Mouse melanoma cell construction method and melanoma cell strain

Abstract

The invention belongs to the technical field of cell construction, and particularly relates to a mouse melanoma cell construction method which is characterized by comprising the following steps: sampling and preparing a sample; sampling; performing secondary screening; performing tissue culture; performing tissue transplantation; tumor formation; the method comprises the following steps: carrying out in-vitro culture, namely selecting a tumor-bearing mouse, killing the tumor-bearing mouse, carrying out super clean bench sterile operation on suspension cells, carrying out in-vitro culture by using DMEM + 20% FBS, observing whether the cells can be stably inherited or not, carrying out in-vitro culture for 7-10 days by using DMEM + 10% FBS culture after the morphology is stable, carrying out stable passage, stable shape and uniform cell morphology, digesting the cells, centrifuging at 3000 revolutions for 5 minutes, collecting the cells, adding PBS for resuspension, and carrying out in-vitro culture by using DMEM + 10% FBS culture after the morphology is stable. Centrifuging the cells at 3000 revolutions for 5 minutes, and discarding the supernatant to obtain a cell precipitate; finally, the cell precipitates are subjected to library building and sequencing for STR identification, subcutaneous tumors are inoculated on C57 mice at the same time, and the subcutaneous tumors expressing melanin are obtained. According to the invention, a completely new mouse melanoma cell line is constructed from another new scheme, and the mouse melanoma cell line is named as an MP306 cell line.

Description

Technical Field

[0001] The present invention relates to the technical field of cell construction, and specifically provides a method for constructing mouse melanoma cells and a melanoma cell line. Background Art

[0002] Cell growth is the basis for the development, growth, reproduction, and metabolism of organisms. In the fields of scientific research and medicine, the study of cell growth is of great significance. Mouse melanoma cells are a commonly used cell line for studying the occurrence, development, and treatment of melanoma.

[0003] B16 is a mouse-derived skin cancer (melanoma) cell line, which is an effective in vitro model for studying human skin cancer and is often used to study the formation of solid tumors and the metastasis of cancer cells.

[0004] The B16 cell line was established in 1954. These cells are derived from C57BL / 6J mice that spontaneously develop tumors in the skin at the Jackson Laboratory in Maine. B16 is a melanin-producing epithelial cell with the ability to metastasize to the spleen, liver, and lungs. However, currently, the internationally available and mature mouse melanoma cells are basically different clone sublines of B16 cells, including B16F0 and B16F10. These sublines are different from B16 cells and retain some specific characteristics.

[0005] However, as described above, for half a century, the mouse melanoma cells used by scientists have mostly been B16-derived cells, which does not reflect the diversity of the objects of scientific research. The single tumor source also limits the research on the anti-tumor pharmacodynamic mechanism, making in vitro tumor research limited to the framework of the B16 line, which also restricts the research and development of anti-tumor drugs invisibly. Summary of the Invention

[0006] The present invention aims to solve one of the technical problems existing in the prior art or related technologies.

[0007] For this reason, the technical solution adopted by the present invention is as follows:

[0008] A method for constructing mouse melanoma cells, comprising the following steps:

[0009] S1: Sampling and sample preparation. After selecting the sample animals, they are fed adaptively for 1 week. Use a razor to remove the excess animal hair, and at the same time use a depilatory cream to remove the hair on the animal's buttocks, exposing the back skin.

[0010] S2: Sample selection. Eliminate the animals with large black spots, and leave the animals with the black spot area accounting for 20-40%.

[0011] S3: Secondary screening. Disinfect the sampled animals by ultraviolet irradiation for 10 - 15 minutes every day. After one week, eliminate the animals with vigorous growth of black-spotted hair and retain the subsequent sample animals.

[0012] S4: Tissue culture. Use laser to irradiate the black-spotted area on the sampled animals for physical stimulation. After repeated irradiations, abnormal tissue hyperplasia occurs at the black-spotted area. Observe the growth of the abnormally hyperplastic tissue.

[0013] S5: Tissue transplantation. Sacrifice the animals and observe whether there are proliferative neoplasms growing in the animals after laser irradiation. Select the tissues with rapid growth and transplant them in vivo into the subcutaneous black-spotted area of normal female C57BL / 6 mice at 5 - 6 weeks old. Retain 10 samples and observe whether the transplanted hyperplastic tissues grow normally after breeding for 5 - 6 weeks.

