A method for extracting primary cancer-associated fibroblasts from renal clear cell carcinoma tissue

Abstract

The application belongs to the technical field of biotechnology, and particularly discloses a method for extracting primary cancer-associated fibroblasts from renal clear cell carcinoma tissue. The method comprises the following steps: treating fresh renal clear cell carcinoma tissue at 0-4 DEG C, and only retaining a region rich in tumor cells and interstitial mixed; low-speed shearing the region rich in tumor cells and interstitial mixed into fragments with a volume of 1-3 mm 3 ; placing the fragments in a collagenase I solution, collecting cell precipitates after digestion is completed; inoculating the cell precipitates into a first culture medium, and replacing the fresh first culture medium every day within the first 3 days after inoculation, and then replacing the first culture medium every 2-3 days. The method overcomes the problems of low cell adhesion rate, serious contamination, low cell activity and low purity of the conventional extraction technology of primary cancer-associated fibroblasts when applied to renal clear cell carcinoma tissue, and can stably obtain primary cancer-associated fibroblasts with high purity and high survival rate within 7-10 days.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for extracting primary cancer-associated fibroblasts from clear cell renal tissue. Background Technology

[0002] Cancer-associated fibroblasts (CAFs) are key stromal cells in the tumor microenvironment, playing a crucial role in promoting tumor progression, angiogenesis, and immunosuppression. In clear cell renal cell carcinoma (ccRCC), the activation status of CAFs is closely related to patient prognosis; therefore, obtaining high-quality primary CAFs is essential for mechanistic research and targeted therapy development. Currently, commonly used methods for primary CAF extraction mainly include tissue block extraction and enzymatic digestion. However, these two conventional fibroblast extraction techniques often suffer from problems such as low cell adhesion, severe contamination, low cell viability, and low purity when applied to ccRCC tissue. Summary of the Invention

[0003] To overcome the problems of low cell adhesion, severe contamination, low cell viability, and low purity encountered when using conventional primary CAF extraction techniques in clear cell renal cell carcinoma (ccRCC) tissues, this invention proposes a method for extracting primary cancer-associated fibroblasts (CAFs) from clear cell renal cell carcinoma (ccRCC) tissues. This method is systematically optimized based on traditional enzymatic digestion, using collagenase I instead of mixed enzymes, and significantly improving extraction efficiency through optimized tissue processing and digestion procedures. Experimental results show that under optimized digestion conditions of 1.5 hours, cell adhesion can reach 75%, and purity reaches 90%. To address common contamination from erythrocytes, immune cells, and lipid droplets during extraction, this invention employs a "daily solution change for the first three days" strategy to effectively remove impurities and avoid hemolysis. Verification showed that this method achieved a success rate of over 85% in 57 ccRCC clinical tissue samples, with the obtained cells exhibiting typical spindle-shaped and radial growth morphology, and immunofluorescence detection revealing an α-SMA positivity rate as high as 90%.

[0004] Based on the above findings, the present invention provides the following technical solution: This invention provides a method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue, comprising the following steps: Fresh clear cell renal cell carcinoma tissue was treated at 0-4℃, preserving only areas rich in a mixture of tumor cells and stroma.

[0005] The region rich in tumor cells and stroma was slowly sheared into pieces with a volume of 1-3 mm. 3 fragments; Place the fragments in a collagenase I solution, and collect the cell precipitate after digestion is complete. The cell pellet was inoculated into the first culture medium. For the first 3 days after inoculation, the first culture medium was replaced with fresh medium daily, and then every 2 to 3 days thereafter.

[0006] In some embodiments of the present invention, the fresh clear cell renal carcinoma tissue is clear cell renal carcinoma tissue that has been ex vivo for less than 1 hour.

[0007] In some embodiments of the present invention, processing fresh clear cell renal cell carcinoma tissue at 0-4°C includes: Wash the clear cell renal carcinoma tissue with buffer solution; Obvious necrotic areas, adipose tissue, and major blood vessels were removed from the clear cell renal cell carcinoma tissue.

[0008] In some embodiments of the present invention, low-speed shearing is performed using scissors and / or a tissue cutter.

[0009] In some embodiments of the present invention, the fragments are placed in a collagenase I solution and digestion is completed, including: placing the fragments in a collagenase I solution with a concentration of 0.8~1.2 mg / mL and digesting for 1~2 hours.

