Method for chemically treating or culturing macrophages and application of macrophages in treatment of fibrotic diseases

Abstract

A novel cell product consisting of chemically treated macrophages for use in the treatment of fibrotic diseases. The cell product protects organs from fibrosis-induced lesions and dysfunctions by reducing collagen content. The macrophages are treated by a mitochondrial respiratory chain inhibitor and are cultured under an anoxic condition. The macrophage can express an anti-fibrosis biomarker with a higher level and reduce the level of a fibrosis-promoting biomarker. The macrophage injection can be applied to pulmonary fibrosis animals through a drug delivery method of tail vein injection or intratracheal delivery. The traditional Chinese medicine composition can also be used for treating patients with idiopathic pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis and renal fibrosis. A novel technique for producing and manufacturing cell products for therapeutic purposes by using tissue culture carriers coated with temperature responsive polymeric materials. Compared with a traditional method, the novel technology has obvious advantages in the aspects of culturing more cells and keeping the integrity of the cells.

Description

[0001] This application claims priority to U.S. Serial No. 63 / 317,291, filed on March 7, 2022. The entire contents and disclosures of the foregoing applications are incorporated herein by reference. Technical Field

[0002] The present invention relates to chemically treated macrophages and their use in treating fibrotic diseases. The present invention also relates to the cultivation of macrophages and other cells and their use as therapeutic products. Background Art

[0003] Fibrosis is a pathological process that refers to the excessive accumulation of collagen and other extracellular matrix (ECM) in organs, resulting in deformation of normal tissue structure and impaired tissue function. Fibrosis can be caused by trauma, iatrogenic injury and disease, and is the result of dysregulated tissue repair response in a variety of tissue injuries, especially chronic inflammatory diseases.

[0004] When tissue is damaged, local fibroblast activation, secretion of inflammatory mediators, and synthesis of extracellular matrix (ECM) (such as collagen and fibronectin) occur, and these changes together initiate the healing response. Under normal circumstances, the physiological repair process is divided into two stages: the first stage is regeneration, in which the cells of the damaged tissue are replaced by new cells of the same type; the second stage is fibrosis, in which connective tissue replaces normal tissue. However, if the injury is severe or recurrent, the repair process will be uncontrolled, resulting in a large amount of ECM deposition, turning normal tissue into permanent tissue scars. ECM components continue to accumulate, leading to structural damage, organ dysfunction and even failure.

[0005] Fibrosis can occur in almost all human organs, damaging the structure and function of tissues. Common diseases associated with fibrosis include cirrhosis, hepatitis, non-alcoholic steatohepatitis (NASH), chronic kidney disease, myocardial infarction, heart failure, diabetes, idiopathic pulmonary fibrosis (IPF) and scleroderma. Globally, pulmonary fibrosis is one of the leading causes of disability and death in a variety of diseases. Deaths caused by pulmonary fibrosis account for 45% of total deaths in industrialized countries, affecting nearly a quarter of the world's population, placing a heavy burden on medical systems and individuals.

[0006] U.S. Patent Publication No. 20210100837A1 describes a novel genetically modified macrophage detection method for treating fibrosis, which can reduce fibrotic lesions in multiple organs such as the liver, lungs and heart. Macrophages are genetically modified to express recombinant targeting proteins and / or recombinant catalytic enzymes. U.S. Patent Publication No. 20200405757A1 describes a detection method for treating cirrhosis using non-polarized monocyte-derived macrophages. The disclosure describes a method for isolating and culturing autologous macrophages from human patients. The disclosure shows the characteristics of the macrophages and evaluates their anti-fibrotic effects in patients with cirrhosis. The present invention reports for the first time that chemically treated macrophages can improve the anti-fibrotic activity of the cells. The novelty of the present invention lies in the steps of the chemical treatment method. There has been no public report that this innovative method can improve the anti-fibrotic effect of macrophages. The main innovative steps of the present invention involve an experimental protocol for treating macrophages with certain chemicals and then culturing macrophages under specific conditions.

[0007] As mentioned above, there are two previous patent disclosures that describe a method of using macrophages to treat fibrotic diseases. One is to use autologous non-polarized macrophages to treat cirrhosis; the other is to use genetically modified macrophages to treat fibrosis in multiple animal models. Compared with the above patent methods, the present invention uses a special chemical substance-mitochondrial respiratory chain inhibitor (MRCi) to treat macrophages, and then places the treated macrophages under very special culture conditions to achieve the purpose of enhancing anti-fibrotic activity. Because the treatment process and culture conditions are very special and do not belong to conventional cell culture processes, it is impossible to discover or achieve the technology described in the present invention through obvious or conventional cell experiments based on the above two prior arts.

[0008] Macrophages, as important immune cells, are widely distributed in many tissues of the human body. They play a very important role in maintaining the homeostasis of the human internal environment, providing homeostasis maintenance and tissue defense. As the first line of defense of the human immune response, they play an important role in both innate and acquired immune responses. Their phagocytic ability is one of the hallmarks of the human body's nonspecific immune function.

[0009] Macrophages are a heterogeneous cell population that participates in maintaining tissue homeostasis, clearing apoptotic cells and cell debris to promote tissue remodeling and repair. This clearance process is an important metabolic contribution, without which the host will not survive; macrophages are also involved in clearing cell debris produced during tissue remodeling and quickly and effectively clear apoptotic cells. In some cases, macrophages enter a pro-inflammatory state, mainly playing a role in defending against external pathogens or participating in the repair of damaged tissues. Therefore, the primary extraction and culture of macrophages is of great significance to human immunology, immune injury, and molecular cell biology.

[0010] In addition to phagocytosis, macrophages also have chemotaxis, secretion and antigen presentation functions, and play a role in all aspects of biological biology. They have multifunctional roles and high plasticity, and therefore have become the subject of research in many fields. Previous studies have shown that macrophages can be a powerful tool for gene and cell therapy for the treatment of a variety of diseases. Among them, macrophages have great potential in developing new therapies for cancer and fibrotic diseases.