[0014] S6: Tumor formation. Select the proliferative neoplasm with stable growth of the transplanted tumor in one animal as the first-generation animal. Sacrifice the animal and transplant the hyperplastic tissue into the subcutaneous area of normal female C57 mice at 5 - 6 weeks old as the second-generation animal. After breeding the animals for 3 - 4 weeks, select the animals without ulceration and with regular shapes in the subcutaneous transplanted tumors of the second-generation animals, extract the tumor contents, grind the tumor contents and resuspend them with PBS, and inoculate them in the subcutaneous area of C57 mice to observe the tumor formation situation.

[0015] S7: In vitro culture. Select 1 tumor-bearing mouse, sacrifice it, and perform aseptic operation to suspend the cells in a laminar flow hood. Culture the cells in vitro using DMEM + 20% FBS and observe whether the cells can be stably inherited. After the cell morphology is stable, culture the cells in vitro using DMEM + 10% FBS for 7 - 10 days. The cells can be stably passaged, with stable shapes and uniform cell morphologies. Then digest the cells, centrifuge them at 3000 rpm for 5 minutes to collect the cells, then add PBS to resuspend the cells, centrifuge the cells at 3000 rpm for 5 minutes, and discard the supernatant to obtain cell pellets.

[0016] Finally, construct a library of the cell pellets for STR identification by sequencing, and at the same time inoculate subcutaneous tumors on C57 mice to obtain subcutaneous tumors expressing melanin.

[0017] Furthermore, the subsequent samples in step S3 are animals with black-spotted areas ranging from 4 mm * 4 mm to 6 mm * 6 mm.

[0018] Furthermore, the tissue culture in step S4 specifically includes the following steps:

[0019] 1), Anesthetize the sampled animals with gas, and then use a laser combined with a thermal imager to irradiate the black-spotted area for physical stimulation of melanin. Observe whether there is ulceration at the irradiated area after 48 hours, and observe the wound healing situation and hair growth situation at the irradiated area after one week.

[0020] (2) For the parts with obvious hair growth at the black spots, perform secondary laser irradiation, and repeat this process. The total cycle is 5 weeks until there is no more hair growth at the black spot area. At 5 - 6 weeks, there is abnormal hyperplasia of black tissue at the black spot.

[0021] Furthermore, the physical stimulation in step S4 includes irradiating the black spot area with a 1 - 5w 808 laser, and then using an infrared thermal imager to control the temperature of the illuminated area at 55°C for 15 minutes. The secondary laser irradiation is to irradiate with a 1 - 5w 808nm laser for 15 minutes.

[0022] Furthermore, the in vitro culture environmental conditions in step S7 are 37°C and 5% CO2.

[0023] Furthermore, a melanoma cell line is constructed according to the above - mentioned method for constructing a mouse melanoma cell line.

[0024] The above - mentioned technical solution of the present invention has the following beneficial technical effects:

[0025] Through the present invention, a completely new mouse melanoma cell line is constructed from a new solution, which we name the MP306 cell line. This provides a broader space for the future research and development of anti - tumor drugs and has great significance in promoting the research and development of anti - tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the detailed identification results of the mouse cell line in an embodiment of the present invention;

[0027] Figure 2 It is a schematic diagram of the detailed sample analysis results of the mouse cell line in an embodiment of the present invention;

[0028] Figure 3 It is a schematic diagram of the detailed identification report of the mouse cell line in an embodiment of the present invention;

[0029] Figure 4 It is a schematic diagram of the preliminary identification results of the mouse cell line in an embodiment of the present invention;

[0030] Figure 5 It is a schematic diagram of the preliminary sample analysis results of the mouse cell line in an embodiment of the present invention;

[0031] Figure 6 It is a schematic diagram of the preliminary identification report of the mouse cell line in an embodiment of the present invention;

[0032] Figure 7 It is a schematic diagram of the cells of the MP306 cell line in a conventional state in an embodiment of the present invention;

[0033] Figure 8Schematic diagram of cells of the MP306 cell line at a magnification of 400 times according to an embodiment of the present invention;

[0034] Figure 9 Schematic diagram of the MP306 cell line according to an embodiment of the present invention after dilution;

[0035] Figure 10 Schematic diagram of the MP306 cell line at a magnification of 400 times after dilution according to an embodiment of the present invention;

[0036] Figure 11 Schematic diagram of the preservation information of the MP306 cells according to an embodiment of the present invention. Detailed implementation manners

[0037] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with specific implementation manners. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0038] It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention.

[0039] The following describes a method for constructing mouse melanoma cells and a melanoma cell line provided by the present invention.

[0040] 1. Sample source information

[0041] Animal source: Comparative Medicine Center of Yangzhou University

[0042] Production license number: SCXK (Jiangsu) 2022-0009

[0043] Animal strain: C57BL / 6 Gender: Female Age: 5-6 weeks.