[0010] In some embodiments of the present invention, collecting the cell pellet includes: Filter the digestive fluid through a sieve and collect the filtrate; After centrifuging the filtrate, the precipitate was collected to obtain cell precipitate.

[0011] In some embodiments of the present invention, the centrifugation rate is 1200~1500 rpm.

[0012] In some embodiments of the present invention, the first culture medium is a complete culture medium containing FBS.

[0013] In some embodiments of the present invention, digestion completion has one or more of the following characteristics: (1) The solution is visibly turbid, large pieces of tissue have basically disappeared, and the digestive juice is no longer viscous; (2) When initially inoculated into a culture dish, the microscope field of view mainly consists of scattered single cells or very small cell clusters, and after adhering to the wall, the cells are spindle-shaped and have good refractive properties.

[0014] In some embodiments of the present invention, this method is applicable to the extraction of primary carcinoma-associated fibroblasts from clear cell renal cell carcinoma tissues at all stages.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Validated by 57 clinical ccRCC samples, the method proposed in this invention can stably obtain primary CAFs within 7-10 days. This method is applicable to early, locally late and late ccRCC tissues, and the overall extraction success rate for each stage is higher than 85%.

[0016] (2) This method uses a single collagenase I for digestion. By precisely controlling the size of the tissue block and the digestion time, the cell membrane is protected while effectively dissociating the tissue. When the digestion time is 1.5 hours, the cell adhesion rate can be as high as 75% and the purity can be as high as 90%.

[0017] (3) To address the problem of severe contamination by erythrocytes, immune cells, and lipid droplets in the samples, this invention employs a strategy of "daily medium change for the first three days." Compared to the group without daily medium change, this strategy significantly improves cell adhesion rate, and the adherent cells exhibit a regular spindle-shaped structure. (The text abruptly ends here, likely due to an incomplete translation or a missing section.) SMA immunofluorescence staining identification showed that the primary CAFs extracted by this method had an average positive rate of up to 90%, confirming their high purity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The present invention provides a schematic flowchart of the method for extracting CAFs from ccRCC tissue; it mainly includes four parts: obtaining ccRCC tissue, cutting the tissue and adding collagenase I for digestion, seeding cells after digestion and changing the medium, screening, and verification of primary CAFs.

[0020] Figure 2 The effect of different digestion times on cell purity and cell adhesion rate; where: the left figure shows the effect of different digestion times on cell purity, with the horizontal axis representing digestion time and the vertical axis representing cell purity, and ns indicating no significant difference. This indicates that p < 0.01. This indicates that p < 0.001. p < 0.0001; the right figure shows the effect of different digestion times on cell adhesion rate, with the horizontal axis representing digestion time and the vertical axis representing cell adhesion rate. ns indicates no statistical significance. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0021] Figure 3 Morphological images of primary CAFs after inoculation into culture dishes for 2-3 days without medium change; these images were obtained through morphological observation under an inverted microscope (100×).

[0022] Figure 4 Morphological images of primary CAFs obtained using the method provided in this invention; these images were acquired and observed under an inverted microscope (100×).

[0023] Figure 5 Immunofluorescence staining results of α-SMA (200×); green fluorescent signal indicates α-SMA positive expression, and blue fluorescent signal indicates DAPI used to label cell nuclei. The scale bar is 50 μm.

[0024] Figure 6 The effect of different types of digestive enzymes on lipid droplet content; where: the left figure shows the lipid droplet content in the suspension after digestion with collagenase IV and hyaluronidase, and the right figure shows the lipid droplet content in the suspension after digestion with collagenase I.

[0025] Figure 7 The effect of different types of digestive enzymes on cell adhesion rate; where: the horizontal axis represents the type of enzyme used in the digestion step, the vertical axis represents the cell adhesion rate, and ns indicates no significant difference. This indicates that p < 0.01. This indicates that p < 0.001. This means p < 0.0001.

[0026] Figure 8 Comparison of the application effects of the method provided by this invention and the tissue block method in ccRCC tissue and adjacent normal tissue.

[0027] Figure 9The method provided by this invention represents the success rate of extracting primary CAFs from ccRCC tissues at different stages; where: the horizontal axis represents the number of samples used, the vertical axis represents ccRCC tissues at different stages, the black part of the bar chart represents the number of cases in which primary CAFs were successfully extracted, and the gray part of the bar chart represents the number of cases in which primary CAFs were not successfully extracted.

