Calcium silicate composite hydrogel, and preparation method and application thereof

Abstract

The application belongs to the field of biomaterials, and particularly relates to a calcium silicate composite hydrogel as well as a preparation method and application thereof. In view of the limitation of the existing hydrogel matrix in supporting the development of organoids, the application provides a calcium silicate composite hydrogel, which comprises calcium silicate, methacrylated gelatin, a dispersion medium and Matrigel; preferably, the volume content of the Matrigel is 0-10 vol%. The calcium silicate composite hydrogel of the application can be used as a culture material for supporting the development of organoids, and the addition of inorganic bioactive materials provides an effective, high-biosafety and low-cost strategy for developing organoid culture additives.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to hydrogel materials supporting organoid culture, and particularly to a method for preparing and applying organoid culture substrates containing the inorganic active material calcium silicate. The organoid culture substrate comprises methacrylamide gelatin, calcium silicate, and matrix gel. The modified organoid culture medium consists of organoid culture medium and calcium silicate. Background Technology

[0002] Organoids are complex three-dimensional (3D) multicellular constructs that are formed by regulating the behavior of stem cells. They can mimic the structure and function of real tissues or organs and have promising biological applications, including as 3D in vitro models to simulate tissue development and homeostasis, drug screening, disease modeling, and in tissue engineering and regenerative medicine.

[0003] Matrix materials are crucial for organoid development. Organoid culture requires a matrix material to provide a 3D microenvironment conducive to stem cell growth and development. Matrigel, a representative of matrix materials, is widely used as a supportive matrix material for organoid development due to its rich extracellular matrix (ECM) content. However, matrix materials have several insurmountable drawbacks, including poor reproducibility of organoids due to batch-to-batch variability, and safety concerns arising from the source of the matrix material for clinical applications. To address these limitations, researchers have developed various three-dimensional matrix materials for organoid culture. Among them, natural hydrogels (such as fibrin and hyaluronic acid) and synthetic hydrogels (such as PEG hydrogels) have been developed for organoid culture. However, these currently developed hydrogel matrices also exhibit some limitations in supporting organoid development, such as the limited bioactivity of natural hydrogels and the complex preparation and cross-linking methods of synthetic hydrogels. Summary of the Invention

[0004] The purpose of this invention is to prepare a calcium silicate-containing matrix material to support the growth and development of organoids. Addressing the problems of existing technologies, this invention provides an optimized calcium silicate composite hydrogel, its preparation method, and its application in preparing materials to support organoid culture. Furthermore, this invention utilizes calcium silicate as an additive to improve the original organoid culture medium, preparing a modified organoid culture medium that further promotes organoid development.

[0005] In a first aspect, the present invention provides a calcium silicate composite hydrogel, wherein the calcium silicate composite hydrogel comprises calcium silicate, methacrylamide gelatin, a dispersion medium and a matrix gel; preferably, the volume content of the matrix gel is 0-10 vol%.

[0006] In this invention, calcium silicate (CS) is selected and is expected to play a positive role in the development of multicellular constructs (organoids) derived from stem cells. Due to its good bioactivity and biodegradability, it has broad prospects for biomedical applications. The released bioactive ions can regulate the self-renewal and pluripotency of stem cells. Methacrylamide gelatin (GelMA) has good biological effects and is widely used. The presence of cell adhesion and matrix metalloproteinase-reactive peptide motifs allows stem cells to proliferate and migrate within GelMA hydrogels. Therefore, in this invention, calcium silicate nanowires rich in bioactive components and a small proportion of matrix gel are incorporated into GelMA hydrogels to construct an optimized calcium silicate composite hydrogel for supporting the growth and development of organoids. Furthermore, the inorganic active material calcium silicate is developed as an additive in organoid culture media to form a modified organoid culture medium to promote organoid development. The calcium silicate-containing matrix material constructed in this patent has strong practical significance in supporting the growth and development of organoids.

[0007] Preferably, the calcium silicate has the morphology of nanowires with a length of 1-10 micrometers and a diameter of 10-50 nm; the content of the calcium silicate does not exceed 4 wt%.

[0008] Preferably, the content of the methacrylamide gelatin is 5-10 wt%.

[0009] Preferably, the dispersion medium is DMEM / F12 culture medium or the original culture medium; the volume content of the dispersion medium is 40-50 vol%.

[0010] Preferably, the main components of the matrix adhesive include laminin and type IV collagen, etc. For example, The brand is Corning, and the product number is 356231.

[0011] In a second aspect, the present invention provides an organoid culture medium, comprising: a dispersion medium, and calcium silicate dispersed in the dispersion medium; preferably, the content of the calcium silicate is not more than 100 μg / mL, and more preferably 50 μg / mL.

[0012] Preferably, the calcium silicate has the morphology of nanowires with a length of 1 to 10 micrometers and a diameter of 10 to 50 nm.

