Fibroblast formulation and use thereof in preparing medicament for preventing hypertrophic spondylitis

Abstract

Provided are a fibroblast formulation and use thereof in preparing a medicament for preventing hypertrophic spondylitis. By separating and culturing skin fibroblasts of a prepuce tissue in vitro, the present invention achieves the expansion of skin fibroblasts and use of the skin fibroblasts in combination with a macromolecular hyaluronic acid for treating and preventing hypertrophic spondylitis caused by intervertebral disc damage. The present invention significantly improves the water content of the damaged intervertebral disc, maintains the stability of the intervertebral disc, and prevents or reduces the formation of intervertebral disc osteophyte or bone spur.

Description

A fibroblast preparation and its application in preparing medicine for preventing hyperplastic spondylitis Technical Field

[0001] The present application belongs to the field of cell therapy technology, and specifically relates to a fibroblast preparation and its application in the preparation of a drug for preventing proliferative spondylitis. Background Art

[0002] Fibroblasts are the primary cellular component of loose connective tissue, differentiated from mesenchymal cells during the embryonic period. Dermal fibroblasts, a type of fibroblast, are the primary cells that make up the dermis. They synthesize and secrete extracellular matrix, including collagen fibers, elastic fibers, reticular fibers, and hyaluronic acid. They play a vital role in maintaining skin strength and elasticity, repairing damage, promoting wound healing, and beautifying the skin. They are a crucial factor in maintaining skin's youthfulness and a crucial component in maintaining skin structural stability.

[0003] Hypertrophic spondylitis, also known as "hypertrophic spondylitis" or "senile spondylitis," is a type of osteoarthritis caused by age and various other factors, characterized by degeneration of spinal joint cartilage and vertebral bone hyperplasia. This primarily involves lumbar and cervical spondylosis. In the cervical and lumbar vertebrae, the intervertebral disc is located between the two vertebrae and consists of three parts: the nucleus pulposus, the annulus fibrosus, and the cartilage endplates. The nucleus pulposus is located in the center of the disc, surrounded by the annulus fibrosus. The cartilage endplates are located in the upper and lower parts, directly connected to the vertebral bone tissue and providing nutrient exchange for the disc. There are many causes of lumbar and cervical bone hyperplasia, including disc degeneration, trauma, chronic strain, long-term weight-bearing, obesity in the elderly, endocrine disorders, and other factors that damage spinal stability. The main cause is long-term pressure and damage to the intervertebral disc, which leads to a decrease in disc water content, a gradual decrease in elasticity, and a destruction of the microenvironment within the disc, the infiltration of tiny blood vessels, and a large number of inflammatory cells. As the disease progresses, the disc structure changes, such as annular fibrosis rupture, disc bulging, herniation, and prolapse. These structural changes reduce the disc's ability to withstand pressure, leading to vertebral instability, which in turn further aggravates its structural damage. The cartilage pads are squeezed out and accumulate at the edges of the vertebrae. Long-term cumulative calcification and growth form osteophytes or bone spurs. Osteophytes or bone spurs themselves are not painful, but when they compress nerves or irritate surrounding soft tissues, they can cause pain, numbness and stiffness in the limbs, and a series of neurological symptoms.

[0004] For proliferative spondylitis, especially the pain, numbness and stiffness of the limbs, and a series of neurological symptoms caused by bone hyperplasia (osteophytes or bone spurs) due to intervertebral disc injury, traditional treatment methods are conservative treatment and surgical treatment. Conservative treatment includes bed rest and taking anti-inflammatory and analgesic drugs, but conservative treatment can only temporarily relieve the patient's clinical symptoms. As the disease progresses, surgical treatment has to be used. Surgical treatment mainly involves removing osteophytes or bone spurs. For intervertebral discs with severe structural damage, intervertebral fusion surgery will be used for treatment. However, this method must sacrifice the patient's lumbar spine mobility, and will inevitably make the adjacent intervertebral discs bear more biomechanical functions, accelerating the degeneration of the adjacent segmental intervertebral discs. At the same time, the probability of recurrence after surgery is very high, and the patient will endure great pain.

[0005] In addition to the traditional treatments mentioned above, growth factor therapy and cell therapy can also be used for the early and mid-term treatment of ankylosing spondylitis. Growth factors, mainly including transforming growth factor-β1 (TGF-β1), platelet-derived growth factor (PDGF), platelet-rich plasma (PRP), etc., have the main mechanism of treatment by improving the microenvironment of the intervertebral disc, increasing the extracellular matrix of nucleus pulposus cells, and delaying the formation of intervertebral disc osteophytes or bone spurs. However, the above treatment effects have certain limitations. Repeated injections of growth factors or combined injections of multiple growth factors are required to achieve the therapeutic effect of alleviating intervertebral disc bone hyperplasia. In addition, growth factors have a very short half-life, are unstable in nature, have a short shelf life, and are not suitable for systemic treatment. This also limits their role in the treatment of ankylosing spondylitis. Cell therapy, mainly mesenchymal stem cell therapy, has the main mechanism of treatment to reduce inflammation of damaged intervertebral discs and relieve pain. However, damaged intervertebral discs have a hypoxic and inflammatory environment, and the survival rate of stem cells in this environment is low. This is also the main challenge currently faced by mesenchymal stem cell therapy.

[0006] With the aging of my country's population, the elderly population is increasing at an annual rate of 5.2%. By 2020, my country's population aged 60 and over exceeded 260 million, making it the only country in the world with over 200 million elderly people. It is projected that by 2050, China's elderly population will reach 480 million, accounting for approximately one-quarter of the global elderly population (http: / / www.stats.gov.cn / ). Related health issues of this aging population, such as angioplasty (APS), will pose a significant challenge to my country's medical system. Furthermore, due to changes in lifestyle, the incidence of APS is increasingly younger. APS, particularly pain (such as low back pain) and nerve compression caused by bone hyperplasia (osteophytes or spurs) due to intervertebral disc injury, is a common clinical symptom with a lifetime prevalence of approximately 60% to 70%. It has become a major cause of life impairment for many patients and a significant reason for surgical intervention, causing significant suffering and economic losses to society.

