%, the supernatant is aspirated, the cells are washed twice with DPBS, digestive enzymes are added, and the cells are digested at 37°C for 3 minutes. Then, tumor cell culture medium is added to stop the digestion. After centrifugation at 800 rpm for 5 minutes, the supernatant is aspirated, the cells are resuspended in tumor culture medium, and then passaged at a 1:2 ratio.

[0012] Furthermore, the pretreatment involves rinsing the tumor tissue sample twice with a tissue rinsing solution to remove fibrous connective tissue, fat, and necrotic tissue, and then cutting it into granules.

[0013] Furthermore, the tissue digestion solution contains 1 mg / ml type II collagenase.

[0014] Furthermore, the tumor cell culture medium is prepared by adding 15% fetal bovine serum and 1% penicillin-streptomycin bispecific antibody to a DMEM / F12 basal culture medium system.

[0015] Furthermore, the method for preparing the tissue irrigation solution is as follows:

[0016] Add 2% penicillin antibody solution to HBSS buffer, mix, and obtain the tissue rinsing solution.

[0017] Furthermore, the method for preparing the digestive enzyme is as follows:

[0018] Dilute the trypsin containing 0.25% EDTA in PBS to a final concentration of 0.05%.

[0019] On the other hand, the present invention also proposes the application of the aforementioned human malignant giant cell tumor cell line in the preparation of a cell model for studying the pathogenesis of malignant giant cell tumor of bone.

[0020] On the other hand, the present invention also proposes the application of the aforementioned human malignant giant cell tumor cell line in the preparation of diagnostic reagents or diagnostic kits for malignant giant cell tumor of bone.

[0021] Compared with existing technologies, the advantages of this invention are as follows: The human malignant giant cell tumor cell line MGCTB-CZ1 provided by this invention is a human-derived single-cell line containing the H3F3A G34W mutation. It can be continuously passaged in vitro while maintaining the characteristics of malignant giant cell tumor cells, making it suitable as a cell material for the research and development of immunotherapies, diagnostics, and drugs related to malignant giant cell tumors. Furthermore, the human malignant giant cell tumor cell line MGCTB-CZ1 of this invention contains the H3F3A G34W mutation, which is believed to drive the occurrence of malignant giant cell tumors and promote tumor cell proliferation. Therefore, this cell line can serve as a tool cell for research on targeted drugs targeting H3F3A. Attached Figure Description

[0022] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0023] Figure 1 Morphological images of different passages of the human malignant giant cell tumor of bone MGCTB-CZ1 cell line provided in the embodiments of the present invention under a light microscope.

[0024] Figure 2 HE staining image of the human malignant giant cell tumor of bone MGCTB-CZ1 provided in an embodiment of the present invention.

[0025] Figure 3 Immunohistochemical staining image of the human malignant giant cell tumor of bone MGCTB-CZ1 (H3.3G34W staining) provided in an embodiment of the present invention.

[0026] Figure 4 The cell growth curve of the human malignant giant cell tumor of bone MGCTB-CZ1 provided in the embodiments of the present invention. Detailed Implementation

[0027] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] On the one hand, in some embodiments of this application, a human malignant giant cell tumor of bone cell line MGCTB-CZ Homosapiens is deposited on April 9, 2025, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan City, Hubei Province, with accession number CCTCC NO:C202539 and classified as human malignant giant cell tumor of bone cell line MGCTB-CZ1.

[0029] In some embodiments of this application, the human malignant giant cell tumor cell line MGCTB-CZ1 contains the H3F3AG34W mutation and can be continuously passaged in vitro while maintaining the characteristics of malignant giant cell tumor cells.

[0030] Specifically, the inventors obtained the MGCTB-CZ1 malignant giant cell tumor cell line from a 31-year-old male patient with malignant giant cell tumor located in the proximal right femur. Postoperative pathology showed recurrence of the malignant giant cell tumor. Tumor tissue samples were taken after surgical resection, and primary culture was successfully established.

[0031] Specifically, the MGCTB-CZ1 cell line of malignant giant cell tumor of bone developed by the inventors exhibits vigorous growth. Most cells are irregularly polygonal, while a small number are spindle-shaped, round, or oval, with prominent nuclei. Under high-density culture conditions, cell aggregation was observed, demonstrating malignant characteristics of loss of contact inhibition. MGCTB-CZ1 cells adhere to the culture medium in a typical epithelial-like manner. After adhesion, they grow rapidly and exhibit good in vitro culture expansion capacity.

