Stem cell gel line, compound containing stem cell gel line, catgut embedding needle and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a stem cell gel line, a compound containing the stem cell gel line, a catgut embedding needle and application. The stem cell gel line is prepared according to the following steps: preparing a suspension from mesenchymal stem cells, and adding a sodium alginate solution to obtain a stem cell-sodium alginate gel solution; sucking the stem cell-sodium alginate gel liquid, extruding the stem cell-sodium alginate gel liquid into a calcium chloride solution with a relative flow rate in a linear manner at a constant speed, and removing the calcium chloride solution to obtain a stem cell gel line; wherein in the stem cell-sodium alginate gel solution, the final concentration of stem cells is (3-6) * 10 < 6 > / mL, and the final concentration of sodium alginate is 1.5-3wt%. The stem cell gel line provided by the invention can remarkably prolong the activity preservation time of stem cells, can be stored in a line embedding needle, and is convenient to apply.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a stem cell gel thread, a composite containing the stem cell gel thread, a thread embedding needle and applications thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have great potential in the treatment of various diseases and are increasingly valued in the clinical field. However, the current clinical promotion and application of MSCs still needs to overcome many problems, two of which need to be urgently solved: (1) After the cells are injected into the body, they often die in large numbers within 24 hours due to sudden changes in environmental conditions and immune cell attacks, resulting in a large difference between the treatment effect and the expected effect. (2) Currently, most products on the market use stem cell saline suspensions, which are easily affected by liquid shear force. Cells are easily killed during transportation. Not only do they need to be stored at a low temperature of 2 to 8°C, but the storage time is generally not more than 10 hours, which seriously limits the promotion and application of stem cells as a product.

[0003] A large number of studies have shown that the use of biomaterials can help maintain the characteristics of MSCs in vitro and in vivo, prolong cell survival, and thus enhance their therapeutic effects. MSCs gel has shown better therapeutic effects than single cell suspension transplantation in many animal experiments and has broad application prospects in the field of clinical medicine.

[0004] Acupuncture point injection therapy is one of the commonly used clinical treatment techniques. Under the guidance of traditional Chinese medicine theory, the needle of the syringe is used instead of the needle to pierce the acupuncture point, combining acupuncture with the penetration treatment of drugs on the acupuncture points to exert comprehensive efficacy, and the treatment direction is very wide. Autologous blood acupuncture point injection is commonly used in clinical practice. Through the stimulation of acupuncture points and the absorption of autologous blood, it can produce significant therapeutic effects on a variety of diseases, including chronic obstructive pulmonary disease, asthma, urticaria, psoriasis, etc. However, its disadvantage is that blood needs to be drawn before each injection, and generally it needs to be injected once every other day. A course of treatment is about 5 times, and the treatment requires several courses of treatment. Compared with the application frequency of stem cells once every few months, the overall process is too cumbersome and seriously consumes the patient's time and energy.

[0005] Acupuncture thread embedding, which also originated from acupuncture treatment, is a treatment method that implants biodegradable materials into specific parts or acupuncture points of the human body, and uses the long-term stimulation of the thread body on the local or meridian points to replace acupuncture stimulation to treat diseases. It is easy to operate, has a definite therapeutic effect, and is highly accepted by patients. Acupuncture thread embedding has both the stimulation of physical therapy on specific acupuncture points and the tissue repair effect of biodegradable materials dissolving in the body. It is a multi-effect composite therapy, but because the biomaterial needs to remain in the body for a period of time, local redness, swelling, allergies, nodules, and bruises often occur as side effects. Summary of the invention

[0006] The present invention provides a stem cell gel thread, which can be perfectly combined with catgut implantation and acupoint injection when used, taking advantage of the advantages and avoiding the disadvantages, thereby reducing toxic and side effects, reducing the frequency of administration, and improving patient compliance, thus solving the problem that traditional single treatment strategies are difficult to meet the multi-faceted requirements of clinical treatment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a stem cell gel thread, which is prepared according to the following steps:

[0009] The mesenchymal stem cells cultured to the P3 to P10 generations are made into a suspension, and mixed with a sodium alginate solution to obtain a stem cell-sodium alginate gel solution;

[0010] The stem cell-sodium alginate gel solution is absorbed, and extruded into a linear shape at a uniform speed in a calcium chloride solution with a mass fraction of 1.5-3% and a relative flow rate of 0.2-1.0 m / s, and the calcium chloride solution is removed, and the stem cell gel line is obtained after washing with PBS;

[0011] The final concentration of stem cells in the stem cell gel line is 3 to 6×10 6 / mL, and the final mass concentration of sodium alginate is 1.5-3%.

[0012] Among them, the relative flow rate is achieved in the following three ways: ① The calcium chloride solution is stationary and the needle moves in the calcium chloride solution; ② The needle remains stationary and the calcium chloride solution flows; ③ Both move and have a relative speed.

[0013] As a preferred embodiment of the present invention, the stem cell-sodium alginate gel solution is extruded into a linear shape at a uniform speed so that the stem cell-sodium alginate gel solution is in a linear shape with a diameter of 0.2 to 0.4 mm after extrusion.

[0014] As a preferred embodiment of the present invention, the stem cell-sodium alginate gel solution is extruded through a flat-head needle with a diameter of 0.1 to 0.3 mm, and the extrusion speed is 0.02 to 0.08 ml / s.

[0015] As a preferred embodiment of the present invention, the stem cell-sodium alginate gel solution is mixed with auxiliary materials, and the auxiliary materials are one or more of hyaluronic acid, collagen, glycerol, propylene glycol, and chitosan. The mass concentration of the auxiliary materials in the mixed system consisting of the stem cell-sodium alginate gel solution and the auxiliary materials is 0.5-10%.

[0016] As a preferred embodiment of the present invention, the stem cell density in the prepared mesenchymal stem cell suspension is 0.8 to 2×10 7 / ml; the mass concentration of the sodium alginate solution is 4-5%.

[0017] In a second aspect, the present invention provides a composite, which is prepared by compounding the stem cell gel thread with a conditioned medium;

[0018] The conditioned medium is prepared according to the following steps:

[0019] Umbilical cord mesenchymal stem cells cultured to P3-P10 are inoculated into a cell factory and cultured until the cell confluence reaches 80-90%. The serum-free culture medium without antibiotics is replaced. After culturing for 3-5 days, the culture medium is centrifuged at 4°C and 150-300g for 5-10 minutes, and the supernatant is collected to obtain the conditioned culture medium.

[0020] As a preferred embodiment of the present invention, the umbilical cord mesenchymal stem cells are mesenchymal stem cells cultured to any generation of P3 to P5.

[0021] As a preferred embodiment of the present invention, the diameter of the stem cell gel thread is 0.2-0.4 mm, the length is 0.5-3 cm, and the usage ratio of the stem cell gel thread to the conditioned medium is 1 thread: 0.5-5 mL.

[0022] In a third aspect, the present invention provides a thread embedding needle containing the stem cell gel thread, wherein the stem cell-sodium alginate gel solution is uniformly extruded through a flat-head needle with a diameter of 0.1 to 0.3 mm at a speed of 0.02 to 0.08 ml / s, and injected into a thread embedding needle with a diameter of 0.5 to 1.2 mm, and the thread embedding needle is suspended in a calcium chloride solution with a mass fraction of 1.5 to 3% and a relative flow rate of 0.2 to 1.0 m / s. The stem cell-sodium alginate gel solution is molded into a thin thread by the flat-head needle, and the calcium chloride solution further solidifies the thin-threaded stem cell-sodium alginate gel, and the thread embedding needle is used as a storage container to store the thin-threaded stem cell-sodium alginate gel.

[0023] As a preferred embodiment of the present invention, the diameter of the flat-head needle is 0.2 mm, and the needle head size of the thread embedding needle is 0.5 mm in diameter and 25 to 120 mm in length.