[0044] 2. Modeling method

[0045] After the animals are adaptively raised for 1 week, a razor is used to remove the excess animal hair, and at the same time, a depilatory cream is used to remove the hair on the animal's buttocks, exposing the back skin;

[0046] # There is spontaneously melanin-enriched skin on the back of C57, which is the so-called black spot. The hair growth rate at the black spot is higher than that of the skin without the black spot;

[0047] Animals with large black spots are removed, and animals with 30% of the black spot area are left;

[0048] Too large a black spot area is not conducive to subsequent laser irradiation;

[0049] The animals are disinfected by ultraviolet irradiation for 10-15 minutes every day;

[0050] Ultraviolet radiation stimulates melanin mutation and accelerates the carcinogenic process;

[0051] One week later, animals with vigorous hair growth on the black spots were excluded. The animals were anesthetized with gas, and 808 laser was used to irradiate the black spots with an area of about 5 mm * 5 mm for 15 minutes at a power of 1 - 5 W. A thermal imager was used to control the temperature of the irradiated area at 55 degrees for 15 minutes;

[0052] Preferably, the 808 laser irradiation uses 1 W;

[0053] The laser is used to destroy the hair follicles. At the same time, the 808 nm laser will cause a local temperature increase, physically stimulating the melanin black spot area;

[0054] After 48 hours, the irradiated area was observed, and ulceration occurred;

[0055] After 808 nm irradiation, since the temperature at the irradiated position will rise to 55 - 60 degrees, ulceration will occur the next day;

[0056] One week later, the wound healing and hair growth conditions of the irradiated area were observed;

[0057] The wound at the light - irradiated ulcerated area will heal in about one week;

[0058] For the areas with obvious hair growth on the black spots, secondary laser irradiation was performed with 808 nm laser for 15 minutes at a power of 1 - 5 W. This was repeated, and the cycle lasted for a total of 5 weeks;

[0059] Repeatedly laser - thermally stimulate the melanin area;

[0060] Until there is no longer hair growth in the black spot area. At 5 - 6 weeks, there is abnormal black tissue hyperplasia at the black spot, and the growth of the abnormal hyperplastic tissue was observed;

[0061] The animals were sacrificed, and it was observed whether there were hyperplastic neoplasms growing in the animals after laser irradiation. The tissue with rapid growth was selected for in - vivo transplantation under the skin of the black spots of normal female 5 - 6 - week - old C57BL / 6 mice;

[0062] Allogeneic transplantation was performed on the suspected mutant tissue; 10 were used. After breeding for 5 - 6 weeks, it was observed whether the transplanted hyperplastic tissue grew normally. The hyperplastic growth with stable growth of the transplanted tumor in one animal was regarded as the first - generation animal;

[0063] Select the suspected carcinogenic tissue for allograft culture;

[0064] The animals were sacrificed, and the hyperplastic tissue was transplanted under the skin of normal female 5 - 6 - week - old C57 mice, regarded as the second - generation animals. After breeding the animals for 3 - 4 weeks;

[0065] Observe whether the proliferative tissue can stably grow in the transplanted mice. Consider the stably growing proliferations as tumors for the next transplantation, and at the same time consider the stably growing proliferations as transplanted tumors.

[0066] Select animals with non-ulcerated and regular-shaped subcutaneous transplanted tumors in the second-generation animals.

[0067] Screen tumors with stable growth and good condition of transplanted tumors for the next experiment.

[0068] Extract the tumor content, grind the tumor content and resuspend it with PBS, inoculate it in C57 subcutaneous animals, observe the tumor formation situation, and select 1 tumor-bearing mouse to sacrifice and perform aseptic operation to suspend the cells in a laminar flow hood.

[0069] Prepare for in vitro culture, and the condition requires aseptic operation.

[0070] Culture in vitro using DMEM + 20% FBS, and observe whether the cells can be stably inherited.

[0071] Wait for the cell morphology to be stable after 2 - 3 days of in vitro culture.

[0072] After the morphology is stable, culture in vitro using DMEM + 10% FBS for 7 - 10 days, and it can be stably passaged, with a stable shape and uniform cell morphology.

[0073] Under this condition, it is considered that the in vitro cells can grow uniformly and stably.

[0074] Digest the cells, centrifuge at 3000 rpm for 5 min to collect the cells, then add PBS to resuspend, centrifuge the cells at 3000 rpm for 5 min, discard the supernatant, obtain the cell pellet, construct a library of the cell pellet for STR identification, and at the same time inoculate subcutaneous tumors on C57 mice to obtain subcutaneous tumors expressing melanin.

