In-vitro directional induction of biconvex lens organoids by stiffness-controllable hydrogel and construction method and application thereof

Abstract

The application discloses a kind of in vitro directional induction lenticular lens organ by relying on stiffness controllable hydrogel and its construction method and application, technical scheme includes the following steps: (1) pluripotent stem cells are directionally induced and differentiated into primary ectoderm cell group;(2) prepare specific stiffness hydrogel and carry out surface cell adhesion treatment;(3) primary ectoderm cell is planted on the polyacrylamide hydrogel after washing and is induced into neural ectoderm cell group;(4) neural ectoderm cell is directionally differentiated into mature lenticular lens organ.The three-dimensional lenticular lens organ model obtained by the method has the top surface and the bottom surface of the spherical surface lenticular morphology, transparent and has capsule, lenticular lens epithelial cell, lenticular lens fiber cell and other structures, bottom surface is also completely wrapped by lenticular lens capsule.Compared with previous method, the formation of bottom surface complete capsule and spherical surface morphology is realized.

Description

Technical Field

[0001] This invention relates to the field of cell biology technology, specifically to a method for constructing and applying a biconvex lens organoid induced in vitro using a stiffness-controllable hydrogel. Background Technology

[0002] In recent years, with the rapid advancements in stem cell technology and regenerative medicine, the use of stem cell-cultured organoids to construct disease models has become a research hotspot with both scientific value and clinical prospects. Organoids are simplified versions of human tissues and organs, constructed in vitro with macroscopic and microscopic three-dimensional structures consistent with human tissues and organs, and even possessing corresponding physiological functions. They can simulate the structure and function of real organs in vivo, providing novel research methods and treatment approaches for precision medicine. Organoid culture generally originates from pluripotent stem cells with broad differentiation potential. These cells, possessing self-renewal and directed differentiation capabilities, can self-grow and assemble into organoids with precise arrangement patterns and spatial structures under specific culture conditions.

[0003] Cataracts are an ophthalmic disease caused by various factors such as aging, genetics, and trauma, resulting in the denaturation and aggregation of lens proteins and clouding of the lens, leading to visual impairment. It is also the leading cause of blindness worldwide. Therefore, there is a need for lens organoids that more closely resemble the structure and function of the natural lens to study the developmental mechanisms of the lens, establish cataract disease models for pathogenic mechanism research and high-throughput drug screening, and even research and achieve cataract stem cell therapy for lens tissue regeneration.

[0004] For a long time, researchers have successfully constructed various lens organoids in vitro using different cell sources and culture methods, which are now known as lens bodies. In 2010, the three-stage culture method was established, pioneering the differentiation and culture of human embryonic stem cells into lens bodies with three-dimensional structures through a combination of various growth factors. This method has also become the technological foundation for the future development and iteration of lens organoid culture methods. Currently, a relatively complete method for constructing lens organoids is the "fried egg method" proposed in 2017 (see Chinese patent CN105238737A, "A Novel Method for Directed Differentiation of Induced Pluripotent Stem Cells into Lens Bodies in Vitro"). This method involves using human urine-derived induced pluripotent stem cells through a more refined three-stage culture and early mechanical screening, resulting in "fried egg-like" cell structures during differentiation. These structures eventually develop into mature, transparent lens bodies by day 25, with a diameter reaching the largest known size of 1-3 mm, and including all the structures of a normal lens, including the capsule. However, this method still has some shortcomings. First, the 25-day culture period is too long. Second, because the lens corpuscles are tightly attached to the rigid culture dish, their bottom surface is flat, with only the top surface exhibiting a spherical convexity, making it impossible to realistically simulate the biconvex shape of the human lens. Third, because the bottom surface of the lens corpuscles is tightly attached to the bottom of the dish, there is no lens capsule to enclose them. These defects limit the realization of the complete structure, physiological, and optical functions of the lens organoids. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing an in vitro directional induction of biconvex lens organoids using a stiffness-controllable hydrogel, along with its construction method and applications.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a method for constructing biconvex lens organoids in vitro by inductively guiding stiffness-controllable hydrogels, comprising the following steps:

[0008] (1) Pluripotent stem cells were seeded into 12-well plates coated with matrix gel solution and filled with mTesR culture medium and cultured. From day 1 of culture, mTesR culture medium containing human growth factor noggin was used until day 4 to 6 of culture, and the mTesR culture medium containing human growth factor noggin was changed daily to obtain primary neuroectodermal cell clusters. The matrix gel solution was formed by dissolving embryonic stem cell matrix gel in DMEM / F12 culture medium, and the volume concentration of the matrix gel solution was 0.8 to 1.2%.

[0009] (2) Prepare polyacrylamide hydrogel with a hardness of 0.5-5 kPa; use ultraviolet light to initiate crosslinking agent to perform ultraviolet crosslinking treatment on polyacrylamide hydrogel, and then coat polyacrylamide hydrogel with matrix gel solution with a volume concentration of 0.8-1.2% to obtain the treated polyacrylamide hydrogel; then wash the treated polyacrylamide hydrogel repeatedly with HEPES buffer and soak it to obtain the cleaned polyacrylamide hydrogel.