[0013] Preferably, the dispersion medium is DMEM / F12 medium or the original medium.

[0014] Thirdly, the present invention provides a method for preparing a calcium silicate composite hydrogel, comprising: (1) Calcium silicate is dispersed in a dispersion medium to obtain a calcium silicate dispersion; (2) Dissolve methacrylamide gelatin in phosphate buffer to obtain solution 1; (3) Mix the calcium silicate dispersion, dispersion medium and matrix gel to obtain solution 2; (4) Mix solution 2 and solution 1 to obtain the precursor solution of the composite hydrogel; (5) The precursor solution of the obtained composite hydrogel is crosslinked to obtain the calcium silicate composite hydrogel.

[0015] Preferably, in step (1), the mass fraction of calcium silicate in the calcium silicate dispersion is 0-16 wt%; the dispersion method is ultrasonic mixing.

[0016] Preferably, in step (2), the phosphate buffer solution is a 0.5 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) phosphate buffer solution with pH = 7.4; the dissolution temperature is 37-60°C, preferably 55°C; and the mass fraction of methacrylamide gelatin in solution 1 is 5-20 wt%, preferably 5-10 wt%.

[0017] Preferably, in step (3), the volume ratio of the calcium silicate dispersion, the dispersion medium and the matrix adhesive is (0-5):(3-8):2, and more preferably 5:3:2.

[0018] Preferably, in step (4), the volume ratio of solution 2 to solution 1 is (1-3):1, and more preferably 1:1.

[0019] Preferably, in step (5), the crosslinking method is to irradiate the precursor liquid of the composite hydrogel with visible light at a wavelength of 405 nm; the irradiation time is 6 to 20 seconds.

[0020] Fourthly, the present invention provides the use of calcium silicate composite hydrogel in the preparation of organoid culture and development materials.

[0021] Fifthly, the present invention provides the use of an organoid culture medium in the preparation of organoid culture and developmental materials.

[0022] The beneficial effects of this invention are: The optimized calcium silicate composite hydrogel of this invention shows great application potential in supporting organoid growth and development, demonstrating the potential of inorganic active materials to support organoid culture and further expanding the application of inorganic biomaterials. The structural and functional characteristics of intestinal and liver organoids cultured using the optimized calcium silicate composite hydrogel are comparable to those cultured using Matrice, indicating its feasibility and applicability as a Matrice alternative. Subsequent research revealed that calcium silicate nanowires have a certain influence on the stiffness of the composite hydrogel, thereby affecting the expression of the mechanosensory factor YAP in organoids at different culture stages. Furthermore, due to the release of bioactive ions from the calcium silicate nanowires, the culture medium containing calcium silicate nanowires can significantly promote the development of intestinal organoids, including promoting proliferation and differentiation. The presence of bioactive ions also promotes the metabolic activity of intestinal organoids. Therefore, this invention demonstrates that the optimized calcium silicate composite hydrogel can serve as a culture material supporting organoid development, and the addition of inorganic bioactive materials provides an effective, biosafe, and low-cost strategy for developing organoid culture medium additives. Attached Figure Description

