Method for detecting degradation capability of human macrophages on liver collagen in vitro

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a method for detecting the degradation capacity of human macrophages to liver collagen in vitro. According to the scheme provided by the invention, the content of CTX I, CTX III, C3M or C1M of the liver type I collagen degradation fragment in the macrophage supernatant is detected through Elisa, the degradation capability of the macrophage on the liver collagen can be accurately reflected, and the macrophage with the liver collagen degradation capability is qualified when the CTX I of the macrophage culture supernatant is greater than 100ng / mL.

Description

[0001] Priority Application This application claims priority to Chinese Invention Patent Application

CN202411938378.6

[0002] The present application belongs to the technical field of biomedical cell biology, and specifically relates to a method for detecting the ability of human macrophages to degrade liver collagen in vitro. BACKGROUND

[0003] Liver fibrosis is a protective mechanism activated by hepatocyte damage, which can be alleviated after the cause is eliminated. However, persistent damage can lead to liver fibrosis evolving into cirrhosis, thereby promoting the development of hepatocellular carcinoma. Macrophages play an important role in the pathological process of liver fibrosis. Cell therapy with macrophages as the main candidate cells is an emerging means for treating liver fibrosis. Although the prospects and feasibility of treating liver fibrosis with macrophages have been reported, existing cell therapies generally have large batch and individual differences.

[0004] The literature “The Role of Macrophages in Liver Fibrosis” discloses that macrophages play an important role in the degradation process of liver fibrosis. Macrophages highly express MMPs (such as MMP-1, MMP-13) that degrade the matrix, promote the degradation of extracellular matrix or collagen, and degrade liver fibrosis. However, this literature only discloses the role of macrophages in the degradation of liver fibrosis, does not give any enlightenment on how to evaluate the reliable and effective collagen degradation ability of macrophages, and cannot guide the quality stability of macrophages between different batches. In summary, it is necessary to develop a method that is efficient, simple, and can evaluate the reliable collagen degradation ability of macrophages in vitro, which has important scientific significance. SUMMARY

[0005] Therefore, the present application aims to provide a method for detecting the ability of human macrophages to degrade liver collagen in vitro, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The present application specifically adopts the following technical solutions.

[0006] In one aspect, the present application provides a method for detecting the ability of human macrophages to degrade liver collagen in vitro and screening qualified macrophages based thereon, thereby providing a cell quality control index for subsequent experiments or clinical applications.

[0007] A method for detecting the ability of macrophages to degrade liver collagen in vitro, comprising the following steps: S01: isolating mononuclear cells from peripheral blood using density gradient method; S02: Isolating CD14+ monocytes by magnetic bead sorting method (technology); S03: Culturing the isolated CD14+ monocytes in a culture medium containing macrophage colony-stimulating factor to obtain a growth-differentiated macrophage culture solution; Those skilled in the art can understand that the culture medium for culturing macrophages used in the present application is a conventional culture medium, which is commercially available; wherein an appropriate amount of macrophage colony-stimulating factor is added to promote the growth and differentiation of macrophages; S04: Collecting a cirrhotic post-transplant liver to prepare a decellularized liver collagen solution; and then co-culturing the decellularized liver collagen solution with the macrophage culture solution obtained in S03; S05: Collecting the co-culture supernatant and detecting the content level of CTX I, CTX III, C3M or C1M in the culture supernatant to evaluate the ability of macrophages to degrade liver collagen; S06: Determining that macrophages with a CTX I content greater than 100 ng / mL have the ability to degrade liver collagen.

[0008] Further, the cell concentration in S03 is 1-3 x 10 6 6 / mL; and the culture condition is 37°C, 5% CO2 for 7 days.

[0009] Further, in S04, the decellularized stiffer liver scaffold is freeze-dried and ground to obtain a decellularized stiffer liver tissue powder; and then the decellularized stiffer liver tissue powder is reacted with pepsin dissolved in an HCl solution at room temperature to obtain a decellularized liver collagen solution.

[0010] Further, the mass ratio of the pepsin to the decellularized stiffer liver tissue powder is 1:10.

[0011] Further, in S04, the decellularized liver collagen solution and the macrophages are co-cultured, and the macrophage culture solution adjusts the protein concentration in the decellularized liver collagen solution to 0.3 mg / mL.