[0032] Specifically, the STR sequencing results of the human malignant giant cell tumor of bone cell line MGCTB-CZ1 were compared with those in cell preservation databases such as ATCC and DSMZ. No identical STR results were found, thus confirming that MGCTB-CZ1 is a human monoculture cell line, originating from the same tissue or individual, and that no cross-contamination with other cell lines occurred during primary culture. Exon sequencing results showed that the human malignant giant cell tumor of bone cell line MGCTB-CZ1 contains the H3F3A G34W mutation.

[0033] The present invention relates to the MGCTB-CZ1 cell line for malignant giant cell tumor of bone, derived from tumor tissue surgically removed from a Chinese patient with malignant giant cell tumor of bone. STR testing confirmed its uniqueness. This cell line can be continuously passaged in vitro while maintaining the characteristics of malignant giant cell tumor cells, making it suitable as a cellular material for the development of immunotherapies, diagnostics, and drugs related to malignant giant cell tumor of bone. The cell line contains the H3F3A G34W mutation, which is believed to drive the development of malignant giant cell tumor of bone by promoting tumor cell proliferation. Therefore, this cell line can serve as a tool for researching targeted drugs targeting H3F3A, providing a new cell model for the study of malignant giant cell tumor of bone in Chinese patients and offering a better platform for the mechanistic study of this tumor.

[0034] In some embodiments of this application, a human malignant giant cell tumor of bone cell line, the method for constructing the human malignant giant cell tumor of bone cell line MGCTB-CZ1, is as follows:

[0035] Tumor tissue samples were obtained from malignant giant cell tumors of bone after surgical resection.

[0036] After pretreatment, the tumor tissue sample was added to DMEM / F12 medium containing 5% fetal bovine serum and tissue digestion solution, and digested at 37°C for 1 hour to obtain a suspension.

[0037] The suspension was filtered sequentially through 70μm and 40μm cell sieves. The filtered suspension was centrifuged at 800 rpm for 5 minutes, and the precipitate was collected to obtain tumor cells. The tumor cells were resuspended in tumor cell culture medium and cultured at 37°C in a 5% CO2 atmosphere. The tumor cell culture medium was changed every 2-3 days during the culture.

[0038] When the cell density reaches 80%-90%, the supernatant is aspirated, the cells are washed twice with DPBS, digestive enzymes are added, and the cells are digested at 37°C for 3 minutes. Then, tumor cell culture medium is added to stop the digestion. After centrifugation at 800 rpm for 5 minutes, the supernatant is aspirated, the cells are resuspended in tumor culture medium, and then passaged at a 1:2 ratio.

[0039] Specifically, all raw materials used in the construction method are commercially available products.

[0040] Specifically, the tumor tissue of the malignant giant cell tumor of bone originated from a patient with malignant giant cell tumor of bone (the patient was a 31-year-old male). The tumor was located in the proximal femur, and postoperative pathology showed that the malignant giant cell tumor of bone had recurred.

[0041] In some embodiments of this application, the pretreatment involves rinsing the tumor tissue sample twice with a tissue rinsing solution to remove fibrous connective tissue, fat, and necrotic tissue, and then cutting it into granules.

[0042] Specifically, the tumor tissue sample was rinsed twice with tissue rinsing solution in a sterile laminar flow hood to remove fibrous connective tissue, fat and necrotic tissue. The tumor tissue sample was then cut into uniform particles of approximately 1 mm × 1 mm × 1 mm using sterile ophthalmic scissors in a 6-well plate.

[0043] In some embodiments of this application, the tissue digestion solution is 1 mg / ml type II collagenase.

[0044] In some embodiments of this application, the tumor cell culture medium is prepared by adding 15% fetal bovine serum and 1% penicillin-streptomycin bispecific antibody to a DMEM / F12 basal culture medium system.

[0045] In some embodiments of this application, the method for preparing the tissue irrigation solution is as follows:

[0046] Add 2% penicillin antibody solution to HBSS buffer, mix, and obtain the tissue rinsing solution.

[0047] In some embodiments of this application, the digestive enzyme is prepared as follows:

[0048] Dilute the trypsin containing 0.25% EDTA in PBS to a final concentration of 0.05%.

[0049] On the other hand, in some embodiments of this application, the application of the human malignant giant cell tumor cell line described above in the preparation of a cell model for studying the pathogenesis of malignant giant cell tumor of bone is also provided.

