Isolation, culture and purification of human gastric cancer primary fibroblast and application thereof

Abstract

The present application relates to a kind of human gastric cancer primary fibroblast separation culture and purification method and its application.The human gastric cancer primary fibroblast separation culture and purification method provided by the present application are simple, efficient.The separation culture method and the purification method provided by the present application can complete tissue dissociation in shorter time, and cooperate multiple immunomagnetic beads to remove miscellaneous cells, so that the gastric cancer primary fibroblast group of stable, relatively consistent morphology adherent growth is obtained, phenotype observation is facilitated, and the interference of miscellaneous cells to subsequent function experiment is reduced.Enriched cells are expanded after culture, and can be used for the subtype research of fibroblast, microtumor environment related function experiment.

Description

Technical Field

[0001] This invention relates to a method for isolating, culturing, and purifying primary human gastric cancer fibroblasts and its application, belonging to the field of biomedicine. Background Technology

[0002] Gastric carcinoma is one of the most common malignant tumors of the digestive system, characterized by high incidence, significant tumor heterogeneity, and poor overall prognosis. With in-depth research into the tumor microenvironment, the role of stromal cells in gastric cancer tissue in tumor progression and treatment response has gradually attracted attention. Among them, tumor-associated fibroblasts participate in tumorigenesis and development through extracellular matrix remodeling, cytokine secretion, and influencing the immune microenvironment. Therefore, obtaining well-derived, viable, and stably passaged human gastric cancer primary fibroblasts from clinical gastric cancer tissue is a crucial foundation for establishing in vitro models and for mechanistic studies and drug evaluation.

[0003] Existing methods for isolating and culturing primary fibroblasts from human gastric cancer tissue mainly include the following two technical routes.

[0004] 1. Tissue block adherent culture method: Fresh tissue is typically cut into small pieces and directly adhered to the bottom of a culture dish. Cells migrate and crawl out from the tissue block to obtain primary fibroblasts from adult gastric cancer. This culture method is relatively simple to operate, but common problems include: long time for cells to migrate out of the tissue block, low initial cell number, and long expansion cycle; at the same time, this culture method is also greatly affected by factors such as the proportion of tissue necrosis, residual hemorrhage and mucus, and microbial contamination, resulting in insufficient reproducibility and stability.

[0005] 2. Enzymatic Digestion: Current techniques often employ a single collagenase or trypsin, or a combination of both, to digest gastric cancer tissue to obtain a single-cell suspension for adherent culture. However, gastric cancer tissue is rich in matrix components and has dense collagen fibers. Under conventional enzymatic digestion conditions, insufficient dissociation and significant tissue residue are common problems. Furthermore, prolonged digestion to increase dissociation can lead to decreased cell viability, thus affecting subsequent culture quality and cell establishment efficiency.

[0006] Furthermore, cell suspensions obtained by enzymatic digestion are typically mixed cell populations, which may simultaneously contain fibroblasts, epithelial cells, immune cells, and vascular endothelial cells. Although primary fibroblasts can be initially enriched using methods such as differential adhesion and differential digestion, a certain proportion of non-fibroblast components may still remain in the resulting primary fibroblast population, leading to fluctuations in the purity of primary fibroblasts in the cell population, which in turn affects the stability and reproducibility of subsequent experimental results.

[0007] In the process of isolating and culturing primary fibroblasts derived from gastric cancer tissue, existing methods generally face the problem of difficulty in simultaneously achieving dissociation efficiency, cell viability, and cell population consistency, mainly in the following aspects.

[0008] (a) Insufficient tissue dissociation and limited cell recovery

[0009] Existing enzymatic digestion methods often employ a single collagenase or trypsin, or a simple combination of both. Because gastric cancer tissue has a dense matrix and abundant collagen fibers, conventional enzymatic digestion often results in tissue residue, numerous cell clumps, and a low proportion of single cells, leading to low cell recovery per unit tissue mass and significant fluctuations between different samples.

[0010] (ii) Increasing the degree of tissue dissociation often comes at the cost of sacrificing cell viability.

[0011] To reduce tissue residue and increase the proportion of single cells, common practices in existing technologies include extending digestion time, increasing enzyme concentration, or increasing pipetting intensity. These operations may lead to increased exposure of cells to the enzyme environment and mechanical stress, resulting in problems such as decreased cell viability, reduced adhesion efficiency, or poor early proliferation status.

[0012] It is evident that existing technologies lack digestion strategies that systematically optimize the extracellular matrix characteristics of gastric cancer tissue, making it difficult to simultaneously meet the requirements of high dissociation efficiency and protection of cell viability.

[0013] (iii) Removal of contaminating cells relies on culture screening, resulting in insufficient purity and stability.

[0014] Cell suspensions obtained from digestion of gastric cancer tissue are typically mixed populations of multiple cells, including epithelial cells, immune cells, and endothelial cells in addition to fibroblasts. Even with differential adhesion or differential digestion for initial enrichment, non-fibroblasts may still remain. Furthermore, the stability of the cell population composition is insufficient due to differences in sample source, adhesion rate, and culture conditions, which affects the reproducibility of subsequent experimental results.

