Human immature teratocarcinoma ascites tumor cell line and its use

Abstract

The present disclosure provides a human immature teratocarcinoma ascites tumor cell strain and its application, and belongs to the technical field of cells. The human IT ascites tumor cell strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO:46310. The human IT ascites tumor cell strain provided by the present disclosure has a large number, can be stably passaged, and is convenient to store and expand. The present disclosure fills the blank of immature teratocarcinoma without circulating tumor cell lines, and provides a powerful tool for in-depth study of the natural occurrence process of circulating tumor cells, and search for new circulating tumor cell markers and drugs.

Description

Technical Field

[0001] This disclosure belongs to the field of cell technology, specifically relating to a human immature teratoma ascites tumor cell line and its application. Background Technology

[0002] Malignant ovarian germ cell tumors (MOGCTs) account for approximately 5% of all ovarian malignancies, and are most common in young adult women and children. Pathological types mainly include dysgerminoma, embryonal carcinoma, immature teratoma (IT), yolk sac tumor, non-pregnant choriocarcinoma, and mixed germ cell tumors. Among these, immature teratomas account for only about one-third of MOGCTs, and their tumor tissue composition is complex, consisting of tissues from the endoderm, mesoderm, and ectoderm, with varying proportions of mature and immature tumor elements, making in vitro culture difficult. Therefore, currently, the only commonly used cell line associated with immature ovarian teratomas is PA-1.

[0003] Therefore, there is an urgent need to develop a stable cell line for immature teratoma and establish in vitro culture methods to advance research on the metastasis and treatment of immature teratomas. Summary of the Invention

[0004] To address at least one of the aforementioned problems, this disclosure provides a human immature teratoma (IT) ascites tumor cell line, enrichment and culture method, and its application.

[0005] According to a first aspect of this disclosure, a human immature teratoma ascites tumor cell line is provided, which was deposited on December 26, 2024, at the China General Microbiological Culture Collection Center with accession number CGMCC NO:46310.

[0006] In some embodiments, the cell line is positive for cytokeratin pan-CK and / or positive for SOX2 protein expression.

[0007] In some embodiments, the cell line is CD45 negative.

[0008] According to another aspect of this disclosure, a method for enriching and culturing the human immature teratoma ascites tumor cell line described herein is provided, the method comprising the following steps: 1) Enrichment of immature teratoma ascites tumor cells from the test sample; 2) Using a selection medium, the enriched immature teratoma ascites tumor cells are resuspended and cultured to obtain a first culture product, wherein the selection medium comprises basal medium and 5 v / v%-20 v / v% serum; 3) Replace the screening medium with a maintenance medium for maintenance culture to obtain a second culture product, wherein the maintenance medium includes basal medium and 5 v / v%-20 v / v% serum substitute.

[0009] In some embodiments, the sample to be tested in step 1) is from a patient with an immature teratoma; and / or The sample to be tested in step 1) is ascites.

[0010] In some embodiments, the sample to be tested in step 1) includes body fluids or derivatives thereof.

[0011] In some embodiments, the sample to be tested in step 1) is selected from one or more of blood, plasma, lymph, saliva, urine, sweat, or ascites.

[0012] In some embodiments, the enrichment can be performed using methods known in the art. For example, cell density-based centrifugation, cell volume-based membrane filtration, magnetic beads targeting circulating tumor cells, or microfluidic technology can be used to enrich circulating tumor cells in the sample.

[0013] In some embodiments, the basal culture medium may be selected from one or more of M199 medium, DMEM / F12 medium, IMDM medium, DMEM medium, H-DMEM medium, high-glucose DMEM medium, or Knockout™-DMEM medium.

[0014] In some embodiments, the screening medium and / or the maintenance medium further contain growth factors.

[0015] In some embodiments, the serum includes one or more of fetal bovine serum, newborn calf serum, calf serum, horse serum, pig serum, rabbit serum, goat serum, or rabbit serum.

[0016] In some embodiments, the serum substitute includes one or more of KnockOut™ serum substitute, N2, or B27.

[0017] In some embodiments, the screening medium and / or the maintenance medium comprises H-DMEM medium.

[0018] In some embodiments, the screening medium contains 5v / v%, 6v / v%, 7v / v%, 8v / v%, 9v / v%, 10v / v%, 11v / v%, 12v / v%, 13v / v%, 14v / v%, 15v / v%, 16v / v%, 17v / v%, 18v / v%, 19v / v%, or 20v / v of fetal bovine serum.

