A human osteosarcoma xenograft cell bank and a construction method and application thereof

Abstract

The present application relates to a human giant cell tumor of bone xenograft cell bank and its construction method and application, the construction method comprises the following steps: S1, obtaining the human giant cell tumor of bone tissue in vitro, and trimming to obtain a human giant cell tumor of bone tissue transplantation block; S2, the puncture needle filled with the human giant cell tumor of bone tissue transplantation block is transplanted into the right subcutaneous tissue of mouse along the tunnel, and the F0 generation human giant cell tumor of bone xenograft mouse model is constructed; S3, the human giant cell tumor of bone tissue in F0 generation model is obtained, and S1 and S2 steps are repeated multiple times, and the Fn generation human giant cell tumor of bone xenograft mouse model is obtained; S4, the human giant cell tumor of bone tissue in Fn generation model is obtained, and the human giant cell tumor of bone xenograft cell bank is obtained by trypsin digestion and freezing storage.Compared with the prior art, the human giant cell tumor of bone xenograft cell bank is creatively constructed, the cell activity and tumor characteristics can be retained by 100%, and the time for obtaining human giant cell tumor of bone is greatly shortened for subsequent human giant cell tumor of bone related research.

Description

Technical Field

[0001] This invention belongs to the field of biological tissue engineering technology, and relates to a human giant cell tumor xenograft cell bank, its construction method and application. Background Technology

[0002] Giant cell tumor of bone is a common bone tumor with a high incidence rate, and its research is of great significance to the field of oncology.

[0003] Patent CN110402895A provides a method for constructing a mouse model of osteosarcoma xenograft. This method involves resuscitating tumor tissue blocks from osteosarcoma patients, preserved in liquid nitrogen, and then transplanting them xenografts into immunodeficient mice to more realistically simulate the tumor growth environment. However, this method primarily focuses on constructing osteosarcoma models and does not adequately consider the specificity and complexity of giant cell tumors of bone.

[0004] Furthermore, the construction of xenograft models for giant cell tumor of bone, especially the construction of human giant cell tumor xenograft cell banks that can preserve tumor activity and consistency, remains a gap in current technology.

[0005] Another patent, CN103911342A, relates to a mouse animal model of human giant cell tumor and its construction method. By injecting primary cells isolated and cultured from human giant cell tumor tissue into the tibia of nude mice, a mouse animal model of human giant cell tumor bone injury was successfully constructed. However, this method has limitations in terms of the model's sustained stability, consistency, and high-throughput application, making it difficult to meet the needs of large-scale research and drug screening.

[0006] Based on the above analysis, although there are reports on the construction of animal models of osteosarcoma and giant cell tumor of bone in the existing technology, there are still technical gaps and challenges in constructing human giant cell tumor xenograft cell banks that can be stably passaged and maintain tumor characteristics. There is an urgent need for innovative methods to fill the needs in this field and promote the in-depth development of research related to giant cell tumor of bone. Summary of the Invention

[0007] The purpose of this invention is to overcome the limitations of the prior art, greatly facilitate the development of tumor-related research, and thus provide a human giant cell tumor xenograft cell bank, its construction method and application.

[0008] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a method for constructing a human giant cell tumor xenograft cell bank, comprising the following steps: S1. Obtain isolated human giant cell tumor tissue, rinse to remove impurities from the surface of the human giant cell tumor tissue, trim to obtain human giant cell tumor tissue transplant block, and pre-fill the puncture needle cannula with the human giant cell tumor tissue transplant block. S2. Use the needle tip to puncture the subcutaneous tissue on the right side of the mouse, expand the tunnel along the subcutaneous tissue, and transplant the puncture needle filled with human giant cell tumor tissue graft block in step S1 along the tunnel to construct the F0 generation human giant cell tumor xenograft mouse model. S3. Monitor and obtain human giant cell tumor tissue in the F0 generation human giant cell tumor xenograft mouse model constructed in step S2. Repeat steps S1 and S2 multiple times to obtain the Fn generation human giant cell tumor xenograft mouse model, where n is the number of repetitions. S4. Monitor and obtain human giant cell tumor tissue from the Fn generation human giant cell tumor xenograft mouse model constructed in step S3. After trypsin digestion, human giant cell tumor cells are obtained and cryopreserved to obtain a human giant cell tumor xenograft cell bank.