[0023] Figure 1 Characterization of the crystal phase and microstructure of calcium silicate. (a) XRD pattern of the prepared calcium silicate nanowires. (b) SEM image of the prepared calcium silicate nanowires. (c) TEM image of the prepared calcium silicate nanowires. The results show that the prepared calcium silicate has a nanowire morphology and good dispersion. Figure 2 The developmental process and statistical results of mouse intestinal organoids cultured in composite hydrogels. GelMA-2CS-10M refers to GelMA containing 2% CS nanowires and 10% matrix gel; other names are similar. (a) Optical images of intestinal organoids cultured in different hydrogels, scale bar: 200 μm. (bd) Statistical results of the formation efficiency of intestinal organoids cultured in different hydrogels for 1, 3, and 5 days. The results show that the optimized calcium silicate composite hydrogel can support the normal growth of organoids. On day 5, the organoid formation efficiency of the GelMA-2CS-10M group was comparable to that of the matrix gel group. Figure 3 To illustrate the differentiation effects of intestinal organoids cultured with different hydrogels. (a) Staining images of differentiated cell types in intestinal organoids cultured with different hydrogels after 5 days of culture, scale bar: 100 μm. (b) Gene expression of four corresponding differentiated cell types in different groups. The results showed that the differentiation capacity of intestinal organoids cultured with the optimized calcium silicate composite hydrogel was comparable to that of the matrix gel group; Figure 4 Bright-field images of intestinal organoids cultured in different hydrogels (GelMA-2CS-10M and Matrigel) after treatment with trichodin. Figure 5 A statistical graph showing the area changes of organoids treated with trichodin before treatment, normalized to the area changes of organoids before trichodin treatment. Figure 6 To enhance the ability of the optimized calcium silicate composite hydrogel to support long-term culture and passage of intestinal organoids. (a) Optical images of intestinal organoids grown in GelMA-2CS-10M hydrogel and Matrigel at passages 0, 2, 4, and 6, scale bar: 200 μm. (bd) Gene expression in intestinal organoids cultured in GelMA-2CS-10M hydrogel and Matrigel at passages 0, 2, and 4. The results show that the optimized calcium silicate composite hydrogel can support long-term expansion and passage of organoids, demonstrating its practicality. Figure 7 YAP expression in intestinal organoids cultured in different hydrogels. (a) YAP staining images in intestinal organoids grown in different hydrogels for 1, 3, and 5 days. Scale bar: 100 μm. (bd) Quantitative statistics of the proportion of nuclear YAP in intestinal organoids. The results show that the proportion of cells with nuclear YAP in intestinal organoids differs due to the change in the hardness of different groups of hydrogels over time; Figure 8 The rheological properties of five groups of hydrogels at different incubation times are shown. (ac) Rheological tests of different hydrogels after soaking in DMEM / F12 medium for 1, 3, and 5 days (37℃). The results show that the addition of a certain proportion of calcium silicate and matrix gel to GelMA reduces its hardness; Figure 9 Ion release from calcium silicate nanowires. (a) Release of calcium silicate nanowires from the composite hydrogel. (b) Ion release of calcium silicate nanowires from the culture medium. The results show that both the calcium silicate composite hydrogel and calcium silicate nanowires can release a certain amount of bioactive ions in DMEM / F12 culture medium; Figure 10 The effects of calcium silicate nanowires on the proliferation and differentiation of intestinal organoids. (a) Proliferation of intestinal organoids cultured with different concentrations of calcium silicate nanowires. (bc) Gene expression of four differentiated cell types in intestinal organoids cultured for 3 and 5 days with medium containing 0, 50 μg / mL, and 100 μg / mL calcium silicate nanowires. (d) Immunofluorescence staining images of four differentiated cell types in intestinal organoids cultured for 3 and 5 days with medium containing 0, 50 μg / mL, and 100 μg / mL calcium silicate nanowires. Scale bar: 100 μm. The results show that the addition of a certain proportion of calcium silicate nanowires to the intestinal organoid culture medium can significantly promote the proliferation and differentiation of organoids; Figure 11To investigate the mechanism by which calcium silicate nanowires promote the development of intestinal organoids. (a) Expression of β-catenin in intestinal organoids cultured for 3 and 5 days with calcium silicate nanowires. Scale bar: 100 μm. (b) Heatmap of gene expression related to the Wnt / β-catenin signaling pathway in intestinal organoids after 3 and 5 days of culture with calcium silicate nanowires. (c) Relative glucose uptake in intestinal organoids cultured for 3 and 5 days with calcium silicate nanowires. (d) Relative ATP production in intestinal organoids cultured for 3 and 5 days with calcium silicate nanowires. The results show that calcium silicate nanowires promote the development of intestinal organoids by acting on the Wnt / β-catenin signaling pathway and influencing cellular energy metabolism. Figure 12 To optimize the developmental effects of liver organoids cultured in calcium silicate composite hydrogels. (a) Bright-field images of liver organoids grown in GelMA-2CS-10M hydrogel and Matrigel for 1, 3, and 5 days. Scale bar: 200 μm. (b) H&E staining images of liver organoids cultured in GelMA-2CS-10M hydrogel and Matrigel for 5 days. Scale bar: 100 μm. (c) PAS and Oil Red O staining of liver organoids cultured in GelMA-2CS-10M hydrogel and Matrigel for 5 days. Scale bar: 100 μm. (d) Representative protein staining (ALB, CK19, and CYP3A4) of liver organoids cultured in both hydrogels for 5 days. Scale bar: 100 μm. The results showed that the optimized calcium silicate composite hydrogel could also support the normal development of liver organoids. The structure, key cell differentiation, glycogen and lipid synthesis of the cultured organoids were comparable to those of the matrix gel group, and it has great application value as a culture substrate material for organoids. Detailed Implementation

[0024] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0025] In this invention, the composite hydrogel is chemically composed of calcium silicate (CS), methacrylamide gelatin (GelMA), and matrigel. The amount of matrigel added does not exceed 10% (v / v). The optimized calcium silicate composite hydrogel designed in this invention can support the normal growth and development of intestinal and liver organoids, demonstrating its feasibility, effectiveness, and low cost as an organoid culture medium material. Furthermore, the bioactive ions released by calcium silicate can promote the proliferation and differentiation of intestinal organoids, serving as an effective and low-cost additive to organoid culture media, enabling the preparation of improved organoid culture media and promoting organoid development.