[0007] Therefore, if we can effectively prevent hyperplastic spondylitis and reduce the formation of osteophytes or bone spurs caused by intervertebral disc damage, it will effectively improve people's health level and quality of life. Summary of the Invention

[0008] 1. Purpose of the Invention

[0009] One of the objectives of the invention of this application is to provide a fibroblast preparation, which contains skin fibroblasts and macromolecular hyaluronic acid.

[0010] The second object of the invention of this application is to provide the use of the above-mentioned fibroblast preparation containing skin fibroblasts and macromolecular hyaluronic acid in the preparation of drugs for preventing proliferative spondylitis.

[0011] 2. Technical solution

[0012] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0013] The present application provides a fibroblast preparation, which includes skin fibroblasts and macromolecular hyaluronic acid. The skin fibroblasts and macromolecular hyaluronic acid are mixed, and the macromolecular hyaluronic acid is used as a carrier of the skin fibroblasts. The macromolecular hyaluronic acid is a type of macromolecular proteoglycan, and its molecular formula is (C 14 H 21 NO 11 ) n It is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine. The macromolecular hyaluronic acid is a non-toxic, non-immunogenic, non-inflammatory biomaterial with viscoelasticity, lubricity and moisturizing properties, and has good biodegradability and biocompatibility.

[0014] Furthermore, the solvent of the fibroblast preparation is physiological saline, phosphate buffered saline (PBS), etc.

[0015] Furthermore, in the above-mentioned fibroblast preparation, the concentration of macromolecular hyaluronic acid is 0.5 to 1.5 mg / mL. Still further, in the above-mentioned fibroblast preparation, the concentration of macromolecular hyaluronic acid is 0.5 mg / mL.

[0016] Furthermore, the number of skin fibroblasts in the above fibroblast preparation is 1×10 6 ~5×10 8 In this application, the number of skin fibroblasts can be adjusted according to the different application subjects. The adjustment method is: adjust the number of skin fibroblasts in the fibroblast preparation based on the number of cells in the effective injection amount and the volume of the fibroblast preparation that can be injected into the application subject. For example, taking the New Zealand rabbit in this application as an example, the effective injection amount of cells is 1×10 6 ~5×10 6 The volume of the fibroblast preparation that can be injected into the intervertebral disc is 40 μL, and the number of skin fibroblasts in the fibroblast preparation is 2.5×10 7 ~1.25×10 8 For example, the effective amount of cells injected into the human body is 1×10 7 ~5×10 7 If the volume of the fibroblast preparation that can be injected into the intervertebral disc is 1 mL, then the number of skin fibroblasts in the fibroblast preparation is 1×10 7 ~5×10 7 pieces / mL.

[0017] Furthermore, the fibroblast preparation described above has skin fibroblasts with positive rates for the fibrosis genes CD90, FSP1, COL1A1, Fibronectin, and Vimentin exceeding 90% and a positive rate for the MHC class II molecules HLA-DR / DP / DQ less than 2%. Furthermore, the skin fibroblasts with positive rates for the fibrosis genes CD90, FSP1, COL1A1, Fibronectin, and Vimentin exceeding 95% and a positive rate for the MHC class II molecules HLA-DR / DP / DQ less than 1%.

[0018] Furthermore, in the above-mentioned fibroblast preparation, the skin fibroblasts are obtained by isolating and culturing skin tissue, and the isolation and culturing method comprises the following steps:

[0019] Obtain skin tissue and clean it;

[0020] Disinfection treatment;

[0021] After overnight digestion with neutral protease, the epidermal tissue was peeled off, leaving the dermal tissue;

[0022] Use scissors to cut the dermis into small pieces as much as possible, and then immerse it in collagenase and incubate it;

[0023] The incubated cell suspension was sieved using a cell strainer with the aid of culture medium, and the filtrate was collected and the mucus and undigested tissue were removed;

[0024] The supernatant was discarded after centrifugation, the cell pellet was collected, and the cell pellet was resuspended in culture medium to obtain the Pr generation skin fibroblasts;

[0025] The Pr generation skin fibroblasts were inoculated and cultured in complete culture medium. When the cell confluence reached 80% to 90%, subculture was performed to obtain skin fibroblasts.

[0026] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the disinfection treatment includes: disinfection treatment with povidone iodine and ethanol. The use of povidone iodine and ethanol instead of antibiotics avoids interference caused by the use of antibiotics.

[0027] Furthermore, the povidone-iodine and ethanol disinfection treatment includes: placing the skin tissue in a disinfectant containing 5% povidone-iodine as an active ingredient, rinsing in a centrifuge tube for 3 to 5 minutes, with the time adjusted according to the size of the tissue block; replacing sterile tweezers, taking the skin tissue into a new cell culture dish, washing it with PBS, and transferring it into a centrifuge tube containing 75% ethanol, and soaking it for 3 to 5 minutes.

[0028] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the neutral protease comprises dispase II neutral protease. Furthermore, the above-mentioned dispase II neutral protease is 2 mg / mL dispase II neutral protease.

[0029] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the overnight digestion includes: overnight digestion for 16 to 20 hours.

[0030] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, mincing includes mincing into pieces of 1 to 2 mm.

[0031] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the collagenase comprises type I collagenase. Furthermore, the above-mentioned type I collagenase is 2 mg / mL type I collagenase.

