Hydrogel based on umbilical cord tissue and chorion tissue and application thereof

Abstract

The invention discloses hydrogel based on umbilical cord tissue and chorion tissue, which is prepared by a method comprising the following steps: (1) taking a placenta, shearing the umbilical cord and separating the chorion; (2) respectively carrying out tissue crushing on the umbilical cord and the chorion, and centrifugally collecting precipitates; mixing, washing, centrifuging and collecting precipitates; (3) freezing with liquid nitrogen, and then melting; repeatedly freezing and thawing; adding a buffer solution containing triton-X-100, and carrying out soaking treatment; rinsing, centrifuging and collecting precipitates; (4) adding urea, oscillating, centrifuging and collecting precipitate; freezing and drying to obtain freeze-dried powder; (5) dissolving the freeze-dried powder in hydrochloric acid, adding pepsin, and reacting; and adjusting the pH value to 7-7.5 to obtain the hydrogel. The hydrogel is applied to culture of cells, tissues or organoids, and is applied to preparation of a culture medium with the effect of promoting angiogenesis. The method is of great significance to development in the fields of regenerative medicine, disease modeling, medicine development and the like.

Description

Technical Field

[0001] This invention relates to a hydrogel based on umbilical cord tissue and chorionic villi tissue and its applications, belonging to the field of biomatrix technology. Background Technology

[0002] With the rapid development of tissue engineering and organoid technology, the demand for high-performance, ethically sound extracellular matrix (ECM) simulation materials is becoming increasingly urgent. Traditional cell culture models mainly rely on two-dimensional environments on culture dishes, which cannot reproduce the complex three-dimensional tissue structures and intercellular interactions in vivo, greatly limiting their application value in disease mechanism research and precise drug testing. With the rise of cutting-edge technologies such as organoids, the demand for in vitro models capable of simulating the complex physiological environments of organs and tumors is growing. An ideal culture medium should be able to simulate the extracellular matrix, providing cells with a three-dimensional growth space and constructing a microenvironment containing physical structures and dynamic biochemical signals, thereby enabling more realistic in vitro disease research and drug testing. However, the core matrix gel materials currently widely used are derived from a single source, mainly from the basement membrane matrix extracted from mouse sarcomas. This animal-derived material poses significant ethical risks; its components have tumor-derived characteristics and inherent limitations in simulating specific tissues. It may not be able to provide a suitable physiological microenvironment for highly specific organoids such as the heart and nerves, and may even introduce culture biases, affecting the reliability of experimental results. Therefore, developing an in vitro culture medium without ethical risks that can more accurately simulate complex physiological environments is of great significance. Summary of the Invention

[0003] In view of the above-mentioned prior art, the present invention provides a hydrogel based on umbilical cord tissue and chorionic villi tissue and its application.

[0004] This invention is achieved through the following technical solution: A method for preparing a hydrogel based on umbilical cord tissue and chorionic villi tissue includes the following steps: (1) Remove the placenta, cut the umbilical cord, and separate the chorion; (2) The umbilical cord and chorion were respectively subjected to tissue disruption, and the precipitate was collected by centrifugation to obtain umbilical cord homogenate and chorion homogenate; the umbilical cord homogenate and chorion homogenate were mixed at a volume ratio of (1~2):1, washed with sodium chloride solution, and the precipitate was collected by centrifugation to obtain mixture A; (3) Mixture A was frozen with liquid nitrogen and then thawed; the freeze-thaw cycle was repeated; after the last thaw, a buffer solution containing Triton-X-100 was added and the mixture was soaked; it was rinsed with sodium chloride solution, and the precipitate was collected by centrifugation to obtain mixture B; (4) Add urea to mixture B, shake at low temperature, centrifuge to collect the precipitate; freeze dry to obtain freeze-dried powder; (5) Dissolve the lyophilized powder in hydrochloric acid, add pepsin, and react; after the reaction, adjust the pH to 7-7.5 to obtain the hydrogel.

[0005] Furthermore, in step (1), after cutting the umbilical cord and separating the chorion, the umbilical cord and chorion are respectively soaked in 75% ethanol solution for sterilization, and then rinsed with deionized water.

[0006] Furthermore, in step (2), the volume ratio of umbilical cord homogenate to chorionic villus homogenate is 1:1 or 2:1; the concentration of sodium chloride solution is 1 mol / L.

[0007] Further, in step (3), the liquid nitrogen freezing time is 30 min; the thawing method is a 37°C water bath; the buffer is PBS, and the concentration of Triton-X-100 in the buffer is 1%; the soaking conditions are: soaking at 4°C for 12 hours; and the concentration of sodium chloride solution is 3 mol / L.

