Method for extracting pig umbilical cord tissue components by using low-oxygen cell culture method
The tissue components of pig umbilical cords were extracted by hypoxic cell culture method, and the problems of low protein concentration, unstable product quality, complex operation and high cost in the umbilical cord extraction method in the prior art were solved, and high purity extraction and efficient production were achieved.
Patent Information
- Application Number
- CN202411619850.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-02-14
AI Technical Summary
In the prior art, the umbilical cord extraction method has problems such as low protein concentration, unstable product quality, complex operation and high cost.
The hypoxic cell culture method (5% oxygen, 5% carbon dioxide) was used to extract pig umbilical cord tissue components, and cell proliferation and bioactive components were optimized through controlled culture conditions.
High-purity ingredient extraction is achieved, the extraction efficiency and effectiveness is improved, the risk of immune response is reduced, and standardized and large-scale production is supported.
Smart Images
Figure CN119432961A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological extraction, and in particular relates to a method for extracting pig umbilical cord tissue components by utilizing a hypoxic cell culture method. Background Art
[0002] The umbilical cord is the link between the fetus and the placenta. The outer layer is the amniotic membrane, and inside it are two umbilical arteries with smaller lumens and thicker walls, and one umbilical vein with a larger lumen and thinner walls. The umbilical blood vessels are surrounded by protective embryonic connective tissue called Wharton's jelly. The umbilical cord is generally about 30 to 70 cm long, with an average of 50 cm. The umbilical cord is an important channel for gas exchange, nutrient supply, and metabolic waste discharge between the mother and the fetus.
[0003] Umbilical cord extract refers to a stable concentrate obtained by extracting and processing human or animal umbilical cord. The main components of umbilical cord extract are: hyaluronic acid, soluble elastin, polypeptides, amino acids, lipids, nucleic acids and their derivatives, which can be mainly used in cosmetic raw materials, biomedicine, beauty, medical treatment, etc.
[0004] The raw material of animal umbilical cord extract is animal umbilical cord tissue, which is usually provided by qualified animal farms. The raw material of human umbilical cord extract is human umbilical cord tissue, which is usually provided by the obstetrics and gynecology department of the hospital after the provider signs an informed consent form.
[0005] According to relevant research, umbilical cord extract has the following major effects: ① It has many skin care effects and can lighten spots; ② It can effectively neutralize active oxygen free radicals that damage the skin and delay skin aging; ③ It can accelerate cell metabolism and promote skin regeneration; ④ Moisturizing effect. Umbilical cord extract is rich in hyaluronic acid, which can maintain skin moisture and make the skin full of tension; ⑤ Promote cell and tissue respiration, activate collagen fibers and elastic fibers, and make the skin full of elasticity; ⑥ Immune activation effect, which can improve allergic dermatitis, etc.
[0006] At present, there are several studies on the extraction of effective active substances from umbilical cord tissue, which are mainly extracted through the following three methods: ⑴ Direct extraction method: After the umbilical cord tissue is washed, minced, an appropriate amount of buffer is added, stirred for 18 hours, and then centrifuged. ⑵ Enzyme digestion method: After the umbilical cord tissue is washed, minced, an appropriate amount of pepsin is added for enzymatic hydrolysis, and after high-temperature inactivation, pancreatic enzyme is added for enzymatic hydrolysis. After inactivation again, ultrafiltration is used for purification to obtain umbilical cord extract. ⑶ Special incubation method: After the umbilical cord tissue is washed, the umbilical cord tissue is first frozen at low temperature, and then special incubation and stirring are performed under respiratory pressure and rewarming to prepare animal umbilical cord tissue extract.
[0007] The protein concentration of umbilical cord extracts extracted by direct extraction is low and of low quality. The umbilical cord extracts extracted by enzyme digestion method are inactivated twice at high temperature. The enzymes are denatured and inactivated, which easily leads to the inactivation of polypeptides, thus affecting the quality of the product. At the same time, two digestive enzymes are introduced during enzyme digestion, which affects the purity of the product. The umbilical cord extracts extracted by special incubation method need to be adjusted in temperature and pressure, which is complicated to operate and has high cost. Summary of the invention
[0008] In view of the shortcomings of the direct extraction method, enzyme digestion method and special incubation method in the prior art, the present invention provides a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method. The present invention uses a hypoxic cell culture method (5% oxygen, 5% carbon dioxide) to extract pig umbilical cord tissue components to achieve high-purity component extraction, optimize cell proliferation and the synthesis of bioactive components through controllable culture conditions, thereby improving extraction efficiency and effectiveness.