[0032] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the incubation includes: incubating at 37° C. for 3 to 5 hours. Furthermore, the incubation includes: incubating at 37° C. for 4 hours.

[0033] Furthermore, the above-mentioned cell filter includes: a 70-micron cell filter.

[0034] Furthermore, the above-mentioned culture medium includes high-glucose DMEM culture medium.

[0035] Furthermore, the above complete culture medium includes: 7.5% hPL+high glucose DMEM+1% b FGF.

[0036] Furthermore, the centrifugation includes: centrifugation at 300 g for 5 minutes.

[0037] Furthermore, the Pr generation skin fibroblasts were seeded at 4×10 4 pieces / cm 2 The density of inoculation was 1.5×10 4 pieces / cm 2 The cells were passaged at a density of 1:1.

[0038] Furthermore, the above-mentioned method for isolating and culturing skin fibroblasts comprises:

[0039] Obtain skin tissue and wash it with sterile PBS;

[0040] Povidone-iodine and ethanol disinfection treatment includes: placing the skin tissue in a disinfectant containing 5% povidone-iodine as the active ingredient and rinsing it in a centrifuge tube for 3 to 5 minutes, with the time adjusted according to the size of the tissue piece; replacing sterile tweezers, taking the skin tissue into a new cell culture dish, washing it with PBS, and transferring it to a centrifuge tube containing 75% ethanol and soaking it for 3 to 5 minutes;

[0041] Immerse in 2 mg / mL dispase II neutral protease and digest overnight for 16 to 20 hours, then peel off the epidermis to leave the dermis;

[0042] Use scissors to cut the dermis into pieces as small as 1-2 mm, soak it in 2 mg / mL type I collagenase, and incubate it at 37°C for 4 h.

[0043] The incubated cell suspension was sieved using a 70-μm cell strainer with the aid of high-glucose DMEM medium, and the filtrate was collected to remove mucus and undigested tissue;

[0044] Centrifuge at 300 g for 5 minutes, discard the supernatant, collect the cell pellet, and resuspend in high-glucose DMEM medium to obtain Pr generation fibroblasts;

[0045] Cell count was calculated as 4 × 10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL in a 6-well plate, and 2 mL of complete culture medium (7.5% hPL + high-glucose DMEM + 1% bFGF) was added to each well, which was the P0 generation.

[0046] When the cell confluence reached 80% to 90%, cells were plated at 1.5 × 10 4 pieces / cm 2 The cells were passaged at a density of 100 nm and the generations were increased in sequence, and skin fibroblasts of each generation were obtained by centrifugation.

[0047] Furthermore, the skin tissue is foreskin tissue. Still further, the skin tissue is foreskin tissue of a child under 18 years old.

[0048] The present application also provides a method for preparing the above-mentioned fibroblast preparation, which comprises:

[0049] A solution containing skin fibroblasts and a solution containing macromolecular hyaluronic acid were prepared separately, and the solution containing skin fibroblasts and the solution containing macromolecular hyaluronic acid were mixed at a volume ratio of 1:1. The skin fibroblasts and macromolecular hyaluronic acid were prepared into solutions separately and then mixed to facilitate better dissolution of the macromolecular hyaluronic acid. The solution containing skin fibroblasts had a high cell concentration and was relatively viscous. If the macromolecular hyaluronic acid was directly added to the solution containing skin fibroblasts, the dissolution effect of the macromolecular hyaluronic acid was poor.

[0050] The present application also provides the use of the above-mentioned fibroblast preparation in the preparation of a drug for preventing proliferative spondylitis. The fibroblast preparation is used to prevent proliferative spondylitis, that is, skin fibroblasts and macromolecular hyaluronic acid are used in combination. The macromolecular hyaluronic acid interacts with the high-affinity hyaluronic acid receptor protein on the surface of skin fibroblasts, which can improve the migration and chemotaxis ability of skin fibroblasts and help skin fibroblasts repair damaged intervertebral discs. The results of this application show that it has a good effect of inhibiting inflammation and improving the water content of intervertebral discs. At the same time, fibroblasts can play a supplementary role in inducing a large amount of extracellular matrix deposition during the repair process, repairing the structure of the damaged intervertebral disc, reducing the pressure on the adjacent intervertebral discs, and restoring the biomechanical function of the responsible segment intervertebral disc, maintaining the stability of the intervertebral disc, and preventing or reducing the formation of osteophytes or bone spurs.

[0051] Furthermore, the above-mentioned proliferative spondylitis includes bone spurs or osteophytes formed by lumbar and / or cervical vertebrae bone hyperplasia.

[0052] Furthermore, the above-mentioned proliferative spondylitis includes bone spurs or osteophytes formed by bone hyperplasia caused by lumbar and / or cervical intervertebral disc damage.

[0053] The present application also provides a method for isolating and culturing skin fibroblasts. The isolated fibroblasts have the characteristics of high purity, low immunogenicity, and good safety, and specifically include the following steps:

[0054] Obtain skin tissue and clean it;

[0055] Disinfection treatment;

[0056] After overnight digestion with neutral protease, the epidermis was peeled off, leaving the dermis;

[0057] Use scissors to cut the dermis into small pieces as much as possible, and then immerse it in collagenase and incubate it;

[0058] The incubated cell suspension was sieved using a cell strainer with the aid of culture medium, and the filtrate was collected and the mucus and undigested tissue were removed;

[0059] The supernatant was discarded after centrifugation, the cell pellet was collected, and the cell pellet was resuspended in culture medium to obtain the Pr generation skin fibroblasts;

[0060] The Pr generation skin fibroblasts are inoculated and cultured in complete medium until the cell confluence reaches 80% to 90%, and then subcultured to obtain skin fibroblasts. Furthermore, in the above-mentioned skin fibroblast isolation and culture method, the disinfection treatment includes: disinfection treatment with povidone iodine and ethanol.