[0026] The following examples illustrate the preparation method of the optimized calcium silicate composite hydrogel and the development of the modified organoid culture medium. The modified organoid culture medium consists of the original organoid culture medium and calcium silicate. The prepared optimized calcium silicate composite hydrogel can support the normal growth and development of intestinal and liver organoids, demonstrating significant advantages as a substrate material for organoid culture. Furthermore, calcium silicate can serve as an effective, biosafe, and low-cost culture medium additive for optimizing the design of organoid culture media.

[0027] The calcium silicate of this invention is synthesized via a hydrothermal method and exhibits a nanowire morphology. For example, the calcium silicate nanowires are approximately a few micrometers in length and approximately 10-50 nm in diameter.

[0028] In one embodiment of the present invention, a composite hydrogel is prepared by mixing GelMA, calcium silicate dispersion, DMEM / F12 culture medium, and matrix gel in different proportions. Different groups are set up according to the different calcium silicate contents and whether or not matrix gel is added: GelMA, GelMA-10M, GelMA-1CS-10M, GelMA-2CS-10M, GelMA-4CS-10M, and GelMA-2CS (where the amount of unlabeled components added is 0). Preferably, the calcium silicate composite hydrogel composition is 5% GelMA (w / v), 2% calcium silicate (w / w), and 10% matrix gel (v / v).

[0029] This invention presents a calcium silicate composite hydrogel that promises to replace matrix gels as a matrix material for supporting organoid culture, thereby improving organoid culture and reducing culture costs. The optimized preparation method of the calcium silicate composite hydrogel is illustrated below using the GelMA-2CS-10M group as an example.

[0030] Calcium silicate powder was ultrasonically dispersed in DMEM / F12 culture medium. The mass fraction of the calcium silicate was 0–16% (w / w), preferably 8% (w / w).

[0031] Photosensitizing GelMA was prepared by introducing methacrylic anhydride into gelatin. Subsequently, the GelMA was dissolved at 55°C in a phosphate buffer solution (PBS, pH 7.4) containing 0.5 wt% phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP). As an example, the GelMA was dissolved in a PBS solution containing 0.5 wt% LAP at 55°C. The mass fraction of the GelMA was 10-20% (w / v), preferably 10% (w / w).

[0032] The precursor solution for the composite hydrogel was prepared by uniformly mixing GelMA solution, calcium silicate powder dispersion, DMEM / F12 culture medium, and matrix gel at a volume ratio of 10:5:3:2. The mass fraction of GelMA was 5-10% (w / v), and the amount of matrix gel added was 10% (v / v).

[0033] Irradiating the precursor solution of the composite hydrogel with visible light at a wavelength of 405 nm for a certain period of time (e.g., 6 s) can crosslink and form an optimized calcium silicate composite hydrogel.

[0034] The following describes the development of a modified organoid culture medium. This modified medium was supplemented with a specific proportion of calcium silicate. Specifically, the original organoid culture medium was supplemented with a certain proportion of calcium silicate, at a concentration of 0–100 μg / mL. The resulting modified organoid culture medium significantly promoted organoid development.

[0035] In this invention, the optimized calcium silicate composite hydrogel and the organoid culture medium containing the inorganic active material calcium silicate are used to support organoid culture and development.

[0036] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0037] Comparative Example 1 Pure GelMA hydrogel was used as a control group. A 10% GelMA solution was mixed with DMEM / F12 medium at a 1:1 ratio, and then the hydrogel precursor solution was irradiated with visible light at a wavelength of 405 nm for 6 seconds to crosslink and form a GelMA hydrogel, denoted as GelMA.

[0038] Comparative Example 2 A GelMA hydrogel containing 10% matrix gel was used as a control group. A 10% GelMA solution, DMEM / F12 medium, and matrix gel were mixed uniformly in a 5:4:1 ratio. The hydrogel precursor solution was then irradiated with visible light at a wavelength of 405 nm for 6 seconds to crosslink and form a hydrogel, which was denoted as GelMA-10M.

[0039] Example 1 (1) GelMA solution (10%, w / v), calcium silicate powder dispersion (8%, w / w), and DMEM / F12 culture medium were mixed uniformly in a volume ratio of 2:1:1 to obtain the precursor solution of the composite hydrogel. Then, the precursor solution of the hydrogel was irradiated with visible light at a wavelength of 405nm for 6s to crosslink and form a calcium silicate composite hydrogel, which was denoted as GelMA-2CS. (2) The differentiation effect of the prepared calcium silicate composite hydrogel on intestinal organoids was analyzed. The results are shown in [reference needed]. Figure 3 ; (3) The expression of YAP in intestinal organoids cultured from the prepared calcium silicate composite hydrogel was investigated. The results are shown in [reference needed]. Figure 7 ; (4) The rheological properties of the prepared calcium silicate composite hydrogel were tested, and the results are shown in [reference]. Figure 8 ; (5) The ion release of the prepared calcium silicate composite hydrogel was investigated, and the results are shown in [reference needed]. Figure 9 ; (6) The mechanism by which the bioactive ions released from the prepared calcium silicate composite hydrogel promote the development of intestinal organoids was investigated. The results are shown in [reference needed]. Figure 11 .