[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for extracting pig umbilical cord tissue components using a hypoxic cell culture method comprises the following steps: The hypoxia: 5% oxygen, 5% carbon dioxide; 1) Primary isolation and culture of porcine umbilical cord mesenchymal stem cells: After the newborn piglets are born, the umbilical cord is cut with sterile surgical instruments and bled under sterile conditions. After the bleeding is complete, the umbilical cord is ligated in sections from the end close to the fetus, and then cut off at intervals. The umbilical cord with both ends ligated is placed in the prepared physiological saline with penicillin and streptomycin, and quickly transported to the laboratory; Under sterile conditions, the umbilical cord tissue was removed and repeatedly rinsed with physiological saline, and then the ligated parts at both ends were discarded, and the ligated inner umbilical cord was taken out and rinsed again. After the rinsing fluid became clear, it was placed in a sterile culture dish; along the longitudinal axis of the umbilical cord, the outer layer of connective tissue was cut open, and two arteries and one vein were extracted from the incision, and the remaining tissue was placed in a sterile culture dish; Glial digestion method: Under sterile conditions, enlarge the incision, turn the umbilical cord tissue cavity in the sterile culture dish in the previous step upwards, and after fixation, gently scrape the gelatinous part of the umbilical cord with a blade, collect the scraped gelatinous material, cut it into 1mm pieces, add 2.0mg / mL collagenase I mixed with 1mg / mL hyaluronidase, digest it at 37℃ for 60min, and then blow it repeatedly; terminate the digestion with culture medium containing 10% (v / v) fetal bovine serum (FBS), filter it with a 200-mesh cell sieve, collect the filtrate, centrifuge it, filter it, resuspend it, and inoculate it into a cell culture bottle, and place it in a cell culture incubator at 37℃ for constant temperature culture; 2) Passaging of porcine umbilical cord mesenchymal stem cells: Under the microscope, when the cells cultured in the cell culture incubator showed 90%-95% fusion at the bottom of the culture dish, the cells were passaged; the culture medium was removed, and PBS was added for continuous rinsing twice; 2 ml of 0.25 wt% trypsin-EDTA solution was added, and the cells were digested at 37°C for 2 min; 2 ml of complete culture medium was added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant was removed; the cell pellet was resuspended with complete culture medium and 8 × 10 3 / cm 2 The number of cells inoculated for subculture; 3) Cryopreservation of porcine umbilical cord mesenchymal stem cells: Reagent preparation, freezing solution: basal medium + 20% fetal bovine serum + 10% DMSO; Under the microscope, when the P1 generation porcine umbilical cord mesenchymal stem cells showed 90%-95% fusion, the cells were cryopreserved; the culture medium was removed, and physiological saline was added for continuous rinsing twice; 2 ml of 0.25 wt% trypsin-EDTA solution was added, and digestion was carried out at 37°C for 2 min; 2 ml of complete culture medium was added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant was removed; the cell pellet was resuspended in the cryopreservation solution at 1E6 cells / ml / tube; the cryopreservation tube was transferred to a pre-cooled program cooling instrument for program cooling; after the cooling was completed, it was transferred to the inspection tank, and after passing the inspection, it was transferred to the liquid nitrogen tank for storage; 4) Recovery of porcine umbilical cord mesenchymal stem cells: Take out one tube of porcine umbilical cord mesenchymal stem cells frozen in liquid nitrogen, quickly place it in a 37°C water bath, shake it, and take it out when the ice in the cryopreserved tube is almost completely melted; transfer the cell suspension in the cryopreserved tube into 10 ml of complete culture medium in an ultra-clean workbench; centrifuge at 400g for 3 minutes; remove the supernatant, resuspend the cells in complete culture medium, and centrifuge at 8×10 3 / cm 2 Cell number for inoculation and culture; 5) Preparation of pig umbilical cord extract: After the pig umbilical cord mesenchymal stem cells were cultured to P5, they were added with complete medium containing serum for 24 hours, the complete medium was removed, and basal medium without serum was added for further 48 hours. The culture supernatant was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris to obtain the pig umbilical cord extract. The obtained pig umbilical cord tissue extract had the following technical indicators: ① Pig umbilical cord tissue extract is a colorless to light yellow transparent liquid with a characteristic odor; ②The protein concentration of pig umbilical cord tissue extract is >6mg / mL.
