Biconvex crystalline lens type organ directionally induced in vitro by rigidity-controllable hydrogel and construction method and application of biconvex crystalline lens type organ

Abstract

The invention discloses a biconvex crystalline lens organ relying on stiffness-controllable hydrogel external directional induction as well as a construction method and application thereof. A crystalline lens organ model provided by the invention is jointly realized by virtue of stiffness-controllable hydrogel and a composite growth factor; and the pluripotent stem cells are rapidly and directionally induced into crystalline lens organs with high quality in vitro. According to the technical scheme, the method comprises the following steps: (1) directionally inducing and differentiating the pluripotent stem cells into primary ectoderm cell clusters; (2) preparing hydrogel with specific rigidity and carrying out surface cell adhesion treatment; (3) planting the primary ectoderm cells on the cleaned polyacrylamide hydrogel and inducing the primary ectoderm cells into a neural ectoderm cell cluster; and (4) inducing the neural ectoderm cells to directionally differentiate into mature crystalline lens organs. The three-dimensional lens organ-like model obtained by the method has a sphere-like biconvex morphology with a top surface and a bottom surface, is transparent and has structures such as capsule membranes, lens epithelial cells and lens fibrocytes, and the bottom surface is also wrapped by a complete lens capsule membrane. Compared with the prior art, the method realizes the formation of a complete capsule film on the bottom surface and a sphere-like form. The obtained crystalline lens organ can be used for research on crystalline lens embryonic development, research on cataract pathogenesis and research, development and screening of cataract drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to an in vitro directional induction of a biconvex lens organoid by means of a rigidity-controllable hydrogel, and a construction method and application thereof. Background Art

[0002] With the rapid development of stem cell technology and regenerative medicine in recent years, the use of stem cells to cultivate organoids and then construct disease models has become a research hotspot with both scientific research value and clinical prospects. Organoids are simplified versions of organs that are constructed in vitro and have the same macroscopic and microscopic three-dimensional structure as human tissues and organs, and even have corresponding physiological functions. They can simulate the structure and function of real organs in the body, providing new research methods and treatment means for precision medicine. The culture of organoids generally comes from pluripotent stem cells with broad differentiation potential. These cells with self-renewal and directed differentiation capabilities can grow and assemble into organoids with precise arrangement patterns and spatial structures under specific culture conditions.

[0003] Cataract is an ophthalmic disease caused by various reasons such as aging, genetics, and trauma, which leads to vision impairment due to denaturation and aggregation of lens proteins and lens opacity. It is also the world's leading cause of blindness. Therefore, it is necessary to use lens organoids that are closer to the structure and function of the natural lens to study the development mechanism of the lens, establish cataract disease models to conduct pathogenic mechanism research and high-throughput drug screening, and even study and achieve cataract stem cell therapy for lens tissue regeneration.

[0004] For a long time, researchers have successfully constructed a variety of lens organoids in vitro through different cell sources and culture methods in the process of continuous exploration, which are now called lens bodies. In 2010, the three-stage culture method was established, and human embryonic stem cells were differentiated and cultured into lens bodies with three-dimensional structures through a combination of multiple growth factors. This method has also become the technical basis for the future iteration of lens organoid culture methods. At present, the relatively complete lens organoid construction method is the "fried egg method" proposed in 2017 (see Chinese patent CN105238737A "New method for in vitro directional differentiation of induced pluripotent stem cells into lens bodies"), that is, human urine-derived induced pluripotent stem cells are cultured through a more complete three-stage culture and early mechanical screening, and a "fried egg-like" cell structure appears during the differentiation process, and finally develops into a mature and transparent lens body on the 25th day, with a diameter of 1-3mm, the largest known so far, and 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, the bottom surface of the lens organoids is flat because they are tightly attached to the hard culture dish, and only the top surface is spherical and convex, which cannot truly simulate the biconvex shape of the human lens; third, the bottom surface of the lens organoids is tightly attached to the bottom of the dish, so there is no lens capsule to wrap it. These defects limit the complete structure and physiological and optical functions of lens organoids. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a method and application of in vitro directional induction of biconvex lens organoids by means of stiffness-controllable hydrogels and their construction.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for constructing biconvex lens organoids by inducing in vitro directional induction of a stiffness-controllable hydrogel, comprising the following steps:

[0008] (1) inoculating pluripotent stem cells onto a 12-well plate coated with a matrigel solution and filled with mTesR culture medium to start culturing; from the first day of culturing, using mTesR culture medium containing human growth factor noggin until the fourth to sixth day of culturing, and replacing the mTesR culture medium containing human growth factor noggin daily to obtain primary neuroectoderm cell clusters; the matrigel solution is formed by dissolving embryonic stem cell matrigel in DMEM / F12 culture medium, and the volume concentration of the matrigel solution is 0.8-1.2%;