[0012] Further, in S04, the decellularized liver collagen solution and the macrophages are co-cultured for 3 days.

[0013] Further, in S04, the culture medium for co-culturing the decellularized liver collagen solution and the macrophages contains TexMACSTM GMP medium, 0.8 mg / mL collagen, and 100 ng / mL M-CSF.

[0014] Further, in S05, the co-culture supernatant is collected, and the content of CTX I in the co-culture supernatant (in the supernatant of the macrophages after co-culture) is detected by ELISA.

[0015] The macrophages with the ability of degrading liver collagen screened by the method for detecting the ability of macrophages degrading liver collagen in vitro have a CTX I content in the supernatant of the co-culture of the macrophages and the liver collagen greater than 100 ng / mL.

[0016] The macrophages with the ability of degrading liver collagen are applied to the preparation of a drug or reagent for liver cirrhosis.

[0017] Beneficial technical effects: The method has guiding significance for evaluating the quality stability of batches of macrophages, and can alleviate the problems of large batch and individual differences in the treatment of liver fibrosis by using macrophages. According to the scheme provided in the application, the content of CTX I, CTX III, C3M or C1M of the liver type collagen degradation fragment in the supernatant of the macrophages is detected by Elisa, which can accurately reflect the degradation ability of the macrophages to the liver collagen, wherein the CTX I in the supernatant of the macrophages is greater than 100 ng / mL, which is the qualified macrophages with the ability of degrading liver collagen. The screened qualified macrophages are reinfused into individuals, which can effectively treat liver fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. In all the drawings, similar elements or parts are generally identified by similar reference signs. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0019] Figure 1 Preparation process of the decellularized liver collagen solution in one of the embodiments of the present application; Figure 2 ELISA detection of the level of CTX I in the supernatant of the induced macrophages in one of the embodiments of the present application; Figure 3 Liver fibrosis effect after the screened macrophages are injected into animals in one of the embodiments of the present application. DETAILED DESCRIPTION

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0022] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0023] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0024] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0025] Example 1 1.1 PBMC separation (D0).

[0026] (1) Take 160 mL of peripheral blood, and take 0.5 mL for five-part differential testing (containing 28 mL of anticoagulant). Set the centrifugation force to 800 g, centrifuge for 8 min, separate the plasma, send one tube for four infectious disease tests, and retain two tubes, 2 mL / tube.

[0027] (2) Add 20 mL of lymphocyte separation solution to a 50 mL centrifuge tube. Dilute the peripheral blood with an equal volume of physiological saline and slowly add it to the top of the Ficoll layer (up to 50 mL), taking care to keep the interface clear. Set the centrifugation force to 400 g at room temperature and centrifuge for 30 min. Set the acceleration and deceleration rate to 1.

[0028] (3) After centrifugation, collect the white membrane layer into a 50 mL centrifuge tube, add physiological saline to the centrifuge tube to 50 mL, set the centrifugation force to 800 g, centrifuge for 8 min, and remove the supernatant. Then resuspend the cells in 40 mL of physiological saline, and take 0.5 mL for five-part differential and flow cytometry detection. Centrifuge the remaining suspension at 800 g for 5 min and remove the supernatant.

[0029] Peripheral blood five-part differential test results: WBC: 5.83×10 6 Cells / mL; Total: 93.28 × 10⁻⁶ 7 PBMC five-part differential diagnosis results: WBC: 8.08 × 10⁻⁶ cells. 6 Cells / mL, Total: 32.32 × 10 7 Cell count: 3.49 × 10⁻⁶. AOPI count result: 3.49 × 10⁻⁶. 6 Cells / mL, viable cell percentage: 98.63%, total cell count: 13.96 × 10⁻⁶ 7 Each cell.

[0030] 1.2 CD14+ cell sorting (D0).

[0031] (1) Take the cell pellet after centrifugation in step (3) of 1.1, and resuspend it in 2600 μL of buffer according to the total number of WBCs (according to a cell concentration of 10). 7 (Resuspended in 80μL buffer volume). The buffer was prepared as MACS BSA Stock Solution and autoMACS™ Rising Solution at a ratio of 1:20, with 0.5% albumin added.