[0015] (iv) The problem of long training process and limited repeatability

[0016] Tissue block adhesion methods often require a long time to obtain a sufficient number of cells; enzymatic digestion methods can also prolong the cell line establishment period if dissociation is insufficient. At the same time, tissues from different patients vary significantly in terms of matrix structure, degree of necrosis, and cellular composition. Existing culture procedures have limited adaptability to sample differences and narrow operating windows, resulting in large fluctuations in cell line establishment success rate, expansion rate, and cell state, which is not conducive to standardization and large-scale reproducibility.

[0017] In summary, existing technologies still have problems in the isolation and culture of primary fibroblasts derived from gastric cancer tissue, such as insufficient tissue dissociation efficiency (leading to low cell recovery), easily affected cell viability, inadequate removal of contaminating cells, unstable purity relying solely on adherence screening, long overall culture cycle, and large fluctuations in results between different samples. It is still difficult to simultaneously meet the requirements of isolation efficiency, cell viability, and cell population consistency.

[0018] Therefore, it is necessary to optimize the digestion system and cell enrichment process of gastric cancer tissue based on the extracellular matrix characteristics of gastric cancer tissue. Under the premise of ensuring cell viability and culture stability, this will improve the dissociation efficiency and cell recovery of gastric cancer tissue, reduce contamination by other cells, and shorten the culture cycle, thereby improving the reproducibility and consistency of primary fibroblast isolation and culture. Summary of the Invention

[0019] The problem to be solved by the invention

[0020] To address common problems in the isolation and culture of primary fibroblasts from gastric cancer tissue in existing technologies, this invention provides a method for isolating and culturing primary fibroblasts from gastric cancer tissue. This method improves tissue dissociation efficiency and cell recovery efficiency while ensuring cell viability and culture status, and reduces the uncertainty caused by contamination with other cells, thereby improving the stability and reproducibility of the process.

[0021] Specifically, the objectives of this invention include:

[0022] (1) Without significantly reducing cell viability, improve the degree of dissociation of gastric cancer tissue, reduce tissue residue, shorten enzyme digestion time, and increase the effective cell recovery per unit tissue mass;

[0023] (2) Reduce the mixing of non-fibroblast components such as epithelial cells, immune cells and endothelial cells, so that the resulting cell population is more uniform and the purification effect is more stable.

[0024] (3) Shorten the preparation cycle of primary fibroblasts, reduce fluctuations caused by sample differences, and improve the consistency and repeatability of the process.

[0025] The solution to the problem

[0026] In view of the problems existing in the prior art, the inventors conducted in-depth research and repeated experiments, employing a combination of techniques including "rapid dissociation with a mixture of type II collagenase and hyaluronidase" and "multiple immunomagnetic beads for negative selection and impurity removal." This resulted in a population of primary human gastric cancer fibroblasts with a clearly defined origin, high purity, good activity, and stable condition, providing a cellular basis for subsequent in vitro functional studies and related applications, thus completing this invention. The invention is described below:

[0027] The first aspect of this invention provides a method for isolating, culturing, and purifying primary human gastric cancer fibroblasts, characterized in that the method comprises the following steps:

[0028] S1) Pretreatment of human gastric cancer tissue samples;

[0029] S2) The pretreated gastric cancer tissue sample is digested using an enzyme mixture, wherein the enzyme mixture contains collagenase and hyaluronidase; in some preferred embodiments, the collagenase is type II collagenase;

[0030] S3) Cell recovery and initial culture;

[0031] S4) Adhesive culture and digestion of cells, and enrichment of primary fibroblasts using multiple immunomagnetic beads; in some preferred embodiments, the multiple immunomagnetic beads include human CD326 immunomagnetic beads, human CD14 immunomagnetic beads and human CD31 immunomagnetic beads.

[0032] In some implementation schemes, step S1) is performed as follows:

[0033] Human gastric cancer tissue samples were transferred to sterile centrifuge tubes and washed with PBS buffer containing penicillin-streptomycin-amphoteric acid B; in some preferred embodiments, the content of penicillin-streptomycin-amphoteric acid B in the PBS buffer was 2%;

[0034] After cleaning, the tissue was transferred to a sterile culture dish, and areas of adipose tissue and necrotic tissue were removed. The tissue was then minced into 1-2 mm pieces. 3 A tissue block of a certain size.

[0035] In some implementation schemes, step S2) is performed as follows:

[0036] The shredded tissue pieces were collected into sterile centrifuge tubes and digested with an enzyme mixture consisting of type II collagenase and hyaluronidase.

[0037] In some preferred embodiments, the working concentration of the type II collagenase is 0.5~2.5 mg / mL; the working concentration of the hyaluronidase is 100~200 U / mL; the volume of the enzyme mixture is 8~12 times the total volume of the tissue block; and the digestion conditions are 35~39℃ for 15~25 min.

[0038] In some preferred embodiments, the working concentration of the type II collagenase is 1.5 mg / mL; the working concentration of the hyaluronidase is 150 U / mL; the volume of the enzyme mixture is 10 times the total volume of the tissue block; and the digestion conditions are 37°C for 20 min.

[0039] In some implementation schemes, step S3) is performed as follows:

[0040] After digestion, the mixture was filtered through a sterile cell filter. The filtrate was collected, centrifuged, and the supernatant was discarded. The cell pellet was collected. The cell pellet was resuspended in complete culture medium and inoculated into culture flasks for culture. After 48 hours of culture, the complete culture medium was replaced, and thereafter the medium was changed every 2 days.