[0011] There are still many technical barriers to developing cell therapies around macrophages. Macrophages are present in most tissues of the human body, and macrophages in tissues are highly heterogeneous and belong to a non-reproductive cell population. They are difficult to survive for a long time, and extraction and enrichment are also relatively difficult. Therefore, it is imperative to establish a system for extracting and culturing macrophages.

[0012] Another major challenge in macrophage culture is the selection of culture containers and separation reagents. The growth characteristics of macrophages determine that they are not easy to harvest, and traditional culture methods and the use of trypsin will not only cause a large number of cell losses during cell harvesting, but also easily affect the state of the cells themselves.

[0013] U.S. Patent No. 5,284,766 describes a novel cell culture bed material that coats tissue culture polystyrene (TCPS) dishes with poly(N-isopropylacrylamide) (PIPAAm). Cells cultured in dishes coated with PIPAAm will detach when the temperature drops below 30°C. Cells harvested using a temperature-responsive method have a higher growth rate compared to conventional cell culture dishes that are not coated with PIPAAm and use an enzyme-based detachment agent.

[0014] The present invention adopts a new method to culture macrophages using temperature-responsive materials, that is, PIPAAm and its copolymers are applied to the surface of TCPS dishes. PIPAAm is considered to be a smart polymer that has reversible temperature-responsive solubility / non-solubility characteristics in aqueous solutions below and above the lower critical solution temperature (LCST) of 32°C. Culturing macrophages on TCPS dishes coated with PIPAAm can avoid the destruction of ECM, cell connections, and cell membrane proteins such as ion channels and growth factor receptors by traditional chelating agents such as EDTA or proteolytic enzymes such as trypsin. The present invention solves the problem of difficulty in harvesting macrophages, and improves cell survival rate in the process while retaining the cell state to the maximum extent. Summary of the invention

[0015] In order to develop an effective and safe method for treating fibrotic diseases, the present invention provides a novel cell product, which protects organs from pathological changes and functional disorders caused by fibrosis, and is composed of chemically treated macrophages. The cell product has the ability to reduce collagen content, is easy to take, and may be generally applicable to the treatment of fibrosis in multiple organs.

[0016] In one aspect of the intervention, the chemically treated macrophages can express higher levels of anti-fibrotic biomarkers. In some aspects, the chemically treated macrophages can also express lower levels of pro-fibrotic biomarkers. In some aspects of embodiments of the present invention, macrophages are treated to reduce fibrosis content in an animal model of pulmonary fibrosis.

[0017] In one embodiment, macrophages isolated from mouse bone marrow for any aspect or embodiment of the present invention are cultured in RPMI-1640 medium containing macrophage colony stimulating factor (M-CSF). On the sixth day of the culture period, cells are treated with mitochondrial respiratory chain inhibitors (MRCi) such as rotenone, norcantharidin, capsaicin or platycodonin, and then cultured under hypoxic conditions. The chemically treated macrophages are characterized by a higher expression of the anti-fibrotic marker CXCL-10 and a lower expression of the pro-fibrotic marker MCP-1.

[0018] In one embodiment, macrophages are chemically treated using MRCi, and then cultured under hypoxic conditions to treat silica and bleomycin-induced pulmonary fibrosis. After the culture process is completed, the macrophages are collected and then injected into mice with silica and bleomycin-induced pulmonary fibrosis by tail vein injection or direct intratracheal delivery. In certain embodiments, hydroxyproline is measured to assess the fibrosis content in lung tissue. Mice with pulmonary fibrosis that receive the macrophages show a decrease in hydroxyproline content relative to mice that receive unpolarized, untreated macrophages.

[0019] In one embodiment of the invention, patients with fibrotic diseases may be treated by administering the macrophages. The unpolarized human macrophages isolated from the autologous body are chemically treated and then administered to patients diagnosed with idiopathic pulmonary fibrosis or IPF after the culture and treatment process. In another aspect, the chemically treated macrophages may also be used to treat patients with liver fibrosis or cirrhosis. In other aspects, the macrophages may also be used to treat patients with myocardial fibrosis caused by acute cardiac injury (such as myocardial ischemia) or chronic heart disease (including hypertension, heart failure and diabetic cardiomyopathy). In some aspects of this intervention, the macrophages may also be used in clinical practice to treat patients with renal fibrosis caused by chronic kidney disease (CKD).

[0020] The present invention reports for the first time that culturing macrophages in TCPS dishes coated with PIPAAm can improve cell survival during the culture and harvesting process. The novelty of the present invention is that there has been no previous report showing the use of this special cell culture material to culture and harvest macrophages to achieve the purpose of cell therapy using macrophages. The key part of the present invention includes using a TCPS dish coated with UpCell TM Surface Nunc TM A method for culturing macrophages in a culture dish, which is produced by Thermo Fisher Scientific and is commercially available. As mentioned above, a prior art describes a method for culturing bovine aortic endothelial cells using a TCPS dish with a PIPAAm coating, which focuses on the temperature response characteristics of PIPAAm. The present invention uses PIPAAm to culture and harvest macrophages. A further difference from the prior art is that the macrophages used in the present invention are therapeutic applications and future clinical treatments for various diseases.

[0021] The present invention describes a new technology for producing and manufacturing cell products for therapeutic purposes, which uses a specific temperature-responsive polymer material, poly (N-isopropylacrylamide) (PIPAAm) and its copolymers, as a coating for tissue culture carriers. Compared with traditional cell culture and harvesting methods, the new technology has obvious advantages in culturing more cells and maintaining cell integrity, which is reflected in higher cell counts and survival rates.

[0022] The growth and harvesting of macrophages has long been a difficult problem in related research. Due to the strong adhesion of cells to traditional culture media, it is considered one of the bottlenecks for large-scale cell manufacturing for potential experiments and clinical practice. To harvest macrophages, classic techniques include mechanical scraping of surface cells, use of enzyme-based lysates such as trypsin-EDTA, and use of enzyme-free agents to preserve surface proteins. However, all of these methods have problems with low cell survival and cell yield loss.