[0009] (2) preparing a polyacrylamide hydrogel with a hardness of 0.5 to 5 kPa; performing ultraviolet crosslinking treatment on the polyacrylamide hydrogel using an ultraviolet light-induced crosslinking agent, and then coating the polyacrylamide hydrogel with a matrix gel solution having a volume concentration of 0.8 to 1.2% to obtain a treated polyacrylamide hydrogel; then repeatedly washing the treated polyacrylamide hydrogel with a HEPES buffer and soaking it to obtain a cleaned polyacrylamide hydrogel;

[0010] (3) First, the washed polyacrylamide hydrogel is placed in a mTesR culture medium containing human growth factor noggin, and then the primary ectoderm cell clusters are dissociated from the 12-well plate using stem cell digestion solution and blown apart, and then 10 to 30 cell clusters that have been blown apart are planted on the washed polyacrylamide hydrogel to start culturing; within 6 to 12 hours after planting and culturing, ROCK inhibitor Y-27632 is added to the culture medium, and the mTesR culture medium containing human growth factor noggin is used until the 6th to 8th day of planting and culturing, and the mTesR culture medium containing human growth factor noggin is replaced daily to obtain neuroectoderm cell clusters;

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

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

[0013] Furthermore, in step (2), the ultraviolet cross-linking treatment is specifically: cross-linking with 365nm ultraviolet light for 20 minutes, energy 9999mJ, rinsing with HEPES buffer, and then placing in a clean bench for UV-C ultraviolet light sterilization for 1 hour.

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

[0015] Furthermore, 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] In the second aspect, the present invention also provides a biconvex lens organoid induced in vitro by stiffness-controllable hydrogel, wherein the biconvex lens organoid induced in vitro by stiffness-controllable hydrogel presents a transparent 3D structure, and both the upper and lower surfaces are spherical protrusions, which are spherical or ellipsoidal, with a diameter of 0.5 to 2 mm, and are composed of a capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells, and the spherical lower surface is wrapped by a complete capsule, expressing the epithelial cell marker capsule cadherin E-cadherin, the lens fiber cell marker γ-crystallin, and the lens capsule marker type 4 collagen Collagen-IV.

[0018] Furthermore, the capsule is a transparent film surrounding the outer layer of the lens, the lens epithelial cells are the only layer of columnar or cubic epithelial cells under the capsule, the primary lens fiber cells are fiber cells differentiated from the lens epithelial cells with some organelles and nuclei beginning to degenerate, and the mature lens fiber cells are cells differentiated from the primary lens fiber cells with intracellular organelles and nuclei completely degenerated.

[0019] In the third aspect, the present invention also provides an application of in vitro directional induction of biconvex lens organoids by relying on stiffness-controllable hydrogels. The in vitro directional induction of biconvex lens organoids by relying on stiffness-controllable hydrogels can be used for the study of the mechanism of lens embryonic development, the study of the pathogenesis of cataracts, and the screening of cataract-related drugs.

[0020] The beneficial effects of the present invention are as follows: the present invention achieves this by regulating the mechanical microenvironment of stem cell differentiation through stiffness-controllable hydrogels, combined with different growth factor combinations, breaking through the previous limitations of lens organoid morphology and structure, obtaining lens organoids with a biconvex 3D structure on the upper and lower surfaces, and successfully forming a complete bottom capsule in addition to being surrounded by a capsule on the top surface. The in vitro lens is closer to the human lens in structure and light transmittance, and the culture time is greatly shortened compared to traditional methods, establishing a more complete and stable in vitro lens development model from the dual perspectives of chemical signals and mechanical signals; therefore, the present method can be used for the study of lens development mechanisms, cataract pathogenesis, cataract treatment drugs, and regenerated lenses, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a morphological diagram of biconvex lens organoids induced in vitro by stiffness-controlled hydrogels, where: Figure 1 (a) is a morphological image of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under stereomicroscope. Figure 1 (b) A morphological image of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under a 4x microscope;

[0022] Figure 2 This is a super-depth three-dimensional imaging image of the top surface of a biconvex lens organoid induced in vitro by means of a controllable stiffness hydrogel;

[0023] Figure 3 This is a three-dimensional immunofluorescence image of the bottom surface of a biconvex lens organoid induced in vitro by a stiffness-controllable hydrogel. Figure 3 (a) A three-dimensional expression diagram of the lens epithelial cell marker E-cadherind on the bottom surface of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels; Figure 3 (b) A three-dimensional expression diagram of the lens capsule marker Collagen-IV on the bottom surface of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels; Figure 3 (c) A three-dimensional expression diagram of the lens fiber marker γ-crystallin on the bottom surface of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels; Figure 3 (d) The three-dimensional distribution of cell nuclei on the bottom surface of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels;

[0024] Figure 4 The morphology of biconvex lens organoid capsule induced in vitro by stiffness-controllable hydrogel under transmission electron microscopy;

[0025] Figure 5 The morphology of the outer epithelial cells of biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under transmission electron microscopy;

[0026] Figure 6 The morphology and arrangement of primary fiber cells in biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under transmission electron microscopy;

[0027] Figure 7 The morphology and arrangement of mature fiber cells in biconvex lens organoids induced in vitro by stiffness-controllable hydrogels under transmission electron microscopy;