[0061] Furthermore, the povidone-iodine and ethanol disinfection treatment includes: placing the skin tissue in a disinfectant containing 5% povidone-iodine as an active ingredient, rinsing in a centrifuge tube for 3 to 5 minutes, with the time adjusted according to the size of the tissue block; replacing sterile tweezers, taking the skin tissue into a new cell culture dish, washing it with PBS, and transferring it into a centrifuge tube containing 75% ethanol, and soaking it for 3 to 5 minutes.

[0062] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the neutral protease comprises dispase II neutral protease. Furthermore, the above-mentioned dispase II neutral protease is 2 mg / mL dispase II neutral protease.

[0063] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the overnight digestion includes: overnight digestion for 16 to 20 hours.

[0064] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, mincing includes mincing into pieces of 1 to 2 mm.

[0065] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the collagenase comprises type I collagenase. Furthermore, the above-mentioned type I collagenase is 2 mg / mL type I collagenase.

[0066] Furthermore, in the above-mentioned method for isolating and culturing skin fibroblasts, the incubation includes: incubating at 37° C. for 3 to 5 hours. Furthermore, the incubation includes: incubating at 37° C. for 4 hours.

[0067] Furthermore, the above-mentioned cell filter includes: a 70-micron cell filter.

[0068] Furthermore, the above-mentioned culture medium includes high-glucose DMEM culture medium.

[0069] Furthermore, the above complete culture medium includes: 7.5% hPL+high glucose DMEM+1% b FGF.

[0070] Furthermore, the centrifugation includes: centrifugation at 300 g for 5 min.

[0071] Furthermore, the Pr generation skin fibroblasts were seeded at 4×10 4 pieces / cm 2 The density of inoculation was 1.5×10 4 pieces / cm 2 The cells were passaged at a density of 1:1.

[0072] Furthermore, the above-mentioned method for isolating and culturing skin fibroblasts comprises:

[0073] Obtain skin tissue and wash it with sterile PBS buffer;

[0074] Povidone-iodine and ethanol disinfection treatment includes: placing the skin tissue in a disinfectant containing 5% povidone-iodine as the active ingredient and rinsing it in a centrifuge tube for 3 to 5 minutes, with the time adjusted according to the size of the tissue piece; replacing sterile tweezers, taking the skin tissue into a new cell culture dish, washing it with PBS, and transferring it to a centrifuge tube containing 75% ethanol and soaking it for 3 to 5 minutes;

[0075] Immerse in 2 mg / mL dispase II neutral protease and digest overnight for 16 to 20 hours, then peel off the epidermis to leave the dermis;

[0076] Use scissors to cut the dermis into pieces as small as 1-2 mm, soak it in 2 mg / mL type I collagenase, and incubate it at 37°C for 4 h.

[0077] The incubated cell suspension was sieved using a 70-μm cell strainer with the aid of high-glucose DMEM medium, and the filtrate was collected to remove mucus and undigested tissue;

[0078] Centrifuge at 300 g for 5 minutes, discard the supernatant, collect the cell pellet, and resuspend in high-glucose DMEM medium to obtain Pr generation fibroblasts;

[0079] The cells were counted and seeded in a 6-well plate at a density of 40,000. 2 mL of complete culture medium (7.5% hPL + high-glucose DMEM + 1% bFGF) was added to each well to define the P0 generation.

[0080] When the cell confluence reaches 80% to 90%, the cells are passaged at a density of 15,000, and the passages are increased in sequence. Skin fibroblasts of each generation are obtained by centrifugation.

[0081] Furthermore, the skin tissue is foreskin tissue. Still further, the skin tissue is foreskin tissue of a child under 18 years old.

[0082] Furthermore, the fibrosis genes CD90, FSP1, COL1A1, Fibronectin, and Vimentin positive rates of the skin fibroblasts are all greater than 90%, and the MHC-II molecule HLA-DR / DP / DQ positive rate is less than 2%. Furthermore, the fibrosis genes CD90, FSP1, COL1A1, Fibronectin, and Vimentin positive rates of the skin fibroblasts are all greater than 95%, and the MHC-II molecule HLA-DR / DP / DQ positive rate is less than 1%.

[0083] The present application also provides a skin fibroblast, which is obtained by the above-mentioned skin fibroblast isolation and culture method.

[0084] 3. Beneficial effects

[0085] Compared with the prior art, the present application has the following advantages:

[0086] (1) The present application provides a fibroblast preparation and its use in the preparation of a drug for preventing proliferative spondylitis. The fibroblast preparation includes skin fibroblasts and macromolecular hyaluronic acid. Macromolecular hyaluronic acid is a non-toxic, non-immunogenic, non-inflammatory biomaterial with good biodegradability and biocompatibility. The fibroblast preparation is used to prevent proliferative spondylitis. Skin fibroblasts and macromolecular hyaluronic acid are used in combination. The macromolecular hyaluronic acid interacts with the high-affinity hyaluronic acid receptor protein on the surface of skin fibroblasts, which can improve the migration and chemotaxis of fibroblasts and help fibroblasts repair damaged intervertebral discs. The results show that it has a good effect of inhibiting inflammation and improving the water content of intervertebral discs. At the same time, fibroblasts induce a large amount of extracellular matrix deposition during the repair process, which can play a supplementary role, repair the structure of damaged intervertebral discs, reduce the pressure of adjacent intervertebral discs, and restore the biomechanical function of the responsible segment intervertebral disc, maintain the stability of the intervertebral disc, and prevent or reduce the formation of osteophytes or bone spurs.