[0019] In some embodiments, the screening medium and / or the maintenance medium comprises Knockout™-DMEM medium.

[0020] In some embodiments, the maintenance culture medium may contain a KnockOut™ serum substitute, such as 5v / v%, 6v / v%, 7v / v%, 8v / v%, 9v / v%, 10v / v%, 11v / v%, 12v / v%, 13v / v%, 14v / v%, 15v / v%, 16v / v%, 17v / v%, 18v / v%, 19v / v%, or 20v / v%.

[0021] In some embodiments, the screening medium and / or the maintenance medium further comprise one or more of the following growth factors: epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), stem cell factor (SCF), insulin-like growth factor (IGF), or transforming growth factor-β (TGF-β).

[0022] In some embodiments, the fibroblast growth factor comprises one or more of the following growth factors: basic fibroblast growth factor (bFGF) or acidic fibroblast growth factor (aFGF).

[0023] In some embodiments, the screening medium and / or the maintenance medium contain 1-10 μg / mL of basic fibroblast growth factor (bFGF).

[0024] In some embodiments, the screening medium and / or the maintenance medium may contain 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL of basic fibroblast growth factor (bFGF).

[0025] In some embodiments, the screening medium and / or the maintenance medium contain 1-10 μg / mL of epidermal growth factor (EGF).

[0026] In some embodiments, the screening medium and / or the maintenance medium may contain 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL of epidermal growth factor (EGF).

[0027] In some embodiments, the screening medium and / or the maintenance medium may also contain antibiotics.

[0028] In some embodiments, the screening medium and / or the maintenance medium may also contain a penicillin-streptomycin mixture.

[0029] In some embodiments, the screening medium and / or the maintenance medium may contain penicillin at concentrations of 60 U / mL, 65 U / mL, 70 U / mL, 75 U / mL, 80 U / mL, 85 U / mL, 90 U / mL, 95 U / mL, 100 U / mL, 105 U / mL, 110 U / mL, 115 U / mL, 120 U / mL, 125 U / mL, 130 U / mL, 135 U / mL, 140 U / mL, 145 U / mL, 150 U / mL, 155 U / mL, or 160 U / mL.

[0030] In some embodiments, the screening medium and / or the maintenance medium may contain streptomycin at concentrations of 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, 120 μg / mL, 125 μg / mL, 130 μg / mL, 135 μg / mL, 140 μg / mL, 145 μg / mL, 150 μg / mL, 155 μg / mL, or 160 μg / mL.

[0031] In some embodiments, the screening medium and / or the maintenance medium may also contain small molecule additives, such as sodium pyruvate, endothelin-1, transferrin, cholesterol, etc.

[0032] In some embodiments, the screening medium and / or the maintenance medium may also contain 60-180 μg / mL of sodium pyruvate.

[0033] In some embodiments, the screening medium and / or the maintenance medium may further comprise sodium pyruvate at concentrations of 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 111 μg / mL, 115 μg / mL, 120 μg / mL, 125 μg / mL, 130 μg / mL, 135 μg / mL, 140 μg / mL, 145 μg / mL, 150 μg / mL, 155 μg / mL, 160 μg / mL, 165 μg / mL, 170 μg / mL, 175 μg / mL, or 180 μg / mL.

[0034] In some embodiments, the screening medium and / or the maintenance medium may also contain amino acids or their derivatives. Exemplary amino acids include, but are not limited to, natural or non-natural amino acids. Natural amino acids include, for example, phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, histidine, arginine, cysteine, glycine, glutamine, proline, serine, tyrosine, alanine, asparagine, aspartic acid, or glutamic acid. Non-natural amino acids include, for example, the D-stereoisomer or β-amino-analyte of a natural amino acid, citrulline, high-citrulline, high-arginine, hydroxyproline, high-proline, ornithine, 4-amino-phenylalanine, cyclohexylalanine, α-aminoisobutyric acid, N-methylalanine, N-methylglycine, ortholeucine, N-methylglutamic acid, tert-butylglycine, α-aminobutyric acid, tert-butylalanine, 2-aminoisobutyric acid, α-aminoisobutyric acid, 2-aminoindan-2-carboxylic acid, selenomethionine, dehydroalanine, lanethionine, or γ-aminobutyric acid.

[0035] In some embodiments, the screening medium and / or the maintenance medium may also contain 120-200 μg / mL of glutamine.