[0041] In some preferred embodiments, the filtration is first performed using a filter with a pore size of 100 μm, and then using a filter with a pore size of 70 μm; the centrifugation conditions are 250 × g for 5 min; and the complete culture medium is DMEM medium + 20% fetal bovine serum (FBS) + 1% penicillin-streptomycin-amphoteric B.

[0042] In some implementation schemes, step S4) is performed as follows:

[0043] When the confluence of adherent cells reaches more than 90%, the adherent cells are digested.

[0044] Human CD326 immunomagnetic beads, human CD14 immunomagnetic beads, and human CD31 immunomagnetic beads were added and incubated at 4°C for 10-20 min. The flow-through cell components that were not labeled by the above magnetic beads were collected under magnetic field sorting conditions to obtain purified primary fibroblast populations.

[0045] In some embodiments, the method of the first aspect of the present invention further includes the following steps:

[0046] S5) Passage the enriched cells.

[0047] In some embodiments, the collagenase described in the method of the first aspect of the present invention is prepared using serum-free DMEM medium, and the hyaluronidase is prepared using PBS buffer.

[0048] The second aspect of the present invention provides the application of human gastric cancer primary fibroblasts obtained by the isolation, culture and purification method of human gastric cancer primary fibroblasts described in the first aspect of the present invention in screening gastric cancer-related drugs.

[0049] In some implementations, the screening method includes the following steps:

[0050] (1) Human gastric cancer primary fibroblasts are cultured using the method described in the first aspect of the present invention;

[0051] (2) Select the drugs to be screened and perform serial dilutions of the drug concentrations;

[0052] (3) Add the graded diluted drug to the primary human gastric cancer fibroblasts cultured in (1);

[0053] (4) Test the cell activity.

[0054] The third aspect of this invention provides the application of human gastric cancer primary fibroblasts obtained by the isolation, culture and purification method of human gastric cancer primary fibroblasts described in the first aspect of this invention in fibroblast subtype studies or microtumor environment function experiments.

[0055] Effects of the invention

[0056] As can be seen from the technical solution of the present invention, compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0057] 1. Gastric cancer tissue matrix is ​​usually quite dense, and conventional digestion often results in a large amount of tissue residue and insufficient cell dissociation. If digestion is prolonged or pipetting is intensified, it can easily affect cell viability and subsequent adhesion and proliferation. In addition, the cultured adherent cell population often contains epithelial cells, immune cells, endothelial cells, etc. If enrichment is achieved solely through differential adhesion and differential digestion, the purity and stability of the cell population are easily affected by sample differences.

[0058] The present invention provides a method for isolating and culturing primary fibroblasts from gastric cancer tissue. By using mixed enzymes to complete tissue dissociation in a short time, and by using multiple immunomagnetic beads for negative selection to remove major contaminating cell components, a stable population of adherent and relatively uniform morphology of fibroblasts can be obtained in a short period of time, reducing the interference of contaminating cells on subsequent research and functional experiments.

[0059] 2. Compared with conventional processing methods in the prior art that only use a single collagenase digestion and mainly rely on differential adhesion and differential digestion for enrichment, the present invention adopts a combination of "rapid dissociation by mixed enzymes + multiple negative magnetic beads sorting for impurity removal and enrichment": the former solves the problems of "difficult tissue dissociation, low cell release, and long dissociation time", while the latter solves the problems of "many mixed cells and large fluctuations between samples". The combination of the two is more conducive to obtaining a stable primary fibroblast population for related research.

[0060] The method for isolating and culturing primary human gastric cancer fibroblasts provided by this invention utilizes type II collagenase, which primarily acts on collagen fiber structures, and hyaluronidase, which primarily acts on matrix components such as hyaluronic acid. The combined use of these two enzymes helps to "loosen" the tissue within a shorter digestion time, reducing tissue residue and cell clumps, thereby increasing cell release and reducing the risk of decreased activity due to over-digestion.

[0061] The method for isolating and culturing primary human gastric cancer fibroblasts provided by this invention employs a multiple immunomagnetic bead negative selection strategy to remove non-target cells. Specifically, magnetic microbeads targeting CD326, CD14, and CD31 antigens are used for negative selection to remove epithelial cells expressing human CD326, monocytes / macrophages expressing human CD14, and endothelial cells expressing human CD31. This retains the cell population not labeled by the aforementioned magnetic beads, reduces the proportion of non-fibroblast contamination, and improves the purity and compositional stability of the resulting cell population, which is beneficial for subsequent phenotypic identification and functional experiments.

[0062] 3. The method for isolating and culturing primary human gastric cancer fibroblasts provided by this invention is simple and fast. When processing samples, the processing should be completed within 3 hours after collection, and the samples should be repeatedly washed with buffer containing compound antibiotics to reduce the probability of contamination and reduce the impact of impurities such as blood and mucus on subsequent digestion and adherent culture, making the primary culture more stable.

[0063] The method employs two-stage filtration and mild conditions for recovery: sequential filtration with 100 μm and 70 μm filters removes incompletely dissociated tissue fragments and larger clumps, resulting in a more homogeneous cell suspension; combined with mild centrifugation conditions (250×g, 5 min), mechanical damage is reduced and recovery efficiency is improved. Attached Figure Description

[0064] Figure 1 Images A and B show representative images comparing the number of cells obtained during the tissue digestion and dissociation phase in the comparative and example cases, respectively.

