Application of hydrogel in in-vitro culture of human tissues and organs

Abstract

The invention discloses an application of hydrogel in in-vitro culture of human tissues and organs, and relates to the technical field of animal organ culture.The hydrogel is prepared from an isocyanate polymer, and cells of the human tissues and organs are planted for in-vitro culture; the hydrogel has the structure and mechanical property characteristics similar to those of collagen, namely fibrous, porous and three-dimensional network structure and semi-flexible and semi-rigid mechanical properties. The characteristics perfectly avoid the defects of conventional animal source materials and synthetic materials, not only effectively realize accurate and controllable components and small batch difference of the material, but also solve the problem of organ subculture, dissipate stress in organoid culture and facilitate subculture, and the material is an ideal material for organoid culture at present.

Description

Technical Field

[0001] The invention relates to the technical field of animal organ culture, and in particular to the application of hydrogel in in vitro culture of human tissue organs. Background Art

[0002] The renal tubule is an important part of the kidney and has multiple key functions. It is essential for maintaining the body's homeostasis and health. In vitro culture of renal tubular organoids can simulate the physiological environment of the renal tubules, which is helpful for studying the mechanism and pathological process of renal tubular related diseases. For example, researchers can use this model to study diseases such as renal tubular injury, chronic kidney disease, and renal tubular acidosis, so as to gain a deeper understanding of the development and treatment of these diseases.

[0003] The biomimetic extracellular matrices currently used for the culture of renal tubular organoids can be roughly classified into animal-derived materials and synthetic materials. Both have their own advantages and disadvantages. For example, animal-derived hydrogels such as Matrigel, which are currently used to culture organoids, come from mouse tumor cells. Although they can provide (stem) cells and tissues with a variety of growth factors and physical support necessary for growth, they have some defects: first, this gel has batch differences, which makes stem cells behave inconsistently; second, it has a high biochemical complexity (composed of nearly 2,000 unique proteins), making it difficult to fine-tune it in order to study the effects of different parameters (such as biomolecules, mechanical properties, etc.) on organoid culture; third, it is very different from the microenvironment in the human body, and may carry pathogens or immunogens that are not suitable for culturing organoids for clinical use.

[0004] In contrast, synthetic materials can be precisely and independently designed for biochemical and biophysical properties, and can also be rationally designed to be used in clinical settings. However, the difficulty with synthetic materials is that they contain too few biologically active components (growth factors and the types and quantities of cell adhesion sites, etc.), cannot accurately simulate the dynamic interactions between cells and the extracellular matrix (lack of spatiotemporal controllability), etc.

[0005] Therefore, constructing a highly biomimetic extracellular matrix that can simulate the dynamic microenvironment of cells, tissues, and organs in the body, thereby providing new platforms and tools for the clinical application of basic biology, pathology, and regenerative medicine has become one of the hot topics and focuses in the biomedical field. Summary of the invention

[0006] The present invention provides a method for applying hydrogel in in vitro culture of human tissues and organs, which is based on a temperature-responsive "reversible" hydrogel prepared from isocyanate polymers, whose structure and mechanical properties are similar to collagen, with a fibrous porous three-dimensional network and semi-flexible and semi-rigid mechanical properties. This is specifically achieved through the following technologies.

[0007] An application of a hydrogel in the in vitro culture of human tissues and organs, using an isocyanate polymer to make a hydrogel, and implanting cells of human tissues and organs for in vitro culture;

[0008] The isocyanate polymer is formed by a cross-linking reaction of polymer units A of formula I,

[0009]

[0010] and a polymer containing a crosslinking unit B of formula II,

[0011]

[0012] Wherein, n=100-10000, m is any natural number, k=0.01-0.05, h=0, 1 or 2;

[0013] Functional group FG is a functional moiety covalently coupled to functional group F1 or F2 of crosslinking unit B;

[0014] The functional groups F1, F2, F and FG capable of providing covalent coupling are one of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, bicyclo[6.1.0]non-4-alkynyl-azide coupling, vinyl sulfone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-streptavidin coupling, amine-carboxylic acid to produce amide bond, alcohol-carboxylic acid coupling to produce ester bond, tetrazine-trans-cyclooctene coupling and NHS-ester (N-hydroxysuccinimide ester)-amine coupling;

[0015] The spacer part is an organic part of the main chain comprising at least two functional parts F1 and F2; the length of the crosslinking unit B is 2.5-12nm, or between 20 and 80 atoms.