[0008] Furthermore, in step (4), the concentration of urea after addition is 3 mol / L; the low-temperature shaking conditions are 10-20℃, 24 rpm / min shaking for 24 hours; the freeze-drying method is: freezing at -80℃ for 12 hours, and vacuum freeze-drying at -40℃ for 24 hours.

[0009] Furthermore, in step (5), the concentration of hydrochloric acid is 0.01 mol / L; the enzymatic hydrolysis reaction time is 6 hours.

[0010] The hydrogel prepared using the above method combines the advantages of the strong angiogenesis-promoting ability of the chorionic membrane and the suitable natural structure of the umbilical cord, and can be used for the culture of cells, tissues and organoids.

[0011] The application of the hydrogel as a culture medium in the culture of cells, tissues or organoids.

[0012] Furthermore, the cells include stem cells and immune cells; the stem cells include mesenchymal stem cells (MSCs) and iPS cells from various tissue sources; the immune cells include mononuclear cells, NK cells, CIK cells, TIL cells, CAR-T cells, etc., derived from umbilical cord blood and peripheral blood.

[0013] The application of the hydrogel in the preparation of a culture medium that promotes angiogenesis.

[0014] The hydrogel of this invention is prepared from umbilical cord tissue and chorionic villus tissue. The placental membranes, as a crucial interface connecting the fetus and mother, are mainly composed of the amnion and chorion. The amnion is close to the fetus, and the chorion surrounds the amnion and is tightly fused with the maternal decidua. This results in a significantly higher content of natural extracellular matrix proteins and growth factors in the chorion compared to the amnion. Furthermore, the chorion contains vascular structures from the decidua, making it more closely resemble natural vascularized tissue and possessing stronger angiogenesis potential compared to other parts of the placenta. The umbilical cord, the channel connecting the placenta and fetus, is composed of Wharton's jelly and two arteries and one vein contained within it. Its extracellular matrix is ​​rich in hyaluronic acid, collagen, glycosaminoglycans, and growth factors. The umbilical cord's vascular wall structure is unique, lacking nerve innervation, and its smooth muscle arrangement results in a contraction mechanism different from that in the body. Its composition and structural characteristics help the umbilical cord form a low-oxygen, low-vascularization three-dimensional gel microenvironment. The decellularized extracellular matrix also retains its three-dimensional porous structure, making it an ideal tissue engineering scaffold material. Both raw materials of this invention have the characteristics of low immunogenicity and their acquisition does not involve ethical disputes.

[0015] This invention innovatively utilizes human placental tissue, a natural biological resource, to prepare a hydrogel by combining Wharton's jelly from the umbilical cord with placental chorionic villi. Wharton's jelly is a water-rich gel-like connective tissue within the umbilical cord, derived from the extraembryonic mesoderm, possessing excellent natural gelling properties and a protective effect on blood vessels. Placental chorionic villi, on the other hand, has a strong ability to promote angiogenesis. This invention combines the two in different proportions, perfectly integrating the superior three-dimensional structural support and shaping capabilities of Wharton's jelly with the bioactivity of chorionic villi in guiding angiogenesis. The humanized hydrogel developed using this design strategy not only avoids ethical and safety issues associated with animal-derived products but also provides a more physiologically relevant and precise in vitro culture system for various tissue-specific organoids (especially tissue models highly dependent on vascular networks) through the synergistic simulation of key elements of the human natural microenvironment (structural support and vascularization). This invention has significant implications for the development of regenerative medicine, disease modeling, and drug development.

[0016] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0017] Figure 1 The results of the gelation test of hydrogels are shown, from left to right: villous hydrogel, mixed hydrogel A, mixed hydrogel B, and mixed hydrogel C.

[0018] Figure 2 Schematic diagram of staining results from angiogenesis assay.

[0019] Figure 3 Schematic diagram of the staining results of calcein AM and propidium iodide. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0021] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0022] Example 1: Preparation of hydrogel The steps are as follows: (1) Collect the placenta, cut the umbilical cord, and separate the chorion. The umbilical cord and chorion are initially sterilized by soaking in 75% ethanol solution (volume percentage), and then rinsed with deionized water.

[0023] (2) Add a small amount of deionized water to the umbilical cord and chorion respectively, use a handheld electric homogenizer to break up the tissue, centrifuge to collect the precipitate, and obtain umbilical cord homogenate and chorion homogenate respectively; mix the umbilical cord homogenate and chorion homogenate in different volume ratios (3 ratios were investigated, namely: 1:2, 1:1, 2:1), wash with 1 mol / L sodium chloride solution, centrifuge to collect the precipitate, and obtain mixture A.

[0024] (3) Mixture A was soaked in liquid nitrogen and frozen for 30 min, and then thawed in a water bath at 37°C; the freeze-thaw cycle was repeated 5 times; after the last thaw, PBS containing 1% triton-X-100 was added, and the mixture was soaked at 4°C for 12 hours; it was rinsed with 3 mol / L sodium chloride solution, and the precipitate was collected by centrifugation to obtain mixture B.