[0010] (3) First, the cleaned polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin. Then, the primary ectodermal cell clusters were dissociated from the 12-well plate and dispersed using stem cell digestion solution. Subsequently, 10 to 30 dispersed cell clusters were seeded onto the cleaned polyacrylamide hydrogel and cultured. ROCK inhibitor Y-27632 was added to the culture medium 6 to 12 hours after seeding. mTesR culture medium containing human growth factor noggin was used until the 6th to 8th day of seeding culture, and the mTesR culture medium containing human growth factor noggin was changed daily to obtain neuroectodermal cell clusters.

[0011] (4) Culture the lens in mTesR medium containing human growth factors bFBF, BMP4 and BMP7 until day 12 to 14 of the implantation culture, and change the mTesR medium containing human growth factors bFBF, BMP4 and BMP7 daily to obtain a biconvex lens organoid composed of a capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells, which is induced in vitro by a stiffness controllable hydrogel.

[0012] Further, in step (1), the concentration of the mTesR culture medium containing human growth factor noggin is 90-110 ng / ml.

[0013] Further, in step (2), the ultraviolet crosslinking treatment specifically involves: crosslinking with 365nm ultraviolet light for 20 minutes at an energy of 9999mJ, rinsing with HEPES buffer, and then sterilizing with UV-C ultraviolet light in a clean bench for 1 hour.

[0014] Furthermore, the concentration of the HEPES buffer is 0.5–5 mmol / L.

[0015] Further, in step (3), the concentration of the mTesR culture medium containing human growth factor noggin is 90-110 ng / ml; and the concentration of the ROCK inhibitor Y-27632 is 10-30 μM.

[0016] Furthermore, in step (4), the mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 contains 17-23 ng / ml of BMP4, 17-23 ng / ml of BMP7 and 80-120 ng / ml of bFGF.

[0017] Secondly, the present invention also provides a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel. The biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel exhibits a transparent 3D structure with both upper and lower surfaces being spherical or ellipsoidal protrusions, 0.5–2 mm in diameter, and composed of a capsule, lens epithelial cells, primary lens fiber cells, and mature lens fiber cells. The spherical lower surface is completely enclosed by a capsule and expresses epithelial cell marker E-cadherin, lens fiber cell marker γ-crystallin, and lens capsule marker type IV collagen.

[0018] Furthermore, the capsule is a transparent membrane surrounding the outer layer of the lens, the lens epithelial cells are the only columnar or cuboidal epithelial cells under the capsule, the primary lens fibroblasts are fibroblasts differentiated from lens epithelial cells in which some organelle nuclei begin to degenerate, and the mature lens fibroblasts are cells differentiated from primary lens fibroblasts in which the nuclei of intracellular organelles have completely degenerated.

[0019] Thirdly, the present invention also provides an application of in vitro directional induction of biconvex lens organoids using stiffness-controllable hydrogels. These in vitro directional inductions of biconvex lens organoids using stiffness-controllable hydrogels can be used for research on the embryonic development mechanism of the lens, the pathogenesis mechanism of cataracts, and the screening of cataract-related drugs.

[0020] The beneficial effects of this invention are as follows: This invention achieves its goal by regulating the mechanical microenvironment of stem cell differentiation through a stiffness-controllable hydrogel, combined with different growth factor combinations. This breaks through the limitations of previous lens organoid morphology and structure, obtaining a lens organoid with a biconvex 3D structure on both upper and lower surfaces. Furthermore, in addition to the top surface being surrounded by a capsule, a complete bottom capsule is successfully formed. In terms of structure and light transmittance, this in vitro lens is closer to the human lens. Compared with traditional methods, it significantly shortens the culture time and establishes a more complete and stable in vitro lens development model from both chemical and mechanical signal perspectives. Therefore, this method can be used for research on lens development mechanisms, cataract pathogenesis, cataract treatment drugs, and regenerative lenses, and has broad application prospects. Attached Figure Description

[0021] Figure 1This is a morphological diagram of a biconvex lens organoid induced in vitro using a stiffness-controllable hydrogel. Figure 1 (a) Morphological image of a biconvex lens organoid induced in vitro by a stereomicroscope using a stiffness-controllable hydrogel. Figure 1 (b) is a morphological diagram of a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel under a microscope at 4x magnification.

[0022] Figure 2 A super-depth three-dimensional stereoscopic image of the top surface of a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel.

[0023] Figure 3 This is an immunofluorescence three-dimensional image of the base of a biconvex lens organoid induced in vitro using a hydrogel with controllable stiffness. Figure 3 (a) A three-dimensional expression diagram of E-cadherind, a marker of lens epithelial cells, on the basal surface of a biconvex lens organoid induced in vitro by directionally inducing the formation of a lens with controllable stiffness using a hydrogel. Figure 3 (b) A three-dimensional representation of Collagen-IV, a marker of the lens capsule on the basal surface of a biconvex lens organoid, induced in vitro by a stiffness-controllable hydrogel. Figure 3 (c) is a three-dimensional expression diagram of γ-crystallin, a marker of lens fibers on the basal surface of a biconvex lens organoid, induced in vitro by a stiffness-controllable hydrogel. Figure 3 (d) is a three-dimensional distribution diagram of cell nuclei on the basal surface of a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel.