[0040] Example 2 (1) GelMA solution (10%, w / v), calcium silicate powder dispersion (8%, w / w), DMEM / F12 culture medium and matrix gel were uniformly mixed in a volume ratio of 10:5:3:2 to obtain the precursor solution of the composite hydrogel. Then, the precursor solution of the hydrogel was irradiated with visible light at a wavelength of 405nm for 6s to crosslink and form an optimized calcium silicate composite hydrogel, which was denoted as GelMA-2CS-10M. (2) The development, differentiation, and luminal fluid secretion regulation capabilities of intestinal organoids cultured from the optimized calcium silicate composite hydrogel were analyzed. The results are shown in [reference needed]. Figure 2 , 3 4; (3) The ability of the optimized calcium silicate composite hydrogel to support the culture and passage of intestinal organoids was investigated. The results are shown in [reference needed]. Figure 6 ; (4) The expression of YAP in intestinal organoids cultured from the optimized calcium silicate composite hydrogel was investigated. The results are shown in [reference needed]. Figure 7 ; (5) The rheological properties of the prepared optimized calcium silicate composite hydrogel were analyzed, and the results are shown in [reference]. Figure 8 ; (6) The ion release of the optimized calcium silicate composite hydrogel was tested, and the results are shown in [reference needed]. Figure 9 ; (7) The mechanism by which the bioactive ions released from the optimized calcium silicate composite hydrogel promote the development of intestinal organoids was investigated. The results are shown in [reference needed]. Figure 11 ; (8) The applicability of the optimized calcium silicate composite hydrogel as an organoid culture material was investigated. The results are shown in [reference needed]. Figure 12 .

[0041] Table 1 shows the composition of the calcium silicate composite hydrogel in this invention: Comparative Example 1 0wt% 5wt% 50% vol 0 vol% Comparative Example 2 0wt% 5wt% 40 vol% 10 vol% Example 1 2wt% 5wt% 50% vol 0 vol% Example 2 2wt% 5wt% 40 vol% 10 vol% .

[0042] Example 3 (1) The original culture medium for intestinal organoids (IntestiCult) TM STEMCELL, Cat#06005, main components: DMEM / F12 medium, 10mM HEPES, 1% Glutamax (Gibco), 1% P / S, 1% N2 (Gibco), 1% B27 (Gibco), 1.25mM N-Acetylcysteine ​​(Sigma), 500ng / mL R-Spondin-1, 100ng / mL Noggin, 100ng / mL EGF, etc.) supplemented with 50μg / mL calcium silicate nanowires as a modified intestinal organoid culture medium; (2) The effect of the modified intestinal organoid culture medium on supporting the proliferation and differentiation of intestinal organoids was investigated. The results are shown in [reference needed]. Figure 10 ; (3) The effects of the modified intestinal organoid culture medium on the key signaling pathway Wnt / β-catenin in intestinal organoid development and its regulation of cellular energy metabolism were investigated. The results are shown in [reference needed]. Figure 11 .

[0043] Example 4 (1) The original culture medium for intestinal organoids (IntestiCult) TM 100 μg / mL of calcium silicate nanowires were added to STEMCELL, Cat#06005 as a modified intestinal organoid culture medium; (2) The effect of the modified intestinal organoid culture medium on supporting the proliferation and differentiation of intestinal organoids was investigated. The results are shown in [reference needed]. Figure 10 ; (3) The mechanism by which the modified intestinal organoid culture medium promotes intestinal organoid development was investigated. The results are shown in [reference needed]. Figure 11 .

[0044] Crystal phase and microstructure of calcium silicate: The crystal phase and morphology of the prepared calcium silicate were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Its composition is Ca6Si6O. 17 (OH)₂, with a morphology of nanowires, several micrometers in length and approximately 10-50 nm in diameter (see [link]). Figure 1 ).

[0045] The optimized calcium silicate composite hydrogel supports the culture of intestinal organoids: Images of intestinal organoid development cultured in different hydrogels at different time points showed that the addition of 10% Matrigel resulted in significant budding of organoids in the composite hydrogel group, indicating improved maturity. Among them, the GelMA-2CS-10M group, containing both 2% CS and 10% Matrigel, exhibited the best culture results. Furthermore, statistical results on intestinal organoid formation efficiency showed no significant difference between the GelMA-2CS-10M group and the Matrigel group on day 5 (see [link to relevant documentation]). Figure 2 ).