[0036] In some embodiments, the 500 mL of the screening medium and / or the maintenance medium may further contain 120 μg / mL, 125 μg / mL, 130 μg / mL, 135 μg / mL, 140 μg / mL, 145 μg / mL, 150 μg / mL, 155 μg / mL, 160 μg / mL, 165 μg / mL, 170 μg / mL, 175 μg / mL, 180 μg / mL, 185 μg / mL, 190 μg / mL, 195 μg / mL, or 200 μg / mL of glutamine.

[0037] In some embodiments, the screening medium and / or the maintenance medium may also contain 160.72 μg / mL of glutamine.

[0038] In some embodiments, in step 3) of the method, after culturing with the screening medium for 10-48 hours, the screening medium is replaced with the maintenance medium for maintenance culture.

[0039] In some embodiments, the screening medium comprises: H-DMEM medium, FBS, FGF, EGF, sodium pyruvate, L-glutamine and / or penicillin-streptomycin mixture; and / or the maintenance medium comprises: knockout™-DMEM medium, KSR solution, bFGF, EGF, sodium pyruvate and / or L-glutamine.

[0040] In some embodiments, the screening medium comprises: H-DMEM medium, 5 v / v%-20 v / v% FBS solution, 1-10 μg / mL bFGF, 1-10 μg / mL EGF, 60-180 μg / mL sodium pyruvate, 120-200 μg / mL L-glutamine, 60-160 U / mL penicillin and / or 60-160 μg / mL streptomycin.

[0041] In some embodiments, the screening medium comprises: H-DMEM medium, 10 v / v% FBS, 2.2 μg / mL FGF, 2.2 μg / mL EGF, 121 μg / mL sodium pyruvate, 160.72 μg / mL L-glutamine, 100 U / mL penicillin and / or 100 μg / mL streptomycin.

[0042] In some embodiments, the maintenance medium comprises: Knockout™-DMEM medium, 6 v / v%-14 v / v% KSR solution, 1-10 μg / mL bFGF, 1-10 μg / mL EGF, 60-180 μg / mL sodium pyruvate and / or 120-200 μg / mL L-glutamine.

[0043] In some embodiments, the maintenance medium comprises: Knockout™-DMEM medium, 10 v / v% KSR solution, 2.2 μg / mL bFGF, 2.2 μg / mL EGF, 121 μg / mL sodium pyruvate and / or 160.72 μg / mL L-glutamine.

[0044] In some implementations, the culture time in step 3) is 10-48 hours.

[0045] In some embodiments, the culture time in step 3) of the method is 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, or 48h.

[0046] In some embodiments, the culture time in step 3) of the method is 24 hours.

[0047] In some embodiments, in step 3) of the method, after culturing with the screening medium for 10-48 hours, the screening medium is replaced with the maintenance medium for maintenance culture.

[0048] In some embodiments, the culture medium used in this disclosure is sterilized by filtration, for example, by using a filter with a pore size of 0.2-0.5 μm.

[0049] In some embodiments, the method further includes the following steps: 5) After the second culture product is digested with trypsin, it is resuspended in the screening medium and cultured. 6) Replace the screening medium with the maintenance medium for maintenance culture; 7) Optionally, repeat steps 5) and 6).

[0050] In some embodiments, the method further includes a cell cryopreservation step.

[0051] In some embodiments, the cryopreservation solution used for cell cryopreservation includes CryoStor CS10 cell cryopreservation solution; In some embodiments, the method further includes a cell resuscitation step.

[0052] In some embodiments, after culturing in the screening medium for 10-48 hours, the screening medium is replaced with the maintenance medium for maintenance culture.

[0053] In some embodiments, in step 5), the culture is carried out for 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, 42h, 44h, 46h, or 48h, and then the screening medium is replaced with the maintenance medium for maintenance culture.

[0054] According to another aspect of this disclosure, the application of the cell lines described herein in the construction of animal models of immature teratomas is provided.

[0055] In some implementations, the animal is a non-human mammal.

[0056] In some embodiments, the animal is selected from mice, rats, dogs, horses, rabbits, sheep, pigs, cattle, or monkeys.

[0057] According to another aspect of this disclosure, the use of the cell lines or animal models described herein in one or more of the following is provided: 1) Screening for drugs to prevent or treat immature teratomas; 2) Screening reagents for the diagnosis or detection of immature teratomas; 3) Research on biomarkers, drug targets, etiology, development mechanism, metastasis mechanism, and tumorigenesis mechanism of immature teratomas.