[0024] In a fourth aspect, the present invention provides a use of the stem cell gel thread or the complex or the catgut embedding needle containing the stem cell gel thread in the preparation of a stem cell product.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The stem cell gel thread prepared by the present invention can be perfectly combined with catgut implantation and acupuncture injection when used, taking advantage of their strengths and avoiding their weaknesses, thereby reducing side effects, reducing the frequency of administration, and improving patient compliance, solving the problem that traditional single treatment strategies are difficult to meet the multi-faceted requirements of clinical treatment, and further improving the efficacy of treatment methods for stem cell-related diseases.

[0027] 2. The present invention utilizes gel-embedded stem cells to prepare a stem cell gel thread. Gel-embedded stem cells can prevent stem cells from being quickly cleared by immune cells and maintain the stability of the stem cell microenvironment, thereby increasing the survival time of stem cells and enabling longer-lasting secretion of cytokines and exosomes, which is equivalent to continuous acupuncture injection and has a more lasting effect.

[0028] 3. The gel material selected in the present invention is sodium alginate, which has good biocompatibility and can be degraded in the body. After contacting with calcium ions, a thin layer of eggshell-like structure is formed on the surface of the gel, which can protect the soft gel inside and ensure the survival of the internal stem cells. It has a certain strength to the outside and can be pushed into human tissues in a linear form along with the stem cell conditioned culture medium and can be stably maintained in a liquid environment or inside the tissue for several months.

[0029] 4. The stem cell conditioned medium provided by the present invention and the cytokines or exosomes obtained by its concentration have good effects in inhibiting inflammation and promoting tissue repair. They are carried out simultaneously with thread embedding, which can effectively reduce the side effects such as redness, swelling, inflammation, and nodules caused by injection and thread embedding operations. As a liquid, it helps to smoothly push the stem cell gel thread out of the needle.

[0030] 5. The stem cell gel thread prepared by the present invention is directly placed in the thread embedding needle, which reduces the step of cutting the thread and placing it in the needle, and improves the convenience of the thread embedding operation.

[0031] 6. The stem cell gel thread prepared by the present invention is directly placed in the needle, and a small amount of isotonic liquid is added and then packaged for transportation, so that the position of the stem cells remains relatively fixed and will not be damaged by the shear force of the liquid. The method of sealing after filling the container eliminates the oxidation effect of air, helps the cells to dormant, and is beneficial to the preservation of cell activity at room temperature.

[0032] 7. One or more other biomaterials, such as hyaluronic acid, collagen, glycerol, propylene glycol, chitosan, etc., can be introduced into the stem cell gel thread according to the subsequent application direction to further enhance and expand the therapeutic effect.

[0033] 8. The materials involved in the present invention are cheap, the overall preparation method is simple, and the requirements for instruments and equipment and professional technicians are low, which is convenient for industrial production and lays a good foundation for large-scale clinical application of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the normal adherent growth state of umbilical cord mesenchymal stem cells obtained by tissue block adherence method;

[0035] Figure 2 The three-dimensional culture state of umbilical cord mesenchymal stem cells in the gel line; A, stem cell gel line (40×); B, stem cell gel line (100×);

[0036] Figure 3 This is a macroscopic picture of the umbilical cord mesenchymal stem cell gel line; A. The prepared stem cell gel line has low hardness and can be bent arbitrarily; B. The diameter measurement of the stem cell gel line;

[0037] Figure 4 It is a stem cell gel thread embedding needle;

[0038] Figure 5 The results of activity detection of single cell suspension and stem cell gel line after being cultured in physiological saline and serum-free medium for different time periods; Group 1: single cell suspension, culture medium is physiological saline; Group 2: stem cell gel line, culture medium is physiological saline; Group 3: single cell suspension, culture medium is serum-free medium; Group 4: stem cell gel line, culture medium is serum-free medium;

[0039] Figure 6 The results of trypan blue staining of stem cells after the stem cell gel lines were cultured in saline for different time periods, with single cell suspension as control; A, stem cell gel lines cultured in saline for 24 hours; B, stem cell gel lines cultured in saline for 72 hours; C, single cell suspension cultured in saline for 24 hours;