[0028] Figure 8 The morphological images of biconvex lens organoids induced in vitro at different time points of maturity by inducing them in vitro with controllable stiffness hydrogel under a 4x microscope;

[0029] Fig. 9 Morphological diagrams of biconvex lens organoids induced on polyacrylamide hydrogels of different stiffness at different time points in vitro by relying on stiffness-controllable hydrogels. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] The purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology, and to provide a biconvex lens organoid induced in vitro by stiffness-controllable hydrogel and a construction method and application thereof. The present invention should be able to form a lens organoid with a spherical convexity on both the top and bottom surfaces, a complete capsule and optical functions, and significantly shorten the culture and maturation time of currently known lens organoids; the obtained lens organoids can be used for the study of the mechanism of lens embryonic development, the study of 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 by inducing in vitro directional induction of a stiffness-controllable hydrogel, comprising the following steps:

[0033] (1) Directed differentiation of pluripotent stem cells into primary ectoderm cell clusters

[0034] The pluripotent stem cells are inoculated onto a 12-well plate coated with a matrix gel solution and filled with an mTesR culture medium to begin culture, which is considered to be the 0th day of culture; from the first day of culture, an mTesR culture medium containing the human growth factor noggin is used until the 4th to 6th day of culture, and the mTesR culture medium containing the human growth factor noggin is replaced daily to obtain a primary neuroectoderm cell mass; the matrix gel solution is formed by dissolving embryonic stem cell matrix gel in a DMEM / F12 culture medium, and the volume concentration of the matrix gel solution is 0.8 to 1.2%.

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

[0036] (2) Preparation of hydrogels with specific stiffness and surface cell adhesion treatment

[0037] A polyacrylamide hydrogel with a hardness of 0.5 to 5 kPa is prepared; a polyacrylamide hydrogel is subjected to ultraviolet crosslinking treatment using an ultraviolet light-initiated crosslinking agent, and then the polyacrylamide hydrogel is coated with a matrix gel solution with a volume concentration of 0.8 to 1.2% to obtain a treated polyacrylamide hydrogel; and then the treated polyacrylamide hydrogel is repeatedly washed and soaked with a HEPES buffer to obtain a cleaned polyacrylamide hydrogel.

[0038] The ultraviolet crosslinking treatment is specifically: using 365nm ultraviolet light for crosslinking for 20 minutes, energy 9999mJ, and placing in a clean bench for UV-C ultraviolet light sterilization for 1 hour after rinsing with HEPES buffer. The concentration of the HEPES buffer is 0.5-5mmol / L.

[0039] (3) Primary ectoderm cells were implanted on the cleaned polyacrylamide hydrogel and induced into neuroectoderm cell clusters

[0040] First, the cleaned polyacrylamide hydrogel is placed in mTesR culture medium containing human growth factor noggin, and then the primary ectoderm cell clusters are dissociated from the 12-well plate using stem cell digestion solution and blown away. Subsequently, 10 to 30 cell clusters that have been blown away are implanted on the cleaned polyacrylamide hydrogel to start culture. Within 6 to 12 hours after implantation and culture, ROCK inhibitor Y-27632 is added to the culture medium, and mTesR culture medium containing human growth factor noggin is used until the 6th to 8th day of implantation and culture, and the mTesR culture medium containing human growth factor noggin is replaced daily to obtain neuroectoderm cell clusters.

[0041] The concentration of the mTesR culture solution 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 with mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 until the 12th to 14th day of implantation and culture, and replacing the mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 daily, biconvex lens organoids composed of capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells can be obtained, which are directionally induced in vitro by stiffness-controllable hydrogels.

[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] In the second aspect, the present invention also provides a biconvex lens organoid induced in vitro by stiffness-controllable hydrogel, wherein the biconvex lens organoid induced in vitro by stiffness-controllable hydrogel presents a transparent 3D structure, and both the upper and lower surfaces are spherical protrusions, which are spherical or ellipsoidal, with a diameter of 0.5 to 2 mm, and are composed of a capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells, and the spherical lower surface is wrapped by a complete capsule, expressing the epithelial cell marker capsule cadherin E-cadherin, the lens fiber cell marker γ-crystallin, and the lens capsule marker type 4 collagen Collagen-IV.

[0046] The capsule is a transparent film surrounding the outer layer of the lens, the lens epithelial cells are the only layer of columnar or cubic epithelial cells under the capsule, the primary lens fiber cells are fiber cells differentiated from the lens epithelial cells with some organelles and nuclei beginning to degenerate, and the mature lens fiber cells are cells differentiated from the primary lens fiber cells with intracellular organelles and nuclei completely degenerated.

[0047] In the third aspect, the present invention also provides an application of in vitro directional induction of biconvex lens organoids by relying on stiffness-controllable hydrogels. The in vitro directional induction of biconvex lens organoids by relying on stiffness-controllable hydrogels can be used for the study of the mechanism of lens embryonic development, the study of the pathogenesis of cataracts, and the screening of cataract-related drugs.