[0058] The human immature teratoma ascites cell line disclosed herein can be stably passaged, expand rapidly, and is easy to preserve and expand. It fills the gap in the lack of circulating tumor cell lines for immature teratomas, and provides a powerful tool for in-depth research into the natural occurrence of circulating tumor cells and for identifying new circulating tumor cell markers and drugs.

[0059] Preservation Information: The human immature teratoma ascites tumor cells provided in this disclosure are named tumor cells B049 and were deposited on December 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC). The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO:46310. Attached Figure Description

[0060] Figure 1 The cell growth curves of the immature teratoma ascites tumor cell line cultured in Example 1 are shown; where the horizontal axis represents the cell generation number and the vertical axis represents the cell count.

[0061] Figure 2 Microscopic images of immature teratoma ascites tumor cell lines at days 4, 7, 11, and 17 of cell culture are shown.

[0062] Figure 3 The image shows a staining observation of the immature teratoma ascites tumor cell line in Example 2.

[0063] Figure 4 The staining observation diagram of the other cells that were filtered out in Example 2 is shown.

[0064] Figure 5 The image shows the results of detecting the germ cell-specific marker SOX2 in the B049 cell line in Example 2. Detailed Implementation

[0065] Immature teratomas account for approximately one-third of malignant ovarian germ cell tumors (MOGCTs). They are often diagnosed at an early stage, predominantly unilateral, with about 10% of patients developing mature cystic teratomas in the contralateral ovary simultaneously or subsequently. These tumors are typically solid, but some areas may exhibit multilocular cystic structures. They may be accompanied by hemorrhage and necrosis. When mature components are present, hair, sebum, teeth, bone, and cartilage may be visible. Immunohistochemically, immature teratomas are positive for SALL4, SOX2, and GPC3.

[0066] Similar to most solid tumors, immature teratomas continuously shed tumor cells. This disclosure collects ascites or peritoneal lavage fluid from clinical IT patients, uses a microfluidic system to enrich and isolate shed tumor cells for culture, and establishes a stable human ovarian IT tumor cell line. This can be used to further advance research on treatment and / or prevention drugs for this rare tumor, particularly those more suitable for people of Asian descent.

[0067] This publication collected tissues from clinical patients with immature teratomas. By isolating tumor cells and culturing them in vitro, a stable human immature teratoma ascites tumor cell line was obtained and classified as: Human IT Ascites Tumor Cell Line B049. It was deposited on December 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, accession number CGMCCNO:46310.

[0068] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description of some embodiments is provided in conjunction with examples. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0069] definition Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0070] In this disclosure, the terms “about,” “approximately,” or “around” generally refer to within 20%, 10%, 5%, 4%, 3%, 2%, or 1% of a given value or range. Unless otherwise specified, the quantities given are approximate values, implying that the terms “about,” “approximately,” or “around” can be derived.

[0071] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0072] In this article, the term "circulating tumor cell" or "CTC" refers to any circulating cancer cells found in a biological sample. Typically, CTCs detach from solid tumors. Therefore, CTCs are usually epithelial cells detached from solid tumors, present in circulation in patients with cancer at very low concentrations.

[0073] When used herein, the term "biological sample" refers to any sample including CTCs. Sources of the sample include whole blood, bone marrow, pleural fluid, peritoneal fluid, central cerebrospinal fluid, metastatic tumors, fresh biopsy samples, urine, saliva, and bronchial lavage fluid. Specifically, the sample is a blood sample or an ascites sample, with blood samples including, for example, whole blood or any fraction or component thereof. Blood samples suitable for this disclosure can be extracted from any known source containing blood cells or components thereof, such as venous blood, arterial blood, peripheral blood, tissue, etc. For example, the sample can be obtained and processed using well-known and conventional clinical methods, such as procedures for drawing and processing whole blood. In a particular embodiment, the sample may be peripheral blood drawn from a subject with cancer.

[0074] In this article, the term "cytokeratin (broad spectrum)" is also known as pan-CK (Cytokeratin Pan), which is a mixture of two antibodies that can label all epithelial cells, including simple epithelium, squamous epithelium, and benign and malignant tumors of urothelial origin.

[0075] In this article, the term " SOX2 "belong SOX The SOX2 family of genes are multifunctional transcription factors that form a trimer complex with OCT4 on DNA, regulating the expression of multiple genes involved in embryonic development (such as YES1, FGF4, UTF1, and ZFP206). They are highly expressed in normal primordial germ cells and embryonic stem cells, playing a central role in maintaining the pluripotency and self-renewal of embryonic stem cells and in early embryonic development. Ovarian malignant germ cells originate from primordial germ cells, and SOX2 is often positively expressed in pathological subtypes such as immature teratomas, embryonal carcinomas, and yolk sac tumors, serving as a marker for auxiliary diagnosis.