[0023] In 1990, Masayuki Yamato and colleagues developed a new TCPS dish using the temperature-responsive polymer PIPAAm. When the ambient temperature is below or above a critical solution temperature (32°C), the polymer coating changes from hydrophobic to hydrophilic. When the TCPS dish is placed at normal tissue culture temperature (37°C), the hydrophobic coating surface allows cells to attach and grow to confluence. When the temperature is below 30°C, i.e., ambient room temperature, the surface becomes hydrophilic and expels cells and allows them to float without damaging cell surface proteins.

[0024] In one embodiment, CD14+ monocytes used in any aspect or embodiment of the present invention can be separated and differentiated from human peripheral blood mononuclear cells (PBMC). After the separation process is completed, CD14+ monocytes are inoculated into TCPS dishes with PIPAAm coatings or traditional TCPS without temperature-responsive coatings, and cultured in TexMACs culture medium containing macrophage colony stimulating factor (M-CSF). By the seventh day of the culture period, cells in both TCPS dishes can be harvested using a variety of methods.

[0025] In one embodiment, macrophages grown on TCPS dishes coated with PIPAAm coating can be harvested by temperature change from 37°C to room temperature, while macrophages grown on TCPS dishes not coated with PIPAAm coating can be harvested by mechanical scraping using a cell lifter or using commercially available enzyme-free cell dissociation buffer. All cells harvested by different methods are tested for cell viability and cell yield is calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1A-1ECompound-treated macrophages were shown to exhibit enhanced anti-fibrotic activity in an in vitro assay with bone marrow-derived macrophages. Figure 1A and 1B Results of ELISA for detecting CXCL10 are shown, where the levels of CXCL10 in bone marrow-derived macrophages (BMDM) treated with 1 μM rotenone or 10 μM deguelin were increased. CXCL10 can inhibit fibroblast migration and is considered a protective factor against the development of fibrosis in pulmonary fibrosis.

[0027] Figure 1C , 1D and 1E show MCP-1 levels in compound-treated and untreated BMDMs. ELISA analysis illustrates the treatment of BMDMs with MRCi. In these conditions, 1 μM rotenone, 5 μM capsaicin, or 0.1 μM pierinocin A were able to individually reduce MCP-1 levels in BMDMs. MCP-1, short for monocyte chemoattractant protein 1, is a chemokine that is upregulated during inflammation and fibrosis. The reduction in MCP-1 levels can be viewed as an enhancement of anti-fibrotic markers. Combining these two results, it can be concluded that treatment with MRCis such as rotenone followed by culturing under hypoxic conditions can significantly enhance the anti-fibrotic effects of BMDMs.

[0028] Figure 2A and 2B This indicates that chemically treated macrophages have enhanced anti-fibrotic activity in vivo. Both figures show the results of biochemical analysis of hydroxyproline. Hydroxyproline is a major component of tissue collagen and is generally used as the most recognized indicator of ECM deposition and severity of fibrotic diseases. N: normoxia; H: hypoxia; R: rotenone.

[0029] Figure 2A showed that tail-injected macrophages reduced hydroxyproline content in mice, and that macrophages cultured under hypoxic conditions after treatment with rotenone reduced hydroxyproline more than untreated cells. However, treating BMDMs with rotenone and then culturing them under normal oxygen did not show the same enhanced anti-fibrotic activity. Figure 2B Similar results were shown for mice that received macrophages by direct intratracheal delivery. These figures show that macrophages cultured under hypoxic conditions after treatment with certain MRCi were more effective at further reducing collagen content in mice with lung fibrosis than macrophages that were not treated with chemical synthetic agents.

[0030] Figure 3Cell growth results for three groups of samples are shown. Macrophages grown in UpCell dishes had the highest cell counts relative to cells grown in regular dishes and harvested using either cell activators or cell dissociation buffer. This result suggests that PIPAAm-coated dishes have a greater ability to yield cells.

[0031] Figure 4 The viability of cells harvested from the two groups is shown. In the UpCell group, the viability of cells harvested by temperature change was 95.92±2.36%, while the viability of cells harvested by gently scraping with a cell lifter or using cell dissociation buffer was only 82.48±3.88% and 76.61±9.57%, respectively.

[0032] Figure 5 Shows hydroxyproline content, where "3M cells" refers to 3 million macrophages injected into mice. DETAILED DESCRIPTION

[0033] The embodiments described herein are directed to chemically treated macrophages with enhanced anti-fibrotic activity in pulmonary fibrosis and other organ fibrosis. The present invention also relates to a series of cell therapy products based on autologous isolated macrophages. In addition, the present invention also relates to new treatment methods for reducing fibrotic content and reconstructing normal tissue in fibrosis of the lung, liver, heart, kidney and other organs by administering chemically treated macrophages.

[0034] It was hypothesized that glycolysis and lactate production may promote the anti-fibrotic effect of BMDM, while MRC is involved in the pro-inflammatory response of BMDM. It was subsequently observed that BMDM treated with MRCi enhanced the anti-fibrotic activity of BMDM under hypoxic conditions. Both in vitro ELISA data and in vivo results support that BMDM itself has a certain degree of anti-fibrotic activity. Treatment of BMDM under normal culture conditions did not change the degree of fibrosis reduction. Treatment of BMDM with MRCi and then cultured under hypoxic conditions did increase the anti-fibrotic activity by 15% to 20%. However, the detailed and exact mechanism of action behind this phenomenon remains unclear.

[0035] The following terms are used to describe the present invention. If no specific definition is given herein, the terms used to describe the present invention shall have the ordinary meaning understood by those of ordinary skill in the art.

[0036] Macrophages are tissue-resident white blood cells that are derived from monocytes, which in turn are derived from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytic cells that participate in innate and cellular immunity. Together with neutrophils, they are the first responders to infection. Macrophages are involved in the recognition, phagocytosis, and degradation of cell debris and pathogens in the form of fixed or free cells, and activate lymphocytes or other immune cells to speed up their response to pathogens. Macrophages can also present antigens to T cells and induce other antigen-presenting cells to express agonist molecules, thereby initiating adaptive immune responses. In addition, macrophages play an important role in the early stages of inflammation by releasing cytokines and chemokines that can recruit other immune cells to the site of inflammation.