[0028] Figure 10 Morphological diagram of primary CAFs isolated from renal pelvis carcinoma tissue by the method provided in this invention.

[0029] Figure 11 The method provided in this invention compares the α-SMA positivity rate of primary CAFs extracted from ccRCC tissue and renal pelvis carcinoma tissue; where: the horizontal axis represents tumor tissue type, the vertical axis represents α-SMA positivity rate, and ns indicates no statistical significance. p means <0.01, p means <0.001, This means p < 0.0001. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] The unique pathological features of clear cell renal cell carcinoma (ccRCC) tissue constitute its complex tumor microenvironment, posing significant challenges to conventional cancer-associated fibroblast (CAF) extraction methods in this tissue. Primary CAFs isolated from ccRCC tissue often retain a large number of lipid droplets, becoming a key limiting factor affecting successful culture. These lipid droplets not only reduce cell viability but also directly inhibit the adhesion and proliferation of primary CAFs. To address this problem caused by tissue-specific pathological features, this invention optimizes the medium change strategy, adjusting the conventional every other day to daily medium change. This strategy, by increasing the frequency of culture system renewal and continuously removing inhibitory components such as lipid droplets, creates a cleaner and more supportive microenvironment for the adherent growth of primary CAFs, ultimately significantly improving the adhesion efficiency of primary CAFs and promoting their subsequent proliferation.

[0032] This invention provides a method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma (ccRCC) tissue. The method mainly includes the following four steps: First, processing fresh ccRCC tissue at 0-4°C, retaining only the region rich in tumor cells and stroma; Second, slowly shearing the region rich in tumor cells and stroma into pieces with a volume of 1-3 mm. 3 The process involves: 1) collecting the cell fragments; 2) placing the fragments in collagenase I solution and collecting the cell pellet after digestion is complete; 3) inoculating the cell pellet into the first culture medium and replacing it with fresh first culture medium daily for the first 3 days after inoculation, and then every 2-3 days thereafter.

[0033] In some implementations, the ex vivo time of clear cell renal cell carcinoma (ccRCC) tissue is less than one hour. Strictly controlling the ex vivo time of tissue samples is crucial for the successful extraction of primary CAFs. Theoretically, shorter ex vivo time is more conducive to maintaining cell viability, but in practice, it is often limited by time costs such as transportation. Furthermore, due to its unique pathological characteristics—rich in lipids, abnormally active metabolism, and abundant blood supply—ccRCC tissue is extremely sensitive to hypoxia and energy depletion. Prolonged ex vivo time drastically accelerates lipid droplet rupture and cell death, leading to the failure of primary CAF extraction; it also exacerbates tissue fibrosis, increasing the difficulty of subsequent enzymatic digestion. Therefore, controlling the ex vivo time to less than one hour is the optimal time point for balancing practical operating conditions with cell viability preservation.

[0034] In some embodiments, fresh clear cell renal cell carcinoma (ccRCC) tissue is processed at 0–4°C, including: firstly, washing the ccRCC tissue with a buffer solution; and secondly, removing obvious necrotic areas, adipose tissue, and major blood vessels from the ccRCC tissue. Preferably, the buffer solution is PBS, which has the main advantages of excellent physiological compatibility and operational economy compared to other buffer solutions. On the one hand, its isotonic properties and strong buffering capacity can maintain cell morphology and pH stability, maximizing sample viability; on the other hand, its components are simple, inexpensive, and easy to prepare aseptically on a large scale. Washing fresh ccRCC tissue samples with PBS is to effectively remove surface blood and adhering substances, obtaining a clearer field of view for subsequent precise removal. Since necrotic areas, adipose tissue, and major blood vessels commonly present in ccRCC tissue can severely hinder the effective digestion of collagenase and contaminate the target cell population, they must be carefully removed, ultimately retaining only areas rich in a mixture of tumor cells and stroma. Regions rich in a mixture of tumor cells and stroma are the primary areas where cancer-associated fibroblasts (CAFs) are activated and recruited by tumor cells. Fibroblasts in these areas are exposed to abundant tumor signals and have been highly activated from a resting state to the CAF phenotype, exhibiting high numbers and active function. Therefore, this region is the most ideal and crucial sampling location for obtaining cancer-associated fibroblasts. The above processing steps are key to ensuring the efficiency of subsequent enzymatic digestion and ultimately obtaining CAFs.