[0076] In this article, the term "short tandem repeats (STRs)," also known as microsatellite DNA, typically refers to a repetitive DNA sequence consisting of 1-6 base units in the genome. Due to the high variability and abundance of the number of core repeat units among individuals, STR loci constitute genetic polymorphism. Cellular STR identification involves establishing the genetic characteristics of a cell line using STR information.

[0077] In this article, the term "DAPI (Diamidino-phenyl-indole)" refers to 4',6-diamidino-2-phenylindole, a fluorescent dye that binds strongly to DNA and is commonly used in fluorescence microscopy. Because DAPI can penetrate intact cell membranes, it can be used for staining both live and fixed cells.

[0078] In this article, the term "CD45," also known as leukocyte common antigen (CLA), is a single-chain transmembrane protein widely distributed on the surface of leukocytes, containing three domains: a cytoplasmic C-terminus, a transmembrane region, and an extracellular glycosylated N-terminal region. CD45 antibodies labeled with fluorescent dyes can specifically bind to the CD45 antigen on the cell surface, thereby giving the cell fluorescein for observation under fluorescence microscopy.

[0079] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description of some embodiments is provided in conjunction with examples. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0080] The following embodiments and accompanying drawings are provided to aid in understanding this disclosure. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of this disclosure is set forth in the claims. It should be understood that any modifications and changes may be made without departing from the spirit of this disclosure.

[0081] Example Experimental materials 1) Preparation of screening medium: Add 50 mL of low endotoxin fetal bovine serum FBS solution (Sijiqing), 1.1 mg bFGF (Sino Biological), 1.1 mg EGF (Sino Biological), 60.5 mg sodium pyruvate (Thermo Fisher Scientific), 80.38 mg L-glutamine (Solarbio), and 10 mL of penicillin-streptomycin mixture (Gibco) (5000 U / mL penicillin, 5000 ug / mL streptomycin) to 440 mL of H-DMEM (Solarbio). After mixing, filter the mixture using a syringe filter with a pore size of 0.22 μm.

[0082] 2) Preparation of maintenance medium: Add 50 mL of KnockOut™ Serum Replacement (KSR) solution (Gibco™, catalog number 10828010), 1.1 mg bFGF (Shenzhou), 1.1 mg EGF (Shenzhou), 60.5 mg sodium pyruvate (Thermo Fisher Scientific), and 80.38 mg L-glutamine (Solepro) to 450 mL Knockout™-DMEM (Gibco). After mixing, filter using a syringe filter with a pore size of 0.22 μm.

[0083] Example 1: Isolation and Culture of Immature Teratoma Ascites Tumor Cells 1. Sample Source The patient's pelvic and abdominal puncture results, combined with the pathological examination of the surgical specimen, revealed an immature teratoma in the left ovary, high-grade / WHO grade 3.

[0084] 2. Isolation and culture of tumor cells 1) Sample collection: Peripheral blood and peritoneal fluid samples were obtained from the above-mentioned patients with immature teratomas. 5-10 mL of peripheral blood and 25-50 mL of peritoneal fluid were collected.

[0085] 2) Sample pretreatment: Peripheral blood and peritoneal fluid samples were treated with human peripheral blood lymphocyte separation medium (Solepro) (800g / 30min / 25℃, ACC: 9, DEC: 0) to obtain nucleated cell layers containing CTCs (or "PBMC solution").

[0086] 3) Add an equal volume of sterile PBS (Sangon Biotech) to the PBMC solution at a 1:1 ratio, mix well and centrifuge again (120g / 10min / 25℃).

[0087] 4) Remove the supernatant to obtain cell pellet, and resuspend in 10 mL of sterile PBS (Sangon Biotech).

[0088] 5) Using a pipette, add 10 mL of sample to the sample slot of the disposable cell separation and enrichment collector (LABYRINTH-K01), and place the collector into the slide compartment of the cell sorter (LABYRINTH-CE01). Click "Start Run". After 15 minutes, wait for the machine to finish running, open the slide compartment, remove the collector, and take out the detachable cell collection tube.

[0089] 6) After enrichment and sorting, the samples are centrifuged and resuspended in screening medium. Cells are then counted. The cells are seeded into six-well plates and cultured (denoted as D0). The six-well plates are placed in an incubator at 37°C and 5% CO2 for 24 hours. At this point, the cells are P1 generation.