[0048] 1. Implementation

[0049] Example 1

[0050] A method for constructing biconvex lens organoids by inducing directionality in vitro using a stiffness-controllable hydrogel comprises the following steps:

[0051] (1) Directed differentiation of pluripotent stem cells into primary ectoderm cell clusters

[0052] First, a 12-well plate was coated with a 0.8% volume concentration of Matrigel solution and placed in a 37°C, 5% CO 2Incubator for 1 hour, then remove the coating solution and quickly wash once with DMEM / F12 medium. Next, pluripotent stem cells were seeded in 12-well plates, which was considered as day 0 of culture; from day 1 of culture, mTesR culture medium containing human growth factor noggin at a concentration of 90 ng / ml was used until day 4 of culture, and the mTesR culture medium containing human growth factor noggin was replaced daily to obtain primary neuroectoderm cell clusters.

[0053] The matrix gel solution is made of embryonic stem cell matrix gel (BD matrigel TM hESC-qualified Matrix) was dissolved in DMEM / F12 culture medium.

[0054] Microscopic observation revealed that the primary ectoderm cell clusters obtained on the fourth day of culture were composed of three types of cells: the outer layer cells were larger, had rich cytoplasm and were loosely arranged; the inner cells were tightly arranged, had 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) Preparation of hydrogels with specific stiffness and surface cell adhesion treatment

[0056] A polyacrylamide hydrogel with a hardness of 0.5 kPa (low stiffness) was prepared. The 24-well plate cell slide and glass slide were treated with affinity silane and separation silane to make the polyacrylamide hydrogel tightly adhere to the cell slide. The polyacrylamide hydrogel was placed in a 37°C oven for 1 hour to promote its solidification. The slide containing the polyacrylamide hydrogel was then transferred to a 24-well plate, washed three times with HEPES buffer and immersed at 4°C for 12 hours to remove the biologically toxic monomer raw materials.

[0057] A layer of liquid film was covered on the surface of the polyacrylamide hydrogel using a 0.5 mmol / L ultraviolet light-initiated crosslinking agent solution, and crosslinked using 365 nm ultraviolet light for 20 minutes at an energy of 9999. The polyacrylamide hydrogel was then coated with a matrix gel solution having a volume concentration of 0.8%, and placed in a 37°C oven for 12 hours to facilitate cell attachment and growth, thereby obtaining a treated polyacrylamide hydrogel. The treated polyacrylamide hydrogel was then rinsed three times with HEPES buffer, and then placed in a clean bench for UV-C ultraviolet light sterilization for 1 hour, and then rinsed again with HEPES buffer once to obtain a cleaned polyacrylamide hydrogel.

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

[0059] (3) Primary ectoderm cells were implanted on the cleaned polyacrylamide hydrogel and induced into neuroectoderm cell clusters

[0060] First, the washed polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin, and then the stem cell digestion solution (ReLeSR TM ) The primary ectoderm cell clusters obtained in step (1) were dissociated from the 12-well plate and blown away, and then 30 cell clusters after blowing away were planted on the washed polyacrylamide hydrogel to start culturing. Within 12 hours after planting and culturing, 1 μL of 10 μM ROCK inhibitor Y-27632 was added to the culture medium to promote the adhesion and survival of the cell clusters, and the mTesR culture medium containing the human growth factor noggin at a concentration of 90 ng / ml was used until the 6th day of planting and culturing, and the mTesR culture medium containing the human growth factor noggin was replaced daily to obtain the neuroectoderm cell clusters.

[0061] Microscopic observation revealed that the neuroectodermal cell clusters obtained on the sixth day of implantation and culture were approximately circular or oval in shape, with smooth and rounded edges, compactly arranged cells, and an arched protrusion of a certain thickness on the top surface.

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

[0063] The neuroectodermal cell clusters obtained in step (3) are continued to be cultured on 24-well plate slides, using mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 and changing it 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 the 7th day of planting and culture, under the microscope, it can be observed that the cell clusters are "red blood cells", that is, the surrounding cells are thicker and the central cells are thinner, presenting a round cake shape with convex surroundings and concave centers. On the 9th day of planting and culture, under the microscope, it can be seen that there are staggered and fused ridge-like protrusions distributed around and in the center of the cell clusters. On the 11th day of planting and culture, under the microscope, a small amount of transparent areas that can transmit light can be seen beginning to appear in the cell clusters. On the 12th day of planting and culture, biconvex lens organoids composed of capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells can be obtained, which rely on stiffness-controllable hydrogels to induce biconvex lens organs in vitro.

[0066] Example 2

[0067] A method for constructing biconvex lens organoids by inducing directionality in vitro using a stiffness-controllable hydrogel comprises the following steps:

[0068] (1) Directed differentiation of pluripotent stem cells into primary ectoderm cell clusters

[0069] First, a 12-well plate was coated with a 1.0% volume concentration of Matrigel solution and placed in a 37°C, 5% CO 2 Incubator for 1 hour, then remove the coating solution and quickly wash once with DMEM / F12 medium. Next, pluripotent stem cells were seeded in 12-well plates, which was considered as day 0 of culture; from day 1 of culture, mTesR culture medium containing human growth factor noggin at a concentration of 100 ng / ml was used until day 5 of culture, and the mTesR culture medium containing human growth factor noggin was replaced daily to obtain primary neuroectoderm cell clusters.