[0037] Macrophages are present in most tissues and therefore have diverse functions. In addition to initiating immune and inflammatory responses to pathogens, macrophages also play a role in maintaining tissue homeostasis as well as tissue repair and remodeling. Unfortunately, this function is associated with many diseases, including metabolic and autoimmune diseases, cancer, infection, obesity, and fibrosis. Therefore, macrophages also appear to play a key role in the tumor microenvironment, especially in matrix remodeling, angiogenesis, metastasis, and tumor progression.

[0038] During routine immune responses, pro-inflammatory macrophages are suppressed, resulting in a reduction in their pro-inflammatory signals. During long-term injury, dysregulated macrophages continue to secrete inflammatory cytokines and recruit other immune cells. These processes maintain chronic inflammation and are thought to play an important role in tumorigenesis and progression. Once a tumor is established, it causes macrophages to differentiate from an immunoactive state to an immunosuppressive state. During wound healing, macrophages can also contribute to fibrosis if the immune response is not adequately controlled. Macrophages also play a key role in other chronic diseases, including atherosclerosis, asthma, inflammatory bowel disease, and rheumatoid arthritis.

[0039] Macrophages can be divided into classically activated macrophages (M1 type) and selectively activated macrophages (M2 type). M1 macrophages participate in proinflammatory responses and play a core role in the host's defense against bacterial and viral infections. M1 macrophages are activated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Activated M1 macrophages secrete a large number of cytokines, such as NO, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6), which have proinflammatory and bactericidal effects. M2a is activated by IL-4 and IL-13; M2b is activated by immune complexes (IC) and Toll-like receptors (TLRs); M2c is activated by IL-10 and glucocorticoids. Although the classification of the above subtypes may not fully represent the complexity of the transition state during macrophage activation, the typing of macrophages is closely related to changes in the microenvironment. In addition, researchers have also discovered other categories of macrophages, such as CD169+ macrophages and T cell antigen receptor T cells (TCR+) macrophages.

[0040] The present invention uses "unpolarized macrophages", i.e. macrophage progenitors or precursors that are fully differentiated from source cells such as monocytes but not yet polarized. In certain embodiments of the present invention, unpolarized macrophages are monocyte-derived cells. Unpolarized macrophages can be obtained from cells such as CD14-positive monocytes in bone marrow samples, and then the monocytes are separated and differentiated to obtain unpolarized macrophages.

[0041] In certain embodiments, non-polarized macrophages are generated from mononuclear cells derived from mouse bone marrow cells according to the culture method and conditions described in the Examples section herein. The bone marrow of mouse tibia and femur is rinsed with PBS, and the red blood cells in the bone marrow cells are then removed by the red blood cell lysis step. The bone marrow cells are then placed in common RPMI1640 culture medium and cultured, and the macrophage colony stimulating factor (M-CSF, R&D Systems, MN) with a concentration of 100ng / ml is added to the culture medium. On the sixth day, cells were treated with mitochondrial respiratory chain inhibitor (MRCi) for two hours, and cultured for another day under hypoxic conditions, as described in Example 1 below.

[0042] Various references or publications are cited in this application. In order to more fully describe the current state of the art involved in the present invention, the entire disclosure of these references or publications is incorporated into this application. It should be noted that the transitional term "comprising" is synonymous with "including", "containing" or "characterized by", and is inclusive or open, and does not exclude other uncited elements or method steps.

[0043] The present invention may be better understood with reference to the following examples, but those skilled in the art will readily appreciate that the specific examples described in detail are only for illustrative purposes and are not meant to limit the invention described herein, which is defined by the following claims.

[0044] Example 1: Enhanced anti-fibrotic effect of chemically treated macrophages in vitro.

[0045] I. Isolation, culture and chemical treatment of macrophages.

[0046] In this embodiment, macrophages are isolated and cultured from mouse bone marrow. Eight-week-old male C57 / BL6 mice (Jackson Laboratory, Michigan, USA) were killed by cervical dislocation. The tibia and femur were separated and the bone marrow was rinsed with ice-cold PBS solution. The bone marrow cells were then resuspended into a single cell suspension and rinsed with ice-cold PBS. The cells were treated with red blood cell lysis buffer (MilliporeSigma, Massachusetts, USA). After treatment, the cell suspension was rinsed with ice-cold PBS and then inoculated with cells on the culture medium.

[0047] The BMDM culture medium used in this example includes RPMI1640 (Thermo Fisher Scientific, Massachusetts, USA), which contains 10% heat-inactivated fetal bovine serum (Gemini Bio, California, USA) and 100 ng / ml recombinant mouse M-CSF protein (R&D Systems, Minnesota, USA). Fresh culture medium was provided on the third and fifth days after isolation. On the sixth day of the culture process, 1 μM rotenone (MilliporeSigma, Massachusetts, USA) in DMSO was added to the culture medium and cultured with the cells for 2 hours. The culture medium was then replaced with fresh culture medium and placed in a hypoxic culture chamber at 37°C with an oxygen content of 1% and a carbon dioxide content of 5%. On the seventh day, the cells were ready for downstream processing. In the untreated group, only the vehicle (DMSO) was added as a control. In addition to rotenone, the following mitochondrial respiratory chain inhibitors (MRCi) were also tested in the same culture process: rotenone (10 μM), pieridin A (0.1 μM) and capsaicin (5 μM).

[0048] II. The enhanced anti-fibrotic effect of compound-treated macrophages was tested in vitro using enzyme-linked immunosorbent assay (ELISA).