[0024] Figure 4 This is a morphological image of the capsule of a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel under a transmission electron microscope.

[0025] Figure 5 Morphological images of outer epithelial cells of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under transmission electron microscopy.

[0026] Figure 6 The image shows the morphology and arrangement of primary fibroblasts in biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under a transmission electron microscope.

[0027] Figure 7 The image shows the morphology and arrangement of mature fibroblasts in biconvex lens organoids induced in vitro by a rigidity-controllable hydrogel under a transmission electron microscope.

[0028] Figure 8 Morphological images of biconvex lens organoids induced to maturity in vitro using stiffness-controllable hydrogels under a 4x microscope at various time points.

[0029] Figure 9 Morphological images of biconvex lens organoids induced in vitro on polyacrylamide hydrogels of different stiffness at various time points. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0031] The purpose of this invention is to overcome the shortcomings of the above-mentioned background technology and provide a method for constructing and applying a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel. This invention should be able to form a lens organoid with both the top and bottom surfaces being spherically convex, having a complete capsule and optical function, and significantly shortening the culture and maturation time of currently known lens organoids. The obtained lens organoids can be used for research on the embryonic development mechanism of the lens, the pathogenesis of cataracts, and the screening of cataract-related drugs.

[0032] In a first aspect, the present invention provides a method for constructing biconvex lens organoids in vitro by inductively guiding stiffness-controllable hydrogels, comprising the following steps:

[0033] (1) Directly induce pluripotent stem cells to differentiate into primary ectodermal cell masses.

[0034] Pluripotent stem cells were seeded into 12-well plates coated with and filled with mTesR culture medium and cultured from day 0. From day 1, mTesR culture medium containing human growth factor noggin was used until day 4-6, with the mTesR culture medium containing human growth factor noggin being changed daily to obtain primary neuroectodermal cell clusters. The matrix gel solution was formed by dissolving embryonic stem cell matrix gel in DMEM / F12 culture medium, and the volume concentration of the matrix gel solution was 0.8-1.2%.

[0035] The concentration of the mTesR culture medium containing human growth factor noggin is 90–110 ng / ml.

[0036] (2) Prepare hydrogels with specific stiffness and perform surface cell adhesion treatment.

[0037] Polyacrylamide hydrogels with a hardness of 0.5–5 kPa were prepared. The polyacrylamide hydrogels were subjected to UV crosslinking treatment using a UV-initiated crosslinking agent, and then coated with a matrix gel solution with a volume concentration of 0.8–1.2% to obtain the treated polyacrylamide hydrogels. The treated polyacrylamide hydrogels were then repeatedly washed and soaked with HEPES buffer to obtain the cleaned polyacrylamide hydrogels.

[0038] The UV crosslinking treatment specifically involves crosslinking with 365nm UV light for 20 minutes at an energy of 9999mJ, rinsing with HEPES buffer, and then sterilizing under UV-C light in a clean bench for 1 hour. The concentration of the HEPES buffer is 0.5–5 mmol / L.

[0039] (3) Primary ectodermal cells were seeded onto washed polyacrylamide hydrogel and induced to form neuroectodermal cell clusters.

[0040] First, the cleaned polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin. Then, the primary ectodermal cell clusters were dissociated from the 12-well plate and dispersed using stem cell digestion solution. Subsequently, 10-30 dispersed cell clusters were seeded onto the cleaned polyacrylamide hydrogel and cultured. 6-12 hours after seeding, the ROCK inhibitor Y-27632 was added to the culture medium. The mTesR culture medium containing human growth factor noggin was used until day 6-8 of seeding culture, and the mTesR culture medium containing human growth factor noggin was changed daily to obtain neuroectodermal cell clusters.

[0041] The concentration of the mTesR culture medium containing human growth factor noggin is 90–110 ng / ml; the concentration of the ROCK inhibitor Y-27632 is 10–30 μM.

[0042] (4) Inducing neuroectodermal cells to differentiate into mature lens organoids

[0043] By culturing the mTesR medium containing human growth factors bFBF, BMP4, and BMP7 to day 12-14 of implantation culture, and changing the mTesR medium containing human growth factors bFBF, BMP4, and BMP7 daily, biconvex lens organoids can be obtained, which are composed of the lens capsule, lens epithelial cells, primary lens fibers, and mature lens fibers, and are oriented in vitro using a stiffness-controllable hydrogel.

[0044] The mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 contains 17-23 ng / ml of BMP4, 17-23 ng / ml of BMP7 and 80-120 ng / ml of bFGF.

[0045] Secondly, the present invention also provides a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel. The biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel exhibits a transparent 3D structure with both upper and lower surfaces being spherical or ellipsoidal protrusions, 0.5–2 mm in diameter, and composed of a capsule, lens epithelial cells, primary lens fiber cells, and mature lens fiber cells. The spherical lower surface is completely enclosed by a capsule and expresses epithelial cell marker E-cadherin, lens fiber cell marker γ-crystallin, and lens capsule marker type IV collagen.