[0009] In some specific embodiments, in step S1, the rinsing process is as follows: human giant cell tumor tissue is immersed in PBS buffer containing penicillin and streptomycin.

[0010] In some specific embodiments, in step S2, the mouse is a 4-6 week old, female NPSG mouse weighing 16-18 g.

[0011] In some specific embodiments, in step S2, the volume of the human giant cell tumor tissue graft is 1-2 mm. 3 .

[0012] In some specific embodiments, in step S3, the volume of the obtained human giant cell tumor tissue is 1000-1500 mm. 3 .

[0013] In some specific implementations, the indicators monitored in step S3 include: (1) Whether the pathological sections of human giant cell tumor tissue in the F0 generation human giant cell tumor xenograft mouse model are consistent with those of human giant cell tumor tissue in step S1; (2) Whether the body weight of the F0 generation human giant cell tumor xenograft mouse model decreased by more than 10%.

[0014] In some specific embodiments, in step S4, the process of trypsin digestion is as follows: human giant cell tumor tissue from F3 generation human giant cell tumor xenograft mouse model is minced, washed with PBS buffer containing double antibodies, centrifuged and the supernatant is discarded, 0.25% trypsin-EDTA digestion solution is added for immersion, digested in a water bath at 37°C for 15-60 min, and after terminating digestion, filtered to obtain human giant cell tumor cells.

[0015] In some specific embodiments, in step S4, the filtration process is as follows: the digested cell suspension is poured into 70... The cells were filtered through a sterile cell sieve, centrifuged, and the precipitate was collected, which was the giant cell tumor cells of human bone.

[0016] The second technical solution of the present invention is to provide a human giant cell tumor xenograft cell bank, which is constructed by the construction method described in one of the above technical solutions.

[0017] The third technical solution of the present invention is to provide an application of the human giant cell tumor xenograft cell bank as described in the second technical solution above in tumor pathogenesis research and drug sensitivity testing.

[0018] Compared with the prior art, the present invention has the following advantages: (1) The present invention provides a method for constructing a human giant cell tumor xenograft cell bank. The technical operation is simple and the construction time of human giant cell tumor is shortened when conducting in vivo and in vitro experiments of human giant cell tumors.

[0019] (2) The cells in the human giant cell tumor xenograft cell bank of the present invention are free from mycoplasma contamination and retain 100% of their activity and tumor characteristics after resuscitation, which facilitates the development of human giant cell tumor related research. Attached Figure Description

[0020] Figure 1 The received human giant cell tumor tissue sample.

[0021] Figure 2 The received human giant cell tumor tissue sample.

[0022] Figure 3 These are human giant cell tumor cells isolated from an F3 generation human giant cell tumor xenograft mouse model.

[0023] Figure 4 This is a schematic diagram of a xenograft cell bank for giant cell tumors of the human bone.

[0024] Figure 5 Microscopic examination of cells after resuscitation in a human giant cell tumor xenograft cell bank.

[0025] Figure 6 The results show the activity of cells after resuscitation in a xenograft cell bank for giant cell tumor of human bone.

[0026] Figure 7 This study aims to detect mycoplasma in cells resuscitated from a human giant cell tumor xenograft cell bank. M represents the DL2000 DNA Marker, 1-6 represent parallel samples, N represents a negative control, and P represents a positive control. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0029] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0030] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0031] This document only specifically discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0032] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument, such as ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C.

[0033] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0034] Example 1: 1. Receiving and processing of human giant cell tumor tissue samples Sample Receipt: We receive human giant cell tumor tissue samples from clinical settings. All samples are pathologically confirmed and meet ethical requirements. Figures 1-2 As shown, the received samples were fresh tissues, which were stored in physiological saline or PBS containing 1× penicillin and streptomycin during the shipping process, transported under refrigeration at 4°C, and the receipt and subsequent processing of the tissue samples were completed within 24 hours.

[0035] Sample cleaning and trimming: In a biosafety cabinet, place fresh tumor tissue into a sterile 6-well plate. Add approximately 3 mL of PBS buffer containing 1× penicillin and streptomycin to the 6-well plate beforehand. Disinfect a 50 mL centrifuge tube containing the tissue with 75% alcohol, then transfer it to the biosafety cabinet and pour it into the 6-well plate. Use forceps to handle the tissue and rinse 3-5 times to remove blood, necrotic tissue, and impurities from the tissue surface. Trim the tissue into uniform small pieces, each approximately 1-2 mm in size, using a sterile scalpel and forceps. 3 This is used for subsequent transplantation, while some tissue blocks are reserved for pathological testing and cell separation.