[0090] (Note: The procedures for peripheral blood and ascites samples are the same from steps 2) to 6). The resulting P1 generation cells can survive and expand in vitro. Subsequent processing of the ascites cell samples begins from step 7). 7) After culturing for 24 hours, remove the waste liquid from the six-well plate and continue culturing using maintenance medium.

[0091] 8) After culturing for 48 hours, passage the cells according to their density. Digest the cells with 0.25% trypsin (Solepro), resuspend the cells in selection medium, and select an appropriate culture flask based on the cell count, such as T25 or T75 flasks, to continue culturing. Count the cells; at this point, the cells are P2 generation.

[0092] 9) After culturing P2 generation cells for 48 hours, use maintenance medium to replace the waste liquid in the culture flask.

[0093] 10) After culturing in maintenance medium for another 72 hours, harvest the cells. At this point, the cells are in generation P3. Count the cells and calculate the fold increase. Dilute the cells appropriately and seed them into multiple T75 culture flasks. Refer to steps 8)-9) for passage methods.

[0094] 11) After subculturing in T75 culture flasks for 48 hours, use maintenance medium to replace the waste liquid in the culture flasks to accelerate cell proliferation.

[0095] 12) After 72 hours, harvest the cells. At this point, the cells are in passage P4. Count the cells and calculate the fold increase. Dilute the cells appropriately and seed them into multiple T75 culture flasks. Refer to steps 8)-9) for passage methods.

[0096] 13) After subculturing in T75 culture flasks for 48 hours, use maintenance medium to replace the waste liquid in the culture flasks to accelerate cell proliferation.

[0097] 14) After 72 hours, harvest the cells. At this point, the cells are in passage P5. Count the cells and calculate the fold increase. Dilute the cells appropriately and seed them into multiple T75 culture flasks. Refer to steps 8)-9) for passage methods.

[0098] Cells from passages 1-5 (P1-P5) were collected and cell counts were performed. The experimental results are shown in Tables 1-2, and the growth curves are shown in [Table 1-2]. Figure 1 .

[0099] Table 1. Cell number statistics per generation

[0100] Table 2 Cell proliferation fold

[0101] Note: Cell proliferation factor = Pn / P(n-1), where n>1.

[0102] Cell cryopreservation: After cell culture, observe the cells under a microscope. When the cells reach 2 / 3 confluence in the flask, digest them with trypsin-EDTA (0.25%), centrifuge (120g / 25℃ / 5min), discard the supernatant, and add an appropriate amount of CryoStor. ® The CS10 cell cryopreservation solution was aliquoted into 1mL cryovials, which were then placed in a gradient cooling box and stored overnight at -80°C in liquid nitrogen.

[0103] Cell resuscitation: The cryovials stored in liquid nitrogen were removed and thawed in a 37°C water bath. 1 mL of selection medium was added to the cryovials, and after centrifugation (120 g / 25°C / 5 min), the supernatant was discarded, the cell suspension was resuspended, and the cells were transferred to culture flasks for further culture. Experimental results showed that the obtained cells could be passaged and cryopreserved normally, and could be stably passaged for more than 5 generations.

[0104] The collected cells were named B049 cells.

[0105] Morphological observations were performed on the immature teratoma tumor cells obtained in Example 1 during passage culture. The results of the cell observations under a microscope are shown below. Figure 2 On day 0, B049 cells were seeded into culture dishes at 168 cells / mL; day 4 showed that the cells had expanded to 70% of the culture dish area; day 7 showed that the cells continued to survive and grow after passage, with few dead cells; day 11 showed that after 3 more days of culture on day 7, the cells had expanded to 80% of the culture dish area; day 17 showed that after another passage, the cells continued to survive and expand, and the circulating tumor cells derived from the peritoneal effusion of patients with immature teratomas showed an epithelial-like appearance after passage.