[0046] The capsule is a transparent membrane surrounding the outer layer of the lens. The lens epithelial cells are the only layer of columnar or cuboidal epithelial cells under the capsule. Primary lens fibroblasts are fibroblasts that differentiate from lens epithelial cells, in which some organelle nuclei begin to degenerate. Mature lens fibroblasts are cells that differentiate from primary lens fibroblasts, in which the nuclei of intracellular organelles have completely degenerated.

[0047] Thirdly, the present invention also provides an application of in vitro directional induction of biconvex lens organoids using stiffness-controllable hydrogels. These in vitro directional inductions of biconvex lens organoids using stiffness-controllable hydrogels can be used for research on the embryonic development mechanism of the lens, the pathogenesis mechanism of cataracts, and the screening of cataract-related drugs.

[0048] I. Implementation Examples

[0049] Example 1

[0050] A method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels, comprising the following steps:

[0051] (1) Directly induce pluripotent stem cells to differentiate into primary ectodermal cell masses.

[0052] First, 12-well plates were coated with a 0.8% (v / v) matrix gel solution and incubated at 37°C with 5% CO2 for 1 hour. The coating solution was then aspirated, and the plates were quickly washed once with DMEM / F12 medium. Next, pluripotent stem cells were seeded into the 12-well plates, marking day 0 of the culture. From day 1, mTesR medium containing 90 ng / ml of human growth factor noggin was used until day 4, with the mTesR medium containing noggin being changed daily to obtain primary neuroectodermal cell clusters.

[0053] The matrix gel solution is composed of embryonic stem cell matrix gel (BD matrix). TM hESC-qualified matrix was prepared by dissolving it in DMEM / F12 culture medium.

[0054] Microscopic observation revealed that the primary ectoderm cell mass obtained on the 4th day of culture consisted of three types of cells: the outer layer cells were larger, with abundant cytoplasm and a relatively loose arrangement; the inner layer cells were tightly arranged, with large nuclei and less cytoplasm, and their morphology was similar to that of pluripotent stem cells; while the middle layer cells had a morphology between the two.

[0055] (2) Prepare hydrogels with specific stiffness and perform surface cell adhesion treatment.

[0056] Polyacrylamide hydrogels with a hardness of 0.5 kPa (low stiffness) were prepared. The polyacrylamide hydrogels were tightly adhered to the cell slides and glass slides of 24-well plates by treating them with affinity silane and separation silane. The polyacrylamide hydrogels were placed in an oven at 37°C for 1 hour to promote their solidification. Subsequently, the cell slides containing polyacrylamide hydrogels were transferred to 24-well plates, washed three times with HEPES buffer, and soaked at 4°C for 12 hours to remove the biotoxic monomeric raw materials.

[0057] A liquid film was applied to the surface of the polyacrylamide hydrogel using a 0.5 mmol / L UV-initiated crosslinking agent solution. Crosslinking was then performed using 365 nm UV light for 20 minutes at an energy of 9999. The hydrogel was then coated with a 0.8% matrix gel solution and placed in a 37°C oven for 12 hours to facilitate cell adhesion and growth, resulting in the treated polyacrylamide hydrogel. The treated hydrogel was then rinsed three times with HEPES buffer, sterilized by UV-C irradiation in a clean bench for 1 hour, and rinsed again with HEPES buffer to obtain the cleaned polyacrylamide hydrogel.

[0058] The UV-initiating crosslinking agent is sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate (Sulfo-SANPAH) dissolved in HEPES buffer.

[0059] (3) Primary ectodermal cells were seeded onto washed polyacrylamide hydrogel and induced to form neuroectodermal cell clusters.

[0060] First, the cleaned polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin, and then stem cell digestion solution (ReLeSR) was used. TM The primary ectodermal cell clusters obtained in step (1) were detached from the 12-well plate and dispersed. Then, 30 dispersed cell clusters were seeded onto a cleaned polyacrylamide hydrogel and cultured. Within 12 hours of seeding, 1 μL of a 10 μM ROCK inhibitor Y-27632 was added to the culture medium to promote cell adhesion and survival. mTesR culture medium containing 90 ng / ml of human growth factor noggin was used until day 6 of seeding culture, with the mTesR culture medium containing human growth factor noggin being changed daily to obtain neuroectodermal cell clusters.

[0061] Microscopic observation revealed that the neuroectodermal cell clusters obtained on the 6th day of seed culture were approximately round or oval, with smooth and rounded edges, compact cell arrangement, and a certain thickness of arched protrusion on the top surface.

[0062] (4) Inducing neuroectodermal cells to differentiate into mature lens organoids

[0063] The neuroectodermal cell clusters obtained in step (3) were further cultured on 24-well plates using mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7, and the medium was changed daily.

[0064] In this embodiment, the mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 contains the following growth factors: 17 ng / ml BMP4 (bone morphogenetic protein 4), 17 ng / ml BMP7 (bone morphogenetic protein 7) and 80 ng / ml bFGF (human fibroblast growth factor).

[0065] On day 7 of culture, the cell clusters appeared "red blood cell" shaped under a microscope, with a thicker ring of cells around the periphery and a thinner central cell, forming a disc-like shape with a raised periphery and a concave center. On day 9, interlaced and fused ridge-like protrusions were visible around and in the center of the cell clusters under a microscope. On day 11, a small number of translucent areas began to appear within the cell clusters under a microscope. On day 12, biconvex lens organoids, composed of the lens capsule, lens epithelial cells, primary lens fibers, and mature lens fibers, were obtained through in vitro directional induction using a stiffness-controllable hydrogel.