[0070] Microscopic observation revealed that the primary ectoderm cell clusters obtained on the 5th day of culture were composed of three types of cells: the outer layer cells were larger in size, rich in cytoplasm and arranged relatively loosely; the inner cells were tightly arranged, with large nuclei and less cytoplasm, and their morphology was similar to pluripotent stem cells, while the morphology of the middle layer cells was between the two.

[0071] (2) Preparation of hydrogels with specific stiffness and surface cell adhesion treatment

[0072] A polyacrylamide hydrogel with a hardness of 1.0 kPa (low stiffness) was prepared. The 24-well plate cell slide and glass slide were treated with affinity silane and separation silane to make the polyacrylamide hydrogel tightly adhere to the cell slide. The polyacrylamide hydrogel was placed in a 37°C oven for 1 hour to promote its solidification. The slide containing the polyacrylamide hydrogel was then transferred to a 24-well plate, washed three times with HEPES buffer and immersed at 4°C for 12 hours to remove the biologically toxic monomer raw materials.

[0073] A layer of liquid film was covered on the surface of the polyacrylamide hydrogel using a 3 mmol / L ultraviolet light-initiated crosslinking agent solution, and crosslinked using 365 nm ultraviolet light for 20 minutes with an energy of 9999. The polyacrylamide hydrogel was then coated with a 1.0% volume concentration of matrix gel solution and placed in a 37°C oven for 12 hours to facilitate cell attachment and growth, thereby obtaining a treated polyacrylamide hydrogel. The treated polyacrylamide hydrogel was then rinsed three times with HEPES buffer, placed in a clean bench for UV-C ultraviolet light sterilization for 1 hour, and rinsed again with HEPES buffer once to obtain a cleaned polyacrylamide hydrogel.

[0074] (3) Primary ectoderm cells were implanted on the cleaned polyacrylamide hydrogel and induced into neuroectoderm cell clusters

[0075] First, the washed polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin, and then the stem cell digestion solution (ReLeSR TM ) The primary ectoderm cell clusters obtained in step (1) were dissociated from the 12-well plate and blown away, and then the 20 blown away cell clusters were planted on the washed polyacrylamide hydrogel to start culturing. Within 9 hours after planting and culturing, 1 μL of 20 μM ROCK inhibitor Y-27632 was added to the culture medium to promote the adhesion and survival of the cell clusters, and the mTesR culture medium containing the human growth factor noggin at a concentration of 100 ng / ml was used until the 7th day of planting and culturing, and the mTesR culture medium containing the human growth factor noggin was replaced daily to obtain the neuroectoderm cell clusters.

[0076] Microscopic observation revealed that the neuroectodermal cell clusters obtained on the 7th day of implantation and culture were approximately circular or elliptical, with smooth and rounded edges, compactly arranged cells, and an arched protrusion of a certain thickness on the top surface.

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

[0078] The neuroectodermal cell clusters obtained in step (3) are continued to be cultured on 24-well plate slides, using mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 and changing it 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 the 8th day of planting and culture, under the microscope, it can be observed that the cell clusters are "red blood cells", that is, the surrounding cells are thicker and the central cells are thinner, presenting a round cake shape with convex surroundings and concave centers. On the 9th day of planting and culture, under the microscope, it can be seen that there are staggered and fused ridge-like protrusions distributed around and in the center of the cell clusters. On the 11th day of planting and culture, under the microscope, a small amount of transparent areas that can transmit light can be seen beginning to appear in the cell clusters. On the 13th day of planting and culture, biconvex lens organoids composed of capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells can be obtained, which rely on stiffness-controllable hydrogels to induce biconvex lens organs in vitro.

[0081] Example 3

[0082] A method for constructing biconvex lens organoids by inducing directionality in vitro using a stiffness-controllable hydrogel comprises the following steps:

[0083] (1) Directed differentiation of pluripotent stem cells into primary ectoderm cell clusters

[0084] First, a 12-well plate was coated with a 1.2% volume concentration of Matrigel solution and placed in a 37°C, 5% CO 2 Incubator for 1 hour, then remove the coating solution and quickly wash once with DMEM / F12 medium. Next, pluripotent stem cells were seeded in 12-well plates, which was considered as day 0 of culture; from day 1 of culture, mTesR culture medium containing human growth factor noggin at a concentration of 110 ng / ml was used until day 6 of culture, and the mTesR culture medium containing human growth factor noggin was replaced daily to obtain primary neuroectoderm cell clusters.

[0085] Microscopic observation revealed that the primary ectoderm cell clusters obtained on the sixth day of culture were composed of three types of cells: the outer layer cells were larger in size, rich in cytoplasm and arranged relatively loosely; the inner cells were tightly arranged, with large nuclei and less cytoplasm, and their morphology was similar to that of pluripotent stem cells, while the morphology of the middle layer cells was between the two.