[0065] Figure 2 Figures A and B show representative images of adherent cells cultured for 48 hours after digestion in the comparative and example samples, respectively, under an inverted microscope.

[0066] Figure 3 Figures A and B show representative images of adherent cells cultured to day 6 after digestion in the comparative and example studies, respectively, under an inverted microscope.

[0067] Figure 4 Figures A and B show representative images of changes in cell populations under a microscope in the comparative example without multiplex immunomagnetic bead enrichment and the example with multiplex immunomagnetic bead enrichment, respectively.

[0068] Figure 5 The results of immunofluorescence staining of α-SMA and CD140b in CD140b-positive primary human gastric cancer fibroblasts are shown.

[0069] Figure 6The results show the expression detection of α-SMA and CD140b proteins in the total population of primary fibroblasts (Total) without CD140b subpopulation sorting and in the primary fibroblast subpopulation (Sub) obtained after CD140b positive sorting. Detailed Implementation

[0070] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0071] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0072] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0073] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0074] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0075] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0076] Example

[0077] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0078] Example 1: Method for isolation, culture and purification of primary human gastric cancer fibroblasts

[0079] [Example]

[0080] (a) Tissue sample pretreatment

[0081] Fresh gastric cancer tissue blocks were immediately placed in tissue preservation / protection solution (Miltenyi, catalog number: 130-100-008) under aseptic conditions and transported to the laboratory. Subsequent processing was completed within 3 hours to minimize the risk of decreased cell viability. The collection and use of fresh gastric cancer tissue block samples involved in this invention were approved by the Medical Ethics Committee of Henan Cancer Hospital (Ethics Approval No.: 2024-KY-0068) and were conducted with the prior written informed consent of the subjects. Specific procedures are as follows:

[0082] Fresh gastric cancer tissue blocks were transferred to sterile centrifuge tubes and washed three times with an appropriate amount of PBS buffer containing 2% penicillin-streptomycin-amphoteric B (triple antibody, Gibco, catalog number: 15240062) to remove blood, mucus and other impurities.

[0083] The cleaned tissue blocks were then transferred to sterile culture dishes. Excess adipose tissue and necrotic tissue areas were removed using sterile tissue scissors and forceps, and the tissue blocks were then cut into pieces of approximately 1–2 mm. 3 Prepare small tissue blocks of the desired size. Keep the tissue blocks moist during the cutting process.

[0084] (ii) Tissue dissociation using enzyme mixture

[0085] The minced tissue pieces were collected into 15 mL sterile centrifuge tubes, and an enzyme mixture consisting of type II collagenase and hyaluronidase was added for digestion and dissociation. The mixed enzyme solution contained:

[0086] Type II collagenase (Sigma-Aldrich, catalog number: C6885) has a working concentration of 1.5 mg / mL; Type II collagenase is prepared using serum-free DMEM medium;

[0087] Hyaluronidase (Sigma-Aldrich, catalog number: H3506) has a working concentration of 150 U / mL; hyaluronidase is prepared using PBS buffer.

[0088] Preparation of mixed enzyme solution: Take appropriate volumes of the two enzymes and mix them thoroughly to prepare a mixed enzyme solution.

[0089] The volume of the mixed enzyme solution is approximately 10 times the total volume of the tissue block.

[0090] The design principle of the mixed enzymatic hydrolysis is as follows: type II collagenase is used to degrade the collagen fiber structure in the tissue block, and hyaluronidase is used to degrade the hyaluronic acid component in the extracellular matrix. The combined action of the two can synergistically destroy the dense matrix structure in gastric cancer tissue, improve cell release efficiency and shorten digestion time.

[0091] The mixture was digested at 37°C for 20 minutes, gently mixed every 5 minutes during digestion to promote tissue dissociation. When the residual tissue volume was significantly reduced and the solution became turbid, complete culture medium was added to terminate the digestion reaction. The complete culture medium consisted of DMEM + 20% fetal bovine serum (FBS) + 1% penicillin-streptomycin-amphoteric B (triple antibodies).

[0092] DMEM: Gibco, Product No.: 11965092;

[0093] FBS: Gibco, Product No.: A5669701;

[0094] Penicillin-Streptomycin-Amphotericin B (Antibiotic-Antimycotic, Triple Antibody), Gibco, Catalog No.: 15240062.

[0095] (III) Cell recovery and preliminary culture

[0096] The digested tissue-cell mixture was filtered through a sterile cell filter (first through a 100 μm filter (Falcon, catalog number: 352360), then through a 70 μm filter (Falcon, catalog number: 352350), for a total of two filtrations) to remove any remaining tissue residue that was not completely dissociated.

[0097] After collecting the filtrate, centrifuge at 250×g for 5 min, discard the supernatant, and collect the cell pellet.

[0098] The cell pellet was resuspended in 4 mL of complete culture medium and seeded into T25 culture flasks and cultured at 37°C and 5% CO2.

[0099] After 48 hours of culture, replace with fresh complete culture medium to remove non-adherent cells and promote adherent cell proliferation. Minimize movement of the flask during culture. Thereafter, perform a simple medium change every 2 days, and proceed to the next step when adherent cell confluence reaches approximately 90%.

