CN-121991886-A - Organoid construction method and application thereof

Abstract

The invention relates to the technical field of biology and new medicines, in particular to a method for constructing a organoid and application thereof. The organoid construction method is based on mechanical and biochemical double factors, takes the spiral polyisonitrile as an extracellular matrix, and realizes effective regulation and control of differentiation paths of cartilage organoids and bone organoids by regulating chain length and RGD modification and carrying out differentiation by matching with cartilage generating cell groups or osteoblast groups.

Inventors

• RUAN CHANGSHUN
• FANG XINYI
• LIU KAIZHENG
• LIU JUAN
• CHEN ZHIGANG

Assignees

• 中国科学院深圳先进技术研究院

Dates

Publication Date

20260508

Application Date

20241107

Claims (8)

1. 1. A method of organoid construction comprising: s1, dissolving the chain length-adjustable spiral polyisonitrile in an induction culture medium to obtain a spiral polyisonitrile hydrogel solution, and S2, embedding a cartilage generating cell group or an osteoblast group into the spiral polyisonitrile hydrogel solution, and obtaining the organoid through gel formation.
2. 2. The organoid construction method according to claim 1, wherein the spiropoly-isonitrile in step S1 is an RGD-functionalized spiropoly-isonitrile.
3. 3. The method according to claim 1, wherein in the step S2, the chondrogenic cell population or the osteoblast cell population comprises human bone marrow mesenchymal stem cells.
4. 4. The method according to claim 3, wherein the induction medium used in the step S1 is a chondrogenic differentiation induction medium; in the step S2, the chondrogenic cell population further includes one or more of chondroprogenitor cells and chondrocytes, and the obtained organoid is a cartilage organoid.
5. 5. The method according to claim 3, wherein the induction medium used in the step S1 is an osteogenic induction medium; In the step S2, the osteoblast population further includes one or more of osteoprogenitor cells and bone cells, and the obtained organoid is a bone organoid.
6. 6. The organoid construction method according to any one of claims 1 to 5, wherein in step S2, the cell embedding density of the chondrogenic cell population or the osteoblastic cell population is 10 5 to 10 7 cells/mL, and the concentration of the helical polyisonitrile hydrogel solution is 1 to 3mg/mL.
7. 7. A organoid obtained by the organoid construction method of any of claims 1-6.
8. 8. Use of the organoid construction method of any of claims 1-6 for disease modeling, drug screening and/or for the preparation of a tissue repair drug.

Description

Organoid construction method and application thereof Technical Field The invention relates to the technical field of biology and new medicines, in particular to a method for constructing a organoid and application thereof. Background Organoids (Organoids) are a class of functional cellular tissue complexes constructed based on in vitro three-dimensional culture techniques, aimed at mimicking the structure and function of natural organs. Compared with the traditional two-dimensional cell culture, the method can provide physiological response and microenvironment which are more similar to those in vivo, and compared with an animal model, the method can better simulate and reflect the specific physiological functions of human beings. Thus, organoids are now widely used in regenerative medicine, disease model construction, and drug screening. Organoids are prepared primarily by differentiation and tissue formation of stem cells in a three-dimensional environment. Specifically, the preparation process generally starts with inducing stem cells such as embryonic stem cells or pluripotent stem cells, and promotes their differentiation into specific cells by adding specific biochemical signals and growth factors. The extracellular matrix (Extracellula r Matrix, ECM) provides a three-dimensional environment for cells to adhere, grow and differentiate, and under appropriate conditions, through its mechanical and biochemical properties, to influence the growth and differentiation behavior of cells, supporting the differentiation of stem cells into specific cells, ultimately forming organoids. In the organoid preparation process, researchers can further subdivide cartilage organoids and bone organoids by adjusting the differentiation path and culture conditions of cells for different tissue types. Cartilage organoids are formed by differentiating stem cells into chondrocytes and further organizing them in a three-dimensional environment, and are often used in cartilage injury repair and joint disease model research. Bone organ is prepared by inducing stem cells to differentiate into osteoblasts, and simulating the generation and mineralization processes of bone tissue for related research such as bone regeneration, osteoporosis and the like. In both organogenesis processes, precise regulation of the direction of cell differentiation is critical, for example to control whether cells differentiate into chondrocytes or osteoblasts. However, this process is highly complex and is often subject to multiple effects of biochemical signals, intercellular interactions, and microenvironment factors. Although the differentiation direction can be guided in part by the addition of growth factors or modulation of signal pathways, there is a high degree of cross-influence between these mechanisms and spatial and temporal precise control of signaling is difficult to achieve in three-dimensional culture systems. Thus, the prior art has failed to achieve effective regulation of the differentiation pathways of cartilage and bone organoids, resulting in these organoids still having limitations in functionality and tissue maturity, which also limits their performance in terms of modeling true tissue function and achieving regenerative capacity. Disclosure of Invention The invention provides a kind of organ construction method and its application, which uses spiral polyisonitrile as extracellular matrix and combines with cartilage generating cell group or osteoblast group to differentiate. Specifically, the invention provides the following technical scheme: In a first aspect of the invention, a method for constructing an organoid is provided, which comprises S1, dissolving a chain length-adjustable spiral polyisonitrile in an induction culture medium to obtain a spiral polyisonitrile hydrogel solution, and S2, embedding a cartilage generating cell population or an osteoblast population in the spiral polyisonitrile hydrogel solution, and obtaining the organoid through gelling. In some embodiments, the spiropolynitrile in step S1 is an RGD functionalized spiropolynitrile. In some embodiments, in step S2, the population of chondrogenic cells or the population of osteoblasts comprises human bone marrow mesenchymal stem cells. In some embodiments, the induction medium used in step S1 is a chondrogenic differentiation induction medium, and in step S2, the chondrogenic cell population further comprises one or more of chondrogenic progenitor cells and chondrocytes, and the organoid obtained is a cartilage organoid. In some embodiments, the induction medium used in step S1 is an osteogenic induction medium, and in step S2, the osteoblast population further comprises one or more of osteoprogenitor cells and bone cells, and the organoid obtained is an osteoorganoid. In some embodiments, in step S2, the population of chondrogenic cells or the population of osteoblasts has a cell embedding density of 10 5 to 10 7 cells/mL and the concentration of the heli