[0046] Effects of optimized calcium silicate composite hydrogels on intestinal organoid differentiation and function: This invention investigated the expression of typical differentiated cell types in intestinal organoids under optimized calcium silicate composite hydrogel culture conditions. Four groups of intestinal organoids cultured on GelMA hydrogels all expressed a certain proportion of the four intestinal epithelial cell types (stem cell marker Lgr5, enteroendocrine cell marker Chromogranin-A, goblet cell marker Mucin-2, and Paneth cell marker lysozyme). Compared with the other three groups (GelMA, GelMA-10M, and GelMA-2CS), the overall expression of the four cell markers was significantly higher in the GelMA-2CS-10M group. Furthermore, this invention evaluated the gene expression of the four cell markers in these five groups. The results showed that the expression of stem cell marker Lgr5, enteroendocrine cell marker Chromogranin-A, and goblet cell marker Mucin-2 in the GelMA-2CS-10M group was significantly higher than that in the Matrigel group, while the expression of Paneth cell marker lysozyme was lower than that in the Matrigel group (see Matrigel). Figure 3 In addition, the function of intestinal organoids is also an important indicator of intestinal organoid development. Figure 4 and Figure 5To investigate the cystic fibrosis protein regulation (CFTR) function of intestinal organoids cultured in different hydrogels (GelMA-2CS-10M and Matrigel), this invention used a trichodin-induced swelling assay to explore the function of cystic fibrosis conductance regulators (CFTR) in intestinal organoids cultured in GelMA-2CS-10M and Matrigel hydrogels. Following trichodin treatment, intestinal organoids in both groups expanded rapidly at similar rates, indicating that intestinal organoids generated in GelMA-2CS-10M hydrogel also possess the ability to regulate luminal fluid secretion (see...). Figure 4 and Figure 5 As a matrix material for organoid culture, calcium silicate composite hydrogels are crucial for maintaining organoid growth duration. This invention evaluated the passage of intestinal organoids in GelMA-2CS-10M hydrogel and Matrigel. Results showed that organoids could grow and be passaged stably in GelMA-2CS-10M hydrogel, with passage numbers reaching 6-8 times. Gene expression results from intestinal organoids at different passage numbers showed normal expression in four typical differentiated cell types (see...). Figure 6 Overall, the optimized calcium silicate composite hydrogel can serve as a matrix material for culturing intestinal organoids, and the resulting organoids exhibit similar structural and functional properties to those produced in Matrigel.

[0047] Mechanism of optimized calcium silicate composite hydrogel in promoting intestinal organoid development: Effect of stiffness on YAP expression in intestinal organoids This invention explores the mechanism by which optimized calcium silicate composite hydrogels promote the development of intestinal organoids. Yes-associated protein (YAP) is a cell mechanosensory factor crucial for the self-renewal and proliferation of intestinal stem cells. This invention first characterized the expression of YAP in intestinal organoids cultured on different hydrogels for 1, 3, and 5 days. Analysis was performed using stained images and statistical results of nuclear YAP. Due to the changing mechanical properties of different hydrogels over time, the proportion of cells with nuclear YAP in intestinal organoids varied. Except for the Matrigel group, the GelMA group showed a higher proportion of cells with nuclear YAP on day 1. When the culture time was 3 days, the proportion of cells with nuclear YAP in the GelMA-2CS-10M group was higher than that in the GelMA, GelMA-2CS, and GelMA-10M groups. Subsequently, on day 5, the proportion of cells with nuclear YAP in the four groups of intestinal organoids cultured on GelMA-based hydrogels was essentially the same. Furthermore, at any time point, the proportion of nuclear YAP in Matrigel was significantly higher than in the other four groups. To explain this phenomenon, this invention measured the storage modulus (G′) and loss modulus (G″) of five hydrogels after different culture times, which can reflect the differences in hydrogel stiffness. The addition of CS nanowires reduced the G′ of GelMA. Furthermore, supplementing with 10% Matrigel further reduced the G′ of the composite hydrogel. The difference in G′ between GelMA and GelMA-2CS-10M was more pronounced on days 1 and 3 (ΔG′). day1 =67Pa; ΔG′ day3 =73Pa), and on the 5th day, the difference in G′ gradually decreased (ΔG day(5′ = 39 Pa). At any time point, the G′ in the Matrigel group was significantly lower than that in the other four groups. Combined with rheological results, the reasons for the differences in the proportion of nuclear YAP cells among the groups can be well analyzed. Many studies have shown that high matrix stiffness in the early stages is conducive to nuclear translocation of YAP. Compared with the other three groups (GelMA, GelMA-10M, and GelMA-2CS), the GelMA group showed the highest G′ and nuclear YAP proportion on day 1. As the culture time increases, maintaining excessively high matrix stiffness may lead to YAP inactivation. Therefore, due to the addition of CS nanowires and the further reduction of G′ by supplementing with 10% Matrigel, the stiffness of GelMA-2CS-10M hydrogel decreased more significantly compared with the other three groups (GelMA, GelMA-10M, and GelMA-2CS), resulting in higher YAP activity than the other three groups, thereby promoting the proliferation and differentiation of intestinal stem cells and forming more budding structures. By day 5 of culture, the differences in G′ among the groups (GelMA, GelMA-10M, GelMA-2CS, and GelMA-2CS-10M) gradually decreased, and the proportion of cells with nuclear YAPs did not differ significantly among the groups. However, due to the significant differences in the rheological properties of Matrigel compared to the other four hydrogel groups, the YAP activity of the cultured intestinal organoids was much higher than that of the other four groups at any culture time. Therefore, the optimized calcium silicate composite hydrogel-regulated YAP activity may not be the primary reason supporting organoid development in achieving comparable culture results. (see Figure 7 , Figure 8 ).