[0035] In some implementations, low-speed shearing is performed using scissors or a tissue cutter. Preferably, sterile ophthalmic scissors are used to cut the tissue. Compared to other shearing methods, this method offers the advantage of achieving more precise and less damaging cuts, which is beneficial for subsequent cell survival and functional preservation.

[0036] In some embodiments, the fragments are placed in a collagenase I solution and digested until complete, including: placing the fragments in a collagenase I solution with a concentration of 0.8-1.2 mg / mL and digesting for 1-2 hours. Preferably, the collagenase I concentration used is 1 mg / mL, and the digestion conditions are digestion at 220 rpm for 1.5 hours in a constant temperature shaker at 37°C. Experiments have shown that primary CAFs obtained with a collagenase I concentration of 1 mg / mL and a shaking digestion time of 1.5 hours have high purity and adhesion rate. Excessive shaking digestion time will lead to the death of all cells, while insufficient time will result in incomplete tissue digestion and extremely low cell yield.

[0037] In some embodiments, collecting the cell pellet includes: filtering the digestion solution through a sieve and collecting the filtrate; centrifuging the filtrate and collecting the pellet to obtain the cell pellet. Preferably, a sieve with a pore size of 40 μm is used to filter the filtrate. A sieve with a pore size of 40 μm is beneficial for trapping large, incompletely digested tissue fragments, cell clusters, and impurities, while allowing target cells to pass through, thereby initially enriching cells and improving the homogeneity of the subsequent seeding suspension.

[0038] In some embodiments, the centrifugation rate is 1200-1500 rpm. Preferably, the centrifugation rate is 1200 rpm, which can minimize the mechanical damage to fragile primary cells caused by centrifugal force while fully precipitating the target cells, thus helping to maintain cell viability and subsequent adhesion rate.

[0039] In some embodiments, the first culture medium is a complete culture medium containing FBS. Preferably, the first culture medium is DMEM medium containing 10% FBS. DMEM medium containing FBS is crucial in the culture of primary CAFs. Its rich and complex components not only meet the basal metabolic needs of cells but also provide a series of paracrine factors, mimicking the growth microenvironment in vivo. This effectively supports the adhesion, extension, and continuous proliferation of newly isolated, fragile primary CAFs, and is key to maintaining their activity and functional phenotype.

[0040] In some implementations, digestion is complete when one or more of the following characteristics are present: the solution is visibly uniformly turbid, large tissue fragments have largely disappeared, and the digestive fluid is no longer viscous; initially, when inoculated into culture dishes, the microscopic field of view mainly shows dispersed single cells or very small cell clusters, and after adhesion, the cells exhibit a spindle-shaped morphology and good refractive properties. Distinguishing digestion stages based on these characteristics offers the direct advantage of achieving precise control over the digestion process. This not only prevents insufficient tissue dissociation and low cell yield due to inadequate digestion but also effectively avoids severe damage to cell viability and increased fragmentation caused by over-digestion, ensuring the final acquisition of a highly active and intact target cell suspension.

[0041] In some embodiments, this method is applicable to the extraction of primary cancer-associated fibroblasts (CAFs) from all stages of clear cell renal cell carcinoma (ccRCC) tissue. By comparing the success rates of the method proposed in this invention in ccRCC tissue at different stages, the applicability of this method to the extraction of primary CAFs from ccRCC tissue at all stages has been confirmed.

[0042] Example: Extraction of primary CAFs from surgical tissue of primary renal clear cell carcinoma lesion (1) Sample preparation Obtain approximately 0.5–1.0 g of fresh, non-necrotic solid ccRCC tissue sample surgically removed, with informed consent from the patient and approval from the hospital ethics committee. Immediately place the sample in a sterile centrifuge tube containing pre-cooled serum-free DMEM and transport on ice. Processing should begin within 30 minutes. In a laminar flow hood, wash the tissue three times with PBS to remove blood. Carefully remove any visible necrotic areas, adipose tissue, and calcifications, preserving the tumor parenchyma. Use sterile ophthalmic scissors to cut the tissue into 1–3 mm³ pieces, avoiding the use of homogenizers or vigorous agitation to prevent mechanical damage to the primary CAFs.