[0066] Example 2

[0067] A method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels, comprising the following steps:

[0068] (1) Directly induce pluripotent stem cells to differentiate into primary ectodermal cell masses.

[0069] First, 12-well plates were coated with a 1.0% (v / v) matrix gel solution and incubated at 37°C with 5% CO2 for 1 hour. The coating solution was then aspirated, and the plates were quickly washed once with DMEM / F12 medium. Next, pluripotent stem cells were seeded into the 12-well plates, which was considered day 0 of culture. From day 1 of culture, mTesR medium containing 100 ng / ml of human growth factor noggin was used until day 5 of culture, with the mTesR medium containing human growth factor noggin being changed daily to obtain primary neuroectodermal cell clusters.

[0070] Microscopic observation revealed that the primary ectoderm cell mass obtained on day 5 of culture consisted of three types of cells: the outer layer cells were large, with abundant cytoplasm and a relatively loose arrangement; the inner layer cells were tightly arranged, with large nuclei and less cytoplasm, and their morphology was similar to that of pluripotent stem cells; while the middle layer cells had a morphology between the two.

[0071] (2) Prepare hydrogels with specific stiffness and perform surface cell adhesion treatment.

[0072] Polyacrylamide hydrogels with a hardness of 1.0 kPa (low stiffness) were prepared. The polyacrylamide hydrogels were tightly adhered to the cell slides and glass slides of 24-well plates by treating them with affinity silane and separation silane. The polyacrylamide hydrogels were placed in an oven at 37°C for 1 hour to promote their solidification. Subsequently, the cell slides containing polyacrylamide hydrogels were transferred to 24-well plates, washed three times with HEPES buffer, and soaked at 4°C for 12 hours to remove the biotoxic monomeric raw materials.

[0073] A liquid film was applied to the surface of the polyacrylamide hydrogel using a 3 mmol / L UV-initiated crosslinking agent solution. Crosslinking was then performed using 365 nm UV light for 20 minutes at an energy of 9999. The hydrogel was then coated with a 1.0% (v / v) matrix gel solution and placed in a 37°C oven for 12 hours to facilitate cell adhesion and growth, resulting in the treated polyacrylamide hydrogel. The treated hydrogel was then rinsed three times with HEPES buffer, sterilized by UV-C irradiation in a clean bench for 1 hour, and rinsed again with HEPES buffer to obtain the cleaned polyacrylamide hydrogel.

[0074] (3) Primary ectodermal cells were seeded onto washed polyacrylamide hydrogel and induced to form neuroectodermal cell clusters.

[0075] First, the cleaned polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin, and then stem cell digestion solution (ReLeSR) was used. TM The primary ectodermal cell clusters obtained in step (1) were detached from the 12-well plate and dispersed. Twenty dispersed cell clusters were then seeded onto a cleaned polyacrylamide hydrogel for incubation. Within 9 hours of seeding, 1 μL of a 20 μM ROCK inhibitor Y-27632 was added to the culture medium to promote cell adhesion and survival. mTesR culture medium containing 100 ng / ml of human growth factor noggin was used until day 7 of seeding, with the mTesR culture medium containing human growth factor noggin being changed daily to obtain neuroectodermal cell clusters.

[0076] Microscopic observation revealed that the neuroectodermal cell clusters obtained on the 7th day of seed culture were approximately round or oval, with smooth and rounded edges, compact cell arrangement, and a certain thickness of arched protrusion on the top surface.

[0077] (4) Inducing neuroectodermal cells to differentiate into mature lens organoids

[0078] The neuroectodermal cell clusters obtained in step (3) were further cultured on 24-well plates using mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7, and the medium was changed daily.

[0079] In this embodiment, the mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 contains the following growth factors: 20 ng / ml BMP4, 20 ng / ml BMP7 and 100 ng / ml bFBF.

[0080] On day 8 of culture, the cell clusters appeared "red blood cell" shaped under a microscope, with a thicker ring of cells around the periphery and a thinner central cell, forming a disc-like shape with a raised periphery and a concave center. On day 9, interlaced and fused ridge-like protrusions were visible around and in the center of the cell clusters under a microscope. On day 11, a small number of translucent areas began to appear within the cell clusters under a microscope. On day 13, biconvex lens organoids, composed of the lens capsule, lens epithelial cells, primary lens fibers, and mature lens fibers, were obtained through in vitro directional induction using a stiffness-controllable hydrogel.

[0081] Example 3

[0082] A method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels, comprising the following steps:

[0083] (1) Directly induce pluripotent stem cells to differentiate into primary ectodermal cell masses.

[0084] First, 12-well plates were coated with a 1.2% (v / v) matrix gel solution and incubated at 37°C with 5% CO2 for 1 hour. The coating solution was then aspirated, and the plates were quickly washed once with DMEM / F12 medium. Next, pluripotent stem cells were seeded into the 12-well plates, marking day 0 of the culture. From day 1, mTesR medium containing 110 ng / ml of human growth factor noggin was used until day 6, with the mTesR medium containing noggin being changed daily to obtain primary neuroectodermal cell clusters.