[0106] Example 2 Identification of immature teratoma ascites tumor cells This embodiment focuses on immature teratoma cells that have undergone pathological diagnosis, verifying the expression of the pan-oncology tumor cell marker pan-CK and the expression of the auxiliary immunohistochemical marker SOX2 in immature teratomas, as detailed below: IT ascites tumor cells (named B049 cells) collected in Example 1 were fixed with 4% PFA (Thermo Fisher Scientific) and diluted to a suitable concentration. Cells filtered out in Example 1 were diluted to a suitable concentration as a control. Boxes were drawn on a slide (Sitaly) using an immunohistochemical pen, and the diluted cell suspension was added dropwise into each box. The slides were dried at 39°C and placed in a humidified chamber for the experiment. 200 μL of 0.2% Triton X-100 (Merck) was added to the slide, and after reacting for 3 min, excess solution was discarded. The slide was then carefully washed three times with PBST (Solepro) using a pipette, allowing it to stand for 3 min each time. Then, 200 μL of PBS containing 10% goat serum (Sijiqing) was carefully added along the edge of the slide, and the slide was blocked at room temperature for 30 min before the serum was removed. Diluted anti-CD45 (Bio-RAD, catalog number: MCA87) was added to the target area of ​​the slide. 25 μL each of the primary antibody and anti-pan-CK (Thermo Fisher Scientific, catalog number: MA191326) were added to the slide, both diluted 1:100. After incubation at room temperature for 1 h, the slides were washed three times with PBST. Then, 25 μL each of the secondary antibodies against CD45 (contamination control group) and pan-CK (experimental group) – goat anti-mouse lgG2a cross-adsorbed, Alexa Fluor® 488 (Thermo Fisher Scientific) and goat anti-mouse lgG1 cross-adsorbed, Alexa Fluor 546 (Thermo Fisher Scientific) – were added to the boxed area of ​​the slide, both diluted 1:100. The slides were incubated at room temperature in the dark for 45 min. After incubation, the slides were washed three times with 200 μL of PBST using a pipette. After washing, one drop of mounting medium solution containing DAPI dye (Abcam) was added, and a coverslip was carefully placed on top. The slides were then observed under a fluorescence microscope.

[0107] The fluorescence identification results of B049 cells are as follows: Figure 3 As shown, the fluorescence identification results of the cells filtered out in Example 1 are as follows: Figure 4 As shown (comparison). According to Figure 3 The results shown indicate that red represents pan-CK staining and blue represents DAPI staining. It can be seen that B049 cells are pan-CK positive (+), DAPI positive (+), and CD45 negative (-). Based on... Figure 4The results show that green represents CD45 staining and blue represents DAPI staining. It can be seen that the filtered cells were pan-CK negative (-), DAPI positive (+), and CD45 positive (+). According to the literature reference (https: / / tlcr.amegroups.org / article / view / 43487 / html), CK+ / CD45- is the standard for identifying tumor cells; therefore, the above test results further confirm that B049 cells are tumor cells.

[0108] The expression of the marker SOX2 in B049 cells was detected using the same procedure as above, except that the pan-CK primary antibody was replaced with an anti-SOX2 monoclonal antibody (Solepro, catalog number: K000326P) to identify the origin of the B049 cell line. This experiment consisted of two groups: ① CK / CD45 (contamination detection control group), ② SOX2 / CK (experimental group). The corresponding anti-SOX2 secondary antibody was a highly cross-adsorbed goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher, Alexa Fluor™ 647, A21245); the anti-CK secondary antibody was a cross-adsorbed goat anti-mouse IgG1 secondary antibody (Thermo Fisher, Alexa Fluor™ 546, A21123); and the anti-CD45 secondary antibody was a cross-adsorbed goat anti-mouse IgG2a secondary antibody (Thermo Fisher, Alexa Fluor™ 488, A21131).

[0109] The results of the identification are as follows Figure 5 As shown, yellow represents SOX2 (germ cell marker) staining, and blue represents DAPI staining. B049 cells were positive (+) for the auxiliary immunohistochemical marker SOX2 and negative (-) for CD45. The identification results were consistent with the pathological diagnosis results.

[0110] Based on the above identification results, it can be concluded that the cells are human-derived IT ascites tumor cells.

[0111] Example 3: STR identification of immature teratoma ascites tumor cells This embodiment utilizes SiFaSTR TM The 23plex identity verification system for forensic science uses a human fluorescently labeled STR multiplex amplification assay (Institute of Forensic Science) to perform multiplex PCR amplification of 21 short tandem repeat (STR) sites and Amelogenin sites in B049 cells and peripheral blood mononuclear cells from the same patient (hereinafter referred to as: B049-PBMC cells); the amplification products are analyzed using ABI Prism... ®Capillary electrophoresis was performed using a 3130 XL gene analyzer (Applied Biosystems, ABI) to generate raw electrophoretic patterns, which were then analyzed using GeneMapper. ® Genotyping was performed using ID v3.2 software (Applied Biosystems, ABI).