[0087] (2) The present application provides a fibroblast preparation and its use in the preparation of a drug for preventing proliferative spondylitis, wherein the fibroblasts are human skin tissue fibroblasts (isolated from foreskin tissue and cultured on a large scale in an in vitro culture system), which are combined with macromolecular hyaluronic acid (using hyaluronic acid as a carrier) and injected in situ into the intervertebral disc of New Zealand rabbits with lumbar spine injury. The results showed that the intervertebral discs in the model group had obvious bone spurs, while the intervertebral discs in the treatment group did not have bone spurs. At the same time, the experimental animals did not show immune rejection reactions, which proved the effectiveness and safety of the allogeneic fibroblast preparation and provided new possibilities for the treatment of intervertebral disc injury diseases such as intervertebral disc bone hyperplasia. Allogeneic fibroblast preparations offer numerous advantages, including: (a) safety and reliability: innovative technology enables rapid in vitro expansion of primary cells without any exogenous genes; (b) high batch yield: cells isolated from a single donor can be expanded, enabling multiple treatments from a single donor; (c) stable and controllable quality and batches: consistent and controllable quality of cell preparations is achieved by adhering to the Drug Administration Law and the requirements for establishing cell banks in accordance with the Pharmacopoeia; and (d) low immunogenicity: cells do not express MHC class II antigens, minimizing the likelihood of immune rejection following in situ cell transplantation. Cell therapy, with its safety, reliability, low immunogenicity, ease of use, minimal surgical difficulty, and low cost, is becoming an emerging treatment option. Currently, there are no cellular therapies for intervertebral disc bone hyperplasia in both the domestic and international markets. Therefore, the commercial prospects for allogeneic fibroblasts in the treatment of intervertebral disc injuries, such as proliferative spondylitis, are promising.

[0088] (3) The present application provides a fibroblast preparation and its application in the preparation of a drug for preventing proliferative spondylitis. The foreskin tissue of a circumcision donor under the age of 18 is obtained clinically (medical waste, ethically compliant, and with sufficient sources). After digestion, separation, and in vitro amplification and culture, skin fibroblasts with high purity (positive rates of fibrosis genes CD90, FSP1, COL1A1, Fibronectin, and Vimentin are all >90%), low immunogenicity (positive rate of MHC-II class molecules HLA-DR / DP / DQ is <2%), and good safety (animal cell therapy, 12 months of observation, no growths were generated at the injection site). By using the compliant tissue provided by the donor, cells are separated and cultured in vitro to achieve large-scale amplification of human skin fibroblasts. A fibroblast classification library is established according to the relevant guidelines for cell therapy, making sufficient preparations for clinical application and providing new hope for the treatment of proliferative spondylitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] FIG1 shows P8 (eighth passage) fibroblasts from donor 1 (10×).

[0090] FIG. 2 shows P8 (eighth passage) fibroblasts from donor 2 (10×).

[0091] FIG3 shows P8 (eighth passage) fibroblasts from donor 3 (10×).

[0092] FIG4 is a graph showing the proliferation curves of fibroblasts isolated from three different donors.

[0093] FIG5 shows the flow cytometry results of P8 fibroblasts from donor 1.

[0094] FIG6 shows the flow cytometry results of P8 fibroblasts from donor 2.

[0095] FIG. 7 shows the flow cytometry results of P8 fibroblasts from donor 3. FIG.

[0096] FIG8 is a schematic diagram of the surgical operation of acupuncture injury to the intervertebral disc of an animal to induce bone spur hyperplasia and cell administration.

[0097] Figure 9 shows the MRI results of the animals 2 weeks after surgery.

[0098] FIG10 shows the MRI examination results of the animals 2 weeks after surgery.

[0099] FIG11 is the statistical result of gray value of intervertebral disc 2 weeks after animal surgery.

[0100] FIG12 is the CT scan result of the animal 3 months after surgery.

[0101] FIG13 is the CT scan result of the animal 3 months after surgery.

[0102] FIG14 shows the statistical results of intervertebral disc bone spur length 3 months after animal surgery.

[0103] FIG15 is the CT scan result of the animal 12 months after surgery.

[0104] FIG16 is the CT scan result of the animal 12 months after surgery.

[0105] FIG17 shows the statistical results of intervertebral disc spur length 12 months after animal surgery. DETAILED DESCRIPTION

[0106] The present application is further described below with reference to specific embodiments.

[0107] It should be noted that the terms such as "upper", "lower", "left", "right", and "middle" cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0109] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0110] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0111] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0112] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0113] Hyaluronic acid (HA) preparations have variable molecular weights, depending on the purification procedure, degree of degradation, and source. In this application, macromolecular hyaluronic acid was purchased from Sangon Biotech (Shanghai) Co., Ltd. with the product number A003907-0010, with a molecular weight range of 1000 kDa to 4000 kDa.

[0114] Example 1

[0115] This embodiment provides a method for isolating and culturing skin fibroblasts and the skin fibroblasts obtained after culture.

[0116] In this embodiment, the skin is the foreskin tissue removed during clinical circumcision. The foreskin tissue comes from a surgical sample of a patient under the age of 18. The patient has no infectious viral infection after medical examination. The patient has not used steroid hormone drugs within 6 months before the operation. The patient is fully informed of the purpose of obtaining the surgical sample before the operation and has signed an informed consent form.

[0117] In this embodiment, the method for isolating and culturing skin fibroblasts includes the following steps:

[0118] Obtain foreskin tissue and wash it with sterile PBS buffer;

[0119] Disinfection with povidone-iodine and ethanol: Place the skin tissue in a disinfectant containing 5% povidone-iodine and rinse in a centrifuge tube for 3–5 minutes, adjusting the time based on the size of the tissue. Replace sterile tweezers and place the skin tissue in a new cell culture dish. Wash with PBS and transfer to a centrifuge tube containing 75% ethanol. Soak for 3–5 minutes (over-disinfection will result in loss of cell viability). Then, soak in 2 mg / mL dispase II (Cat. No. D4693, Sigma). After overnight digestion for 16–20 hours, use tweezers to peel off the epidermis, leaving the dermis.