[0025] (4) Add urea to mixture B to make its concentration 3 mol / L, shake at low temperature (15℃) (24 rpm / min) for 24 hours, centrifuge to collect the precipitate; freeze at deep low temperature (-80℃) for 12 hours, freeze dry in vacuum (-40℃) for 24 hours to obtain freeze-dried powder.

[0026] (5) Take 50 mg of dry powder, dissolve it in 5 ml of hydrochloric acid (concentration of 0.01 mol / L), add pepsin (pepsin enzyme efficiency 1:10000, dissolve it in 0.01 mol / L hydrochloric acid beforehand to prepare 5 mg / ml pepsin working solution), stir magnetically for 6 hours; adjust the pH to 7-7.5 with hydrochloric acid or sodium hydroxide solution to obtain hydrogel, and let it stand at 37℃ for 30-60 min to form a gel.

[0027] In the above centrifugation to collect the precipitate, the centrifugation parameters were: 700 g, acceleration rate 9, deceleration rate 5, and centrifugation time 5 min.

[0028] Experiment 1: Hydrogel gelation test The mixed hydrogels (the three hydrogels prepared in Example 1, which were prepared with different ratios of umbilical cord tissue to chorionic villus tissue, wherein the mixed hydrogel with chorionic villus:umbilical cord = 2:1 is called mixed hydrogel A, the mixed hydrogel with chorionic villus:umbilical cord = 1:1 is called mixed hydrogel B, and the mixed hydrogel with chorionic villus:umbilical cord = 1:2 is called mixed hydrogel C; the same applies below) and chorionic hydrogels (the preparation method of chorionic hydrogels is the same as in Example 1, except that in step 2, they are not mixed with umbilical cord homogenate) were transferred to 1.5 mL centrifuge tubes, labeled, and frozen in a -20°C freezer for 48 hours.

[0029] Remove the frozen hydrogel sample and thaw it in a 4°C freezer for 4 hours. After the sample is completely thawed, incubate it in a 37°C water bath for 2 hours and observe whether the sample has formed a gel. The entire experiment was conducted under sterile, low-temperature conditions.

[0030] Result: As Figure 1 As shown, individual villous hydrogels and mixed hydrogel A cannot form a gel, while mixed hydrogels B and C can form relatively strong hydrogels.

[0031] Experiment 2 Protein Component Analysis The protein composition of the mixed hydrogels (three hydrogels prepared in Example 1: mixed hydrogel A, mixed hydrogel B, and mixed hydrogel C) and the villous hydrogels was analyzed, and the results are shown in Table 1.

[0032] Table 1 project Measurement methods vellum hydrogel Mixed hydrogel A Mixed hydrogel B Mixed hydrogel C Total protein Biuret method 750 μg / mg dry weight 730 μg / mg dry weight 700 μg / mg dry weight 650 μg / mg dry weight Collagen Hydroxyproline assay 448 μg / mg dry weight 426 μg / mg dry weight 400 μg / mg dry weight 360μg / mg dry weight Sulfated glycosaminoglycans 1,9-Dimethylmethylene blue method 4μg / mg dry weight 6μg / mg dry weight 8μg / mg dry weight 11 μg / mg dry weight Laminin Enzyme-linked immunosorbent assay (ELISA) 2512 pg / mg dry weight 2256 pg / mg dry weight 2130 pg / mg dry weight 1500 pg / mg dry weight Vascular endothelial growth factor (VEGF) Enzyme-linked immunosorbent assay (ELISA) 14 pg / mg dry weight 12 pg / mg dry weight 10 pg / mg dry weight 2 pg / mg dry weight

[0034] The experimental results showed that hydrogels with higher chorionic content had higher total protein, collagen, and laminin content, and lower sulfated glycosaminoglycan content than hydrogels with lower chorionic content. In terms of VEGF content, chorionic hydrogels had the highest content, while mixed hydrogels had the lowest C content.

[0035] Experiment 3 Angiogenesis Experiment 5 mL of human umbilical vein endothelial cell (HUVECs) culture medium (cell density 4 × 10⁻⁶) was added. 6 Mix 5 mL of hydrogel B with 10 mL of FBS (Gibco) to ensure uniform cell distribution. Then add 10 mL of cell culture medium (M199 medium supplemented with 10% FBS (Gibco)) and culture under standard conditions (37℃, 5% CO2), changing the medium every 2 days. The experiment was conducted at multiple time points (3 days, 7 days, 14 days) to assess the dynamic process of vascular network formation and its long-term stability. At each predetermined time point, the hydrogel was removed and fixed with formaldehyde solution. Immunofluorescence staining was performed: CD31 (red), a key protein at the junction of endothelial cells, was labeled with a specific antibody; the actin backbone of all cells was labeled with Phalloidin (green); and the cell nuclei were labeled with DAPI (blue). The stained hydrogel was scanned three-dimensionally using a laser confocal microscope to obtain high-resolution images.