[0043] (2) Enzyme digestion The sample fragments were transferred to a sterile centrifuge tube containing 10 mL of digestion solution (PBS + 1 mg / mL collagenase I) and digested at 37°C and 220 rpm for 1.5 hours with shaking. Immediately after digestion, 10 mL of complete DMEM medium (containing 10% FBS) was added to terminate the reaction. The resulting mixture was filtered through a 40 μm cell sieve, and the filtrate was collected in a 50 mL centrifuge tube. The tube was centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was obtained. Furthermore, quantitative analysis showed that when the digestion time was 1.5 hours, the cell adhesion rate was as high as 75%, and the purity was as high as 90%, significantly higher than the adhesion rate and purity at other digestion times. Figure 2 ).

[0044] (3) Cell culture Add 5 mL of complete DMEM medium to the cell pellet obtained above and gently resuspend the cells. Then, seed the cell suspension into T25 culture flasks and incubate at 37°C with 5% CO2. During the initial culture period (days 1-3), change the medium daily with fresh complete DMEM. From day 4 onwards, adjust the medium change frequency to once every 2-3 days. After 7 days of culture, typical spindle-shaped cells are observed adhering to the culture surface, and their morphology is mostly radial or whorled. Figure 4 When cell confluence reached 80% or higher, cells were digested and passaged using trypsin, designated as generation P1. Additionally, during culture, it was observed that cells without daily medium changes had low adhesion rates, and even those that adhered showed difficulty in observing regular spindle-shaped structures. Figure 3 ).

[0045] (4) Verification of primary CAF phenotype by α-SMA immunofluorescence staining Considering that α-SMA is a typical marker of CAFs derived from ccRCC, the above-mentioned P1 generation cells were subjected to α-SMA immunofluorescence staining to verify their CAF characteristics. First, after cell smears were fixed, permeabilized, and blocked, anti-α-SMA primary antibody diluted 1:200 was added, and the cells were incubated overnight at 4°C. Subsequently, unbound primary antibody was washed away, and FITC-labeled secondary antibody working solution was added, and the cells were incubated at room temperature in the dark for 1 hour. Finally, the cell nuclei were counterstained with DAPI, and the images were observed and acquired under a fluorescence microscope. Figure 5 The results show that primary CAFs expressing α-SMA and possessing good activity were successfully obtained using the method provided in this invention.

[0046] Comparative Example 1: Collagenase IV + hyaluronidase was used to treat ccRCC tissue instead of collagenase I alone. Comparative Example 1 followed essentially the same procedures as the Examples, except that the enzymes used in the enzymatic digestion step were collagenase IV and hyaluronidase, instead of collagenase I alone. The results showed that a large number of lipid droplets appeared in the suspension after digestion with collagenase IV and hyaluronidase. Figure 6 Furthermore, the cells have difficulty adhering to the wall and exhibit abnormal morphology. Figure 7 ).

[0047] Comparative Example 2: Comparison of the application effects of the method of the present invention and the tissue block method in ccRCC tissue and its adjacent normal tissue. Comparative Example 2 follows essentially the same operational steps as the Examples, except that it includes a dual control group: ccRCC tissue and its paired adjacent normal tissue are treated using both the method proposed in this invention and the tissue block method. The specific grouping process is as follows: (1) The method and tissue block method of the present invention are used to process fresh ccRCC tissues respectively; (2) The method and tissue block method of the present invention are used to process the normal kidney tissue adjacent to the paired ccRCC cancer.

[0048] The results showed that in ccRCC tissue, samples using the traditional tissue block method did not show typical CAF migration, or only a few irregularly shaped, arrested cells were observed in a very few samples, and these cells could not be passaged; while the method proposed in this invention successfully obtained high-purity, spindle-shaped, well-adherent primary cells in all 5 samples. In paired adjacent normal kidney tissue, the traditional tissue block method showed typical CAF migration, and the cells possessed proliferative capacity; while the method of this invention only isolated a small number of CAFs, which were few in number and difficult to proliferate. Figure 8 ).