[0085] Microscopic observation revealed that the primary ectoderm cell mass obtained on day 6 of culture consisted of three types of cells: the outer layer cells were large, with abundant cytoplasm and a relatively loose arrangement; the inner layer cells were tightly arranged, with large nuclei and less cytoplasm, and their morphology was similar to that of pluripotent stem cells; while the middle layer cells had a morphology between the two.

[0086] (2) Prepare hydrogels with specific stiffness and perform surface cell adhesion treatment.

[0087] Polyacrylamide hydrogels with a hardness of 5.0 kPa (low stiffness) were prepared. The polyacrylamide hydrogels were tightly adhered to the cell crawling slides and glass slides of 24-well plates by treating them with affinity silane and separation silane. The polyacrylamide hydrogels were placed in an oven at 37°C for 1 hour to promote their solidification. Subsequently, the crawling slides containing polyacrylamide hydrogels were transferred to 24-well plates, washed three times with HEPES buffer, and soaked at 4°C for 12 hours to remove the biotoxic monomeric raw materials.

[0088] A liquid film was applied to the surface of the polyacrylamide hydrogel using a 5 mmol / L UV-initiated crosslinking agent solution. Crosslinking was then performed using 365 nm UV light for 20 minutes at an energy of 9999. The hydrogel was then coated with a 1.2% (v / v) matrix gel solution and placed in a 37°C oven for 12 hours to facilitate cell adhesion and growth, resulting in the treated polyacrylamide hydrogel. The hydrogel was then rinsed three times with HEPES buffer, sterilized by UV-C irradiation in a clean bench for 1 hour, and rinsed again with HEPES buffer to obtain the cleaned polyacrylamide hydrogel.

[0089] (3) Primary ectodermal cells were seeded onto washed polyacrylamide hydrogel and induced to form neuroectodermal cell clusters.

[0090] First, the cleaned polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin, and then stem cell digestion solution (ReLeSR) was used. TM The primary ectodermal cell clusters obtained in step (1) were detached from the 12-well plate and dispersed. Ten dispersed cell clusters were then seeded onto a cleaned polyacrylamide hydrogel and cultured. Within 6 hours after seeding, 1 μL of 30 μM ROCK inhibitor Y-27632 was added to the culture medium to promote cell cluster adhesion and survival. mTesR culture medium containing 110 ng / ml human growth factor noggin was used until day 8 of seeding culture, and the mTesR culture medium containing human growth factor noggin was changed daily to obtain neuroectodermal cell clusters.

[0091] (4) Inducing neuroectodermal cells to differentiate into mature lens organoids

[0092] The neuroectodermal cell clusters obtained in step (3) were further cultured on 24-well plates using mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7, and the medium was changed daily.

[0093] In this embodiment, the mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 contains the following growth factors: 23 ng / ml BMP4, 23 ng / ml BMP7 and 120 ng / ml bFBF.

[0094] On day 9 of culture, the cell clusters appeared "red blood cell" shaped under a microscope, with a thicker ring of cells around the periphery and a thinner central cell, forming a disc-like shape with a raised periphery and a concave center. On day 10, interlaced and fused ridge-like protrusions were visible around and in the center of the cell clusters under a microscope. On day 12, a small number of translucent areas began to appear within the cell clusters under a microscope. On day 14, biconvex lens organoids, composed of the lens capsule, lens epithelial cells, primary lens fibers, and mature lens fibers, were obtained through in vitro directional induction using a stiffness-controllable hydrogel.

[0095] II. Obtained lens organoids (see...) Figures 1 to 2 )

[0096] The obtained biconvex lens organoids, induced in vitro by stiffness-controllable hydrogels, exhibit transparent 3D structures (such as...). Figure 1 (a) and Figure 1 (b) shows that they are circular or elliptical, with a diameter of approximately 1-2 mm. Based on the top surface morphology of the organoids captured by a super-depth-of-field 3D microscope (e.g., ...). Figure 2 As shown in the image, the top surface exposed to the culture medium is spherically convex. Furthermore, and more remarkably, the bottom surface of the lens organoid in contact with the low-stiffness polyacrylamide hydrogel is also spherically convex. Immunofluorescence staining, three-dimensional imaging, and transmission electron microscopy reveal that the lens organoid possesses structures such as a capsule, lens epithelial cells, primary lens fibers, and mature lens fibers. The capsule completely encloses the bottom surface of the lens organoid, making it even more similar to the structure of a normal human lens.

[0097] The capsule is a transparent membrane that surrounds the outer layer of the lens. The lens body epithelial cells are the only layer of columnar or cuboidal epithelial cells under the capsule. Primary lens fibroblasts are fibroblasts that differentiate from lens epithelial cells, in which some organelle nuclei begin to degenerate. Mature lens fibroblasts are cells that differentiate from primary lens fibroblasts, in which the nuclei of intracellular organelles have completely degenerated.