[0036] In this invention, tumor tissue is trimmed to a volume of 1-2 mm. 3 On the one hand, it can reduce immune rejection and make it easier for the tumor to colonize and survive; on the other hand, with this size, the host blood vessels can quickly supply oxygen and nutrients to the tumor, greatly increasing the tumor formation rate; furthermore, it can avoid central necrosis, which would prevent tumor formation; in addition, it can be fitted with catheters, making the operation convenient.

[0037] Tissue preservation and thawing (for frozen tissues): For frozen tumor tissues, after removing them from the -80°C freezer or liquid nitrogen tank, quickly place them in a 37°C constant temperature water bath for rapid thawing. After the tissues are completely thawed, rinse them three times with PBS buffer containing 1× penicillin and streptomycin. Subsequent treatment is the same as for fresh tissues.

[0038] 2. Construction of F0 generation human giant cell tumor xenograft mouse model Subcutaneous inoculation of tumor tissue: First, cut the tumor tissue specimen into pieces with a diameter of approximately 1-2 mm. 3 Prepare small pieces of tissue by pre-filling the cannula of the puncture needle. Anesthetize female NPSG mice aged 4-6 weeks and weighing 16-18 g. Disinfect the skin on the right side of the mouse (specifically, the area slightly posterior to the scapular region on the right side of the back when the mouse is lying down). Use the puncture needle to make a subcutaneous puncture on the right side of the mouse, creating a tunnel along the subcutaneous surface. Then, insert the prepared tumor tissue piece into the right subcutaneous tissue for transplantation through the tunnel using the puncture needle. Withdraw the puncture needle and disinfect the insertion site to prevent infection.

[0039] In this invention, NPSG mice are mice lacking T cells and with almost no activity of B cells and NK cells. Compared with other types of mice that completely lack T cells, B cells and NK cells, NPSG mice can significantly reduce or eliminate xenotransplantation reactions in mice.

[0040] In this invention, the selected inoculation site causes minimal damage to mice, has a low mortality rate, facilitates tumor growth stability, and is easy to observe and measure.

[0041] Postoperative care and observation: Mice transplanted should be returned to SPF-grade cages, housed individually or in groups (no more than 5 mice per cage). Maintain stable ambient temperature (20-26℃) and humidity (30%-70%), with a 12-hour light / 12-hour dark cycle. Allow free access to food and water. Observe the mice daily post-surgery for their mental state, activity level, food and water intake, and wound healing. If signs of infection such as redness, swelling, or oozing are observed, treat with antibiotic ointment promptly.

[0042] 3. Monitoring and identification of F0 generation human giant cell tumor xenograft mouse model Tumor growth monitoring: Starting from day 7 post-transplantation, the long diameter (L) and short diameter (W) of the tumor were measured weekly using calipers. The tumor volume (V) was calculated using the formula: Tumor volume (V) = 0.5 × L × W. 2 Calculate tumor volume and plot tumor growth curves. Simultaneously, weigh the mice weekly and observe weight changes. If a mouse's weight decreases by more than 10% or exhibits significant cachexia, terminate the experiment immediately.

[0043] Mouse condition: The mice were in good overall condition after transplantation, with normal activity levels and no significant impact on their diet or water intake. Within one week post-surgery, all wounds healed well, with no signs of infection such as redness, swelling, or oozing. During tumor growth, some tumors became quite large (exceeding 1200 mm). 3 Mice showed a slight decrease in body weight (3%-5%), but did not exhibit obvious cachexia; the body weight of the remaining mice remained stable.

[0044] Pathological morphological identification: When the tumor volume reaches 1000-1500 mm 3 In this study, some mice were euthanized (cervical dislocation method), and tumor tissue was removed. After being fixed with 4% paraformaldehyde for 24 h, the tissue was embedded in paraffin, sectioned (4-5 μm thick), and then stained with hematoxylin and eosin (HE). The pathological morphological characteristics of the tumor tissue were observed under an optical microscope and compared with the pathological sections of the original patient's tumor tissue to determine the consistency of the tumor tissue.