[0040] Figures 7-10 It is a comparison of the secretion of different factors between single cell suspension of stem cells and stem cell gel line cultured in normal saline for 6h to 72h; Group 1: single cell suspension, culture medium is normal saline; Group 2: stem cell gel line, culture medium is normal saline;

[0041] Fig.11 This is a schematic diagram of the preparation and application process of the implantation needle containing stem cell gel thread; 1. Stem cell-sodium alginate gel solution; 2. Calcium chloride solution; 3. Relative flow direction of the liquid in the container; 4. Injection solution (stem cell conditioned medium / exosomes / cytokines); 5. Stem cell gel thread injected into the body; 6. Injection solution diffused into the body; DETAILED DESCRIPTION

[0042] Herein, unless otherwise specified, “a” or “1” before a name, quantifier or unit indicates that there is at least one in number, thus encompassing a plural meaning.

[0043] Herein, unless otherwise specified, the various technical features described herein, such as components, contents, steps, condition parameters, parameter values, assemblies, connection relationships, working relationships, etc., their combination is not limited to the various implementation methods and examples specifically described herein, and any other arbitrary combination of them also falls within the scope of the present invention.

[0044] Herein, unless otherwise specified, a numerical range expressed as two end values ​​is deemed to specifically disclose all real numbers between the two end values ​​and the numerical range formed by any combination thereof. In addition, unless otherwise specified, when multiple optional numerical ranges or values ​​are specifically described for the same parameter, these end values ​​and values ​​may be arbitrarily combined, and the ranges derived therefrom, as long as they are included in the maximum continuous range specifically described, belong to the scope of the present invention.

[0045] Herein, unless otherwise specified, the numerical values, whether preceded by the word "about" or not, include a range of ±10% of the numerical value, or a range of ±5%, ±3%, ±2%, ±1% or ±0.5%.

[0046] Herein, unless otherwise specified, the meanings of all scientific and technical terms are consistent with the well-known or known meanings in the art, especially in the field of stem cells and mesenchymal stem cells, such as the meanings recorded in textbooks, experimental manuals or prior art documents.

[0047] The present invention will be further described in detail below in conjunction with examples. It should be understood that these examples are only for illustrative purposes and are not intended to limit the scope of the present invention.

[0048] Example 1

[0049] Preparation of stem cell gel threads

[0050] 1. Preparation of mesenchymal stem cell suspension:

[0051] Fresh human umbilical cord isolated from clinical sources was used to obtain umbilical cord mesenchymal stem cells by tissue block adherence method. The obtained cell adherence culture state is shown in Figure 1 The cells were cultured and expanded to P3-P10; washed once with PBS, and 0.25% trypsin was added to digest the cells. Under microscope, most of the cells became round, and serum-containing medium was added to terminate the digestion step. The cells were dispersed by blowing, and all the liquid was collected. The cells were separated by centrifugation at 100g for 5 min; the cells were washed 3 times with PBS or saline to remove serum residues, and then resuspended to a cell density of 1.0×10 7 Single cell suspension at 100 cells / ml.

[0052] As a preferred experiment of the present invention, mesenchymal stem cells of any generation from P3 to P5 are preferred.

[0053] 2. Collection of stem cell conditioned medium, which can be further concentrated and separated into cytokines and exosomes

[0054] The P5 umbilical cord mesenchymal stem cells obtained above were inoculated into a 10-layer cell factory (brand: Jie Te, item number: UCF011010) and cultured normally for 2 days. When the cell confluence reached 80%, fresh serum-free medium without antibiotics was replaced. After culturing for 5 days, the cell culture supernatant was collected and centrifuged at 4°C and 300g for 10 min. The precipitated impurities were discarded and the upper liquid, i.e., the conditioned medium, was collected.

[0055] The above liquid was sterilized by filtering with a 0.22 μm sterile filter and set aside.