[0010] In the present invention: In step 1), the physiological saline with penicillin and streptomycin added thereto comprises 2% (v / v) penicillin and 2% (v / v) streptomycin.
[0011] The pig umbilical cord tissue extract obtained by the above method of extracting pig umbilical cord tissue components by using the hypoxic cell culture method has the following technical indicators: ① Pig umbilical cord tissue extract is a colorless to light yellow transparent liquid with a characteristic odor; ②The protein concentration of pig umbilical cord tissue extract is >6mg / mL.
[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of extracting pig umbilical cord tissue components by hypoxic cell culture method described in the present invention can achieve high-purity component extraction by extracting pig umbilical cord tissue components by cell culture method, and optimize cell proliferation and synthesis of bioactive components through controllable culture conditions, thereby improving extraction efficiency and effectiveness. In addition, the method reduces the risk of immune response and supports standardized and large-scale production.
[0013] 2. The method of extracting pig umbilical cord tissue components using a hypoxic cell culture method described in the present invention can simulate the in vivo microenvironment, promote the proliferation and differentiation of mesenchymal stem cells, protect cell activity, reduce oxidative stress, and improve the synthesis and extraction efficiency of bioactive components, thereby improving the extraction efficiency and effectiveness of umbilical cord extracts. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a cell morphology diagram of DMEM P5-D03 in a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method according to an embodiment of the present invention; Figure 2 This is a cell morphology diagram of Fenghui P5-D03 in a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method as described in an embodiment of the present invention; Figure 3 This is a cell morphology diagram of T4 P5-D03 in a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method as described in an embodiment of the present invention; Figure 4 This is a graph showing the protein content of a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method according to an embodiment of the present invention; Figure 5 This is a cell morphology diagram of hypoxic P5-D03 in a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method according to an embodiment of the present invention; Figure 6This is a cell morphology diagram of normoxic P5-D03 in a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method according to an embodiment of the present invention; Figure 7 This is a graph showing the protein content of a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method as described in an embodiment of the present invention. DETAILED DESCRIPTION
[0015] The present invention will be more fully understood through the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are only exemplary of the present invention, which can be embodied in various forms. Therefore, the specific functional details disclosed herein should not be interpreted as limiting, but only as the basis of the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in different ways in virtually any appropriate detailed embodiment.
[0016] The present invention is further described in detail below by way of examples, but these examples should not be considered as limiting the present invention.
[0017] Example: A method for extracting pig umbilical cord tissue components using a hypoxic cell culture method comprises the following steps: 1) Primary isolation and culture of porcine umbilical cord mesenchymal stem cells: After the newborn piglets are born, the umbilical cord is cut with sterile surgical instruments and bled under sterile conditions. After the bleeding is complete, the umbilical cord is ligated in sections from the end close to the fetus, and then cut off at intervals. The umbilical cord with both ends ligated is placed in the prepared physiological saline with penicillin and streptomycin, and quickly transported to the laboratory; Under sterile conditions, the umbilical cord tissue was removed and repeatedly rinsed with physiological saline, and then the ligated parts at both ends were discarded, and the ligated inner umbilical cord was taken out and rinsed again. After the rinsing fluid became clear, it was placed in a sterile culture dish; along the longitudinal axis of the umbilical cord, the outer layer of connective tissue was cut open, and two arteries and one vein were extracted from the incision, and the remaining tissue was placed in a sterile culture dish; Glial digestion method: Under sterile conditions, enlarge the incision, turn the umbilical cord tissue cavity in the sterile culture dish in the previous step upward, fix it, gently scrape the gelatinous part of the umbilical cord with a blade, collect the scraped gelatinous, cut it into 1mm size, add 