[0032] (2) Mix 2600 μL of cell suspension with 650 μL of CD14 magnetic beads (20 μL / 10) 7 Mix (10 cells) and incubate at 4°C in the dark for 15 minutes to obtain the incubation solution.

[0033] (3) Based on the total number of cells, add 40 mL of buffer (per 10 cells) to the incubation medium. 7 Add 1-2 mL of buffer to each cell, set the centrifugation force to 800 g, and centrifuge for 5 min.

[0034] (4) After centrifugation, discard the supernatant and resuspend the cells in 4 mL of buffer until the cell density is no higher than 2 × 10⁻⁶. 8 per mL.

[0035] (5) Assemble the sorting column, sorter and sorting rack. Add 3 mL of buffer to the LS sorting column for rinsing, and then add the cell suspension from step (4) to the LS sorting column (the filtrate is CD14-cells).

[0036] (6) Add 3 mL of buffer three times to wash away the unadsorbed cells in the sorting column.

[0037] (7) Remove the sorting column and place it in the collection tube. Wash the sorting column with 5 mL of buffer to obtain washing solution (CD14+ cells). Take 0.2 mL for five-part differential cell counting and flow cytometry.

[0038] Five-category classification result: 13.35 × 10⁻⁶ 6 Cells / mL; Total: 66.75 × 10⁻⁶ 6 Each cell.

[0039] AOPI count result: 7.25 × 10 6 Cells / mL; Viable cell percentage: 98.46%; Total cell count: 36.25 × 10⁻⁶ 7 Each cell.

[0040] 1.3 Macrophage culture.

[0041] (1) Take the remaining cells separated from 1.1 and resuspend the cells in a medium containing M-CSF (cell concentration approximately 2 × 10⁻⁶). 6 (cells / mL). Add the above cell suspension to two low-adsorption T25 flasks. Incubate at 37°C and 5% CO2 for 7 days.

[0042] (2) On Day 2, 13 mL of the remaining culture medium in the T25 flask was taken out and centrifuged at 500 g for 5 min. The supernatant was stored at -80℃ (D2 supernatant). The cell pellet was then collected and resuspended in 13 mL of culture medium containing special cytokines. The cell suspension was added back to the original T25 flask, leaving a total of 26 mL.

[0043] (3) Day 4: Repeat the operation of Day 2. Take 13 mL of the remaining 26 mL of culture medium, set the centrifugation force to 500 g, and centrifuge for 5 min. Store the supernatant after centrifugation at -80℃ (D4 supernatant), then collect the cell pellet, resuspend the cells in 13 mL of culture medium containing cell growth-promoting cytokines, and add the suspension back to the original T25 flask. Take 0.2 mL of cell suspension in a 15 mL centrifuge tube, add physiological saline to 16 mL for bacterial detection; take 0.1 mL of cell suspension in a 15 mL centrifuge tube, add physiological saline to 1 mL for mycoplasma detection; take 0.3 mL of cell suspension in the same EP tube for endotoxin detection; the remaining volume in the T25 flask is 25.4 mL.

[0044] (4) On Day 7, aspirate the cell suspension from the T25 flask and transfer it to a 50 mL centrifuge tube. Use physiological saline containing 0.5% albumin to blow off the adherent cells, and then transfer them to the same 50 mL centrifuge tube. Set the centrifugation force to 500 g and centrifuge for 5 min. After centrifugation, store the supernatant at -80℃ (D7 supernatant). Resuspend the cell pellet in 5 mL of physiological saline containing 0.5% albumin, and then take 0.5 mL for five-part differential analysis, trypan blue counting, and flow cytometry.

[0045] Five-category classification result: 4.51 × 10⁻⁶ 6 Cells / mL; Total: 22.55 × 10⁻⁶ 6 Each cell.

[0046] AOPI count result: 4.12 × 10 6 Cells / mL, viable cell percentage: 98.8%; total cell count: 20.6 × 10⁻⁶ 6 Each cell.

[0047] Furthermore, a dose of 2 million cells per mouse was injected into nude mice. Seven days later, tissue samples were collected from the nude mice to assess the degree of liver cirrhosis following the injection of macrophages.