[0056] 3. Preparation of stem cell gel line

[0057] The mesenchymal stem cell suspension prepared in step 1 was mixed evenly with a sodium alginate solution having a mass fraction of 4% to obtain a stem cell-sodium alginate gel solution. The final cell concentration in the stem cell-sodium alginate gel solution was 3×10 6 / mL, and the final concentration of sodium alginate was 1.5wt%.

[0058] The needle head was suspended and fixed in the container, and the needle head was covered with a 2% calcium chloride solution by mass, 5mm higher, and the needle tip was filled with the calcium chloride solution. The stem cell-sodium alginate gel solution was sucked with a 1mL syringe, and extruded at a uniform speed in the 2% calcium chloride solution through a flat-head needle with a diameter of 0.2mm and a length of 1.0mm, and entered into the needle head with a diameter of 0.8mm and a length of 45mm. The positions of the needle head and the flat-head needle were relatively fixed, and the relative speed with the calcium chloride solution in the container was 0.5m / s, and a gel line with uniform thickness was obtained. The calcium chloride solution was removed, and physiological saline was added to the container for washing 3 times, and the needle was immersed in physiological saline, serum-free culture medium or stem cell conditioned culture medium for storage, so as to obtain a needle head containing a stem cell gel line, and the surface of the stem cell gel line was completely calcified, wrapping the stem cells inside. Figure 2 The stem cell gel lines are magnified 40 times and 100 times respectively, and it can be seen that the stem cells are evenly distributed in the gel line.

[0059] Macroscopic diagram of stem cell gel line is shown in Figure 3 The stem cell gel line prepared in this embodiment has a low hardness and can be bent arbitrarily. The measured diameter of the gel line is about 0.25 mm.

[0060] Figure 4 The invention is a needle embedding needle containing stem cell gel thread. According to different extrusion lengths and diameters of extrusion channels, gel threads of different diameters and lengths can be prepared. When used, the gel threads correspond to different parts such as the face with thin subcutaneous tissue and the abdomen with thick subcutaneous tissue.

[0061] According to subsequent application requirements, other components can be compounded in the stem cell-sodium alginate gel solution, including one or more of hyaluronic acid, collagen, glycerol, propylene glycol, and chitosan, with the final added concentration being 0.5 to 10 wt%.

[0062] The above ingredients are prepared into liquid and used after irradiation sterilization.

[0063] Example 2

[0064] Preparation of stem cell gel threads

[0065] The only difference from Example 1 is that the final cell density of the mesenchymal stem cell suspension is 2×10 7 Pieces / ml.

[0066] Example 3

[0067] Preparation of stem cell gel threads

[0068] The only difference from Example 1 is that the final cell density of the mesenchymal stem cell suspension is 0.8×10 7 Pieces / ml.

[0069] Example 4

[0070] Preparation of stem cell gel threads

[0071] The only difference from Example 1 is the preparation process of stem cell conditioned medium, specifically:

[0072] The obtained P3 generation umbilical cord mesenchymal stem cells were inoculated into a 10-layer cell factory (brand: Jie Te, item number: UCF011010) and cultured normally for 1 day. When the cell confluence reached 90%, fresh serum-free medium without antibiotics was replaced. After culturing for 3 days, the cell culture supernatant was collected and centrifuged at 4°C and 150g for 5 minutes. The precipitated impurities were discarded and the upper liquid, i.e., the conditioned medium, was collected.

[0073] Example 5

[0074] Preparation of stem cell gel threads

[0075] The only difference from Example 1 is the preparation process of stem cell conditioned medium, specifically:

[0076] The obtained P10 umbilical cord mesenchymal stem cells were inoculated into a 10-layer cell factory (brand: Jie Te, item number: UCF011010) and cultured normally for 3 days. When the cell confluence reached 85%, fresh serum-free medium without antibiotics was replaced. After culturing for 5 days, the cell culture supernatant was collected and centrifuged at 4°C and 200g for 8 minutes. The precipitated impurities were discarded and the upper liquid, i.e., the conditioned medium, was collected.