2.0mg / mL collagenase I mixed with 1mg / mL hyaluronidase, digest it at 37℃ for 60min, and then blow it repeatedly; terminate the digestion with culture medium containing 10% (v / v) FBS, filter it with a 200-mesh cell sieve, collect the filtrate, centrifuge it, filter it, resuspend it and inoculate it into a cell culture bottle, and place it in a cell culture incubator at 37℃ for constant temperature culture; 2) Passaging of porcine umbilical cord mesenchymal stem cells: Observe under a microscope. When the cells cultured in the cell culture incubator show 90%-95% fusion at the bottom of the culture dish, cell passage is performed. Remove the culture medium and add PBS to rinse twice continuously. Add 2 ml of 0.25 wt% trypsin-EDTA solution and digest at 37°C for 2 min. Add an appropriate amount of complete culture medium to terminate the digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 400 g for 3 min. Remove the supernatant. Resuspend the cell pellet with complete culture medium and centrifuge at 8 × 10 3 / cm 2 The number of cells inoculated for subculture; 3) Cryopreservation of porcine umbilical cord mesenchymal stem cells: Reagent preparation, freezing solution: basal medium + 20% fetal bovine serum + 10% DMSO; Under the microscope, when the P1 generation porcine umbilical cord mesenchymal stem cells showed 90%-95% fusion, the cells were cryopreserved; the culture medium was removed, and physiological saline was added for continuous rinsing twice; 2 ml of 0.25 wt% trypsin-EDTA solution was added, and digestion was carried out at 37°C for 2 min; an appropriate amount of complete culture medium was added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant was removed; the cell pellet was resuspended in the cryopreservation solution at 1E6 cells / ml / tube; the cryopreservation tube was transferred to a pre-cooled program cooling instrument for program cooling; after the cooling was completed, it was transferred to the inspection tank, and after the inspection was qualified, it was transferred to the liquid nitrogen tank for storage; 4) Recovery of porcine umbilical cord mesenchymal stem cells: Take out one tube of porcine umbilical cord mesenchymal stem cells frozen in liquid nitrogen, quickly place it in a 37°C water bath, shake it, and take it out when the ice in the cryopreserved tube is almost completely melted; transfer the cell suspension in the cryopreserved tube into 10 ml of complete culture medium in an ultra-clean workbench; centrifuge at 400g for 3 minutes; remove the supernatant, resuspend the cells in complete culture medium, and centrifuge at 8×10 3 / cm 2 Cell number for inoculation and culture; 5) Preparation of pig umbilical cord extract: After the pig umbilical cord mesenchymal stem cells were cultured to P5, they were added with complete medium containing serum and cultured for 24 hours, then the complete medium was removed and serum-free basal medium was added to continue culturing for 48 hours. The culture supernatant was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris to obtain the pig umbilical cord extract.
[0018] Experimental Example 1: BCA protein concentration assay kit detection method: Prepare BSA standard system: Use standard diluent to dilute the standard in series.
[0019] Prepare BCA working solution: Prepare BCA working solution: add 1 volume of BCA reagent B to 50 volumes of BCA reagent A (A:B=50:1) and mix thoroughly.
[0020] Take 25 μL of each standard and sample to be tested and add them to the microplate.
[0021] Add 200 μl of BCA working solution to each well, shake for 30 seconds to mix thoroughly, cover the microplate, and incubate at 37°C for 30 minutes.
[0022] After cooling to room temperature, the absorbance was measured on a microplate reader at a wavelength range of 540-595 nm, with 562 nm being the best.
[0023] The sample protein concentration was calculated based on the standard curve and the sample OD value.
[0024] Experimental Example 2: A process study on a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method described in this application 1. Compare the protein content of umbilical cord extracts extracted from the cell supernatants of porcine umbilical cord mesenchymal stem cells cultured in different culture media, and determine which culture medium can culture more cells and extract higher concentrations of umbilical cord extracts; then select this culture medium to study whether hypoxia or normoxia is better.
[0025] The primary porcine umbilical cord mesenchymal stem cells digested from the same porcine umbilical cord were divided into three parts, cultured and passaged to the P5 generation using DMEM medium, T4 medium, and Fenghui Biological Umbilical Cord Mesenchymal Stem Cell Medium, respectively. The preparation was then completed according to the above-mentioned porcine umbilical cord extract preparation method, and the protein concentrations of the porcine umbilical cord extracts extracted using the three culture media were detected using a microspectrophotometer.