[0048] Example 2 Triton X-100 and SDS working solutions with a volume and mass fraction of 1% were prepared using double-distilled water. Prior to perfusion, liver samples (source: cirrhotic patients) underwent three freeze-thaw cycles at 4°C and -20°C. Portal vein perfusion was used for decellularization, with a continuous flow rate of 200 mL / min at 4°C. The perfusion procedure was as follows: Triton X-100 working solution perfusion for 3 h; double-distilled water perfusion for 3 h; SDS working solution perfusion for 3 h; double-distilled water perfusion for 3 h; Triton X-100 working solution perfusion for 3 h; double-distilled water perfusion for 6 h.

[0049] Cell-free scaffolds of cirrhotic liver samples were prepared, then thoroughly dried in a freeze dryer for 72 h, and finally shredded and ground using a ball mill. DLEM was prepared by pepsin hydrolysis: the enzymatic concentration of DLS powder was 10 mg / mL; the mass ratio of pepsin to cirrhotic liver sample scaffold powder was 1:10; the mixture was continuously stirred in 0.01 M HCl solution for 2 h until no visible particles were observed. The entire process was carried out at room temperature to obtain a decellularized liver collagen solution. The pH of the decellularized liver collagen solution was adjusted to 7.2-7.4 using 0.1 M NaOH solution, and the entire process was performed on ice, followed by storage at 4°C. The protein content in the decellularized liver collagen solution was then measured using a NanoDrop spectrophotometer, yielding a result of 8.63 mg / mL. The protein concentration in the decellularized liver collagen solution was adjusted to 0.3 mg / mL using the macrophage culture medium from section 1.3 of Example 1. It was then co-cultured with macrophages.

[0050] After 3 days of co-culture, the co-culture supernatant was collected and CTX I was detected by ELISA to evaluate the macrophages' ability to degrade liver collagen, thereby screening out qualified macrophages for subsequent cell therapy.

[0051] The results of this embodiment showed that a CTX I content greater than 100 ng / mL was considered to have a therapeutic effect. (See attached results). Figure 3 .

[0052] Experimental methods: Liver tissue was collected from nude mice and stained with Sirius red. The positive area was then semi-quantitatively analyzed. Results showed that the area of ​​liver fibrosis in nude mice treated with re-infusion was significantly reduced, and the degree of liver fibrosis was significantly alleviated.

[0053] The present invention also relates to an assessment of the collagen degradation capacity of macrophages.

[0054] Example 3 This study provides an alternative method for in vitro detection of the ability of human macrophages to degrade liver collagen and for screening qualified macrophages based on this method.

[0055] S01: Separation of PBMCs: Peripheral blood was collected, anticoagulant was added, mixed well, and centrifuged (800g, 8min) to separate the plasma. The plasma was then tested and retained.

[0056] S02: Peripheral blood was diluted with Ficoll and physiological saline and then subjected to density gradient centrifugation (400g, 30min). The white membrane layer was collected, and the cells were further washed and resuspended for five-part differential and flow cytometry analysis. The results were used to record cell number and percentage, as well as cell viability and other information.

[0057] S03: Sorting of CD14+ cells: Cells were resuspended in buffer, mixed with CD14 magnetic beads, and incubated in the dark. CD14+ mononuclear cells were then separated using magnetic bead sorting technology.

[0058] S04: Repeated washing removes unadsorbed cells, and CD14+ monocyte suspension is collected for cell counting and viability testing. The remaining cells are used for subsequent culture.

[0059] S05: Culture of macrophages: The sorted CD14+ cells were cultured in a medium containing M-CSF (macrophage colony-stimulating factor). The medium was changed regularly and the M-CSF concentration was adjusted to promote cell growth and differentiation. The culture time was 7 days.

[0060] S06: A portion of macrophages were used to degrade collagen solution: Preparation of decellularized liver collagen solution: Liver tissue was collected after clinical cirrhosis transplantation, and the entire liver was perfused via the portal vein with Triton-SDS-Triton to prepare a decellularized liver scaffold. After decellularization, the liver tissue appeared white and transparent, indicating that cells were effectively removed; at the same time, the extracellular matrix (ECM) component was successfully preserved. Then, through freeze-drying, pulverization, and enzymatic hydrolysis, a decellularized liver collagen solution was successfully prepared. An appropriate amount of the decellularized liver collagen solution was added to macrophage culture medium for co-culturing with macrophages. The control group received only macrophage culture medium containing the decellularized liver collagen solution, without the addition of macrophages.