[0049] In order to evaluate the anti-fibrotic activity enhanced by chemically treated BMDM, we used commercially available detection kits to perform ELISA detection. The present embodiment uses mouse MCP-1 ELISA kit and mouse IP-10 ELISA kit (CXCL10) (both from Abcam plc, Cambridge, UK). These two kits are used for detection according to the instructions provided by Abcam. In brief, the cell culture supernatant is taken out from the culture plate, the cell debris is removed by centrifugation, and the diluted supernatant is then added to the microwell strip. Then the prepared cocktail antibody is mixed with the culture supernatant and incubated according to the instructions. After the cultivation is completed, a color developing reagent and a stop reagent are added, and then the microwell plate is placed in a spectrometer to record the OD value set.

[0050] The results showed that chemically treated macrophages had enhanced anti-fibrotic activity compared with untreated cells, as reflected by an increase in the anti-fibrotic marker CXCL10 and a decrease in the pro-fibrotic marker MCP-1.

[0051] Example 2: Use of chemically treated macrophages to reduce fibrosis in a mouse lung fibrosis model.

[0052] I. Establishment of mouse pulmonary fibrosis model.

[0053] In this example, a silica-induced mouse pulmonary fibrosis model was established to evaluate the anti-fibrotic activity of chemically treated macrophages. The silica-induced pulmonary fibrosis model is one of the most widely used animal models for studying pulmonary fibrosis diseases. Eight-week-old male C57 / BL6 mice were used in this study. Mice were anesthetized by inhalation of isoflurane and then exposed to 10 mg of silica (MilliporeSigma, Massachusetts, USA) dissolved in 40 μl PBS.

[0054] II. Transplantation and biochemical analysis of chemically treated macrophages.

[0055] Three weeks after the initial silica exposure, BMDM were isolated and treated with rotenone and then cultured under normoxic and hypoxic conditions as described in Example 1. On the seventh day of the culture process, compound-treated and untreated macrophage injections were injected into mice exposed to silica by tail vein injection or direct intratracheal delivery. BMDM cultured under normoxic conditions served as a control group. Mice were sacrificed seven days after macrophage inoculation. Lung tissue was collected for subsequent testing.

[0056] The hydroxyproline colorimetric assay kit (BioVision Inc.) was used. Tissues were homogenized and hydrolyzed with 12N HCl at 120°C, then mixed with the reagents and incubated as indicated. The final concentration of hydroxyproline was calculated by comparing the OD reading at 560 nm with the standard curve.

[0057] Hydroxyproline quantification is the most commonly used method to assess tissue collagen deposition. The results of the hydroxyproline test in this study showed that while both chemically treated and untreated macrophages reduced collagen content in silica-induced mouse lung tissue, the chemically treated macrophage group had a static advantage in reducing fibrosis relative to untreated cells. This phenomenon occurred in both mice administered via tail vein and mice delivered directly intratracheally.

[0058] Example 3: Treatment of fibrosis using chemically treated macrophages.

[0059] Below is a prophetic example of how chemically manipulated macrophages can be used to treat fibrosis in human patients.

[0060] I. Fibrosis

[0061] Fibrotic disease is a disease progression characterized by an increase in fibrotic connective tissue and a decrease in normal cells in tissues and organs, with the characteristics of difficult early diagnosis, complex pathogenesis and poor prognosis. Injured tissues usually heal by secreting collagen, but in cases of severe or repeated injury and dysregulated wound healing response, extracellular matrix (ECM) components, including collagen and fibronectin, accumulate excessively in or around inflamed or damaged tissues and form permanent scars on tissues and organs, ultimately leading to organ deformation and functional failure. Injuries associated with various organ diseases can trigger complex cellular and molecular cascades that lead to the occurrence of fibrotic diseases.

[0062] Organ fibrosis is mainly divided into four stages. The first stage is the fibrosis process induced by primary organ damage, the second stage is the activation of effector cells related to the fibrosis process, the third stage is the subtle changes in the extracellular matrix, and the fourth stage is the dynamic deposition of the extracellular matrix. The irreversible progression of these four stages continues to promote the fibrosis process, ultimately leading to organ structural destruction and dysfunction, and even organ failure. At present, the treatment methods for fibrotic diseases are very limited.

[0063] II. Cell product preparation.

[0064] The macrophages used in certain embodiments described herein can be derived from peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from the patient's peripheral blood by gradient centrifugation in Ficoll centrifuge tubes. After PBMCs are screened out by leukocyte apheresis or from a whole blood sample, CD14+ monocytes are screened out using a human classic monocyte isolation kit (Miltenyi Biotec, CA), and then the CD14+ cells are cultured in TexMACs medium (Miltenyi Biotec, CA) containing 100ng / ml human M-CSF. On the sixth day of the culture process, the cells are treated with MRCi for two hours, and then the cells are placed under hypoxic conditions with fresh culture medium and continue to be cultured for one day. On the seventh day, the chemically treated macrophages are collected and cryopreserved using cGMP-grade DMSO, and the autologous product is then stored in cGMP-grade vials for transportation to the hospital. III. Idiopathic Pulmonary Fibrosis (IPF)

[0065] Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) characterized by scarring of the lung interstitium, which ultimately leads to damage to the lung parenchyma and loss of normal respiratory function. It mainly affects men aged 60-75 years. The average survival after diagnosis of idiopathic pulmonary fibrosis is 2.8 years, and the 5-year survival rate is even lower than that of patients with tumors such as lung cancer and pancreatic cancer, which is only 30%. Therefore, idiopathic pulmonary fibrosis is a neoplastic disease that is even more life-threatening than some tumors. Studies have shown that repeated exposure to unknown damaging stimuli can lead to abnormal alveolar epithelial function, excessive wound healing response, myofibroblast activation, and excessive extracellular matrix (ECM) deposition. Recent studies have emphasized the role of alveolar epithelial cells in the pathogenesis of the disease, and therefore, IPF is considered a multifactorial disease characterized by alveolar epithelial damage and alveolar collapse, alveolar epithelial cell type II pancytopenia, and alveolar stem cell failure. Smoking, viral infection and environmental pollution, as well as genetic susceptibility, are considered potential pathogenic factors.