[0048] Ion release from calcium silicate nanowires: Calcium silicate, as an inorganic bioactive material, exhibits superior bioactivity, and the released bioactive ions provide valuable guidance for stem cell behavior. Ion release results show that both calcium silicate composite hydrogels and calcium silicate nanowires can release a certain amount of bioactive ions in DMEM / F12 culture medium (see [link to DMEM / F12]). Figure 9 ).

[0049] The effects of calcium silicate nanowires on the proliferation and differentiation of intestinal organoids: This invention discovers that bioactive ions released from calcium silicate composite hydrogels are crucial for promoting the development of intestinal organoids. To verify this hypothesis, intestinal organoids were cultured in conditioned medium containing different proportions of calcium silicate nanowires to investigate the role of the released bioactive ions. First, the effect of calcium silicate nanowires on intestinal organoid proliferation showed that the experimental group cultured in medium containing 50 μg / mL calcium silicate nanowires significantly promoted intestinal organoid proliferation on day 5. Subsequently, this invention selected three different calcium silicate ratios of 0, 50 μg / mL, and 100 μg / mL to explore the differentiation effect of calcium silicate nanowires on intestinal organoids. Gene expression results showed that after 3 days of culture, the expression of four typical differentiation cell markers in the two groups of organoids containing calcium silicate nanowires significantly increased. After 5 days of culture, in the presence of 100 μg / mL calcium silicate nanowires, the expression of intestinal stem cell markers and enteroendocrine cell markers significantly increased. Furthermore, protein staining results of four typical cell markers in intestinal organoids cultured for 3 and 5 days, respectively, showed that calcium silicate nanowires added to the culture medium could promote organoid differentiation (see...). Figure 10 ).

[0050] Mechanism by which calcium silicate nanowires promote the development of intestinal organoids. Next, this invention explores the mechanism by which bioactive ions promote the development of intestinal organoids. The Wnt / β-catenin signaling pathway is crucial for the proliferation and differentiation of intestinal stem cells and is an indispensable regulatory pathway for crypt-village structure formation. High expression of β-catenin can demonstrate the activation of Wnt / β-catenin signaling. Therefore, this invention first evaluated the expression of β-catenin in intestinal organoids cultured in media containing different proportions of calcium silicate nanowires. The results showed that β-catenin expression was significantly enhanced in intestinal organoids co-cultured with calcium silicate nanowires. In addition, under the influence of calcium silicate nanowires, genes related to the Wnt / β-catenin signaling pathway were also significantly overexpressed, including Wnt3, LRP6, R-spondin1, R-spondin2, and R-spondin3. Furthermore, the presence of calcium silicate nanowires can significantly promote glucose uptake and increase ATP activity in organoids, which is beneficial for promoting the proliferation and differentiation of intestinal organoids (see [link to relevant documentation]). Figure 11 ).

[0051] Optimized calcium silicate composite hydrogels support the culture of liver organoids. To demonstrate the universality and applicability of the optimized calcium silicate composite hydrogel as a substrate material for organoid culture, this invention constructed a mouse liver organoid culture model. Studies showed that liver organoids cultured in GelMA-2CS-10M hydrogel and Matrigel for 1, 3, and 5 days, respectively, exhibited good development. H&E staining results indicated that both groups of cultured liver organoids displayed typical central lumen and peripheral epithelial structures. Next, this invention assessed the expression of mature hepatocyte markers albumin (ALB and CYP3A4) and the bile duct cell-specific marker CK19 using protein staining. Protein staining images showed that, compared to the Matrigel group, liver organoids cultured in GelMA-2CS-10M hydrogel also normally expressed ALB, CK19, and CYP3A4. Subsequently, this invention further explored the functional characteristics of the cultured liver organoids, including glycogen and lipid synthesis. Results showed that organoids cultured in GelMA-2CS-10M hydrogel and Matrigel were able to synthesize and accumulate glycogen and lipids. The above results indicate that the optimized calcium silicate composite hydrogel can not only support the development of liver organoids, but also that the cultured liver organoids can differentiate into various hepatocyte types that perform liver functions (see [link to original text]). Figure 12 ).