[0045] Tumor tissue passage: The extracted tumor tissue was used for pathological and immunohistochemical identification, and the remaining part was processed according to steps (1) and (2) and then subcutaneously transplanted into the next generation of NPSG mice to establish a passage system for the PDX model. The stability of tumor growth characteristics and biological characteristics during passage was observed.

[0046] 4. Obtaining cells from human giant cell tumor tissue in F3 generation human giant cell tumor xenograft mouse models (1) Tumor tissue acquisition and pretreatment (operation in biosafety cabinet) The F3 generation human giant cell tumor xenograft mouse model was euthanized by cervical dislocation, and then the mice were immersed in 75% ethanol for 3-5 minutes for surface disinfection.

[0047] After disinfection, the mice were transferred to a sterile dissecting tray in the laminar flow hood. The skin of the tumor site was lifted with sterile forceps, and the tumor tissue was carefully dissected along the edge of the skin with surgical scissors (avoiding damage to the tumor capsule and reducing the mixing of necrotic tissue). The tumor tissue was then placed in a 10 cm culture dish containing 5 mL of sterile PBS (containing antibiotics).

[0048] (2) Cleaning and mincing of tumor tissue Carefully pick up the tumor tissue with tweezers and place it in a 6-well plate containing PBS (with double antibodies) beforehand. Rinse the tumor tissue 3-5 times to remove any residual blood, skin tissue, or other impurities from the surface. If there are necrotic areas at the edges of the tumor tissue, remove the necrotic parts with sterile tweezers, leaving the active tumor tissue that is uniform in texture and has a reddish color.

[0049] Use sterile surgical scissors to cut the tumor tissue into 1-2 mm pieces. 3 Cut the tissue into small pieces (as small as possible to increase the contact area with pancreatic enzymes and improve digestion efficiency). Do not cut the tissue for too long to prevent it from drying out.

[0050] (3) Pancreatic enzyme digestion and cell release Transfer the shredded tumor tissue to a 15 mL sterile centrifuge tube, rinse with PBS (containing antibiotics), and transfer again to a 15 mL sterile centrifuge tube to reduce tissue loss. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and add 3-5 mL of preheated 0.25% trypsin-EDTA digestion solution (the volume of the digestion solution should be enough to completely submerge the tissue block). Gently pipette and mix thoroughly to ensure that the tissue block and digestion solution are fully mixed.

[0051] Place the centrifuge tubes in a 37°C constant temperature water bath. Remove the centrifuge tubes every 5 minutes and gently tap the tube walls 10-15 times to observe the digestion status of the tissue blocks (if the tissue blocks gradually loosen and the solution becomes slightly turbid, it indicates that digestion is in progress).

[0052] The total digestion time should be controlled between 15 and 60 minutes (the specific time needs to be adjusted according to the tissue stiffness to avoid over-digestion and cell damage). When most of the tissue blocks have disappeared and the solution is obviously turbid, immediately add about 10 mL of RPMI-1640 complete culture medium containing 10% FBS, and gently invert the centrifuge tube 5 times to stop the trypsin digestion reaction.

[0053] (4) Cell filtration and purification Place a 70 μm sterile cell sieve on a 50 mL sterile centrifuge tube, slowly pour the digested cell suspension into the sieve, and at the same time rinse the inner wall of the 15 mL centrifuge tube twice with 1 mL sterile PBS, pouring the remaining cell suspension into the sieve as well.

[0054] Remove the 70 μm cell sieve, place the 50 mL centrifuge tube containing the collected single-cell suspension into a centrifuge, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and retain the cell pellet at the bottom of the tube.

[0055] Add 2 mL of RPMI-1640 complete culture medium containing 10% FBS to the washed cell pellet, and gently pipette 10-15 times to prepare a uniform single-cell suspension.

[0056] Subsequent culture and identification confirmed that the suspension contained the required human giant cell tumor cells of bone (e.g., Figure 3 As shown in the figure, the cells possessed adherent growth ability and tumor cell morphology characteristics, indicating that the target cell isolation experiment in the human giant cell tumor tissue of the F3 generation human giant cell tumor xenograft mouse model was successful.

[0057] (5) Preserve human giant cell tumor cells in liquid nitrogen, such as... Figure 4 The diagram shown is a schematic of the human giant cell tumor xenograft cell bank in this embodiment.