[0098] III. Immunofluorescence staining of the lens organoid capsule, lens epithelium, and lens fibers (see [reference]). Figure 3 (a) to Figure 3 (d))

[0099] The culture medium for biconvex lens organoids induced in vitro using a stiffness-controllable hydrogel was aspirated, washed three times with PBS, and aspirated dry. The cells were fixed with 4% (M / V) PFA (Sigma-Aldrich) solution at room temperature for 15 minutes, aspirated dry, washed three times with PBS for 5 minutes each time, and aspirated dry. The cells were treated with 0.4% (V / V) Triton X (in PBS) for 20 minutes to penetrate the cell membrane. The cells were blocked with 10% (V / V) goat serum (in 0.1% Triton X) at room temperature for 1 hour with slow vortexing. The corresponding primary antibody diluted with 0.1% Triton X (all purchased from Santa Cruz Biotechnology) was added to the cell samples and incubated overnight at 4°C in a humidified chamber. After overnight incubation, the cells were washed four times with 0.1% Triton X for 8 minutes each time and aspirated dry. The secondary antibody diluted with 0.1% Triton X (Cell Signaling Technology) was added to the cell samples and the cells were incubated with slow vortexing in the dark for 1 hour. Wash five times with 0.1% Triton X, 8 minutes each time, and aspirate dry. Add 5 μg / ml DAPI (Sigma-Aldrich) and stain with low-speed shaking in the dark for 30 minutes. Wash four times with 0.1% Triton X, 5 minutes each time, aspirate dry, and then add PBS to prevent cell drying. Observe under a confocal fluorescence microscope, perform field-of-view stitching and Z-axis multi-slice scanning at 10x magnification to obtain an immunofluorescence three-dimensional image of the basal surface of the biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel, as shown below. Figure 3 As shown.

[0100] like Figure 3 (a) Figure 3 (b) Figure 3 (c) and Figure 3 As shown in (d), the immunofluorescence three-dimensional imaging results revealed that the basal surface of the lens organoid is spherically convex, with nucleated cells covering the outer periphery, while nucleated cells are absent inside. The spherical basal surface of the lens organoid is covered with lens epithelial-like cells expressing the epithelial cell marker E-cadherin, and the spherical basal surface is completely encapsulated by lens capsule-like structures expressing collagen-IV. Lens fiber-like cells expressing the lens fiber marker γ-crystallin are present near the outer surface. This suggests that this biconvex lens organoid possesses all the substructures of the natural lens.

[0101] IV. Electron Microscopic Observation of Lens Organoids (See...) Figures 4-8 )

[0102] Lens organoids were fixed overnight in 2.5% glutaraldehyde solution at 4°C, and then the samples were processed as follows: the fixative was discarded, and the samples were rinsed three times with 0.1M, pH 7.0 phosphate buffer for 15 minutes each time; the samples were fixed with 1% osmium tetroxide solution for 1-2 hours; the osmium tetroxide waste solution was carefully removed, and the samples were rinsed three times with 0.1M, pH 7.0 phosphate buffer for 15 minutes each time; the samples were dehydrated with ethanol solutions of gradient concentrations (including five concentrations: 30%, 50%, 70%, 80%, 90%, and 95%) for 15 minutes at each concentration, followed by treatment with 100% ethanol for 20 minutes; finally, the samples were treated with pure acetone for 20 minutes. Samples were treated with a mixture of embedding agent and acetone (V / V = 1 / 1) for 1 hour; then with a mixture of embedding agent and acetone (V / V = 3 / 1) for 3 hours; and finally with pure embedding agent overnight. The infiltrated samples were then embedded and heated at 70°C overnight to obtain the embedded samples. Samples were sectioned using a LEICAEMUC7 ultramicrotome to obtain sections of 70-90 nm. The sections were stained with lead citrate solution and 50% ethanol saturated solution of uranium acetate for 5-10 minutes each, and then observed under a Hitachi H-7650 transmission electron microscope. Glutaraldehyde, buffer solution, ethanol, acetone, lead citrate, and methylene blue were all purchased from Sinopharm Chemical Reagent Company. Osmium tetroxide, Spurr embedding agent, and uranium acetate were all purchased from SPI-CHEM.

[0103] Electron microscopy results showed that the lens organoid possesses structures such as a capsule, lens epithelial cells, primary lens fiber cells, and mature lens fiber cells, similar to the structure of a normal human lens. The capsule is a transparent membrane surrounding the outer layer of the lens. The lens epithelial cells are the only layer of columnar or cuboidal epithelial cells beneath the capsule. Primary lens fiber cells are fiber cells differentiated from lens epithelial cells, in which some organelle nuclei begin to degenerate. Mature lens fiber cells are cells differentiated from primary lens fiber cells, in which the nuclei of intracellular organelle cells have completely degenerated.

[0104] V. The Culture Process of Biconvex Lens Organoids (see...) Figure 8 )

[0105] Throughout the process of culturing mature biconvex lens organoids on 0.5-5 kPa ultra-low stiffness hydrogels, starting from the 5th day of culture, the cell clusters were photographed daily using a microscope to record the growth process of the lens organoids and their key morphological and structural features.