[0077] Example 6

[0078] Preparation of stem cell gel threads

[0079] The only difference from Example 1 is that the conditioned medium collected in Example 1 is ultrafiltered and concentrated through a 100 kd filter membrane to remove small molecules and obtain an exosome solution.

[0080] Example 7

[0081] Preparation of stem cell gel threads

[0082] The only difference from Example 1 is that the conditioned medium collected in Example 1 is filtered through a 3kd filter membrane to remove small molecules and filtered through a 50kd filter membrane to remove large molecules, thereby enriching factors with intermediate molecular weights to obtain a cytokine solution.

[0083] Example 8

[0084] Preparation of stem cell gel threads

[0085] The only difference from Example 2 is the preparation process of the stem cell gel line, specifically:

[0086] The mesenchymal stem cell suspension prepared in step 1 was mixed evenly with a 5% sodium alginate solution to obtain a stem cell-sodium alginate gel solution. The final cell concentration in the stem cell-sodium alginate gel solution was 6×10 6 / mL, and the final concentration of sodium alginate was 3wt%.

[0087] Example 9

[0088] Preparation of stem cell gel threads

[0089] The only difference from Example 1 is the preparation process of the stem cell gel line, specifically:

[0090] The mesenchymal stem cell suspension prepared in step 1 was mixed evenly with a 5% sodium alginate solution to obtain a stem cell-sodium alginate gel solution. The final cell concentration in the stem cell-sodium alginate gel solution was 4×10 6 / mL, and the final concentration of sodium alginate was 2wt%.

[0091] Example 10

[0092] Preparation of stem cell gel threads

[0093] The only difference from Example 1 is the preparation process of the stem cell gel line, specifically:

[0094] The thread embedding needle was suspended and fixed in a container, and a 1.5% calcium chloride solution was used to cover the needle and 5mm above the needle tip. The calcium chloride solution was filled in the needle tip. The stem cell-sodium alginate gel solution was aspirated with a 1mL syringe, and extruded at a uniform speed in the 1.5% calcium chloride solution through a flat-headed needle with a diameter of 0.1mm and a length of 0.5mm, and entered into a thread embedding needle with a diameter of 0.5mm and a length of 25mm. The positions of the thread embedding needle and the flat-headed needle were relatively fixed, and the relative speed with the calcium chloride solution in the container was 0.2 m / s, so that a gel line of uniform thickness was obtained, the calcium chloride solution was removed, and physiological saline was added to the container for washing 3 times, and the needle was immersed in physiological saline, serum-free culture medium or stem cell conditioned culture medium for storage, so as to obtain a thread embedding needle containing a stem cell gel line.

[0095] Embodiment 11

[0096] Preparation of stem cell gel threads

[0097] The only difference from Example 1 is the preparation process of the stem cell gel line, specifically:

[0098] The thread embedding needle was suspended and fixed in a container, and a 3% calcium chloride solution was used to cover the needle and protrude 5mm. The needle tip was filled with the calcium chloride solution. The stem cell-sodium alginate gel solution was aspirated with a 1mL syringe, and extruded at a uniform speed in the 3% calcium chloride solution through a flat-headed needle with a diameter of 0.3mm and a length of 1.5mm, and entered into a thread embedding needle with a diameter of 1.2mm and a length of 120mm. The positions of the thread embedding needle and the flat-headed needle were relatively fixed, and the relative speed with the calcium chloride solution in the container was 1.0 m / s, and a gel line of uniform thickness was obtained. The calcium chloride solution was removed, and physiological saline was added to the container for washing 3 times, and the needle was immersed in physiological saline, serum-free culture medium or stem cell conditioned culture medium for storage, thus obtaining a thread embedding needle containing a stem cell gel line.

[0099] Since the activities of the stem cell gel threads prepared in Examples 1 to 3 and Examples 8 to 11 are basically similar, the present invention will hereinafter only use the stem cell gel thread prepared in Example 1 as an example to illustrate the effects.