[0086] (2) Preparation of hydrogels with specific stiffness and surface cell adhesion treatment

[0087] A polyacrylamide hydrogel with a hardness of 5.0 kPa (low stiffness) was prepared. The 24-well plate cell slide and glass slide were treated with affinity silane and separation silane to make the polyacrylamide hydrogel tightly adhere to the cell slide. The polyacrylamide hydrogel was placed in a 37°C oven for 1 hour to promote its solidification. The slide containing the polyacrylamide hydrogel was then transferred to a 24-well plate, washed three times with HEPES buffer and immersed at 4°C for 12 hours to remove the biologically toxic monomer raw materials.

[0088] A layer of liquid film was covered on the surface of the polyacrylamide hydrogel using a 5 mmol / L ultraviolet light-initiated crosslinker solution, and crosslinked using 365 nm ultraviolet light for 20 minutes with an energy of 9999. The polyacrylamide hydrogel was then coated with a 1.2% volume concentration of matrix gel solution and placed in a 37°C oven for 12 hours to facilitate cell attachment and growth, thereby obtaining a treated polyacrylamide hydrogel. The hydrogel was then rinsed three times with HEPES buffer, sterilized with UV-C light in a clean bench for 1 hour, and rinsed again with HEPES buffer once to obtain a cleaned polyacrylamide hydrogel.

[0089] (3) Primary ectoderm cells were implanted on the cleaned polyacrylamide hydrogel and induced into neuroectoderm cell clusters

[0090] First, the washed polyacrylamide hydrogel was placed in mTesR culture medium containing human growth factor noggin, and then the stem cell digestion solution (ReLeSR TM ) The primary ectoderm cell clusters obtained in step (1) were dissociated from the 12-well plate and blown away, and then the 10 blown away cell clusters were planted on the washed polyacrylamide hydrogel to start culture. Within 6 hours after planting and culture, 1 μL of 30 μM ROCK inhibitor Y-27632 was added to the culture medium to promote the adhesion and survival of the cell clusters, and the mTesR culture medium containing the human growth factor noggin at a concentration of 110 ng / ml was used until the 8th day of planting and culture, and the mTesR culture medium containing the human growth factor noggin was replaced daily to obtain the neuroectoderm cell clusters.

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

[0092] The neuroectodermal cell clusters obtained in step (3) are continued to be cultured on 24-well plate slides, using mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 and changing it 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 the 9th day of planting and culture, under the microscope, it can be observed that the cell clusters are "red blood cells", that is, the surrounding cells are thicker and the central cells are thinner, presenting a round cake shape with convex surroundings and concave centers. On the 10th day of planting and culture, under the microscope, it can be seen that there are staggered and fused ridge-like protrusions distributed around and in the center of the cell clusters. On the 12th day of planting and culture, under the microscope, a small amount of transparent areas that can transmit light can be seen beginning to appear in the cell clusters. On the 14th day of planting and culture, biconvex lens organoids composed of capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells can be obtained, which rely on stiffness-controlled hydrogels to induce biconvex lens organs in vitro.

[0095] 2. Obtained Lens Organoids (see Figure 1 to Figure 2 )

[0096] The obtained biconvex lens organoids induced in vitro by stiffness-controlled hydrogels present transparent 3D structures (such as Figure 1 (a) and Figure 1 (b)), which is round or oval with a diameter of about 1-2 mm. According to the top surface morphology of the organoids photographed by the ultra-depth 3D microscope (as shown in Figure 2 The top surface exposed to the culture medium is spherical and convex. What is even more rare is that the bottom surface of the lens organoid in contact with the low-rigidity polyacrylamide hydrogel is also spherical and convex. Under immunofluorescence staining three-dimensional imaging and projection electron microscopy, it can be observed that the lens organoid has structures such as capsule, lens epithelial cells, primary lens fiber cells, and mature lens fiber cells. The capsule completely wraps the bottom surface of the lens organoid, which is more similar to the normal human lens structure.

[0097] The capsule is a transparent film 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 fiber cells are fiber cells differentiated from lens epithelial cells with some organelles and nuclei beginning to degenerate. Mature lens fiber cells are cells differentiated from primary lens fiber cells with intracellular organelles and nuclei completely degenerated.

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

[0099] The culture medium of biconvex lens organoids induced in vitro by stiffness-controlled hydrogel was aspirated, washed 3 times with PBS, and aspirated. The cells were fixed with 4% (M / V) PFA (purchased from Sigma-Aldrich) solution at room temperature for 15 minutes, the PFA was aspirated, washed three times with PBS, 5 minutes each time, and aspirated. 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 low-speed shaking. The corresponding primary antibodies (all purchased from Santa Cruz Biotechnology) diluted with 0.1% Triton X were added to the cell samples and placed in a humidified box at 4°C overnight. After overnight, the cells were washed four times with 0.1% Triton X, 8 minutes each time, and aspirated. The secondary antibodies (purchased from Cell Signaling Technology) diluted with 0.1% Triton X were added to the cell samples and agitated at low speed for 1 hour in the dark. Wash five times with 0.1% Triton X, 8 minutes each time, and blot dry; add 5ug / ml DAPI (purchased from Sigma-Aldrich) and stain at low speed for 30 minutes in the dark. Wash four times with 0.1% Triton X, 5 minutes each time, blot dry, and then add PBS to prevent the cells from drying. Observe under a confocal fluorescence microscope, perform field stitching and Z-axis multi-layer scanning at 10x magnification, and obtain immunofluorescence three-dimensional stereoscopic images of the bottom surface of the biconvex lens organoid induced in vitro by stiffness-controllable hydrogel, as shown in the figure. Figure 3 shown.