[0016] Preferably, the hardness of the hydrogel at 37°C is 100-300 Pa.

[0017] Further optionally, the hardness of the hydrogel at 37° C. is generally 10 3 Pa.

[0018] Preferably, the human tissue organ is one of cancer tissue, kidney, stem cell and nerve.

[0019] It should be noted that the human tissues and organs mentioned in the present invention include not only conventional tissues and organs, but also the corresponding cells that make up the tissues and organs, and organoids. Organoids refer to tissue analogs with a certain spatial structure formed by three-dimensional culture of adult stem cells or pluripotent stem cells in vitro.

[0020] Based on the above application, the present invention provides a method for in vitro culture of human tissues and organs, using isocyanate polymers to make hydrogels, inoculating cells of human tissues and organs for in vitro culture;

[0021] The isocyanate polymer is formed by a cross-linking reaction of polymer units A of formula I,

[0022]

[0023] and a polymer containing a crosslinking unit B of formula II,

[0024]

[0025] Wherein, n=100-10000, m is any natural number, k=0.01-0.05, h=0, 1 or 2;

[0026] Functional group FG is a functional moiety covalently coupled to functional group F1 or F2;

[0027] Functional groups F1, F2, F and FG are one of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, bicyclo[6.1.0]non-4-alkynyl-azide coupling, vinyl sulfone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-streptavidin coupling, amine-carboxylic acid to produce amide bond, alcohol-carboxylic acid coupling to produce ester bond, tetrazine-trans-cyclooctene coupling and NHS-ester (N-hydroxysuccinimide ester)-amine coupling;

[0028] The spacer part is an organic part of the main chain comprising at least two functional parts F1 and F2; the length of the crosslinking unit B is 2.5-12nm, or between 20 and 80 atoms.

[0029] Preferably, the above in vitro culture method comprises the following steps:

[0030] The isocyanate polymer was placed in a 4° C. PBS buffer and allowed to stand, and was shaken vigorously until a transparent hydrogel solution was obtained;

[0031] Mixing a suspension of cells of human tissues and organs to be cultured with a hydrogel solution, and inoculating and incubating the mixture until gelation occurs;

[0032] A cell culture medium is added for culturing, and the cell culture medium is periodically renewed.

[0033] Preferably, the concentration of the suspension of cells of human tissues and organs to be cultured is 50-100 organoids / 50 μl.

[0034] Preferably, the mass fraction of the isocyanate polymer in the hydrogel solution is 0.01-1%.

[0035] Preferably, the mixed solution is inoculated on the culture plate at 30 μl / drop.

[0036] Preferably, the incubation conditions are: incubation at 37° C. for 30-60 min.

[0037] Preferably, the conditions for adding cell culture medium for culturing are: 37° C., CO 2 concentration 5%.

[0038] Through this bionic hydrogel, a three-dimensional cell complex similar to the renal tubules in the body was successfully constructed, providing an ideal material for the in vitro culture of renal tubular organoids.

[0039] Compared with the prior art, the present invention is beneficial in that:

[0040] 1. The present invention uses a hydrogel prepared from an isocyanate polymer to culture human tissues and organs in vitro. The hydrogel has a structure and mechanical properties similar to collagen, namely a fibrous, porous, three-dimensional network structure and semi-flexible and semi-rigid mechanical properties. These characteristics perfectly avoid the drawbacks of conventional animal-derived materials and synthetic materials, and can be designed into a model that is closer to the in vivo environment of cells, more accurately simulating the three-dimensional structure, cell-cell interaction and cell-matrix interaction of organoids, which helps to more realistically reproduce the physiological state of renal tubules.

[0041] 2. The hydrogel used in this method not only achieves precise control of the physical and chemical properties of its components (such as pore size, stiffness, biochemical properties, etc.), allowing researchers to better control the growth and arrangement of cells in the gel and achieve more precise experimental design; it also solves the problem of organ subculture, dissipates the stress in organoid culture, and makes subculture more convenient. It is currently an ideal material for organoid culture.

[0042] 3. The batch differences of organoids cultured using this method are small. Compared with the matrix gel material of animal origin, the composition and properties of the hydrogel used in the present invention can be better standardized, thereby improving the consistency and repeatability of the experiment.

[0043] 4. The organoids cultured by this method can be used for drug screening and evaluation of drug effects and toxicity; they can be used for disease research, such as studying renal tubule-related disease mechanisms and drug treatment strategies.