[0100] (iv) Enrichment of primary fibroblasts based on adherent culture and multiple immunomagnetic beads

[0101] Magnetic beads negative selection enriches fibroblasts

[0102] When the adherent cells reached a confluence of approximately 90%, the adherent cells were digested, recovered, and counted. Subsequently, a multiple immunomagnetic bead negative selection strategy was used to remove non-fibroblast components, thereby enriching and obtaining primary fibroblasts.

[0103] The method for isolating and culturing primary human gastric cancer fibroblasts provided by this invention employs a multiple immunomagnetic bead negative selection strategy to remove non-target cells / non-fibroblast components in order to enrich primary fibroblasts.

[0104] Specifically, the multiplex immunomagnetic bead negative selection strategy involves using magnetic microbeads targeting CD326, CD14, and CD31 antigens to label epithelial cells expressing human CD326, monocytes / macrophages expressing human CD14, and endothelial cells expressing human CD31, respectively, thereby achieving negative selection of target cells. This operation reduces the proportion of non-fibroblast components while retaining the unlabeled cell population, thus improving the purity and homogeneity of the resulting cell population, which is beneficial for subsequent phenotypic analysis and functional studies.

[0105] Human CD326 MicroBeads (Miltenyi, Product No.: 130-061-101);

[0106] Human CD14 MicroBead (Miltenyi, Product No.: 130-050-201);

[0107] Human CD31 MicroBead (Miltenyi, Product No.: 130-091-935).

[0108] Adherent cells were digested, recovered, and counted, at a rate of 1 × 10⁻⁶ cells per cell. 7 Calculate the required volume of immunomagnetic beads by adding 20 μL of immunomagnetic beads to each cell, or add the corresponding volume of immunomagnetic beads to the cell suspension according to the ratio recommended in the product instructions. Gently mix and incubate at 4°C for about 15 min. Then place the mixture in a magnetic field sorting device for separation, discard the cells bound to the magnetic beads, and collect the flow-through cell fraction that was not bound to the magnetic beads, thereby obtaining enriched primary fibroblasts with high purity.

[0109] The specific procedure for digesting adherent cells is as follows: When the adherent cells reach approximately 90% confluence, discard the old culture medium and digest the adherent cells for approximately 3 minutes at 37°C using 0.25% trypsin-EDTA digestion solution (Gibco, catalog number: 25200056) to induce detachment. Then, add complete culture medium to terminate the digestion reaction. Collect the cell suspension, centrifuge to obtain the cell pellet, resuspend, and count the cells for later use.

[0110] (v) Subculture and application

[0111] The enriched cells were further cultured and expanded for subsequent fibroblast phenotype identification, subtype studies, and tumor microenvironment-related functional experiments. The specific procedures are as follows:

[0112] Cells enriched through negative selection with magnetic beads were seeded into complete culture medium (DMEM + 20% fetal bovine serum (FBS) + 1% penicillin-streptomycin-amphoteric B (triple antibody)) and cultured for further expansion at 37°C and 5% CO2. The resulting primary fibroblasts were used for subsequent phenotypic identification and related biological function analysis.

[0113] [Comparative Example]

[0114] To evaluate the effectiveness of the "rapid dissociation with mixed enzymes" combined with "multiple negative magnetic bead sorting for impurity removal and enrichment" technique used in the method provided by this invention, a comparative example was also provided. The difference between the examples and the comparative example lies only in some steps and parameter conditions, as detailed below:

[0115] (1) Fresh gastric cancer tissue blocks were dissociated by digestion with type II collagenase only, without the addition of hyaluronidase; the working concentration of type II collagenase was 1 mg / mL, digestion was carried out at 37℃ for 40 min, and the mixture was gently mixed once every 5 min during the process; the other filtration and centrifugation conditions were the same.

[0116] (2) Negative selection of human CD326 / human CD14 / human CD31 immunomagnetic beads was not performed; instead, differential adhesion combined with differential digestion was used to culture and enrich fibroblasts. The specific operation was as follows: after the primary adherent cells were cultured to a confluence of about 90%, they were passaged. The traditional differential adhesion combined with differential digestion method was used, and the cell composition was optimized by controlling the pancreatic enzyme digestion time and the cell adhesion time to improve the purity of primary human gastric cancer-related fibroblasts. The other culture conditions were the same.

[0117] The operation of differential wall adhesion and differential digestion methods is briefly explained below:

[0118] Differential adhesion method: The single-cell suspension obtained from digestion is inoculated into a culture flask and cultured for a short time at 37°C and 5% CO2 to enrich the cells by utilizing their rapid adhesion.

[0119] Differential digestion method: When the adherent cells grow to about 80%~90% confluence, use 0.25% trypsin for short-term digestion to cause some of the weaker adherent cells to detach preferentially. After collecting the cell suspension, stop digestion, then continue to digest the remaining cells and collect them. The resulting cells are then cultured.

[0120] During the culture process, the purity of fibroblasts was assessed by morphological observation, and cells with a high proportion of fibroblasts and stable expansion were selected for subsequent passage culture.

[0121] [Experimental Results]

[0122] 1. Experimental results in the cell recovery and preliminary culture steps

[0123] (1) Observe the cells under a microscope

[0124] Comparative images of cell suspensions obtained after digestion and cessation of enzymatic digestion, under the same tissue volume and observation conditions. (Example:) Figure 1 As shown, the number of cells obtained in the examples is greater than that in the comparative examples.