[0100] like Figure 3 (a) Figure 3 (b) Figure 3 (c) and Figure 3 As shown in (d), the results of immunofluorescence three-dimensional imaging show that the bottom surface of the lens organoid is spherical and convex, and the outer periphery of the bottom surface is covered with cells with nuclei, while there are no cells with nuclei inside. The spherical bottom surface of the lens organoid is covered with lens epithelial-like cells expressing the epithelial cell marker capsule cadherin E-cadherin, and the spherical bottom surface is completely covered with lens capsule-like structures expressing collagen-IV. There are lens fiber-like cells expressing the lens fiber marker γ-crystallin near the outer surface. This suggests that the biconvex lens organoid has all the substructures of the natural lens.

[0101] 4. Electron microscopy observation of lens organoids (see Figure 4-Figure 8 )

[0102] Lens organoids were fixed in 2.5% glutaraldehyde solution at 4°C overnight, and then the samples were processed according to the following steps: 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 acid solution for 1-2 hours; the osmium acid 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 gradient concentrations of ethanol solution (including five concentrations of 30%, 50%, 70%, 80%, 90% and 95%), each concentration was treated for 15 minutes, and then treated with 100% ethanol for 20 minutes; finally, transitioned to pure acetone for 20 minutes. Treat the sample with a mixture of embedding agent and acetone (V / V=1 / 1) for 1 hour; treat the sample with a mixture of embedding agent and acetone (V / V=3 / 1) for 3 hours; treat the sample with pure embedding agent overnight; embed the sample after infiltration treatment and heat it at 70℃ overnight to obtain the embedded sample. The sample is sliced ​​in a LEICAEMUC7 ultrathin slicer to obtain 70-90nm slices, and the slices are stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10 minutes each, and then observed in a HitachiH-7650 transmission electron microscope. Glutaraldehyde, buffer, ethanol, acetone, lead citrate, and methylene blue were all purchased from Sinopharm Chemical Reagent Company. Osmic acid, Spurr embedding agent, and uranyl acetate were all purchased from SPI-CHEM.

[0103] Electron microscopic results showed that the lens organoids have structures such as capsule, lens epithelial cells, primary lens fiber cells, and mature lens fiber cells, which are similar to the normal human lens structure. The capsule is a transparent film surrounding the outer layer of the lens, the lens epithelial cells are the only columnar or cubic epithelial cells under the capsule, the primary lens fiber cells are fiber cells differentiated from lens epithelial cells with some organelles and nuclei beginning to degenerate, and the mature lens fiber cells are cells differentiated from primary lens fiber cells with completely degenerated intracellular organelles and nuclei.

[0104] 5. Cultivation process of biconvex lens organoids (see Figure 8 )

[0105] During the entire process of culturing mature biconvex lens organoids on 0.5-5kPa extremely low stiffness hydrogels, cell clusters were photographed under a microscope every day starting from the 5th day of culture to record the growth process of lens organoids and their key morphological and structural characteristics.

[0106] Under the microscope, on the 7th day of culture, the cell clusters can be observed to be "red blood cells", that is, the surrounding cells are thicker and the central cells are thinner, showing a round cake shape with convex surroundings and concave centers. On the 9th day of culture, ridge-like protrusions that are staggered and fused can be seen under the microscope around and in the center of the cell clusters. On the 10th-11th day of culture, a small amount of transparent areas that can transmit light can be seen under the microscope in the cell clusters. After 11-13 days of culture, transparent lens organoids with a biconvex surface composed of capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells can be obtained.

[0107] VI. Differences in the development of lens organoids on hydrogels of different stiffness (see Fig. 9 )

[0108] The method described in the patent was used to induce pluripotent stem cells into lens organoids on hydrogels with different stiffness of 1, 5, 20, and 100 kPa. During the 13-day process, starting from the fifth day of culture, the cell clusters were photographed every day under a microscope to record the growth process of the lens organoids and the effects of different stiffness substrates on the growth rate and morphological structure.

[0109] Under the microscope, it can be seen that the maturation rate of lens organoids on 1kPa and 5kPa hydrogels is significantly faster than that on 20kPa and 100kPa. After the 11th day, the rapid and massive production of transparent-like tissue in the 1kPa and 5kPa groups can be observed under the microscope, and the volume of the organoids on 1kPa is larger than that on 5kPa, suggesting that the relatively soft mechanical microenvironment is conducive to the directional and rapid differentiation into mature lens fiber-like cells.