[0106] Under a microscope, on day 7 of culture, the cell clusters appear as "red blood cells," with a thicker ring of cells around the periphery and a thinner central cell, presenting a disc-like shape with a raised periphery and a concave center. On day 9, interlaced and fused ridge-like protrusions are visible around and in the center of the cell clusters. On days 10-11, a small number of translucent areas begin to appear within the cell clusters. After 11-13 days of culture, a biconvex, transparent lens organoid composed of the lens capsule, lens epithelial cells, primary lens fibers, and mature lens fibers is obtained.

[0107] VI. Differences in the developmental process of lens organoids on hydrogels of different stiffness (see...) Figure 9 )

[0108] The method described in this patent was used to induce pluripotent stem cells into lens organoids on hydrogels with different stiffnesses of 1, 5, 20, and 100 kPa. Throughout the 13-day process, starting from day 5 of culture, the cell clusters were photographed daily using a microscope to record the growth process of the lens organoids and the influence of different substrate stiffnesses on the growth rate and morphological structure.

[0109] Under a microscope, the maturation rate of lens organoids on 1 kPa and 5 kPa hydrogels was significantly faster than that on 20 kPa and 100 kPa hydrogels. After 11 days, rapid and large-scale production of clear tissues in the 1 kPa and 5 kPa groups could be observed under a microscope. Furthermore, the volume of organoids on 1 kPa hydrogels was relatively larger than that on 5 kPa hydrogels, suggesting that the softer mechanical microenvironment is conducive to the directional and rapid differentiation into mature lens fibroblast-like cells.

[0110] The manufacturers and product numbers of the reagents mentioned in this article are as follows:

[0111] Noggin: Manufactured by R&D (R&D Systems, USA), part number 6057-NG.

[0112] bFGF: Manufactured by PeproTech (USA), part number 100-18B.

[0113] BMP4: Manufactured by R&D (R&D Systems, USA), part number 314-BP-010.

[0114] BMP7: Manufactured by R&D (R&D Systems, USA), part number 354-BP-010.

[0115] Y-27632: Manufactured by R&D (R&D Systems, USA), part number 1254-10.

[0116] Sulfo-SANPAH: Manufactured by Sigma-Aldrich (USA), part number 803332.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels, comprising the following steps: (1) Pluripotent stem cells were seeded into 12-well plates coated with matrix gel solution and filled with mTesR culture medium and cultured. From day 1 of culture, mTesR culture medium containing human growth factor noggin was used until day 4-6 of culture, and the mTesR culture medium containing human growth factor noggin was changed daily to obtain primary neuroectodermal cell clusters. The matrix gel solution was formed by dissolving embryonic stem cell matrix gel in DMEM / F12 culture medium, and the volume concentration of the matrix gel solution was 0.8-1.2%. (2) Polyacrylamide hydrogel with a hardness of 0.5-5 kPa was prepared. The polyacrylamide hydrogel was subjected to UV crosslinking treatment using a UV-initiated crosslinking agent, and then coated with matrix gel solution with a volume concentration of 0.8-1.2% to obtain the treated polyacrylamide hydrogel. The treated polyacrylamide hydrogel was then repeatedly washed and soaked with HEPES buffer to obtain the cleaned polyacrylamide hydrogel. (3) First, place the cleaned polyacrylamide hydrogel in mTesR culture medium containing human growth factor noggin. Then, use stem cell digestion solution to detach the primary neuroectodermal cell clusters from the 12-well plate and disperse them. Subsequently, seed 10-30 dispersed cell clusters onto the cleaned polyacrylamide hydrogel and start culturing. 6-12 hours after seeding, add the ROCK inhibitor Y-27632 to the culture medium. Use mTesR culture medium containing human growth factor noggin until day 6-8 of seeding culture, changing the mTesR culture medium containing human growth factor noggin daily to obtain neuroectodermal cell clusters. (4) Culture in mTesR culture medium containing human growth factors bFGF, BMP4, and BMP7 until day 12-14 of seeding culture. Furthermore, by changing the mTesR culture medium containing human growth factors bFGF, BMP4, and BMP7 daily, biconvex lens organoids composed of the lens capsule, lens epithelial cells, primary lens fibers, and mature lens fibers can be obtained through in vitro directional induction using a stiffness-controllable hydrogel.

2. The method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels according to claim 1, characterized in that, In step (1), the concentration of the mTesR culture medium containing human growth factor noggin is 90-110 ng / ml.

3. The method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels according to claim 1, characterized in that, In step (2), the ultraviolet crosslinking treatment specifically involves: crosslinking with 365nm ultraviolet light for 20 minutes at an energy of 9999mJ, rinsing with HEPES buffer, and then sterilizing with UV-C ultraviolet light in a clean bench for 1 hour.

4. The method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels according to claim 3, characterized in that, The concentration of the HEPES buffer solution is 0.5–5 mmol / L.

5. The method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels according to claim 1, characterized in that, In step (3), the concentration of the mTesR culture medium containing human growth factor noggin is 90-110 ng / ml; the concentration of the ROCK inhibitor Y-27632 is 10-30 μM.

6. The method for constructing biconvex lens organoids by in vitro directional induction using stiffness-controllable hydrogels according to claim 1, characterized in that, In step (4), the mTesR culture medium containing human growth factors bFGF, BMP4 and BMP7 contains 17-23 ng / ml of BMP4, 17-23 ng / ml of BMP7 and 80-120 ng / ml of bFGF.

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