[0125] (2) Comparison of cell viability and live cell yield

[0126] Experimental groups: In the examples and comparative examples, tissue blocks weighing 700mg, 750mg, and 900mg were selected as experimental samples, respectively; and they were named experimental group 1, control group 1; experimental group 2, control group 2; experimental group 3, control group 3.

[0127] Cell viability (%) and viable cell yield (cells / g) were measured in experimental groups 1-3 and control groups 1-3. Cell viability (%) was determined using trypan blue staining exclusion method; viable cell yield (cells / g) was calculated as "viable cell count / tissue mass (g)". Specific results are shown in Table 1.

[0128] Table 1 Comparison of live cell yield and cell viability after tissue dissociation between the examples and the comparative examples.

[0129]

[0130] (3) Comparison of cell morphology at different culture times

[0131] Cells were seeded and cultured for 48 h. The morphology of adherent cells in the examples and comparative examples was photographed under the same culture conditions and microscope parameters.

[0132] like Figure 2 As shown, the number of adherent cells in the embodiment is higher, and more spindle-shaped, extended fibroblast-like cell morphologies are visible, while the number of adherent cells in the comparative example is relatively smaller.

[0133] Then, on the fourth day, a simple medium change was performed, minimizing any significant shaking of the bottle. The old culture medium was discarded, and fresh complete culture medium was added for continued culturing.

[0134] On day six, a simple medium change was performed again. Cell density and morphology were recorded under the same observation conditions on day six, for both the example and comparative cases.

[0135] like Figure 3 As shown, compared to the comparative example, a greater number of adherent cells, including spindle-shaped and fibroblast-like adherent cells, are visible in the embodiments.

[0136] 2. Experimental results in the step of combining immunomagnetic beads with enrichment of primary fibroblasts

[0137] The primary fibroblasts with high purity were cultured for 48 h and photographed to observe the microscopic images of the changes in the cell population after enrichment treatment.

[0138] like Figure 4 As shown, after enrichment treatment, compared with the control group that did not undergo multiple immunomagnetic bead enrichment treatment, the cell population uniformity of the examples that underwent multiple immunomagnetic bead enrichment treatment was significantly improved, the proportion of spindle-shaped and extended fibroblast-like cells was significantly increased, and the mixed cell components were significantly reduced.

[0139] Example 2: Magnetic bead sorting and phenotypic verification of CD140b-positive subsets in primary fibroblasts.

[0140] Information on experimental materials, consumables, equipment, and culture media is as follows:

[0141] Cell source: The primary fibroblasts prepared in the [Example] above were in the logarithmic growth phase and showed good adherence and growth.

[0142] Buffer solution (freshly prepared, pre-cooled, use at 4°C):

[0143] Antibody staining buffer (Buffer1): PBS + BSA, with a final BSA concentration of 0.1%-0.5% (preferably 0.2%).

[0144] Magnetic bead sorting buffer (Buffer2): PBS + BSA + EDTA, with a final BSA concentration of 0.2%-1.0% (preferably 0.5%) and EDTA concentration of 1-5 mM (preferably 2 mM).

[0145] Antibody: Anti-CD140b-PE antibody (i.e., antibody that binds to CD140b and is labeled with PE, BioLegend, 323606).

[0146] Magnetic beads: Anti-PE MicroBeads (i.e., magnetic microbeads bonded to PE, Miltenyi, 130-048-801).

[0147] Consumables and equipment: LS column, magnetic sorting rack, 50 mL centrifuge tubes, 15 mL centrifuge tubes, T25 culture flasks, 4℃ centrifuge.

[0148] Culture medium: Complete culture medium, specifically DMEM + 20% fetal bovine serum (FBS) + 1% penicillin-streptomycin-amphoteric B (triple antibody).

[0149] Primary fibroblasts with good growth status and approximately 90% confluence obtained in [Example] were washed with PBS buffer and digested with 0.25% trypsin-EDTA digestion solution for 3 min. Complete culture medium was added to terminate the digestion, and the cells were centrifuged at 250×g for 5 min to collect the cell pellet. The pellet was resuspended in PBS buffer and counted.

[0150] After centrifuging the cells again and discarding the supernatant, each 1×10⁶ cells were centrifuged. 7 Each cell was resuspended in 90 μL of antibody staining buffer (PBS buffer containing 0.2% BSA), and 10 μL of anti-CD140b-PE antibody was added. The cells were incubated at 4°C in the dark for 10 min. Then, the cells were washed with 2 mL of magnetic bead sorting buffer (PBS buffer containing 0.5% BSA and 2 mM EDTA), centrifuged at 250×g for 10 min, and the supernatant was discarded.

[0151] Each 1×10 7 Each cell was resuspended in 80 μL of magnetic bead sorting buffer, and 20 μL of Anti-PE MicroBeads was added. The cells were incubated at 4°C in the dark for 15 min. After washing with 2 mL of magnetic bead sorting buffer, the cells were centrifuged at 250×g for 10 min, the supernatant was discarded, and the cells were resuspended in 500 μL of magnetic bead sorting buffer to obtain a cell suspension.