[0075] Cell culture conditions: DMEM + 10% FBS

[0076] Culture conditions: 5% CO2; 37 °C

[0077] Cryopreservation conditions:

[0078] Passage ratio: 1:2 - 1:4

[0079] Doubling time: 48 - 72 h.

[0080] In order to identify the authenticity of the above cells, we conducted the following tests and preservation.

[0081] Under STR identification, we conducted preliminary tests and detailed tests.

[0082] Submitting unit: Nanjing Pusheng Biomedicine

[0083] Name of the sample: Cell line

[0084] Testing unit: Shanghai YeeGene Applied Biotechnology Co., Ltd.

[0085] Report date: 2024 / 03 / 11

[0086] Among them, the preliminary identification results are as follows:

[0087] (I) Basic situation of the inspection

[0088]

[0089] Test results of sample genotype

[0090] Multi-allele refers to the phenomenon of three or more alleles.

[0091] The typing results of each cell in this test are good.

[0092] (II) Descriptions of each sample

[0093] 20240311-01: The identification result of this cell line is a mouse cell line. No valid matching cell line was found in the cell line search for DNA typing. The highest match in the EXPASY database is MB49, with a match degree of 89%. This result is for reference only and cannot fully confirm that it is MB49. It can be confirmed that the sample is not from B16. No multi-allele was found in this cell line, and there is no human source contamination, as Figure 4 shown.

[0094] Note: The STR data of the cell line to be tested matches the STR data in the ATCC, DSMZ (DSMZ has STR data of 2,490 cell lines from ATCC, DSMZ, JCRB, and RIKEN, etc.), and ExPASy cell bank (ExPASy has STR data of approximately 8,000 human cell lines from databases such as ATCC, DSMZ, JCRB, ECACC, and Riken). Cells not included in the above cell banks cannot be matched. According to the identification standard of the ATCC Standards Committee (ANSI / ATCC ASN-0002-2022), a match degree of EV≥80% is considered to have a correlation and may be derived from a common ancestral cell; between 55%-80% of the match degree, other methods need to be combined for further identification and certification of its correlation. Among the following loci, D4S2408 is a human source locus, which is used to detect whether there is human source contamination in this cell.

[0095] (III) Sample analysis results are as Figure 5 .

[0096] Other instructions:

[0097] (I) Typing scheme and locus distribution

[0098]

[0099] Experimental scheme and loci

[0100] (II) The identification report is Figure 6 。

[0101] The specific details of the second detailed test are as follows:

[0102] Submitting unit: Nanjing Pusheng Biomedicine

[0103] Name of the sample for inspection: Cell line

[0104] Testing unit: Shanghai YeeGene Applied Biotechnology Co., Ltd.

[0105] Report date: 2024 / 03 / 15

[0106] The test results are as follows:

[0107] (I) Basic situation of the inspection

[0108]

[0109] Test results of sample genotypes

[0110] Multi-allele refers to the phenomenon of three or more alleles.

[0111] The typing results of each cell in this test are good.

[0112] (II) Descriptions of each sample

[0113] 20240311-01: The identification result of this cell line is a mouse cell line, and no matching cell line was found in the cell line search for DNA typing. In this cell line, no multi-alleles were found in this test. This cell strain is a single-source mouse-derived cell, and no human source contamination was found, as Figure 1 shown.

[0114] Note: The STR data of the cell line to be tested matches the STR data in the ATCC, DSMZ (DSMZ has STR data of 2,490 cell lines from ATCC, DSMZ, JCRB, and RIKEN, etc.), and ExPASy cell bank (ExPASy has STR data of approximately 8,000 human cell lines from databases such as ATCC, DSMZ, JCRB, ECACC, and Riken, etc.). Cells not included in the above cell banks cannot be matched. According to the identification criteria of the ATCC Standards Committee (ANSI / ATCC ASN-0002-2022), a match degree EV≥80% is considered to have a correlation and may be derived from a common ancestral cell; when the match degree is between 55%-80%, other methods need to be combined for further identification and authentication of its correlation. Among the following loci, TH01 is a human locus, which is used to detect whether there is human contamination in the cell.

[0115] The sample analysis results are as Figure 2 .

[0116] Other instructions:

[0117] (I) Typing scheme and locus distribution

[0118]

[0119] Experimental scheme and loci

[0120] The identification report is Figure 3 .

[0121] According to the content of the specification, the content of this application is true and valid. We constructed a completely new mouse melanoma cell line from another new scheme, and we named it the MP306 cell line, which provides a broader space for the future research and development of anti-tumor drugs and has great significance in promoting the research and development of anti-tumor drugs.