[0066] In certain embodiments of the present disclosure, chemically treated autologous macrophages can be used to treat IPF patients. PBMCs are isolated from the peripheral blood of patients receiving treatment. The macrophages are differentiated and treated with MRCi according to the methods described above. The frozen autologous cell product is then thawed and the autologous cell product injection is administered to the patient via an intravenous delivery method.

[0067] IV. Liver fibrosis and cirrhosis

[0068] Liver fibrosis is a chronic disease that occurs in the liver. Its main pathological manifestation is the excessive accumulation of extracellular matrix proteins, including collagen, which is accompanied by the destruction of normal liver tissue structure. Most chronic liver injuries will lead to changes in liver fibrosis, including viral infections (hepatitis A, B and C), alcoholic liver damage and non-alcoholic steatohepatitis (NASH). If liver fibrosis is not treated in time, it will develop into cirrhosis, leading to dysfunction of liver cells and obstructed blood flow, followed by liver failure and portal hypertension, which directly threatens the patient's life.

[0069] There is still a lack of clinical tools and drugs to effectively treat liver fibrosis and cirrhosis. Some non-specific treatments targeting pathogenic factors, including anti-inflammatory, antiviral, and lifestyle changes, can alleviate the progression of liver fibrosis to a certain extent, but to date, most people still believe that liver fibrosis is an "irreversible" disease and liver transplantation is the only treatment that can fundamentally solve liver fibrosis and cirrhosis.

[0070] In some embodiments of the present disclosure, chemically treated autologous macrophages can be used to treat patients with liver fibrosis or cirrhosis. The patient receiving treatment will first provide PBMCs. The macrophage product is then developed according to the protocol described above. After the MRCi-treated macrophages are successfully developed, the frozen cells can be thawed and then injected into the patient's body through the vein or portal vein using a fiber optic device.

[0071] V. Myocardial fibrosis

[0072] Heart disease is the leading cause of death in developed countries, with approximately 800,000 deaths from heart disease each year in the United States alone. Cardiovascular disease occurs in a variety of forms with varying pathological manifestations. Most heart diseases are associated with cardiac fibrosis, which refers to an abnormal scarring process of the heart valves caused by inappropriate proliferation of myofibroblasts and excessive deposition of extracellular matrix (ECM) proteins in the myocardium. Myofibroblasts are the main factor causing excessive fibrotic ECM deposition. The activation of cardiac fibrosis has been extensively studied over the past few decades. In the event of acute heart injury such as ischemia or myocardial infarction or chronic diseases such as hypertension and diabetic cardiomyopathy, cardiac fibroblasts (CFs) in the cardiac connective tissue are activated and transformed into myofibroblasts, inducing excessive deposition of extracellular matrix (ECM).

[0073] There are two most common types of cardiac fibrosis, reactive interstitial fibrosis (RIF) and replacement fibrosis (RF). Reactive interstitial fibrosis is usually induced by one or more progressive chronic diseases (such as diabetes and hypertension) and is characterized by diffuse deposition of collagen (a type of ECM) and increased interstitial volume. Replacement fibrosis occurs after acute injury, and the expansion of the ECM and increased deposition of collagen I replace apoptotic cardiomyocytes to prevent rupture of the infarcted myocardium. In general, the progression of cardiac fibrosis leads to distortions in organ structure and function, leading to heart failure. In the pathological process of cardiac fibrosis, necrotic and apoptotic cardiomyocytes trigger excessive accumulation of ECM proteins in RIF and RF.

[0074] In some embodiments of the present disclosure, chemically treated autologous macrophages are used to treat patients with myocardial fibrosis (both RIF and RF subtype patients). First, the patient receiving treatment will provide PBMC as a source of chemically treated macrophages. Then, the cell therapy product is developed according to the method described above. For patients with RF type myocardial fibrosis, the macrophage product is injected into the myocardium around the boundary of the fibrotic site through an optical fiber device. For patients with RIF type myocardial fibrosis, the cell injection is administered by intravenous delivery.

[0075] VI. Renal fibrosis and chronic kidney disease (CKD)

[0076] Similar to the fibrotic pathological process of other organs, renal fibrosis is characterized by abnormal deposition of extracellular matrix (ECM). As a pathophysiological change, renal fibrosis is a gradual process, and renal function changes from healthy to injured and then to impaired, until loss of function. Under the stimulation of multiple pathogenic factors such as trauma, infection, inflammation, blood circulation disorders, and immune response, the kidney's intrinsic cells are damaged, and in the later stages, a large amount of collagen is deposited and accumulated, causing the renal parenchyma to gradually harden and scar, until the kidney completely loses its organ function. The process of fibrosis and hardening of the kidney's intrinsic cells is the process of renal fibrosis.

[0077] Renal fibrosis is also a common manifestation and hallmark of many types of chronic kidney disease (CKD), often presenting in different morphological patterns. In some cases, patients' kidneys will have extensive scarring visible to the naked eye, which is usually caused by severe focal damage and complete parenchymal damage. In addition, chronic glomerular damage leads to corresponding tubular atrophy and degeneration of specific nephrons, which will eventually lead to renal interstitial fibrosis and tubular atrophy (IF / TA). In contrast, diffuse fibrosis unrelated to tubular atrophy and focal replacement scarring caused by glomeruli appear to be different pathogenic processes. Renal fibrosis seems to develop in a segment-specific manner, but whether focal and diffuse fibrosis have distinct features associated with other glomerular or tubulointerstitial lesions remains difficult to determine. The disease progression of patients with chronic kidney disease and secondary renal fibrosis is relatively slow, but if targeted diagnosis and treatment are not carried out in a timely manner, the disease can easily worsen and develop into chronic renal insufficiency, which may eventually lead to end-stage renal disease or even uremia.

[0078] In some embodiments of the present disclosure, chemically treated autologous macrophages can be used to treat patients with renal fibrosis or chronic kidney disease. PBMCs are isolated from patients receiving cell therapy and then used as a source of cultured macrophages as described above. Once the cells are available for clinical treatment, they can be administered by intravenous injection or local injection in the kidney through a subcutaneous renal fiberoscope.