[0152] Place the LS column on a magnetic sorting rack and pre-wash with 3 mL of magnetic bead sorting buffer. Slowly add the cell suspension to the LS column and wash the column three times with 3 mL of magnetic bead sorting buffer to improve purity. Then remove the LS column from the rack and place it on a new centrifuge tube. Add 3 mL of magnetic bead sorting buffer and elute by pushing with the plunger. Collect the eluent. Centrifuge the eluent at 250×g for 5 min, discard the supernatant, resuspend in complete culture medium, and inoculate to obtain a CD140b-positive primary fibroblast subset.

[0153] When the cell density approaches 80%, collect the cell pellet. Place three sterile round glass slides into each well of a 24-well plate, add 0.5 mL of complete culture medium to each well, and seed cells at a density of 2 × 10⁶ cells / well. 5 Cells were cultured overnight at 37°C in a 5% CO2 incubator to allow them to adhere and grow.

[0154] Discard the culture medium and wash once with PBS buffer; add 300 μL of 4% paraformaldehyde and fix at room temperature for 15 min; wash three times with PBS buffer, 5 min each time. Add 300 μL of PBS mixture containing 0.1% Triton X-100 and 5% BSA and incubate at room temperature for 1 h; wash gently three times with PBS buffer.

[0155] The primary antibody was diluted using universal antibody diluent (Synthetamide, WB500D). 50 μL of the diluted primary antibody was added to a glass slide and incubated overnight in a humidified chamber at 4°C. The following day, the slide was washed three times with PBS buffer, 5 min each time.

[0156] The primary antibodies used were: α-SMA (1:150, CST, 19245S) and CD140b (1:200, Abcam, ab69506).

[0157] The fluorescent secondary antibodies (Abcam, ab150077, ab150115) were diluted with PBS buffer at a ratio of 1:1000 and incubated at room temperature in the dark for 1 h; they were then washed 3 times with PBS buffer for 5 min each time.

[0158] Place one drop of anti-fluorescence decay mounting medium containing DAPI (Solepro, S2110) in the center of the slide, then invert the slide with the cell-containing side facing up onto the slide to avoid air bubbles, and complete the mounting.

[0159] Imaging observation was performed using a confocal laser scanning microscope.

[0160] Experimental results: Figure 5 The immunofluorescence staining results of α-SMA and CD140b in CD140b-positive primary human gastric cancer fibroblasts are shown. The results indicate that the isolated CD140b-positive primary fibroblasts simultaneously exhibit positive expression signals for both CD140b and α-SMA, with a clear signal distribution. This demonstrates that the isolation, culture, and purification method provided by this invention can stably obtain primary fibroblast subpopulations expressing specific phenotypes of both CD140b and α-SMA, providing a reliable cell source for subsequent phenotypic analysis and functional studies of different fibroblast subtypes.

[0161] Example 3: Western blot detection of CD140b-positive fibroblast subset marker protein expression

[0162] Primary fibroblasts in logarithmic growth phase that were not sorted for CD140b subsets (Total) and primary fibroblast subsets obtained through CD140b positive sorting (Sub) were collected as primary fibroblast samples. After discarding the old culture medium, the cells were washed three times with PBS buffer, and an appropriate amount of protein lysis buffer was added. The cells were lysed on ice for 15 min. The cells were scraped off with a cell scraper and transferred to 1.5 mL EP tubes. The tubes were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected as the total protein sample.

[0163] Protein quantification was performed using the BCA method. Based on the calculated protein concentration, 5× SDS-PAGE protein loading buffer was added to achieve a final concentration of 1×. After thorough mixing, the mixture was denatured in a 95℃ metal bath for 10 min. The mixture was then briefly centrifuged for 10 s, cooled, and stored at -20℃ for later use.

[0164] Prepare an SDS-PAGE gel. After the gel has completely solidified and no leakage has been detected, add 10 μL of protein sample and 5 μL of protein molecular weight marker to the sample well. Electrophoresis is first performed at 80 V until the sample enters the separating gel, at which point bromophenol blue will form a straight line. Then, the voltage is increased to 120 V and electrophoresis is continued for about 1 hour until bromophenol blue is close to the bottom of the gel.

[0165] After electrophoresis, wet transfer was performed. The gel and PVDF membrane were assembled in the transfer apparatus, air bubbles were removed, and rapid transfer buffer (New Semiconductor, WB4600) was added. Transfer was performed at a constant current of 400 mA for 30 min.

[0166] After transfer, the membrane was placed in 5% skim milk (prepared with TBST buffer) and blocked at room temperature for 1-2 h. It was then washed three times with TBST buffer for 10 min each time. Primary antibody solution was then added and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST buffer for 10 min each time. The primary antibodies used were: CD140b (1:1000, Abcam, ab69506), α-SMA (1:1000, CST, 19245S), and GAPDH (1:1000, CST, 97166S). The corresponding HRP-labeled secondary antibodies (1:5000, CST, 7074S-Rabbit, 7076S-Mouse) were added and incubated at room temperature for 1 h. After secondary antibody incubation, the membrane was washed three times with TBST buffer for 10 min each time. Mix HRP chemiluminescence reagent solution A and solution B in a 1:1 ratio, and drop them evenly onto the membrane surface. Use a chemiluminescence imaging system for exposure imaging, and adjust the exposure time according to the strip intensity.

[0167] The experimental results showed that, compared with the total primary fibroblast population (Total) that was not sorted by CD140b subsets, the expression levels of α-SMA and CD140b proteins were significantly increased in the primary fibroblast subset (Sub) obtained by CD140b positive sorting.