[0061] The macrophage culture medium containing decellularized liver collagen solution comprises TexMACS™ GMP medium, 0.8 mg / mL collagen, and 100 ng / mL M-CSF.

[0062] S07: During the culture process, collect the culture supernatant at different time points periodically for subsequent analysis. For example, on day 7, day 8, day 9, or day 10.

[0063] In some preferred embodiments, after culturing macrophages for 7 days (to complete macrophage induction), they are co-cultured with the decellularized liver collagen solution for 3 days. The supernatant is collected on day 10 for subsequent analysis.

[0064] S08: A portion of the macrophages were used for treatment in nude mice: 2 million macrophages per mouse were injected into nude mice. Seven days later, tissue samples were collected from the mice to assess the degree of liver cirrhosis following macrophage induction. This was to demonstrate that the in vitro collagen degradation experiment reflects the macrophages' ability to treat liver cirrhosis in vivo, and that there is a correlation between the two (i.e., the same batch of cells was subjected to both in vitro and in vivo experiments). Based on the in vivo degradation effect in nude mice, a cutoff value for the effectiveness of the in vitro collagen degradation experiment was selected as a quality control line for subsequent macrophage collagen degradation capacity.

[0065] It should be noted that, in this document, 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. Unless otherwise specified, 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 that element.

[0066] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for detecting the ability of macrophages to degrade liver collagen in vitro, characterized in that, Includes the following steps: S01: Mononuclear cells were isolated from peripheral blood using the density gradient method; S02: CD14+ mononuclear cells were separated by magnetic bead sorting. S03: The isolated CD14+ monocytes were cultured in a medium containing macrophage colony-stimulating factor to obtain a culture medium for macrophage growth and differentiation. S04: Collect livers after liver cirrhosis transplantation and prepare decellularized liver collagen solution; then co-culture with macrophage culture medium obtained in S03; S05: Collect the co-culture supernatant and detect the levels of CTX I, CTX III, C3M or C1M in the culture supernatant to assess the liver collagen degradation capacity of macrophages; S06: Macrophages with CTX I content greater than 100 ng / mL were determined to have the ability to degrade liver collagen.

2. The method as described in claim 1, characterized in that, The cell concentration cultured in S03 was 1-3 × 10⁻⁶. 6 Cells / mL; culture conditions were 37℃ and 5% CO2 for 7 days.

3. The method as described in claim 1, characterized in that, In S04, the cirrhotic liver is decellularized and then freeze-dried and ground to obtain cirrhotic liver tissue powder with decellularized scaffold; subsequently, it is dissolved in HCl solution with pepsin and reacted fully at room temperature to obtain decellularized liver collagen solution.

4. The method as described in claim 3, characterized in that, The mass ratio of the pepsin to the sclerotic liver tissue powder from the decellularized scaffold is 1:

10.

5. The method as described in claim 1, characterized in that, The decellularized liver collagen solution described in S04 is co-cultured with the macrophages, and the macrophage culture medium is used to adjust the protein concentration in the decellularized liver collagen solution to 0.3 mg / mL.

6. The method as described in claim 1, characterized in that, The decellularized liver collagen solution and the macrophages described in S04 are co-cultured for 3 days.

7. The method as described in claim 1, characterized in that, The decellularized liver collagen solution described in S04 and the culture medium for co-culturing the macrophages contain TexMACS™ GMP medium, 0.8 mg / mL collagen and 100 ng / mL M-CSF.

8. The method as described in claim 1, characterized in that, Collect the co-culture supernatant in S05 and detect the CTX I content in the co-culture supernatant using ELISA.

9. Macrophages capable of degrading liver collagen screened using the method for detecting the ability of macrophages to degrade liver collagen in vitro according to any one of claims 1-8.

10. The use of the macrophages with the ability to degrade liver collagen as described in claim 9 in the preparation of drugs or reagents for liver cirrhosis.

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