[0110] The production companies and product numbers of the reagents described in this article are as follows:

[0111] Noggin: Produced by R&D (R&D Systems USA), the product number is 6057-NG.

[0112] bFGF: produced by PeproTech (USA), product number is 100-18B.

[0113] BMP4: Produced by R&D (R&D Systems USA), the product number is 314-BP-010.

[0114] BMP7: Produced by R&D (R&D Systems USA), the product number is 354-BP-010.

[0115] Y-27632: Produced by R&D (R&D Systems USA), the product number is 1254-10.

[0116] Sulfo-SANPAH: produced by Sigma-Aldrich (USA), product number is 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 in the scope of protection of the present invention.

Claims

1. A method for constructing biconvex lens organoids by in vitro directional induction of stiffness-controllable hydrogels, comprising the following steps: (1) inoculating pluripotent stem cells onto a 12-well plate coated with a matrigel solution and filled with mTesR culture medium to start culturing; from the first day of culturing, using mTesR culture medium containing human growth factor noggin until the fourth to sixth day of culturing, and replacing the mTesR culture medium containing human growth factor noggin daily to obtain primary neuroectoderm cell clusters; the matrigel solution is formed by dissolving embryonic stem cell matrigel in DMEM / F12 culture medium, and the volume concentration of the matrigel solution is 0.8-1.2%; (2) preparing a polyacrylamide hydrogel with a hardness of 0.5 to 5 kPa; performing ultraviolet crosslinking treatment on the polyacrylamide hydrogel using an ultraviolet light-induced crosslinking agent, and then coating the polyacrylamide hydrogel with a matrix gel solution having a volume concentration of 0.8 to 1.2% to obtain a treated polyacrylamide hydrogel; then repeatedly washing the treated polyacrylamide hydrogel with a HEPES buffer and soaking it to obtain a cleaned polyacrylamide hydrogel; (3) First, the washed polyacrylamide hydrogel is placed in a mTesR culture medium containing human growth factor noggin, and then the primary ectoderm cell clusters are dissociated from the 12-well plate using stem cell digestion solution and blown apart, and then 10 to 30 cell clusters that have been blown apart are planted on the washed polyacrylamide hydrogel to start culturing; within 6 to 12 hours after planting and culturing, ROCK inhibitor Y-27632 is added to the culture medium, and the mTesR culture medium containing human growth factor noggin is used until the 6th to 8th day of planting and culturing, and the mTesR culture medium containing human growth factor noggin is replaced daily to obtain neuroectoderm cell clusters; (4) Cultivating the cells with mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 until the 12th to 14th day of implantation and culture, and replacing the mTesR culture medium containing human growth factors bFBF, BMP4 and BMP7 daily, a biconvex lens organoid composed of capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells can be obtained, which is induced in vitro by stiffness-controllable hydrogel.

2. The method for constructing biconvex lens organoids by in vitro directional induction of stiffness-controllable hydrogel 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 of stiffness-controllable hydrogel according to claim 1, characterized in that: In step (2), the ultraviolet cross-linking treatment is specifically as follows: cross-linking with 365 nm ultraviolet light for 20 minutes, with an energy of 9999 mJ, and after rinsing with HEPES buffer, 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 of stiffness-controllable hydrogel according to claim 3, characterized in that: The concentration of the HEPES buffer is 0.5-5 mmol / L.

5. The method for constructing biconvex lens organoids by in vitro directional induction of stiffness-controllable hydrogel 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 of stiffness-controllable hydrogel according to claim 1, characterized in that: 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.

7. A method for in vitro directional induction of biconvex lens organoids by a stiffness-controllable hydrogel prepared according to any one of the construction methods of claims 1-6, characterized in that: The biconvex lens organoid induced in vitro by stiffness-controllable hydrogel presents a transparent 3D structure, with spherical or ellipsoidal protrusions on both the upper and lower surfaces, and is spherical or ellipsoidal with a diameter of 0.5 to 2 mm. It is composed of a capsule, lens epithelial cells, lens primary fiber cells and lens mature fiber cells, and the spherical lower surface is wrapped by a complete capsule, expressing the epithelial cell marker capsule cadherin E-cadherin, the lens fiber cell marker γ-crystallin, and the lens capsule marker type 4 collagen Collagen-IV.

8. The method of in vitro directional induction of biconvex lens organoids by means of a stiffness-controllable hydrogel according to claim 7, characterized in that: The capsule is a transparent film surrounding the outer layer of the lens, the lens epithelial cells are the only layer of columnar or cubic epithelial cells under the capsule, the primary lens fiber cells are fiber cells differentiated from the lens epithelial cells with some organelles and nuclei beginning to degenerate, and the mature lens fiber cells are cells differentiated from the primary lens fiber cells with intracellular organelles and nuclei completely degenerated.

9. A use of the method of claim 7 or 8 for inducing biconvex lens organoids in vitro by means of a stiffness-controllable hydrogel, characterized in that: The in vitro directional induction of biconvex lens organoids by means of stiffness-controllable hydrogels can be used for the study of lens embryonic development mechanisms, the study of cataract pathogenesis, and the screening of cataract-related drugs.

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