[0112] STR identification results showed that B049 cells and B049-PBMC cells had 100% similarity, confirming that the submitted sample atlas was human and did not match any atlas in the ExPASy database. This indicates that B049 cells are a new immature teratoma ascites tumor cell line. The specific results are shown in Table 3.

[0113] Table 3. Genotyping results of STR and Amelogenin loci in B049 cells and B049-PBMC cells

[0114] STR identification results of B049 cells were compared with the Cellosaurous database from the Swiss Institute of Bioinformatics to confirm cell identity. According to the cell STR identification criteria (issued by Suzhou Jianda Biotechnology Co., Ltd.), a cell line matching degree ≥80% is considered related, meaning they originate from a common ancestral cell; a matching degree between 70% and 79% requires further verification of the correlation; less than 70% indicates no correlation. The identification results (Table 3) show that the matching degree between B049 cells and the Cellosaurous database is 68.18%, and no cells with a matching degree greater than 80.00% were found in the Cellosaurous database. Using a similar method, STR identification was performed on 21 short tandem repeat (STR) sites and Amelogenin sites of B049 cells and B049-PBMCs from the same patient. The results showed that the matching degree between B049 cells and PBMCs in the patient's peripheral blood was greater than 90%, and the likelihood ratio (LR) was as high as 4.5680 × 10⁻⁶. 27 This confirms that B049 cells and B049-PBMCs originated from the same human individual, that B049 cells are human-derived cells, and that there is no other human gene contamination.

[0115] Based on the above results, it can be determined that the isolated cell B049 is a new immature teratoma ascites tumor cell line.

[0116] While some exemplary embodiments of this application have been described and illustrated, this application is not limited to the disclosed embodiments. Rather, those skilled in the art will recognize that modifications and changes may be made to the described embodiments without departing from the spirit and scope of this application as described in the appended claims.

Claims

1. A human immature teratoma ascites tumor cell line, characterized in that, The cell line was deposited on December 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO:46310.

2. A method for enriching and culturing the human immature teratoma ascites tumor cell line of claim 1, characterized in that, The method includes the following steps: 1) Enrichment of immature teratoma ascites tumor cells from the test sample; 2) Using a selection medium, the enriched immature teratoma ascites tumor cells are resuspended and cultured to obtain a first culture product, wherein the selection medium comprises basal medium and 5 v / v%-20 v / v% serum; 3) Replace the screening medium with a maintenance medium for maintenance culture to obtain a second culture product, wherein the maintenance medium comprises basal medium and 5 v / v%-20 v / v% serum substitute.

3. The method according to claim 2, characterized in that, The sample to be tested in step 1) is from a patient with an immature teratoma; and / or The sample to be tested in step 1) is ascites.

4. The method according to claim 2, characterized in that, The serum includes one or more of fetal bovine serum, newborn calf serum, calf serum, horse serum, swine serum, rabbit serum, goat serum, or rabbit serum; and / or The serum substitutes include one or more of KnockOut™ serum substitutes, N2, or B27.

5. The method according to claim 2, characterized in that, In step 3) of the method, after culturing with the screening medium for 10-48 hours, the screening medium is replaced with the maintenance medium for maintenance culture.

6. The method according to claim 2, characterized in that, The selection medium and / or the maintenance medium further comprise one or more of the following growth factors: epidermal growth factor, nerve growth factor, brain-derived neurotrophic factor, fibroblast growth factor, stem cell factor, insulin-like growth factor, or transforming growth factor-β; and / or The fibroblast growth factor includes one or more of the following growth factors: basic fibroblast growth factor or acidic fibroblast growth factor.

7. The method according to claim 2, characterized in that, The screening medium comprises: H-DMEM medium, FBS, FGF, EGF, sodium pyruvate, L-glutamine and / or a penicillin-streptomycin mixture; and / or The maintenance medium comprises: Knockout™-DMEM medium, KSR solution, bFGF, EGF, sodium pyruvate and / or L-glutamine.

8. The application of the cell line described in claim 1 in constructing an animal model of immature teratoma.

9. The application according to claim 8, characterized in that, The animal is a non-human mammal; and / or The animals are selected from mice, rats, dogs, horses, rabbits, sheep, pigs, cattle, or monkeys.

10. The use of the cell line of claim 1 or the animal model of claim 8 in one or more of the following: 1) Screening for drugs to prevent or treat immature teratomas; 2) Screening reagents for the diagnosis or detection of immature teratomas; 3) Research on biomarkers, drug targets, etiology, development mechanism, metastasis mechanism, and tumorigenesis mechanism of immature teratomas.

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