Soft scaffold suspension self-assembly system for alveolar organoid culture and application thereof

Abstract

The present disclosure belongs to the field of cell engineering, and particularly relates to a soft scaffold suspension self-assembly system for alveolar organoid culture and application thereof. The present disclosure provides a soft scaffold suspension self-assembly system for alveolar organoid culture, which comprises a low-adsorption culture plate for containing a differentiation culture medium, and a Matrigel added into the differentiation culture medium for providing a polarity signal and a growth factor and maintaining a suspension culture environment, wherein the Matrigel and the differentiation culture medium constitute a differentiation culture system, and the volume ratio of the Matrigel to the differentiation culture system is (1-2):100. The present disclosure also provides a culture method for inducing and differentiating an alveolar organoid from human embryonic stem cells, which is based on the soft scaffold suspension self-assembly system and successfully induces and differentiates human pluripotent stem cells into a plurality of key cell types including proximal airway epithelial cells (such as basal cells and goblet cells) and distal alveolar epithelial cells (including functional type 1 and type 2 alveolar cells) within 5 weeks.

Description

Technical Field

[0001] This disclosure pertains to the field of cell engineering, and specifically relates to a soft scaffold suspension self-assembly system for alveolar organoid culture and its application. Background Technology

[0002] Current lung research faces a dual dilemma of model distortion: on the one hand, simplified 2D cell cultures, lacking key tissue structures and cell diversity, struggle to simulate the complex functions of the human lung. The extremely flat and large morphology of AT1 cells is difficult to replicate in vitro, and the transdifferentiation process from AT2 to AT1 is complex with unclear regulatory signals. On the other hand, animal models, due to inherent species differences, frequently exhibit biased predictive results in drug development. These limitations stemming from the inherent defects of the models themselves severely restrict breakthroughs in related fields. Against this backdrop, constructing an advanced in vitro model that can accurately simulate the physiological characteristics of the human lung and effectively overcome the shortcomings of existing models is an extremely urgent practical need in the current biomedical field.

[0003] Human embryonic stem cells, due to their unlimited self-renewal capacity and multi-directional differentiation potential, provide ideal seed cells for constructing alveolar organoids—three-dimensional structures that mimic human lung tissue—in vitro. They can highly simulate real lung tissue in fields such as lung development, disease modeling, and drug screening. In 2009, Rock et al. achieved a technological breakthrough, successfully culturing pseudostratified epithelial structures containing basal cells and ciliated cells in matrix gel for the first time using basal cells isolated from the trachea of ​​adult mice. However, this approach is based on specific adult stem cells, and its technical path and applicable scope differ from the induced differentiation strategies based on human embryonic stem cells. In 2023, Hans-Willem Snoeck's team developed a method to directionally differentiate human pluripotent stem cells (hPSCs) into lung organoids with branching morphology; however, SFTPC+ AT2 cells were only detected on day 42, and PDPN+ AT1 cells were still not detected until day 170. Therefore, alveolar organoids face the bottleneck of the excessively long maturation cycle of AT1 cells. Most optimized methods still require at least 60 days to obtain a mature AT1 cell population, which severely restricts the application of this model in high-throughput drug screening and rapid modeling of acute lung injury.