[0050] Specifically, the study compared the human malignant giant cell tumor of bone (MGCTB-CZ1) cell line with peripheral blood from the same patient, analyzing differences in transcriptomics, epigenetics, and proteomics. Furthermore, the expression of genes and signaling pathways in the MGCTB-CZ1 cell line was regulated using various techniques (such as CRISPR-Cas9, lentiviral transfection, and inhibitor treatment). Changes in the morphology and function of the MGCTB-CZ1 cell line were then examined. Cells were inoculated into immunodeficient animals, and tumor size, survival time, and metastasis were observed in both groups to identify the molecular mechanisms regulating the development and progression of human malignant giant cell tumors of bone.

[0051] It is understood that the human malignant giant cell tumor cell line MGCTB-CZ1 of the present invention was established from Chinese people, and the establishment time is relatively short and the characteristics are stable. Using this malignant giant cell tumor cell line as a research model, by analyzing the cell morphology, function and genetic information of this cell line, it is of great help to understand the pathogenesis mechanism of primary malignant giant cell tumor of bone in Chinese people.

[0052] On the other hand, in some embodiments of this application, the application of the human malignant giant cell tumor cell line described herein is also provided in the preparation of diagnostic reagents or diagnostic kits for malignant giant cell tumor of bone.

[0053] Specifically, since the human malignant giant cell tumor of bone has specific tumor markers, diagnostic reagents or diagnostic kits for detecting the occurrence or development of malignant giant cell tumor of bone can be developed based on these tumor markers.

[0054] Specifically, by comparing human malignant giant cell tumor of bone cell lines with normal osteoblasts and other types of cancer cells, and analyzing differences in transcriptomics, epigenetics, and proteomics, specific molecular markers for malignant giant cell tumor of bone can be discovered. These molecular markers can then be used for subsequent disease detection and drug development.

[0055] Specifically, once a tumor marker specific to malignant giant cell tumor of bone is discovered, diagnostic reagents or kits can be developed based on this tumor marker to detect the occurrence or development of malignant giant cell tumor of bone. By detecting the expression of this tumor marker in human peripheral blood, sputum, or biopsy tissue, it is possible to help diagnose the occurrence and development of malignant giant cell tumor of bone, providing guidance and basis for clinical treatment.

[0056] Specifically, firstly, by adding different drugs to the culture medium of the human malignant giant cell tumor of bone, and detecting changes in cell state and function, effective candidate drugs targeting human malignant giant cell tumor of bone are obtained. The human malignant giant cell tumor of bone cell line can serve as a tool for preclinical research on drug therapy for human malignant giant cell tumor of bone.

[0057] Example 1

[0058] S1. Preparation of tissue digestion solution: The tissue digestion solution contains 1 mg / ml type II collagenase;

[0059] S2. Preparation of tumor cell culture medium: In the DMEM / F12 basal culture medium system, supplement with 15% fetal bovine serum and 1% penicillin-streptomycin antibiotics, and mix thoroughly.

[0060] S3. Preparation of tissue rinsing solution: Add 2% penicillin antibody solution to HBSS buffer, mix well and the tissue rinsing solution is obtained.

[0061] S4. Preparation of digestive enzyme: Add 100ml of trypsin containing 0.25% EDTA to 20ml of PBS for dilution to obtain a final concentration of 0.05% digestive enzyme. After aliquoting, store in a -20℃ refrigerator for later use.

[0062] S5. Sample Processing: Tumor tissue samples were collected from a 31-year-old male patient with malignant giant cell tumor of bone (the tumor was located in the proximal femur, and postoperative pathology showed recurrence of malignant giant cell tumor of bone). The samples were transferred to the laboratory as soon as possible from an ice bath. The tumor tissue samples were washed twice with tissue rinsing solution in a sterile laminar flow hood to remove fibrous connective tissue, fat, and necrotic tissue. The tumor tissue samples were then cut into uniform particles of approximately 1 mm × 1 mm × 1 mm in a 6-well plate using sterile ophthalmic scissors. 9 mL of DMEM / F12 medium containing 5% fetal bovine serum and 1 mL of tissue digestion solution were added and the plate was incubated at 37°C. After 1 hour, the suspension was filtered through 70 μm and 40 μm cell sieves. The filtered suspension was centrifuged at 800 rpm for 5 minutes, the precipitate was collected, and the cells were resuspended in tumor cell culture medium and seeded into T25 culture flasks and incubated at 37°C in a 5% CO2 incubator.