[0044] 5. The use of synthetic hydrogels to culture organ models can reduce dependence on animal experiments, reduce the use of animals, and comply with ethical and animal welfare requirements.

[0045] 6. If the patient's cells are used for culture, personalized medicine and drug customization can be achieved, the disease mechanism of specific patients can be studied, and a basis for personalized medicine and drug customization can be provided.

[0046] 7. Organoid culture can be used to study the development, differentiation, function and interaction of various tissues and organs in the human body, and expand the understanding of the biology of various tissues and organs in the human body.

[0047] In summary, the in vitro organoid culture method provided by the present invention can provide a more realistic, controllable and standardized model, which will help promote the progress of scientific research and medical applications in multiple fields. The present invention is expected to provide new platforms and tools for clinical applications in the fields of basic biology, disease studies and regenerative medicine, and promote innovative progress in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The mechanical properties test results of the 1% concentration hydrogel solution synthesized in Example 1;

[0049] Figure 2 , 3 This is a photo of the renal tubular organoids finally cultured in Example 3.

[0050] Figure 4 , 5 These are photos of the renal tubular organoids cultured in Example 3 on days 3 and 5. DETAILED DESCRIPTION

[0051] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] The hydrogel raw materials, isocyanate polymers, used in the following examples and comparative examples are all referenced from US Patent No. 11053349B2 “Biomimetic networks comprising polyisocyanopeptide hydrogels”.

[0053] The cells to be cultured used in the following examples and comparative examples are human renal tubular cells, which are directly sourced from human biopsy samples from Nanjing Drum Tower Hospital. The human renal tubular cells have undergone ethical review.

[0054] The in vitro culture method provided by the present invention, in addition to targeting human renal tubular cells, has also been studied for cells of human tissues and organs such as neural tube organoids, intestinal organoids, and liver, and can obtain ideal organoid in vitro culture results; it can also be applied to cancerous cells of various tissues and organs of the human body, such as lung cancer and liver cancer cells. After analysis, it was found that this is because the three-dimensional network structure of the hydrogel provided by the present invention has an ultra-high water content, and the polypeptide biomimetic fragments of the polymer skeleton are similar to the cell tissue microenvironment in the body.

[0055] Example 1: Preparation of hydrogel

[0056] The isocyanate polymer is cut into pieces and placed in a cold PBS buffer (common commodity, purchased from the market, generally 4°C in this embodiment) overnight, and shaken vigorously on the second day until a transparent hydrogel solution is obtained. At this time, the isocyanate polymer in the solution is completely dissolved and can be used directly. The mass fraction of the isocyanate polymer is 0.01-1%, and the following embodiments all use a 1% concentration hydrogel solution.

[0057] The mechanical properties of the synthesized 1% hydrogel solution were tested, such as Figure 1 As shown, the results show that at 37°C, the hardness of the biomimetic hydrogel solution is 103Pa.

[0058] When storage is required, the obtained hydrogel solution is divided and stored in a freezer (the freezing condition is generally -80°C to -20°C). When it is needed, the hydrogel solution is taken out of the refrigerator and slowly thawed on ice or in a refrigerator at 4°C to avoid overheating.

[0059] Example 2: Cell seeding

[0060] (1) preparing cells to be cultured, such as human renal tubular cells, and preparing a cell suspension with a concentration of 2,000-20,000 cells / 100 μl of hydrogel;

[0061] (2) mixing the cell suspension and the thawed hydrogel solution evenly;

[0062] (3) Add the cell-gel mixture to the culture plate at 30-50 μl / drop (optionally 50 μl / drop);

[0063] (4) Incubate the culture plate in a 37°C incubator for 30-60 min, depending on the gelation time.

[0064] Example 3: Cell culture, maintenance and harvesting

[0065] Cell culture medium (common product purchased from Gibco) was added to the culture plate, which was placed in a cell culture incubator and cultured at 37° C. and 5% CO 2 concentration.

[0066] During the culture period, the cell culture medium is replaced regularly (usually every 2-3 days) to ensure the growth and health of the cells.

[0067] During the culture process, the growth and morphological changes of cells in the hydrogel were observed and recorded. Figure 4 This is the shape of the 3rd day. Figure 5 This is the growth status on the 5th day.

[0068] When the cells are harvested (cold PBS can be blown onto the hydrogel containing the organoids), the hydrogel solution and the organoid suspension are recovered together and centrifuged (the optional parameter is 2000 rpm) to obtain the organoids.