[0036] Result: As Figure 2 As shown, on day 3, HUVECs had not yet formed obvious tubular structures in the mixed hydrogel, and CD31 signaling was dispersed; at this time, the cells were in the adaptation and migration phase. On day 7, it could be clearly observed that HUVECs had self-organized into well-connected, branched tubular structures (CD31 was distributed in a continuous linear pattern). On day 14, it was evident that the mixed hydrogel provided a long-term, supportive microenvironment for angiogenesis.

[0037] Experiment 4 Cytotoxicity Experiment Add 5 mL of human umbilical cord mesenchymal stem cell culture medium (cell density 5 × 10⁶ cells / mL) 6 Mix 5 mL of hydrogel B with 1 mL of calcein AM (for live cells, green) and 5 mL of hydrogel B, and culture for 6 days under standard conditions (37°C, 5% CO2). After culture, staining was performed using calcein AM (for live cells, green) and propidium iodide (for dead cells, red), and the results were observed under a fluorescence microscope. Figure 3 As shown, the vast majority of human umbilical cord mesenchymal stem cells survived, with only a small number of dead cells. This indicates that the hydrogel of the present invention has excellent cell compatibility and a high cell survival rate.

[0038] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A method for preparing a hydrogel based on umbilical cord tissue and chorionic villi tissue, characterized in that, Includes the following steps: (1) Remove the placenta, cut the umbilical cord, and separate the chorion; (2) The umbilical cord and chorion were respectively subjected to tissue disruption, centrifugation to collect the precipitate, and umbilical cord homogenate and chorion homogenate were obtained; the umbilical cord homogenate and chorion homogenate were mixed at a volume ratio of (1~2):1, washed, centrifuged to collect the precipitate, and mixture A was obtained. (3) Mixture A was frozen with liquid nitrogen and then thawed; the freeze-thaw cycle was repeated; after the last thaw, a buffer solution containing Triton-X-100 was added and the mixture was soaked. Rinse, centrifuge to collect the precipitate, and obtain mixture B; (4) Add urea to mixture B, shake, centrifuge to collect the precipitate; freeze dry to obtain freeze-dried powder; (5) Dissolve the lyophilized powder in hydrochloric acid, add pepsin, and react; after the reaction, adjust the pH to 7-7.5 to obtain the hydrogel.

2. The method for preparing hydrogels based on umbilical cord tissue and chorionic villi tissue according to claim 1, characterized in that: In step (1), after cutting the umbilical cord and separating the chorionic membrane, the cord is sterilized by soaking in 75% ethanol solution and then rinsed with deionized water.

3. The method for preparing hydrogels based on umbilical cord tissue and chorionic villi tissue according to claim 1, characterized in that: In step (2), the volume ratio of umbilical cord homogenate to chorionic villus homogenate is 1:1 or 2:1; rinsing is performed using a sodium chloride solution with a concentration of 1 mol / L.

4. The method for preparing hydrogel based on umbilical cord tissue and chorionic villi tissue according to claim 1, characterized in that: In step (3), the liquid nitrogen freezing time is 30 min; the thawing method is a 37°C water bath; the number of freeze-thaw cycles is 5; the buffer is PBS, and the concentration of Triton-X-100 in the buffer is 1%; the soaking conditions are: soaking at 4°C for 12 hours; rinsing is done with a 3 mol / L sodium chloride solution.

5. The method for preparing hydrogel based on umbilical cord tissue and chorionic villi tissue according to claim 1, characterized in that: In step (4), the concentration of urea after addition is 3 mol / L; the low-temperature shaking conditions are 10-20℃, 24 rpm / min shaking for 24 hours; the freeze-drying method is: freezing at -80℃ for 12 hours, and vacuum freeze-drying at -40℃ for 24 hours.

6. The method for preparing hydrogel based on umbilical cord tissue and chorionic villi tissue according to claim 1, characterized in that: In step (5), the concentration of hydrochloric acid is 0.01 mol / L; the enzymatic hydrolysis reaction time is 6 hours.

7. A hydrogel prepared by the method for preparing hydrogels based on umbilical cord tissue and chorionic villi tissue according to any one of claims 1 to 6.

8. The use of the hydrogel of claim 7 in culturing cells, tissues or organoids.

9. The application according to claim 8, characterized in that: The cells are selected from stem cells or immune cells.

10. The use of the hydrogel of claim 7 in the preparation of a culture medium with angiogenesis-promoting effects.

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