[0120] The dermal tissue was then cut into pieces as small as 1-2 mm using scissors, immersed in 2 mg / mL type I collagenase (abs47048000, Aibixin Biotechnology Co., Ltd.), and incubated at 37°C for 4 h.

[0121] The incubated cell suspension was sieved using a 70-μm cell strainer with the aid of high-glucose DMEM medium (L110KJ, source culture), and the filtrate was collected to remove mucus and undigested tissue;

[0122] Centrifuge at 300 g for 5 minutes, discard the supernatant, collect the cell pellet, and resuspend in high-glucose DMEM medium to obtain Pr generation fibroblasts;

[0123] Cell count was calculated as 4 × 10 4 pieces / cm 2 The cells were seeded at a density of 100 μg / mL in a 6-well plate, and 2 mL of complete culture medium (formula of complete culture medium: 7.5% hPL + high-glucose DMEM + 1% bFGF) was added to each well, which was the P0 generation.

[0124] When the cell confluence reached 80% to 90%, cells were plated at 1.5 × 10 4 pieces / cm 2 The density of the cells was increased, and the generations were passed on in increasing order.

[0125] Result analysis:

[0126] In this example, foreskin tissues were obtained from three donors, namely Donor 1, Donor 2, and Donor 3.

[0127] (1) Morphological observation of skin fibroblasts

[0128] The P8 (eighth generation) fibroblasts of donor 1, donor 2, and donor 3 are shown in Figures 1, 2, and 3, respectively. They have uniform morphology, clear boundaries, and are long spindle-shaped.

[0129] (2) Proliferation ability of skin fibroblasts

[0130] The proliferation curves of fibroblasts isolated from three different donors are shown in Figure 4. Skin fibroblasts have a very strong proliferation ability in vitro and good cell stability. The cells can be stably cultured for more than 20 generations, and the total number of cell proliferation can reach 10 20 The large-scale expansion of fibroblasts in vitro can be achieved by orders of magnitude, which can be fully prepared for clinical applications.

[0131] (3) Fibroblast flow cytometry

[0132] Flow cytometry was performed on P8 fibroblasts from three donors, including:

[0133] (a) Surface marker flow detection steps:

[0134] For P8 fibroblasts, aspirate the culture medium and rinse with 5 mL of sterile PBS. Then, add 2 mL of trypsin to the culture dish to digest the cells to obtain a cell mixture. Place the cell mixture in a 15 mL centrifuge tube and centrifuge at 300 g for 5 minutes. Discard the supernatant to obtain a cell pellet. Resuspend the cell pellet in 700 μL of staining buffer and transfer the resuspended cell pellet to six 1.5 mL centrifuge tubes (100 μL / tube). Add 5 μL of the flow cytometry antibody to be tested to each of the six 1.5 mL centrifuge tubes and mix thoroughly by pipetting. After the centrifuge tubes are refrigerated at 2-8°C for 30 minutes, add 800 μL of sterile PBS to each tube. The tubes are then centrifuged at 300 g for 5 minutes. After centrifugation, discard the supernatant and add 400 μL of staining buffer to each centrifuge tube to resuspend the cell pellet. Then transfer the cell pellet to a flow cytometry tube and perform flow cytometry on the cell mixture. The antibodies used for flow cytometry detection of surface markers are: CD90, HLA-DR / DP / DQ, including CD90 (BD, 555596) and HLA-DR / DP / DQ (BD, 555811).

[0135] (b) Flow cytometric detection of intracellular markers:

[0136] For P8 fibroblasts, aspirate the culture medium and rinse with 5 mL of sterile PBS. Then, add 2 mL of trypsin to the culture dish to digest the cells to obtain a cell mixture. Place the cell mixture in a 15 mL centrifuge tube and centrifuge at 300 g for 5 minutes. Discard the supernatant to obtain a cell pellet. Add 1 mL of fixative transmembrane buffer to the cell pellet. Refrigerate the tube at 2-8°C for 50 minutes. Add 2 mL of sterile PBS to the tube and centrifuge at 300 g for 5 minutes. After centrifugation, resuspend the cell pellet in 500 μL of staining buffer. Transfer the resuspended cell pellet to four 1.5 mL centrifuge tubes, with a 100 μL / tube size. Add 5 μL of the flow cytometric antibody to be tested to each of the four 1.5 mL centrifuge tubes, pipette to mix thoroughly, and place the tubes in a 37°C incubator with 5% CO₂ for 30 minutes. After incubation, add 800 μL of sterile PBS buffer to each tube. Centrifuge the tubes at 300 g for 5 minutes. After centrifugation, discard the supernatant and resuspend the cell pellet in 400 μL of staining buffer to each tube. Transfer the pellet to a flow cytometer and perform flow cytometric analysis of intracellular markers. The names of the antibodies used in the flow cytometric detection of intracellular markers are: FSP1, COL1A1, Fibronectin, and Vimentin, among which FSP1 (Proteintech, CL488-16105), COL1A1 (LSBio, LS-C721218-100), Fibronectin (R&D Systems, IC1918P), and Vimentin (BD, 562337).

[0137] The results are shown in Figures 5-7. The positive rates of CD90, FSP1, COL1A1, Fibronectin, and Vimentin are all above 95%, and the positive rate of HLA-DR / DP / DQ related to immunogenic MHC class II molecules is below 1%, indicating that the fibroblasts isolated and cultured in this application have very good purity and low immunogenicity.