[0004] While traditional low-adsorption suspension culture technology can utilize gravity and intercellular affinity to promote the aggregation of single cells into spheres, achieving scaffold-free 3D culture, the lack of polar signals and physical anchoring provided by the extracellular matrix (ECM) often results in disordered cell aggregation, forming loosely structured micro-tissue spheres lacking functional zones. The suspension environment lacks the growth factors (such as bFGF and EGF) and extracellular matrix proteins (such as laminin and collagen) abundant in the matrix gel, leading to low efficiency in maintaining stem cell stemness or directed differentiation, resulting in low organoid maturity and function far inferior to in vivo tissues. Without matrix support, cell spheres are prone to overgrowth, hindering the permeation of internal nutrients, making core cells highly susceptible to hypoxia and apoptosis, severely affecting the stability of experimental data. While existing high-concentration matrix gel (typically ≥50%) embedding methods are commonly used for organoid culture, matrix gel, as an extract derived from mouse sarcoma, has an extremely complex composition and exhibits significant batch-to-batch variability. At high concentrations, these differences are amplified, leading to significant fluctuations in organoid growth efficiency and morphology across different experimental batches, severely impacting experimental reproducibility and making standardization difficult. The dense gel network formed by high-concentration gels creates a rigid physical shell around the organoids, and this three-dimensional embedding structure is unfavorable for the formation of flattened AT1 cells. In drug screening applications, this shell severely hinders the diffusion of test compounds (especially macromolecular drugs), preventing effective drug contact with the organoid surface and resulting in false-negative results. The gel's hardness transmits abnormal mechanical signals to cells, potentially interfering with their natural physiological state. Passaging and sampling are difficult because the organoids are firmly encased within the gel droplets. Passaging, medium changes, or downstream analyses (such as flow cytometry and confocal imaging) require the use of specialized matrix gel recovery solutions to disrupt the gel structure. This process is not only cumbersome but also highly susceptible to irreversible physical damage to the fragile organoid structure. Furthermore, the high cost of matrix gels hinders large-scale production and clinical applications.

[0005] Therefore, the urgent need to find an economical, practical, efficient, and stable method for inducing differentiation of lung organoids has become a pressing technical problem to be solved in this field. Summary of the Invention

[0006] This disclosure provides a soft scaffold suspension self-assembly system for alveolar organoid culture. By using a low-adsorption culture plate, a suspension culture environment with a physical barrier is created, eliminating hard contact between cells and substrate. Matrix gel is added to the differentiation medium at a volume ratio of matrix gel:(Matrix gel + differentiation medium) (1-2):100. The extremely low gel concentration avoids the formation of a dense gel shell while maintaining a suspension state, solving the problems of difficult drug penetration, large passage damage, and abnormal mechanical signals.

[0007] The objective of this disclosure is achieved through the following technical solution: In a first aspect of this disclosure, a soft scaffold suspension self-assembly system for alveolar organoid culture is provided, comprising: a low-adsorption culture plate for containing differentiation culture medium, and a matrix gel added to the differentiation culture medium for providing polar signals and growth factors and maintaining a suspension culture environment, wherein the matrix gel and the differentiation culture medium constitute a differentiation culture system, and the volume ratio of the matrix gel to the differentiation culture system is (1-2):100. In some specific embodiments of this disclosure, the alveolar organoids are induced to differentiate from human embryonic stem cells. The human embryonic stem cells are induced to differentiate sequentially into a fixed endoderm, foregut endoderm, ventral foregut endoderm, lung bud organoids, and alveolar organoids. The soft scaffold suspension self-assembly system is applied in the culture stages of the ventral foregut endoderm, the lung bud organoids, and the alveolar organoids.

[0008] In some specific embodiments of this disclosure, the differentiation culture medium includes at least one of a culture medium for inducing the differentiation of the foregut endoderm into the ventral foregut endoderm, a culture medium for inducing the differentiation of the ventral foregut endoderm into the lung bud organoids, and a culture medium for inducing the differentiation of the lung bud organoids into the alveolar organoids.

[0009] In a second aspect, this disclosure provides a method for culturing alveolar organoids induced to differentiate from human embryonic stem cells, the method comprising the aforementioned soft scaffold suspension self-assembly system.