[0069] The final photo of the renal tubular organoids cultured is shown in the figure below. Figure 2 , 3 As shown in the figure, it can be seen that the renal tubular cells / tissues grow into a hollow ring-shaped structure, which is consistent with the morphology in the gold standard hydrogel-Matrigel. It can be seen that the in vitro culture method of the present invention can successfully culture human tissue organoids and obtain ideal culture effects.

[0070] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Application of a hydrogel in in vitro culture of human tissues and organs, characterized in that: Using isocyanate polymers to make hydrogels, cells of human tissues and organs are planted for in vitro culture; The isocyanate polymer is formed by a cross-linking reaction of polymer units A of formula I, and a polymer containing a crosslinking unit B of formula II, Wherein, n=100-10000, m is any natural number, k=0.01-0.05, h=0, 1 or 2; Functional group FG is a functional moiety covalently coupled to functional group F1 or F2 of crosslinking unit B; The functional groups F1, F2, F and FG capable of providing covalent coupling are one of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, bicyclo[6.1.0]non-4-alkynyl-azide coupling, vinyl sulfone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-streptavidin coupling, amine-carboxylic acid to produce amide bond, alcohol-carboxylic acid coupling to produce ester bond, tetrazine-trans-cyclooctene coupling and NHS-ester (N-hydroxysuccinimide ester)-amine coupling; The spacer part is an organic part of the main chain comprising at least two functional parts F1 and F2; the length of the crosslinking unit B is 2.5-12nm, or between 20 and 80 atoms.

2. The use of the hydrogel according to claim 1 in in vitro culture of human tissues and organs, characterized in that: The hardness of the hydrogel at 37° C. is 100-300 Pa.

3. Use of the hydrogel according to claim 1 or 2 in in vitro culture of human tissues and organs, characterized in that: The human tissue organ is one of cancer tissue, kidney, stem cell and nerve.

4. A method for culturing human tissues and organs in vitro, characterized in that: Using isocyanate polymers to make hydrogels, inoculated with cells from human tissues and organs for in vitro culture; The isocyanate polymer is formed by a cross-linking reaction of polymer units A of formula I, and a polymer containing a crosslinking unit B of formula II, Wherein, n=100-10000, m is any natural number, k=0.01-0.05, h=0, 1 or 2; Functional group FG is a functional moiety covalently coupled to functional group F1 or F2; Functional groups F1, F2, F and FG are one of alkyne-azide coupling, dibenzocyclooctyne-azide coupling, bicyclo[6.1.0]non-4-alkynyl-azide coupling, vinyl sulfone-thiol coupling, maleimide-thiol coupling, methyl methacrylate-thiol coupling, ether coupling, thioether coupling, biotin-streptavidin coupling, amine-carboxylic acid to produce amide bond, alcohol-carboxylic acid coupling to produce ester bond, tetrazine-trans-cyclooctene coupling and NHS-ester (N-hydroxysuccinimide ester)-amine coupling; The spacer part is an organic part of the main chain comprising at least two functional parts F1 and F2; the length of the crosslinking unit B is 2.5-12nm, or between 20 and 80 atoms.

5. The method for in vitro culture of human tissues and organs according to claim 4, characterized in that: The following steps are involved: The isocyanate polymer was placed in a 4° C. PBS buffer and allowed to stand, and was shaken vigorously until a transparent hydrogel solution was obtained; Mixing a suspension of cells of human tissues and organs to be cultured with a hydrogel solution, and inoculating and incubating the mixture until gelation occurs; A cell culture medium is added for culturing, and the cell culture medium is periodically renewed.

6. The method for in vitro culture of human tissues and organs according to claim 4, characterized in that: The concentration of the suspension of cells of human tissues and organs to be cultured is 50-100 organoids / 50 μl.

7. The method for in vitro culture of human tissues and organs according to claim 4, characterized in that: The mass fraction of the isocyanate polymer in the hydrogel solution is 0.01-1%.

8. The method for in vitro culture of human tissues and organs according to claim 4, characterized in that: The mixed solution was inoculated on the culture plate at 30 μl / drop.

9. The method for in vitro culture of human tissues and organs according to claim 4, characterized in that: The incubation conditions are: 37°C for 30-60 min.

10. The method for in vitro culture of human tissues and organs according to claim 4, characterized in that: The conditions for culturing with the addition of cell culture medium were: 37°C, CO2 concentration 5%.

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