[0026] Experimental results: Cell morphology: Figure 1 Cell morphology of DMEM P5-D03; Figure 2 Cell morphology of Fenghui P5-D03; Figure 3 Cell morphology of T4 P5-D03; Figure 4 A graph showing the protein content of a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method as described in an embodiment;
[0027] The experimental results show that the pig umbilical cord mesenchymal stem cells cultured with Fenghui Biological Umbilical Cord Mesenchymal Stem Cell Culture Medium proliferate the fastest, followed by those cultured with T4 culture medium, and the slowest by those cultured with DMEM culture medium; at the same time, the umbilical cord extract extracted from the supernatant of Fenghui Biological Umbilical Cord Mesenchymal Stem Cell Culture Medium has the highest content, followed by that extracted from the supernatant of T4 culture medium, and the lowest by that extracted from the supernatant of DMEM culture medium.
[0028] 2. Comparison of the protein content of umbilical cord extracts extracted from the cell supernatants of porcine umbilical cord mesenchymal stem cells cultured in different culture methods (normoxia, hypoxia): Normoxia: 20% oxygen, 5% carbon dioxide; Hypoxia: 5% oxygen, 5% carbon dioxide.
[0029] The primary porcine umbilical cord mesenchymal stem cells digested from the same porcine umbilical cord were divided into two parts, cultured in normoxia and hypoxia with Fenghui Biological Umbilical Cord Mesenchymal Stem Cell Culture Medium to the P5 generation, and then prepared according to the above-mentioned porcine umbilical cord extract preparation method. The protein concentration of the porcine umbilical cord extracts extracted using the two different culture methods was detected using a microspectrophotometer.
[0030] Experimental results: Cell morphology: Figure 5 Cell morphology of hypoxic P5-D03; Figure 6 Cell morphology of normoxic P5-D03; Figure 7 A graph showing the protein content of a method for extracting pig umbilical cord tissue components using a hypoxic cell culture method as described in an embodiment;
[0031] The experimental results showed that the proliferation rate of porcine umbilical cord mesenchymal stem cells cultured using hypoxic culture method was slightly faster than that of porcine umbilical cord mesenchymal stem cells cultured using normoxic culture method; the concentration of umbilical cord extract extracted from the culture supernatant using hypoxic culture method was higher than that extracted from the culture supernatant using normoxic culture method.
[0032] Results and Discussion: The present invention utilizes a hypoxic cell culture method to extract components of pig umbilical cord tissue, which can achieve high-purity component extraction, optimize cell proliferation and synthesis of bioactive components through controllable culture conditions, thereby improving extraction efficiency and effectiveness. In addition, the method reduces the risk of immune response and supports standardized and large-scale production. At the same time, the hypoxic environment can simulate the in vivo microenvironment, promote the proliferation and differentiation of mesenchymal stem cells, protect cell activity, reduce oxidative stress, and improve the synthesis and extraction efficiency of bioactive components, thereby improving the extraction efficiency and effectiveness of umbilical cord extracts.
[0033] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.