[0100] Experimental Example 1

[0101] Verification of cell activity in stem cell gel lines

[0102] The stem cell gel line of Example 1 was placed in a cell culture environment, i.e., 37°C, 5% CO2 environment, and cultured in physiological saline or stem cell conditioned medium for 24 h and 72 h, respectively, and then taken out. The gel line was lysed with 55 mmol / L sodium citrate to release the cells, and the cell activity was detected by MTT experiment. The number of dead cells was confirmed by trypan blue staining and compared with the single cell suspension.

[0103] The results of MTT cell activity assay are shown in Figure 5 , Group 1 is a single cell suspension cultured in saline; Group 2 is a stem cell gel line cultured in saline; Group 3 is a single cell suspension cultured in serum-free medium; Group 4 is a stem cell gel line cultured in serum-free medium; the cell activity after 24h and 72h of culture was compared, and it can be seen that the activity of stem cells placed in the gel line can be maintained longer compared with single cell suspension, whether cultured in saline or serum-free medium. After 24h of culture, the cell activity in the stem cell gel line is twice that of single cells, and after 72h of culture, the cell activity in the stem cell gel line is nearly 10 times that of single cells. The activity of stem cells in the gel line placed in serum-free medium is better than that in saline.

[0104] like Figure 6 As shown, trypan blue staining shows the number of dead cells. Figure A shows the stem cell gel line cultured in saline for 24 hours; Figure B shows the stem cell gel line cultured in saline for 72 hours; Figure C shows a single cell cultured in saline for 24 hours. Figure 6 It can be seen that when the stem cell gel line was cultured in physiological saline for 24 hours, only a very small number of cells were stained positive. After 72 hours of culture, half of the cells were stained positive. When a single cell was cultured in physiological saline for 24 hours, more than 90% of the cells were stained positive, indicating that the vast majority of cells had died.

[0105] The above results indicate that encapsulating stem cells in the form of gel threads can significantly increase the survival time of stem cells, confirming that the cell activity within the stem cell gel threads is preserved for a longer period of time.

[0106] Figures 7-10 The factor secretion of single cell suspension and stem cell gel line of umbilical cord mesenchymal stem cells cultured in normal saline for 6 to 72 hours is compared. Group 1 is a single cell suspension cultured in normal saline; Group 2 is a stem cell gel line cultured in normal saline. Since stem cells can survive longer in the gel line, the content of cytokines secreted by them can be maintained at a higher level for a longer time. This example detects the content of 8 stem cell factors, and the results show that the secretion of stem cells in the gel line is about 1.5 to 2 times the secretion of cells in the single cell suspension.

[0107] Experimental Example 2

[0108] Application method of stem cell gel thread in improving stem cell efficacy

[0109] The stem cell gel thread prepared in Example 1 is used as a thread embedding material, and acupoint thread embedding and acupoint injection are used simultaneously, and the steps are as follows:

[0110] Use a 1-5 ml syringe to absorb the conditioned medium prepared in Example 1 (the effects of the conditioned medium prepared in Example 1 and Example 4-7 are basically the same, so this application example only uses the conditioned medium prepared in Example 1 as an example for illustration), or further concentrate the obtained exosome or cytokine solution, connect the needle head containing the stem cell gel line, determine the required thread embedding acupoints according to the needs of disease treatment through traditional Chinese medicine, and select the specifications of the stem cell gel line according to the actual situation of the application location. For example, the thickness and length of the gel line are selected according to the location of the acupoints, the thickness of the subcutaneous tissue and the individual situation. Generally, when embedding the line in the limbs, 1-1.5 cm and a diameter of 0.2-0.3 mm are selected, and 2-3 cm and a diameter of 0.3-0.4 mm are selected for the abdomen, back and buttocks. The head and face are relatively thin in subcutaneous tissue and exposed to the outside, so they are used as little as possible and the line length is selected to be 0.5-1 cm and a diameter of 0.1-0.2 mm.