[0049] The above results confirm from both positive and negative perspectives that the single collagenase I method proposed in this invention successfully extracts primary CAFs from ccRCC tissue, relying on the unique pathological microenvironment of ccRCC and exhibiting high specificity. Firstly, the soft tissue structure of ccRCC makes effective adhesion difficult using traditional tissue block methods, thus hindering the capture of primary CAFs. However, the method of this invention, through a targeted enzymatic digestion system and culture strategy, can efficiently dissociate and enrich activated CAFs within the tissue. Secondly, in paired adjacent normal tissues, due to their firm texture, traditional tissue block methods can effectively separate and obtain primary CAFs with adhesion and proliferation capabilities. In summary, this invention does not provide a general CAFs isolation technique, but rather a specific extraction scheme targeting the ccRCC tumor microenvironment and its activated CAFs.

[0050] Comparative Example 3: Extraction success rate of the method of the present invention in different ccRCC phases Comparative Example 3 follows essentially the same operational steps as the Example, except that the samples selected for Comparative Example 3 were early ccRCC tissue (n=34), locally late ccRCC tissue (n=16), and late ccRCC tissue (n=7). The results show that the extraction success rate of the method of the present invention in different ccRCC stages is higher than 85%, with a success rate of 94.1% (32 / 34) for early ccRCC tissue, 87.5% (14 / 16) for locally late ccRCC tissue, and 85.7% (6 / 7) for late ccRCC tissue. Figure 9 This result demonstrates that the method of the present invention is applicable to the extraction of primary CAFs from ccRCC tissues of all stages.

[0051] Comparative Example 4: Comparison of the application effects of the method of the present invention in other types of tumor tissues The operating steps of Comparative Example 4 were basically the same as those of the Examples, except that the samples selected for Comparative Example 4 were fresh renal pelvis cancer tissue (n=5). The results showed that in renal pelvis cancer tissue, three samples developed CAFs on days 3-5 of culture after using the method of this invention, but these were accompanied by extensive contamination with epithelial cells and immune cells, and the spindle cell morphology was atypical. Figure 10 Compared to ccRCC tissue, its α-SMA positivity rate is also relatively low. Figure 11 (This requires additional flow cytometry sorting or multiple passages to obtain relatively pure primary CAFs.) The above results further demonstrate that the method proposed in this invention is not a general CAF extraction scheme, but rather optimized for the unique tissue microenvironment of ccRCC, exhibiting significant tissue specificity and technical adaptability advantages.

[0052] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0053] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0054] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features of the invention herein.

Claims

1. A method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue, characterized in that, Includes the following steps: Fresh clear cell renal cell carcinoma tissue was treated at 0-4℃, preserving only the areas rich in a mixture of tumor cells and stroma; The region rich in tumor cells and stroma was slowly sheared into pieces with a volume of 1-3 mm. 3 fragments; Place the fragments in a collagenase I solution, and collect the cell precipitate after digestion is complete. The cell pellet was inoculated into the first culture medium. For the first 3 days after inoculation, the first culture medium was replaced with fresh medium daily, and then every 2 to 3 days thereafter.

2. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The fresh clear cell renal cell carcinoma tissue refers to clear cell renal cell carcinoma tissue that has been excised within 1 hour.

3. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The treatment of fresh clear cell renal cell carcinoma tissue at 0-4°C includes: Wash the clear cell renal carcinoma tissue with buffer solution; Obvious necrotic areas, adipose tissue, and major blood vessels were removed from the clear cell renal cell carcinoma tissue.

4. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The low-speed shearing is performed using scissors and / or a tissue cutter.

5. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The step of placing the fragments in a collagenase I solution and waiting for digestion to complete includes: placing the fragments in a collagenase I solution with a concentration of 0.8~1.2 mg / mL and digesting for 1~2 hours.

6. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The collection of cell pellets includes: Filter the digestive fluid through a sieve and collect the filtrate; After centrifuging the filtrate, the precipitate was collected to obtain cell precipitate.

7. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 6, characterized in that: The centrifugation rate is 1200~1500 rpm.

8. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The first culture medium is a complete culture medium containing FBS.

9. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The digestion process is complete and has one or more of the following characteristics: (1) The solution is visibly turbid, large pieces of tissue have basically disappeared, and the digestive juice is no longer viscous; (2) When initially inoculated into a culture dish, the microscope field of view mainly consists of scattered single cells or very small cell clusters, and after adhering to the wall, the cells are spindle-shaped and have good refractive properties.

10. The method for extracting primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissue according to claim 1, characterized in that: The method is applicable to the extraction of primary cancer-associated fibroblasts from clear cell renal cell carcinoma tissues at all stages.

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