[0052] In summary, the objective of this invention is to prepare an organoid culture material containing the inorganic active material calcium silicate, capable of supporting the normal growth and development of organoids. The composite hydrogel described herein is prepared by uniformly mixing bioactive silicate and biocompatible GelMA hydrogel, and then adding a small proportion of matrix gel. This composite hydrogel can support the normal development of organoids and is expected to replace matrix gel as a matrix material for organoid culture. The bioactive component calcium silicate added to the culture medium can promote the proliferation and differentiation of organoids, providing an additive for the development of improved organoid culture media.

Claims

1. The use of a calcium silicate composite hydrogel in the preparation of organoid culture and development materials, characterized in that, The calcium silicate composite hydrogel comprises calcium silicate, methacrylamide gelatin, DMEM / F12 culture medium as dispersion medium and a matrix gel whose main components include laminin and type IV collagen, which are cross-linked to obtain the calcium silicate composite hydrogel; the volume content of the matrix gel is 10 vol.

2. The use of the calcium silicate composite hydrogel according to claim 1 in the preparation of organoid culture and development materials, characterized in that, The calcium silicate has the morphology of nanowires with a length of 1-10 micrometers and a diameter of 10-50 nm; the content of the calcium silicate does not exceed 4 wt%.

3. The use of the calcium silicate composite hydrogel according to claim 1 or 2 in the preparation of organoid culture and development materials, characterized in that, The content of the methacrylamide gelatin is 5-10 wt%.

4. The use of the calcium silicate composite hydrogel according to claim 1 in the preparation of organoid culture and development materials, characterized in that, The volume content of the dispersion medium is 40-50 vol.

5. An organoid culture medium comprising the calcium silicate composite hydrogel according to any one of claims 1-4, characterized in that, The calcium silicate composite hydrogel, as an additive, includes: a dispersion medium, and calcium silicate dispersed in the dispersion medium.

6. The organoid culture medium according to claim 5, characterized in that, The content of calcium silicate does not exceed 100 μg / mL.

7. The organoid culture medium according to claim 5, characterized in that, The calcium silicate content is 50 μg / mL.

8. The organoid culture medium according to claim 5, characterized in that, The calcium silicate has the morphology of nanowires with a length of 1–10 micrometers and a diameter of 10–50 nm.

9. The organoid culture medium according to claim 5, characterized in that, The dispersion medium is DMEM / F12 culture medium.

10. A method for preparing a calcium silicate composite hydrogel as described in any one of claims 1-4, characterized in that, include: (1) Calcium silicate is dispersed in a dispersion medium to obtain a calcium silicate dispersion; (2) Dissolve methacrylamide gelatin in phosphate buffer to obtain solution 1; (3) Mix the calcium silicate dispersion, dispersion medium and matrix gel to obtain solution 2; (4) Mix solution 2 and solution 1 to obtain the precursor solution of the composite hydrogel; (5) The precursor solution of the obtained composite hydrogel is crosslinked to obtain the calcium silicate composite hydrogel.

11. The preparation method according to claim 10, characterized in that, In step (1), the mass fraction of calcium silicate in the calcium silicate dispersion is 8-16 wt%; the dispersion method is ultrasonic mixing.

12. The preparation method according to claim 10, characterized in that, In step (2), the phosphate buffer solution is a 0.5 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) phosphate buffer solution with pH = 7.4; the dissolution temperature is 37–60 °C. The mass fraction of methacrylamide gelatin in solution 1 is 10-20 wt%.

13. The preparation method according to claim 12, characterized in that, The melting temperature is 55°C.

14. The preparation method according to claim 12, characterized in that, The mass fraction of methacrylamide gelatin in solution 1 is 10-15 wt%.

15. The preparation method according to claim 10, characterized in that, In step (3), the volume ratio of the calcium silicate dispersion, the dispersion medium and the matrix adhesive is 5:(3-8):

2.

16. The preparation method according to claim 15, characterized in that, The volume ratio of the calcium silicate dispersion, the dispersion medium, and the matrix adhesive is 5:3:

2.

17. The preparation method according to claim 10, characterized in that, In step (4), the volume ratio of solution 2 to solution 1 is (1-3):

1.

18. The preparation method according to claim 17, characterized in that, The volume ratio of solution 2 to solution 1 is 1:

1.

19. The preparation method according to claim 10, characterized in that, In step (4), the crosslinking method is to irradiate the precursor liquid of the composite hydrogel with visible light at a wavelength of 405nm; the irradiation time is 6 to 20 seconds.

20. Use of the organoid culture medium according to any one of claims 5-9 in the preparation of organoid culture and development materials.

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