[0063] S6. Cell passage: Change the tumor cell culture medium every 2-3 days. When the cell density reaches 80%-90%, aspirate the supernatant, wash twice with DPBS, add 1 mL of digestive enzyme, digest at 37°C for 3 minutes, then add tumor cell culture medium to stop digestion, centrifuge at 800 rpm for 5 minutes, aspirate the supernatant, resuspend the cells in tumor culture medium, and passage at a 1:2 ratio.

[0064] During passage culture, the different sensitivities of fibroblasts and tumor cells to trypsin and the differences in adhesion ability of different cell types to culture medium and solid surfaces were utilized. Fibroblasts were continuously removed using a combination of differential digestion and differential adhesion. After multiple passages, fibroblasts were no longer visible to the naked eye in the culture dish, leaving only single stromal cells that continued to grow and passage. Currently, the in vitro culture time has exceeded six months, and the cell passages have exceeded 100. After successful establishment, the cell line was named the human malignant giant cell tumor of bone cell line MGCTB-CZ1.

[0065] Effect detection

[0066] 1. Observe the growth of the human malignant giant cell tumor of bone MGCTB-CZ1 cell line described in Example 1 directly under an optical microscope.

[0067] like Figure 1 As shown, Figure 1 Morphological images of different passages of human malignant giant cell tumor of bone MGCTB-CZ1 cells observed under a light microscope, including (a) p5; (b) p10; (c) p15; (d) p20; (e) p50; and (f) p100. The scale bar is 100 μm.

[0068] As can be seen, the human malignant giant cell tumor of bone cell line MGCTB-CZ1 of this invention exhibits vigorous growth, with MGCTB-CZ1 cells adhering to the culture vessel in a typical epithelial-like manner. Most cells are irregularly polygonal, while a small number are spindle-shaped, round, or oval, and all have prominent nuclei. Each generation of cells maintains vigorous metabolic activity and stable cell morphology; even at the 20th passage, the cell morphology and growth rate remain unchanged. Under high-density culture conditions, cell aggregation was observed, exhibiting malignant characteristics due to loss of contact inhibition.

[0069] Subsequently, HE staining was performed on human malignant giant cell tumor of bone using an HE staining kit. After cell adhesion to the slides, staining was performed strictly according to standard operating parameters. First, the cell slides were washed twice with distilled water for 2 minutes each time. The samples were then subjected to hematoxylin nuclear staining (5 minutes), rapid separation with hydrochloric acid and ethanol (30 seconds), and eosin cytoplasmic counterstaining (120 seconds). Gradient alcohol dehydration and xylene clearing were then performed. Finally, resin mounting and image acquisition were performed. The results are shown below. Figure 2 As shown, the cells exhibited typical characteristics of malignant tumors, with varying cell morphologies, including spindle-shaped, short spindle-shaped, and polygonal shapes, and round or oval nuclei. The nuclei were round or oval, with some exhibiting a curved shape, and a few nucleoli were visible within them. Furthermore, the nuclei displayed atypical proliferative features, such as uneven and coarse chromatin aggregation, thickened nuclear membrane, and the presence of large nucleoli.

[0070] 2. Detection of marker protein in the malignant giant cell tumor of bone MGCTB-CZ1 cell line.

[0071] Immunohistochemical staining was performed on the human malignant giant cell tumor of bone cell line MGCTB-CZ1 from Example 1. MGCTB-CZ1 cells in logarithmic growth phase were selected and spread onto sterile glass slides 24 hours in advance. The slides were then subjected to standardized pretreatment procedures including washing three times with PBS, fixation (immersion in 4% paraformaldehyde at room temperature for 30 min), and permeabilization (permeabilization with 0.5% Triton X-100 for 20 min). The cell slides were then sent to the Department of Pathology, Shanghai Changzheng Hospital, for professional immunohistochemical staining. The results are as follows: Figure 3 As shown, the H3.3 G34W protein in the nucleus of tumor cells showed diffuse positive expression and stained brown.

[0072] The above results indicate that the human malignant giant cell tumor of bone cell line MGCTB-CZ1 cells grow vigorously. Based on histopathological characteristics and verification of specific molecular markers, the MGCTB-CZ1 cell line is a cell line derived from malignant giant cell tumor of bone, and retains the key molecular and malignant phenotypic characteristics of the primary tumor.