[0079] The embodiments described herein are related to the use of temperature-responsive cell culture surfaces to culture and harvest macrophages for cell therapy purposes. More specifically, the material is poly(N-isopropylacrylamide) (PIPAAm). Under normal cell culture conditions, i.e., at 37°C and 5% carbon dioxide, macrophages cultured on surfaces coated with PIPAAm can grow to confluence. When the culture process reaches the harvest point, the culture environment temperature is reduced to 20-25°C, and the macrophages will detach from the TCPS without the use of traditional enzyme-based proteolytic agents such as trypsin-EDTA.

[0080] Poly(N-isopropylacrylamide) (PIPAAm) is a polymer first synthesized in the 1950s. It has the intelligent property of changing its structure with temperature and has been applied to many aspects of cell biology practice, including tissue engineering and controlled drug delivery. When the ambient temperature changes, PIPAAm undergoes a reversible phase transition between hydrophobic and hydrophilic near the lower critical solution temperature (LCST) of 32°C.

[0081] Under normal cell culture conditions at 37°C, PIPAAm is hydrophobic and the polymer forms a collapsed spherical shape, allowing macrophages to attach and proliferate, similar to macrophages in normal culture dishes. Once the ambient temperature is below the LCST, such as around room temperature, PIPAAm becomes hydrophilic and soluble, and the polymer becomes an extended coil shape. Once the hydrophobic to hydrophilic transition occurs, macrophages will automatically detach and float freely without the use of proteolytic enzymes or chelating agents. Traditional cell harvesting methods using trypsin or chelating agents usually damage cell surface proteins and extracellular matrix by cleaving various membrane-associated proteins, which will reduce cell survival and cause unexpected characteristic damage to cells. Culturing macrophages on TCPS coated with PIPAAm can avoid the drawbacks of traditional methods and maintain cell integrity to the greatest extent.

[0082] The present invention uses macrophage progenitor cells or precursor cells that have been completely differentiated from monocytes or other source macrophages. These cells are mature but not yet polarized macrophages. In certain embodiments of the present invention, unpolarized macrophages can be obtained from CD14-positive monocytes such as peripheral blood mononuclear cells (PBMCs), and then separated and cultured on TCPS with PIPAAm coating using macrophage colony stimulating factor (M-CSF).

[0083] In certain embodiments, CD14+ monocytes are isolated from human PBMCs using the CliniMACS CD14 separation system (Miltenyi Biotec BV&Co.KG, Germany) according to the culture methods and conditions described in Example 4 herein to generate non-polarized macrophages. The CD14+ monocytes are then cultured in TexMACS medium (Miltenyi Biotec BV&Co.KG, Germany) to which macrophage colony stimulating factor (M-CSF, R&D Systems, MN) is added at a concentration of 100 ng / ml. The cells are cultured in a medium with UpCell TM Surface Nunc TM The cells were cultured in culture dishes (Thermo Fisher Scientific, Waltham, MA) with PIPAAm coating or in plain cell culture dishes produced by Corning Incorporated (Tewksbury, MA) as controls. After the culture process, the cells were harvested by different methods, and the number and survival rate of cells in each group were compared to illustrate the advantages of PIPAAm-coated culture dishes in culturing macrophages.

[0084] The present invention shows that the tissue culture carrier coated with PIPAAm has obvious advantages in growing and culturing macrophages, which can increase the number of cells and maintain the integrity of cells.

[0085] Example 4: Culturing macrophages on PIPAAm-coated surfaces can increase cell number and survival rate.

[0086] I. Isolation of CD14+ monocytes and differentiation of macrophages.

[0087] In this example, macrophages were isolated and cultured from fresh human PBMCs. Fresh whole blood was diluted with Hank's 1X Balanced Salt Solution (HBSS) (Marlborough, MA) (Thermo Fisher Scientific, Waltham, MA) containing 2 mM EDTA and then transferred to LeucoSep TM The cells were separated in a 4% paraformaldehyde (2% paraformaldehyde) tube (Greiner Bio-One International GmbH, Monroe, North Carolina). After centrifugation at 800 g for 15 minutes, PBMCs were harvested from the second layer at the top. The cells were then purified three times using HBSS / EDTA. Red blood cell lysis buffer (MilliporeSigma, Massachusetts, USA) was added to the cells and incubated at room temperature for five minutes. After the above treatment, the cells were rinsed with HBSS / EDTA and prepared for subsequent CD14+ cell separation.

[0088] Prepare a separation buffer for use with the CliniMACS CD14 separation system (Miltenyi Biotec BV & Co. KG, Germany). The buffer consists of phosphate buffered saline, 0.1% endotoxin-free bovine serum albumin, and 2mM EDTA (all from MilliporeSigma, Massachusetts, USA). Resuspend the cell pellet in the separation buffer and gently mix with the CliniMACS CD14+ microbeads at 4°C for 15 minutes. Place the LS column of the CliniMACS CD14 separation system on a magnetic rack and rinse with the separation buffer, then load the cell / microbead mixture into the column and rinse three times with the separation buffer. After washing, collect the cells for subsequent procedures.

[0089] CD14+ cells were divided into three groups. One group was treated with UpCell TM Surface Nunc TMThe cells were cultured in two groups in a tissue culture dish (Thermo Fisher Scientific, Waltham, MA), and the other two groups were cultured in a conventional tissue culture dish produced by Corning (Tewksbury, MA) and different harvesting methods were used. All groups were cultured in TexMACS medium (Miltenyi Biotec B.V. & Co. KG, Germany) and 100 ng / ml M-CSF protein (R&D Systems, MN, USA) at 37°C and 5% carbon dioxide for seven days.

[0090] II. Harvesting of macrophages and measuring cell viability.

[0091] To evaluate the advantages of TCPS coated with PIPAAm for harvesting macrophages, different harvesting methods were used for the three groups of cells at the end of the seven-day culture process. Cells from all groups were removed from the incubator.