[0058] The reason for selecting tumor cells from the F3 generation model for cryopreservation to construct a human giant cell tumor xenograft cell bank in this invention is as follows: a. F1 / F2 generation tumors are susceptible to heterogeneity of the primary lesion and residual immune responses in mice, resulting in unstable characteristics; b. After multiple in vivo acclimatizations, tumor proliferation rate, pathological classification, and gene phenotype are basically finalized, closely resembling the characteristics of the original tumor. c. Stepwise purification of heterogeneity; d. The cells exhibit stable proliferation and invasion capabilities, resulting in good reproducibility for subsequent experiments; e. Excessive inoculation generations can easily lead to gene drift and phenotypic mutations, deviating from the original tumor characteristics.

[0059] (6) Cell viability detection after cell resuscitation Take 10 μL of the resuscitated single-cell suspension and mix it thoroughly with 10 μL of 0.4% trypan blue staining solution. Add the mixture to a cell counting plate and use a cell counter to count cell density, cell viability, and cell size. Simultaneously, observe the sample under an inverted microscope: live cells appear colorless and transparent, while dead cells are stained blue by trypan blue. Figure 5 and 6 As shown, the cell survival rate is 100%.

[0060] (7) Quality inspection after cell resuscitation Collect the supernatant from the resuscitated single-cell suspension for mycoplasma nucleic acid testing, such as... Figure 7 As shown, no mycoplasma infection was observed.

[0061] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for constructing a human giant cell tumor xenograft cell bank, characterized in that, Includes the following steps: S1. Obtain isolated human giant cell tumor tissue, rinse to remove impurities from the surface of the human giant cell tumor tissue, trim to obtain human giant cell tumor tissue transplant block, and pre-fill the puncture needle cannula with the human giant cell tumor tissue transplant block. S2. Use the needle tip to puncture the subcutaneous tissue on the right side of the mouse, expand the tunnel along the subcutaneous tissue, and transplant the puncture needle filled with human giant cell tumor tissue graft block in step S1 along the tunnel to construct the F0 generation human giant cell tumor xenograft mouse model. S3. Monitor and obtain human giant cell tumor tissue in the F0 generation human giant cell tumor xenograft mouse model constructed in step S2. Repeat steps S1 and S2 multiple times to obtain the Fn generation human giant cell tumor xenograft mouse model, where n is the number of repetitions. S4. Monitor and obtain human giant cell tumor tissue from the Fn generation human giant cell tumor xenograft mouse model constructed in step S3. After trypsin digestion, human giant cell tumor cells are obtained and cryopreserved to obtain a human giant cell tumor xenograft cell bank.

2. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, In step S1, the rinsing process is as follows: human giant cell tumor tissue is immersed in PBS buffer containing penicillin and streptomycin.

3. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, In step S2, the mice are 4-6 week old, female NPSG mice weighing 16-18 g.

4. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, At S2, the volume of the human giant cell tumor of bone tissue graft block was 1-2 mm 3 .

5. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, In step S3, the volume of the obtained human giant cell tumor tissue is 1000-1500 mm. 3 .

6. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, In step S3, the monitored indicators include: (1) Whether the pathological sections of human giant cell tumor tissue in the F0 generation human giant cell tumor xenograft mouse model are consistent with those of human giant cell tumor tissue in step S1; (2) Whether the body weight of the F0 generation human giant cell tumor xenograft mouse model decreased by more than 10%.

7. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, In step S4, the process of trypsin digestion is as follows: human giant cell tumor tissue from F3 generation human giant cell tumor xenograft mouse model is minced, washed with PBS buffer containing double antibodies, centrifuged and the supernatant is discarded, 0.25% trypsin-EDTA digestion solution is added and immersed, digested in a water bath at 37℃ for 15~60 min, and after digestion is terminated, the human giant cell tumor cells are obtained by filtration.

8. The method for constructing a human giant cell tumor xenograft cell bank according to claim 1, characterized in that, In step S4, the filtration process is as follows: the digested cell suspension is poured into 70... The cells are filtered through a sterile cell sieve, centrifuged, and the precipitate is collected to obtain human giant cell tumor cells. These cells are then cryopreserved to obtain a human giant cell tumor xenograft cell bank.

9. A human giant cell tumor xenograft cell bank, characterized in that, It is constructed by the construction method described in any one of claims 1 to 8.

10. The application of a human giant cell tumor xenograft cell bank as described in claim 9 in tumor pathogenesis research and drug sensitivity testing.

Images