[0073] 3. STR (Short Tandem Repeat) gene loci consist of short tandem repeat sequences of 3 to 7 base pairs. These sequences are ubiquitous in the human genome and are widely used in cellular applications due to their high polymorphism.

[0074] DNA fingerprinting is performed at the cellular level, while STR identification is the preferred method for human cell line authentication. Currently, STR genotyping has been recognized by authoritative organizations such as the International Committee on Identification and Certification of Cell Lines (ICLAC) and the American Academy of Cell Banks (ATCC), becoming the gold standard for cell identification. The human malignant giant cell tumor of bone cell line MGCTB-CZ1, cultured in Example 1, was sent to a professional institution for STR testing. The origin of the cells was identified through STR analysis. Based on STR typing technology, this invention systematically analyzed MGCTB-CZ1 cells and their original tumor tissue at 21 loci (see Table 1). The sequences were compared with STR data of cell lines included in the ATCC, DSMZ, JCRB, and RIKEN databases. No identical STR test results were found, thus proving its uniqueness and confirming that no cross-contamination with other cell lines occurred during primary culture.

[0075] Table 1 Comparison of STR analysis results of MGCTB-CZ1 and primary tumor tissues

[0076]

[0077] 4. Plotting cell growth curves and cell population doubling time.

[0078] CellCounting Kit-8, or CCK-8 for short, is a rapid and highly sensitive detection kit based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) that is widely used for the detection of cell proliferation and cytotoxicity.

[0079] The logarithmically growing human giant cell tumor of bone cell line MGCTB-CZ1 from Example 1 was used. Cells were digested with trypsin, centrifuged at 1000 rpm for 5 minutes to collect the cell pellet, resuspended in tumor cell culture medium, and counted. Cells were seeded at a density of 3 × 10³ cells / well in 96-well plates at five time points (0, 24, 48, 72, 96 h), with nine technical replicates per well, for a total of 45 wells. Cells were cultured at 37°C in a 5% CO2 incubator. Two hours before the end of each time point, 100 μL of 10% CK-8 reagent was added to the corresponding well, and the cells were incubated in the dark. The absorbance at 450 nm was measured using a microplate reader. Cell growth curves were plotted using GraphPad Prism 9.5.1 software, with culture time on the x-axis and OD value on the y-axis. Cell population doubling time was calculated using an online doubling time calculation platform (https: / / doublingtime.com / ). Results are as follows: Figure 4 As shown, the cell line doubling time calculated from the growth curve is approximately 40.36 hours.

[0080] 5. Exon sequencing of the human malignant giant cell tumor of bone cell line MGCTB-CZ1.

[0081] DNA was extracted from the logarithmically growing human giant cell tumor of bone cell line MGCTB-CZ1 (Example 1) and peripheral blood from the same patient, and exon sequencing and analysis were performed. The genome sequence of the human giant cell tumor of bone cell line MGCTB-CZ1 was successfully aligned 100% with the human reference genome (NCBI GRCh38), proving it is a human cell line. Furthermore, the human giant cell tumor of bone cell line MGCTB-CZ1 contains the H3F3A G34W mutation.

[0082] It should be noted that:

[0083] Numerous specific details are set forth in the specification provided herein. However, it will be understood that embodiments of this application may be practiced without these specific details. In some instances, well-known structures and techniques have not been shown in detail so as not to obscure the understanding of this specification.

[0084] Similarly, it should be understood that, in order to simplify this application and aid in understanding one or more of the various aspects of the invention, in the above description of exemplary embodiments of this application, various features of this application are sometimes grouped together in a single embodiment, figure, or description thereof. However, the system disclosed herein should not be construed as reflecting a schematic diagram in which the claimed application requires more features than are expressly recited in each claim.

[0085] More precisely, as reflected in the following claims, the inventive aspect lies in fewer than all the features of the single embodiment disclosed above. Therefore, the claims following the detailed description are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of this application.

[0086] Furthermore, those skilled in the art will understand that although some embodiments described herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of this application and form different embodiments.

[0087] For example, any of the claimed embodiments can be used in any combination as described in the following claims.

[0088] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A human malignant giant cell tumor of bone cell line, characterized in that, The human malignant giant cell tumor of bone cell line was deposited on April 9, 2025, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, with accession number CCTCC NO:C202539 and classified as human malignant giant cell tumor of bone cell line MGCTB-CZ1.

2. The application of the human malignant giant cell tumor cell line as described in claim 1 in the preparation of a cell model for studying the pathogenesis of malignant giant cell tumor of bone.

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