Claims
1. A method for extracting pig umbilical cord tissue components using a hypoxic cell culture method, characterized in that: The following steps are involved: The hypoxia described: 5% oxygen, 5% carbon dioxide; 1) Primary isolation and culture of porcine umbilical cord mesenchymal stem cells: After the newborn piglets are born, the umbilical cord is cut with sterile surgical instruments and bled under sterile conditions. After the bleeding is complete, the umbilical cord is ligated in sections from the end close to the fetus, and then cut off at intervals. The umbilical cord with both ends ligated is placed in the prepared physiological saline with penicillin and streptomycin, and quickly transported to the laboratory; Under sterile conditions, the umbilical cord tissue was removed and repeatedly rinsed with physiological saline, and then the ligated parts at both ends were discarded, and the ligated inner umbilical cord was taken out and rinsed again. After the rinsing fluid became clear, it was placed in a sterile culture dish; along the longitudinal axis of the umbilical cord, the outer layer of connective tissue was cut open, and two arteries and one vein were extracted from the incision, and the remaining tissue was placed in a sterile culture dish; Gel digestion method: Under sterile conditions, enlarge the incision, place the umbilical cord tissue cavity in the sterile culture dish in the previous step facing up, and after fixation, gently scrape the gelatinous part of the umbilical cord with a blade, collect the scraped gelatinous material, cut it into 1 mm size, add 2.0 mg / mL collagenase I mixed with 1 mg / mL hyaluronidase, digest at 37°C for 60 minutes, and then repeatedly blow and beat; terminate the digestion with culture medium containing 10% v / v fetal bovine serum, filter with a 200-mesh cell sieve, collect the filtrate, centrifuge, filter, resuspend and inoculate into a cell culture bottle, and place it in a cell culture incubator at 37°C for constant temperature culture; 2) Passaging of porcine umbilical cord mesenchymal stem cells: Under microscope observation, when the cells cultured in the cell culture incubator showed 90%-95% fusion at the bottom of the culture dish, the cells were passaged; the culture medium was removed, and PBS was added for continuous rinsing twice; 2 ml of 0.25 wt% trypsin-EDTA solution was added, and the cells were digested at 37°C for 2 min; 2 ml of complete culture medium was added to terminate the digestion, and the cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant was removed; the cell pellet was resuspended with complete culture medium and centrifuged at 8 × 10 3 / cm 2 The number of cells inoculated for subculture; 3) Cryopreservation of porcine umbilical cord mesenchymal stem cells: Reagent preparation, freezing solution: basal medium + 20% fetal bovine serum + 10% DMSO; Under a microscope, when the P1 generation porcine umbilical cord mesenchymal stem cells show 90%-95% fusion, the cells are cryopreserved; the culture medium is removed, and physiological saline is added for continuous rinsing twice; 2 ml of 0.25wt% trypsin-EDTA solution is added, and digestion is carried out at 37°C for 2 min; 2 ml of complete culture medium is added to terminate digestion, and the cell suspension is transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant is removed; the cell pellet is resuspended in the cryopreservation solution at 1E6 cells / ml / tube; the cryopreservation tube is transferred to a pre-cooled program cooling instrument for program cooling; after the cooling is completed, it is transferred to the inspection tank, and after the inspection is qualified, it is transferred to the liquid nitrogen tank for storage; 4) Resuscitation of porcine umbilical cord mesenchymal stem cells: Take out one tube of porcine umbilical cord mesenchymal stem cells frozen in liquid nitrogen, quickly place it in a 37°C water bath, shake it, and take it out when the ice in the cryopreserved tube is almost completely melted; transfer the cell suspension in the cryopreserved tube into 10 ml of complete culture medium in an ultra-clean workbench; centrifuge at 400g for 3 minutes; remove the supernatant, resuspend the cells in complete culture medium, and centrifuge at 8×10 3 / cm 2 Cell number for inoculation and culture; 5) Preparation of pig umbilical cord extract: After the pig umbilical cord mesenchymal stem cells were cultured to P5, they were added with complete medium containing serum for 24 hours, the complete medium was removed, and basal medium without serum was added for further 48 hours. The culture supernatant was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris to obtain the pig umbilical cord extract. The obtained pig umbilical cord tissue extract had the following technical indicators: ① Pig umbilical cord tissue extract is a colorless to light yellow transparent liquid with a characteristic odor; ②The protein concentration of pig umbilical cord tissue extract is >6mg / mL.
2. The method for extracting pig umbilical cord tissue components by hypoxic cell culture method according to claim 1, characterized in that: In the physiological saline added with penicillin and streptomycin in step 1), the amount of penicillin added is 2% v / v, and the amount of streptomycin added is 2% v / v.
3. A porcine umbilical cord tissue extract obtained by a method for extracting porcine umbilical cord tissue components using a hypoxic cell culture method, characterized in that: The method for extracting pig umbilical cord tissue components by using a hypoxic cell culture method according to claim 1 or 2 is used to obtain the pig umbilical cord tissue extract, which has the following technical indicators: ① Pig umbilical cord tissue extract is a colorless to light yellow transparent liquid with a characteristic odor; ②The protein concentration of pig umbilical cord tissue extract is >6mg / mL.