[0111] Use the thumb and index finger of your left hand to tighten or pinch the skin at the injection site, hold the needle with your right hand, and after inserting it to the required depth, push the syringe and withdraw the needle tube to implant the stem cell gel thread in the subcutaneous tissue or muscle layer of the acupuncture point. At the same time, the stem cell conditioned medium is also injected into the acupuncture point, and press the needle hole with a sterilized cotton swab for a while.

[0112] The schematic diagram of the whole process of preparation and application of the stem cell gel thread embedding needle provided by the present invention is as follows Fig.11 As shown, 1 is the stem cell-sodium alginate hydrogel solution; 2 is the relative flow direction of the liquid in the container; 3 is the calcium chloride solution; 4 is the injection solution, that is, the stem cell conditioned medium prepared by the present invention or the exosome or cytokine solution obtained by further concentration; 5 is the stem cell gel line injected into the body; 6 is the injection solution component injected into the body and diffused.

[0113] Although the present invention has been described with specific examples, it is obvious to those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Therefore, the appended claims cover all such changes within the scope of the present invention.

Claims

1. A stem cell gel thread, characterized in that: It is prepared according to the following steps: The mesenchymal stem cells cultured to the P3 to P10 generations are made into a suspension, and a sodium alginate solution is added to obtain a stem cell-sodium alginate gel solution; The stem cell-sodium alginate gel solution is absorbed, and extruded into a linear shape at a uniform speed in a calcium chloride solution with a relative flow rate of 0.2 to 1.0 m / s and a mass fraction of 1.5 to 3%, and the calcium chloride solution is removed to obtain the stem cell gel line; Wherein, the final concentration of stem cells in the stem cell-sodium alginate gel solution is 3-6×10 6 / mL, and the final concentration of sodium alginate is 1.5-3wt%.

2. The stem cell gel thread according to claim 1, characterized in that: The stem cell-sodium alginate gel solution is extruded into a linear shape at a uniform speed so that the stem cell-sodium alginate gel solution is in a linear shape with a diameter of 0.2 to 0.4 mm after extrusion.

3. The stem cell gel thread according to claim 2, characterized in that: The extrusion speed of the stem cell-sodium alginate gel solution is 0.02-0.08 ml / s.

4. The stem cell gel thread according to claim 1, characterized in that: The stem cell-sodium alginate gel solution is mixed with auxiliary materials, which are one or more of hyaluronic acid, collagen, glycerol, propylene glycol, and chitosan. The mass concentration of the auxiliary materials in the mixed system is 0.5-10%.

5. A composite, characterized in that The stem cell gel line according to claim 1 is compounded with a conditioned medium, and the conditioned medium is prepared according to the following steps: Umbilical cord mesenchymal stem cells cultured to P3-P10 generations are cultured until the cell confluence reaches 80-90%, and the serum-free medium without antibiotics is replaced. After culturing for 3-5 days, the culture medium is centrifuged at 4°C and 150-300g for 5-10 minutes, and the supernatant is collected to obtain the conditioned medium.

6. The composite according to claim 5, characterized in that The diameter of a single stem cell gel line is 0.2-0.4 mm, and the length is 0.5-3 cm. The usage ratio of the stem cell gel line to the conditioned medium is 1 line: 0.5-5 mL.

7. The composite according to claim 5, characterized in that The mesenchymal stem cells are mesenchymal stem cells cultured to any generation of P3 to P5.

8. A thread embedding needle containing the stem cell gel thread according to claim 1, characterized in that: The method is obtained by injecting the stem cell-sodium alginate gel solution in a linear form into a thread embedding needle soaked in a calcium chloride solution with a mass fraction of 1.5 to 3%.

9. The thread embedding needle according to claim 8, characterized in that: The needle head of the thread embedding needle has a diameter of 0.5 to 1.2 mm and a length of 25 to 120 mm.

10. Use of the stem cell gel thread according to claim 1, the complex according to claim 5, or the catgut embedding needle containing the stem cell gel thread according to claim 8 in the preparation of stem cell related products.

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