[0168] The results indicate that the CD140b-positive sorting method used in this invention can effectively enrich primary fibroblast subpopulations with high CD140b expression. The sorted primary fibroblast subpopulations show significant differences from the unsorted cell population in terms of α-SMA and CD140b protein expression levels.

[0169] Therefore, the method for isolating, culturing, and purifying primary human gastric cancer fibroblasts provided by this invention can yield fibroblast populations with distinct phenotypic characteristics in terms of molecular expression. Furthermore, the cell populations obtained using the method described in this invention can be used for molecular characterization studies of fibroblast subtypes, as well as for constructing and analyzing experimental systems related to the gastric cancer microtumor environment, thereby providing a stable and reproducible experimental cell model for tumor matrix function research and related mechanism exploration.

[0170] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0171] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for isolating, culturing, and purifying primary human gastric cancer fibroblasts, characterized in that, The method includes the following steps: S1) Pretreatment of human gastric cancer tissue samples; S2) Digesting the pretreated gastric cancer tissue sample using an enzyme mixture, wherein the enzyme mixture contains collagenase and hyaluronidase; preferably, the collagenase is type II collagenase; S3) Cell recovery and initial culture; S4) The cells are cultured and digested to adhere to the culture vessel, and primary fibroblasts are enriched using multiple immunomagnetic beads; preferably, the multiple immunomagnetic beads include human CD326 immunomagnetic beads, human CD14 immunomagnetic beads and human CD31 immunomagnetic beads.

2. The method for isolating, culturing, and purifying primary human gastric cancer fibroblasts according to claim 1, characterized in that, The operation method for step S1) is as follows: Human gastric cancer tissue samples were transferred to sterile centrifuge tubes and washed with PBS buffer containing penicillin-streptomycin-amphoteric acid B; preferably, the content of penicillin-streptomycin-amphoteric acid B in the PBS buffer was 2%; After cleaning, the tissue was transferred to a sterile culture dish, and areas of adipose tissue and necrotic tissue were removed. The tissue was then minced into 1-2 mm pieces. 3 A tissue block of a certain size.

3. The method for isolating, culturing, and purifying primary human gastric cancer fibroblasts according to claim 1, characterized in that, The operation method for step S2) is as follows: The shredded tissue pieces were collected into sterile centrifuge tubes and digested with an enzyme mixture consisting of type II collagenase and hyaluronidase. Preferably, the working concentration of the type II collagenase is 0.5~2.5 mg / mL; the working concentration of the hyaluronidase is 100~200 U / mL; the volume of the enzyme mixture is 8~12 times the total volume of the tissue block; and the digestion conditions are 35~39℃ for 15~25 min. More preferably, the working concentration of the type II collagenase is 1.5 mg / mL; the working concentration of the hyaluronidase is 150 U / mL; the volume of the enzyme mixture is 10 times the total volume of the tissue block; and the digestion conditions are 37°C for 20 min.

4. The method for isolating, culturing, and purifying primary human gastric cancer fibroblasts according to claim 1, characterized in that, The operation method for step S3) is as follows: After digestion, the mixture was filtered through a sterile cell filter. The filtrate was collected, centrifuged, and the supernatant was discarded. The cell pellet was collected. The cell pellet was resuspended in complete culture medium and inoculated into culture flasks for culture. After 48 hours of culture, the complete culture medium was replaced, and thereafter the medium was changed every 2 days. Preferably, the filtration is first performed using a filter with a pore size of 100 μm, and then using a filter with a pore size of 70 μm; the centrifugation conditions are 250×g for 5 min; the complete culture medium is DMEM medium + 20% fetal bovine serum (FBS) + 1% penicillin-streptomycin-amphoteric B.

5. The method for isolating, culturing, and purifying primary human gastric cancer fibroblasts according to claim 1, characterized in that, The operation method for step S4) is as follows: When the confluence of adherent cells reaches more than 90%, the adherent cells are digested. Human CD326 immunomagnetic beads, human CD14 immunomagnetic beads, and human CD31 immunomagnetic beads were added and incubated at 4°C for 10-20 min. The flow-through cell components that were not labeled by the above magnetic beads were collected under magnetic field sorting conditions to obtain purified primary fibroblast populations.

6. The method for isolating, culturing, and purifying primary human gastric cancer fibroblasts according to claim 1, characterized in that, The method further includes the following steps: S5) Passage the enriched cells.

7. The method for isolating, culturing, and purifying primary human gastric cancer fibroblasts according to any one of claims 1 to 6, characterized in that, The collagenase was prepared using serum-free DMEM medium, and the hyaluronidase was prepared using PBS buffer.

8. The use of human gastric cancer primary fibroblasts obtained by the isolation, culture and purification method of human gastric cancer primary fibroblasts according to any one of claims 1 to 7 in screening gastric cancer-related drugs.

9. The application according to claim 8, characterized in that, The screening method includes the following steps: (1) Culture human gastric cancer primary fibroblasts using the method described in any one of claims 1 to 7; (2) Select the drugs to be screened and perform serial dilutions of the drug concentrations; (3) Add the graded diluted drug to the primary human gastric cancer fibroblasts cultured in (1); (4) Test the cell activity.

10. The application of human gastric cancer primary fibroblasts obtained by the isolation, culture and purification method of any one of claims 1 to 7 in fibroblast subtype studies or microtumor environment function experiments.

Images