[0122] More importantly, as Figure 7 shown, we provide a cell schematic diagram of the MP306 cell line under normal conditions, as Figure 8 shown is the cell schematic diagram magnified 400 times.

[0123] As Figures 9 - 10 shown, we provide a cell schematic diagram of the MP306 cell line after dilution and a cell schematic diagram magnified 400 times after dilution.

[0124] As Figure 11 shown, we deposited the cells.

[0125] The mouse melanoma cell line MP306, Mus musculus, of the present invention was deposited at the China Center for Type Culture Collection (CCTCC) on August 13, 2024, with the deposit number: CCTCC NO: 2024254.

[0126] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for constructing mouse melanoma cells, characterized in that: The following steps are involved: S1: Sampling and sample preparation: After selecting the sample animals, they were placed in the field for adaptive feeding for 1 week. Excess animal hair was removed with a razor, and hair removal cream was used to remove the hair on the animal's buttocks, exposing the back skin; S2: Sampling, removing animals with large black spots, leaving only animals with black spots accounting for 20-40% of the area; S3: Secondary screening: disinfect the sample animals with ultraviolet irradiation for 10-15 minutes every day, and remove the animals with black spots and vigorous hair growth after one week, leaving the subsequent sample animals; S4: Tissue culture, using laser to irradiate the black spot area on the sampled animal for physical stimulation. After repeated exposure, abnormal tissue will proliferate in the black spot, and the growth of abnormal proliferating tissue will be observed; S5: Tissue transplantation: animals were sacrificed and observed for growth of hyperplastic vegetation after laser irradiation. Rapidly growing tissues were selected and transplanted into subcutaneous black spots of normal female 5-6-week-old C57BL / 6 mice. Ten samples were kept and observed for normal growth of transplanted hyperplastic tissues after 5-6 weeks of feeding. S6: Tumor formation: one animal with a stably growing transplanted tumor was selected as the first generation animal. The animal was killed and the proliferative tissue was transplanted subcutaneously into a normal female 5-6 week old C57 mouse, which was regarded as the second generation animal. After the animals were raised for 3-4 weeks, the second generation animal with a subcutaneous transplanted tumor that was free of ulceration and had a regular shape was selected, and the tumor contents were extracted. The tumor contents were ground and resuspended in PBS, and inoculated into the C57 subcutaneous animal to observe the tumor formation. S7: In vitro culture, select a tumor-bearing mouse and kill it, then aseptically operate the suspended cells in a clean bench, and culture them in vitro with DMEM + 20% FBS to observe whether the cells can be genetically stable. After the morphology is stable, use DMEM + 10% FBS to culture in vitro for 7-10 days. The cells can be stably passaged, the shape is stable, and the cell morphology is uniform. Then, the cells are digested and centrifuged at 3000 rpm for 5 minutes to collect the cells, and then PBS is added to resuspend them, and the cells are centrifuged at 3000 rpm for 5 minutes. The supernatant is discarded to obtain the cell pellet; Finally, the cell pellet library was constructed and sequenced for STR identification, and subcutaneous tumors were inoculated in C57 mice to obtain subcutaneous tumors expressing melanin.

2. A method for constructing mouse melanoma cells according to claim 1, characterized in that: The subsequent samples in step S3 are animals with black spot areas ranging from 4mm*4mm to 6mm*6mm.

3. A method for constructing mouse melanoma cells according to claim 1, characterized in that: The tissue culture in step S4 specifically includes the following steps: 1) The animals were anesthetized with gas, and then the dark spot area was irradiated with a laser and a thermal imager to physically stimulate the melanin. Ulceration was observed at the irradiated site 48 hours later, and wound healing and hair growth at the irradiated site were observed one week later; 2) Perform a second laser irradiation on the areas where there is obvious hair growth on the dark spots. Repeat this process for a total of 5 weeks until there is no more hair growth on the dark spots and abnormal black tissue proliferation is observed on the dark spots after 5-6 weeks.

4. A method for constructing mouse melanoma cells according to claim 3, characterized in that: The physical stimulation in step S4 includes irradiating the dark spot area with 1-5w 808 laser, and then using a thermal imager to control the temperature of the illuminated area at 55°C for 15 minutes. The secondary laser irradiation is 1-5w 808nm laser irradiation for 15 minutes.

5. A method for constructing mouse melanoma cells according to claim 1, characterized in that: The in vitro culture environment conditions in step S7 are 37° C. and 5% CO 2 .

6. A melanoma tumor cell line constructed according to the mouse melanoma cell construction method according to any one of claims 1-5.

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