[0092] The macrophages cultured in the UpCell dish were placed at room temperature for 25 minutes, and then the culture medium containing the detached cells was collected into a 50 ml centrifuge tube.

[0093] For a group of macrophages cultured in regular tissue culture dishes, the culture medium was removed and replaced with ice-cold PBS. The cells were then placed at 4°C for 10 min. After the 10-min incubation, the cells on the surface of the dish were gently scraped off using a Fisherbrand cell harvester (ThermoFisher Scientific, Waltham, MA) and transferred to a 50-ml centrifuge tube.

[0094] For another group of macrophages cultured in ordinary tissue culture dishes, Gibco cell dissociation buffer was used to harvest the cells. This buffer is a membrane-filtered, isotonic, enzyme-free salt solution and chelating agent, which is said to be suitable for dissociating mammalian cells while retaining cell surface proteins. The culture medium was removed and rinsed with PBS, and then the cells were incubated with cell dissociation buffer at 37°C for 10 minutes. After the incubation period, the buffer containing the cells was collected into a 50 ml test tube.

[0095] The cell number and viability of the three groups were measured using a Cellometer Auto T4 bright field cell counter (Nexcelom Bioscience, Lawrence, MA).

[0096] Example 5: Isolation and processing of PBMC-derived macrophages.

[0097] In this example, CD14 positive cells were isolated from fresh human PBMC using a classic monocyte isolation kit (Miltenyi Biotec Bergisch Gladbach, Germany). The cells were then cultured for seven days in a culture medium containing 100ng / ml human M-CSF (Miltenyi Biotec Bergisch Gladbach, Germany). On the seventh day, 1μm rotenone (Sigma-Aldrich, Missouri, USA) was added to the culture medium and cultured for two hours. After the two-hour culture, the culture medium was replaced with a fresh culture medium without rotenone, and then the culture medium was placed in an anaerobic box containing 1% oxygen and cultured for different lengths as described in the animal treatment section. The cells were then collected and administered to bleomycin-induced BALB / c nude mice IPF by a drug delivery method of tail vein injection.

[0098] I. Animal Treatment (3, 6 and 12 hours of Hypoxia)

[0099] Six-week-old BALB / c nude mice were randomly divided into five groups: Group 1 received only 4U / kg bleomycin delivered intratracheally; Group 2 received 4U / kg bleomycin and PBMC-derived macrophage injections administered by intravenous delivery; Groups 3, 4, and 5 received 4U / kg bleomycin and PBMC-derived macrophage injections administered by intravenous delivery, and these macrophages were treated with rotenone (1 μM) and cultured in hypoxia (1% oxygen) for 3, 6, or 12 hours before injection. Two weeks after the injection of bleomycin, PBMC-derived macrophage injections with or without rotenone / hypoxia treatment were administered by tail vein injection to mice in Groups 2, 3, 4, and 5. Mice in Group 1 were administered PBS by tail vein injection as a control.

[0100] II. Hydroxyproline Measurement.

[0101] One week after macrophage transplantation, mice were sacrificed, and lung hydroxyproline content was measured using a hydroxyproline kit (Nanjing Jiancheng Bioengineering Institute, China).

[0102] III. Results

[0103] Treatment with PBMC-derived macrophages significantly reduced lung hydroxyproline content, an indicator of the extent of lung fibrosis. Hydroxyproline content was significantly reduced in all groups treated with macrophages compared with the bleomycin plus PBS group. The decrease in hydroxyproline content was greater in the macrophage groups treated with hypoxia and rotenone, with the greatest decrease in hydroxyproline content in the 12-hour hypoxia group, suggesting that this treatment may be particularly effective in treating lung fibrosis. These results suggest that treatment with PBMC-derived macrophages has the potential to effectively attenuate bleomycin-induced lung fibrosis in mice, with hypoxia and rotenone treatments being particularly promising.

Claims

1. A method for treating fibrosis in a subject, characterized in that: The method comprises administering macrophages to the subject, wherein the macrophages are treated with a mitochondrial respiratory chain inhibitor (MRCi) selected from rotenone, capsaicin or pieridin A and cultured for a period of time under hypoxic conditions, and the macrophages exhibit enhanced anti-fibrotic activity after treatment.

2. The method according to claim 1, characterized in that: The macrophages are selected from bone marrow-derived macrophages or fresh human PBMCs.

3. The method according to claim 1, characterized in that: The fibrosis is pulmonary fibrosis, liver fibrosis, myocardial fibrosis or kidney fibrosis.

4. The method according to claim 1, characterized in that: The macrophages are administered to the subject by tail vein injection, intravenous injection, portal vein injection, intracardiac injection, or intratracheal delivery.

5. The method according to claim 1, characterized in that: The hypoxic condition includes a low oxygen environment.

6. The method according to claim 1, characterized in that: After the treatment, the hydroxyproline content in the fibrotic tissue of the subject is reduced when detected using a hydroxyproline colorimetric assay kit.

7. The method according to claim 1, characterized in that: The macrophages were treated with rotenone and cultured under hypoxic conditions for 12 hours.

8. A method for increasing cell yield and survival rate, characterized in that: The method involves culturing cells in UpCell dishes coated with PIPAAm.

9. The method according to claim 8, characterized in that: The cells are macrophages.

10. The method according to claim 8, characterized in that: The cells harvested by the method have a higher yield and a higher survival rate than cells cultured by traditional methods.

11. A method for producing macrophages with enhanced anti-fibrotic activity, characterized in that: The method comprises the following steps: (a) A macrophage sample in culture treated with a mitochondrial respiratory chain inhibitor (MRCi); (b) culturing the treated macrophage sample under hypoxic conditions for a period of time; and (c) Harvesting macrophages with enhanced anti-fibrotic activity.

12. The method according to claim 11, characterized in that: The macrophages are selected from bone marrow-derived macrophages or fresh human PBMCs.

13. The method according to claim 11, characterized in that: The MRCi is rotenone, capsaicin or pieridin A.

14. The method according to claim 11, characterized in that: The treated macrophage samples were cultured under hypoxic conditions for up to 12 hours.

Images