[0138] The cell morphology, proliferation curves and flow cytometry results of the above three donors showed that the quality of fibroblasts isolated and cultured from different donors using the skin fibroblast isolation and culture method of the present application was consistent, and skin fibroblasts with high purity, low immunogenicity and good safety were obtained through digestion, separation and in vitro expansion culture.

[0139] Example 2

[0140] This embodiment provides a fibroblast preparation (skin fibroblasts combined with macromolecular hyaluronic acid) and its use in preventing hypertrophic spondylitis.

[0141] This example uses New Zealand rabbits as subjects to construct a New Zealand rabbit model of lumbar intervertebral disc injury. Skin fibroblasts with hyaluronic acid as a carrier are injected into the injured intervertebral disc to verify its effectiveness in preventing proliferative spondylitis. Specifically, it includes:

[0142] (1) Animal experimental segmental disc grouping

[0143] In this example, the injectable volume of the New Zealand rabbit intervertebral disc is 40 μL, and the volume of the injected 1×10 6 ~5×10 6 The effective amount is 2.5×10 cells. Therefore, in the fibroblast preparation of this embodiment, the number of skin fibroblasts is 2.5×10 7 ~1.25×10 8 / mL, and the concentration of macromolecular hyaluronic acid is 0.5mg / mL.

[0144] In this embodiment, the preparation of the fibroblast preparation includes:

[0145] The skin fibroblasts obtained from donor 1 in Example 1 were resuspended in physiological saline, wherein the number of skin fibroblasts was 5×10 7 ~2.5×10 8 / mL, and a solution containing skin fibroblasts was obtained;

[0146] Take 1 mg of macromolecular hyaluronic acid and dissolve it in 1 mL of normal saline to obtain a macromolecular hyaluronic acid solution;

[0147] 20 μL of the prepared solution containing skin fibroblasts and 20 μL of the prepared solution containing macromolecular hyaluronic acid were taken and mixed to obtain 40 μL of a fibroblast preparation.

[0148] (2) Animal modeling

[0149] After anesthesia, New Zealand rabbits were placed in the right lateral decubitus position. A 5 cm incision was made on the iliac spine. After palpating the lumbar transverse process, an incision was made along the intermuscular space to expose the transverse process. The vertebral body was then exposed along the anterior edge of the transverse process. The level of the iliac spine was defined as the inferior intervertebral space, and the space two intervertebral discs above was defined as the superior intervertebral space. For the experimental groups (cell therapy group and model group), a 20G syringe was used to puncture the intervertebral disc 4 mm, rotate it 360°, and then withdraw it for modeling.

[0150] (3) Animal cell therapy

[0151] After modeling, the cell therapy group received a microinjection (not the original injection site) with 40 μL of the fibroblast preparation, pipetting and mixing each time before aspirating the cells. The model group received a similar injection of 40 μL of a 0.5 mg / mL solution of macromolecular hyaluronic acid. Following the injections, the animals' muscles and skin were sutured with conventional surgical closure.

[0152] The schematic diagram of the surgical operation of acupuncture injury to the animal intervertebral disc to induce bone spur hyperplasia and cell administration is shown in Figure 8.

[0153] (4) Result analysis

[0154] The intervertebral disc water content and bone spur formation of the experimental group (cell therapy group and model group) and the control group were observed by MRI or CT scan of the rabbit lumbar spine 2 weeks, 3 months and 12 months after the animal surgery.

[0155] MRI test results of animals 2 weeks after surgery:

[0156] As shown in Figures 9 and 10, MRI examination 2 weeks after animal surgery showed that the intervertebral disc signal (water content) of the experimental groups (cell therapy group and model group) was significantly lower than that of the control group, indicating that the model was successful. The intervertebral disc signal of the cell therapy group was significantly stronger than that of the model group. At the same time, the grayscale value of each intervertebral disc was statistically analyzed (Table 1 and Figure 11). The larger the grayscale value, the more water content. The analysis showed that the grayscale value of the cell therapy group was significantly greater than that of the model group, indicating that fibroblast therapy can significantly improve the water content of degenerative intervertebral discs.

[0157] Table 1

[0158] CT scan results of animals 3 months after surgery:

[0159] CT scans were performed on the animals three months after surgery. The results are shown in Figures 12 and 13. Significant bone hyperplasia (formation of osteophytes or bone spurs) was observed in the intervertebral discs of the model group, while this phenomenon did not occur in the cell therapy group. At the same time, the length (mm) of the hyperplastic bone spurs was statistically analyzed (Table 2 and Figure 14). The analysis showed that the length of the bone spurs in the cell therapy group was significantly smaller than that in the model group, indicating that fibroblasts can significantly prevent or reduce the formation of osteophytes or bone spurs.

[0160] Table 2

[0161] CT scan results of animals 12 months after surgery:

[0162] CT scans of the animals 12 months after surgery: The results are shown in Figures 15 and 16. It was observed that the model group had increased intervertebral disc bone hyperplasia (formation of osteophytes or bone spurs), while the cell therapy group did not have this phenomenon or had only mild bone spur formation. At the same time, the length (mm) of the hyperplastic bone spurs was statistically analyzed (Table 3 and Figure 17). The analysis showed that the length of the bone spurs in the cell therapy group was significantly smaller than that in the model group, indicating that fibroblasts can significantly prevent or reduce the formation of osteophytes or bone spurs.

[0163] Table 3

[0164] In summary, skin fibroblasts combined with hyaluronic acid can improve the water content of the intervertebral disc and reduce intervertebral disc inflammation, thereby repairing the damaged intervertebral disc structure, maintaining intervertebral disc stability, and preventing or reducing the formation of osteophytes or bone spurs.

Claims

1. A fibroblast preparation, characterized in that The fibroblast preparation comprises skin fibroblasts and macromolecular hyaluronic acid.

2. A fibroblast preparation according to claim 1, characterized in that In the fibroblast preparation, the concentration of macromolecular hyaluronic acid is 0.5-1.5 mg / mL.

3. The fibroblast preparation according to claim 2, characterized in that The number of skin fibroblasts in the fibroblast preparation is 1×10 6 ~5×10 8 pieces / mL.

4. A fibroblast preparation according to claim 2 or 3, characterized in that: The skin fibroblasts are obtained by isolating and culturing skin tissue, and the isolation and culturing method comprises the following steps: Obtain skin tissue and clean it; Disinfection treatment; After overnight digestion with neutral protease, the epidermis was peeled off, leaving the dermis; Use scissors to cut the dermis into small pieces as much as possible, and then immerse it in collagenase and incubate it; The incubated cell suspension was sieved using a cell strainer with the aid of culture medium, and the filtrate was collected and the mucus and undigested tissue were removed; The supernatant was discarded after centrifugation, the cell pellet was collected, and the cell pellet was resuspended in culture medium to obtain the Pr generation skin fibroblasts; The Pr generation skin fibroblasts were inoculated and cultured in complete culture medium. When the cell confluence reached 80% to 90%, subculture was performed to obtain skin fibroblasts.

5. The fibroblast preparation according to claim 4, characterized in that The disinfection treatment includes: disinfection with povidone iodine and ethanol; the disinfection with povidone iodine and ethanol includes: placing the skin tissue in a disinfectant containing 5% povidone iodine as an active ingredient, rinsing in a centrifuge tube for 3 to 5 minutes, the time being adjusted according to the size of the tissue block; replacing sterile tweezers, taking the skin tissue into a new cell culture dish, washing with PBS, and transferring it into a centrifuge tube containing 75% ethanol, and soaking it for 3 to 5 minutes; and / or the neutral protease comprises: dispase II neutral protease; and / or the overnight digestion comprises: overnight digestion for 16 to 20 hours; and / or the collagenase comprises type I collagenase; and / or the incubation comprises: incubating at 37° C. for 4 h; and / or the cell filter comprises: a 70 micron cell filter; and / or the culture medium comprises high-glucose DMEM medium; And / or the complete culture medium includes: 7.5% hPL + high glucose DMEM + 1% b FGF; and / or Pr generation skin fibroblasts were seeded at 4×10 4 pieces / cm 2 Density inoculation; And / or the centrifugation comprises: centrifugation at 300 g for 5 min; and / or subculture at 1.5×10 4 pieces / cm 2 The cells were passaged at a density of 1:

1.

6. The fibroblast preparation according to claim 5, characterized in that The skin tissue is foreskin tissue.

7. The method for preparing a fibroblast preparation according to any one of claims 1 to 5, characterized in that: The method comprises: A solution containing skin fibroblasts and a solution containing macromolecular hyaluronic acid were prepared separately, and then the solution containing skin fibroblasts and the solution containing macromolecular hyaluronic acid were mixed at a volume ratio of 1:

1.

8. Use of the fibroblast preparation according to any one of claims 1 to 5 in the preparation of a medicament for preventing proliferative spondylitis.

9. The use according to claim 8, characterized in that The proliferative spondylitis includes bone spurs or osteophytes formed by lumbar and / or cervical vertebrae bone hyperplasia.

10. A method for isolating and culturing skin fibroblasts, characterized in that: The steps include: Obtain skin tissue and clean it; Disinfection treatment; After overnight digestion with neutral protease, the epidermis was peeled off, leaving the dermis; Use scissors to cut the dermis into small pieces as much as possible, and then immerse it in collagenase and incubate it; The incubated cell suspension was sieved using a cell strainer with the aid of culture medium, and the filtrate was collected and the mucus and undigested tissue were removed; The supernatant was discarded after centrifugation, the cell pellet was collected, and the cell pellet was resuspended in culture medium to obtain the Pr generation skin fibroblasts; The Pr generation skin fibroblasts were inoculated and cultured in complete culture medium. When the cell confluence reached 80% to 90%, subculture was performed to obtain skin fibroblasts.

11. The method for isolating and culturing skin fibroblasts according to claim 10, characterized in that: The disinfection treatment includes: disinfection with povidone iodine and ethanol; the disinfection with povidone iodine and ethanol includes: placing the skin tissue in a disinfectant containing 5% povidone iodine as an active ingredient, rinsing in a centrifuge tube for 3 to 5 minutes, the time being adjusted according to the size of the tissue block; replacing sterile tweezers, taking the skin tissue into a new cell culture dish, washing with PBS, and transferring it into a centrifuge tube containing 75% ethanol, and soaking it for 3 to 5 minutes; and / or the neutral protease comprises: dispase II neutral protease; and / or the overnight digestion comprises: overnight digestion for 16 to 20 hours; and / or the collagenase comprises type I collagenase; and / or the incubation comprises: incubating at 37° C. for 4 h; and / or the cell filter comprises: a 70 micron cell filter; and / or the culture medium comprises high-glucose DMEM medium; And / or the complete culture medium includes: 7.5% hPL + high glucose DMEM + 1% b FGF; and / or Pr generation skin fibroblasts were seeded at 4×10 4 pieces / cm 2 Density inoculation; And / or the centrifugation comprises: centrifugation at 300 g for 5 min; and / or subculture at 1.5×10 4 pieces / cm 2 The cells were passaged at a density of 1:

1.

12. A skin fibroblast, characterized in that: The skin fibroblasts are obtained by the isolation and culture method of claim 10 or 11, wherein the positive rates of fibrosis genes CD90, FSP1, COL1A1, Fibronectin, and Vimentin of the skin fibroblasts are all greater than 90%; and the positive rate of MHC-II class molecules HLA-DR / DP / DQ is less than 2%.

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