[0010] In some embodiments of this disclosure, the cultivation method includes: Culture of the finalized endoderm: From day 1 to day 4, the revived human embryonic stem cells were cultured in multi-well plates using the first-stage induction medium to induce the differentiation of human embryonic stem cells into finalized endoderm cells. The medium was changed daily. Culture of foregut endoderm: From day 4 to day 8, cultured in multi-well plates using stage 2 induction medium to induce the differentiation of shaped endoderm cells into foregut endoderm cells, with the medium changed daily; Culture of ventral foregut endoderm: From day 8 to day 14, foregut endoderm cells were digested and transferred to low-absorption culture plates. Matrix gel was added to the third-stage induction medium at a volume ratio of Matrix gel:(Matrix gel + third-stage induction medium) (1-2):100 to induce differentiation of foregut endoderm cells into ventral foregut endoderm cells. The medium was changed every 2-3 days. Culture of lung bud organoids: From day 14 to day 21, replace with a new low-absorption culture plate and add matrix gel to the fourth-stage induction medium at a volume ratio of matrix gel:(matrix gel + fourth-stage induction medium) (1-2):100 to induce differentiation of ventral foregut endoderm cells into lung bud organoids. Change the medium every 2-3 days. Culture of alveolar organoids: From day 21 to day 35, replace with a new low-adsorption culture plate. Add matrix gel to the fifth-stage induction medium at a volume ratio of matrix gel:(matrix gel and fifth-stage induction medium) (1-2):100 to induce lung bud organoids to differentiate into alveolar organoids. Change the medium every 2-3 days. Replace the low-adsorption plate after 7-8 days of continuous culture.

[0011] In some embodiments of this disclosure, the induction medium for the first stage is as follows: On the first day, 100 ng / mL of recombinant Activin A protein is added to RPMI1640 medium; on the second day, the old medium from the first day is discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 0.2% fetal bovine serum; on the third day, the old medium from the second day is discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 2% fetal bovine serum. The second-stage induction medium was: Advanced DMEM / F12 medium supplemented with 200 ng / mL Noggin recombinant protein, 500 ng / mL FGF4, 2 μmol / L CHIR 99021 and 10 μmol / L SB431542; The third-stage induction medium was: DMEM / F12 medium supplemented with recombinant protein 1. 100 ng / ml BMP4, 0.5 μmol / L all-trans retinoic acid, 3.5 μmol / L CHIR, 1% Glutamax and 2% B27; The fourth-stage induction medium was: Advanced DMEM / F12 medium supplemented with 3 μmol / L CHIR, 10 ng / mL FGF10, 10 ng / mL KGF, 20 μmol / L DAPT, 1% B27, and 1% N2. The fifth-stage induction medium consisted of Ham's F12 medium supplemented with 50 nmol / L dexamethasone, 100 nmol / L 8-Br-cAMP, 100 nmol / L 3-isobutyl-1-methylxanthine, 10 ng / mL KGF, 3 μmol / L CHIR, 10 μmol / L SB431542, 1% ITS supplement, 1% B-27, and 0.25% BSA.

[0012] In a third aspect, this disclosure provides an in vitro lung model constructed from the aforementioned soft scaffold suspension self-assembly system or the aforementioned culture method.

[0013] In a fourth aspect, this disclosure provides a use of the aforementioned in vitro lung model, comprising at least one of the following: (1) Drug detection and / or drug screening; further, the drugs include small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs; (2) Regenerative medicine and in vitro non-therapeutic scientific research; (3) Establish in vitro lung airway or alveolar system disease models to study the corresponding pathogenic mechanisms and drug action mechanisms; (4) Test the toxicity of inhaled substances or the effectiveness of inhaled medications; (5) Detect the effects of pathogen infection on lung tissue; (6) As a screening model for biomarkers and therapeutic targets during acute lung injury and repair.

[0014] In a fifth aspect of this disclosure, a culture medium composition is provided, the culture medium composition comprising: the aforementioned first-stage induction medium, second-stage induction medium, third-stage induction medium, fourth-stage induction medium and fifth-stage induction medium.

[0015] In some embodiments of this disclosure, the induction medium for the first stage is as follows: On the first day, 100 ng / mL of recombinant Activin A protein is added to RPMI1640 medium; on the second day, the old medium from the first day is discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 0.2% fetal bovine serum; on the third day, the old medium from the second day is discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 2% fetal bovine serum. The second-stage induction medium was: Advanced DMEM / F12 medium supplemented with 200 ng / mL Noggin recombinant protein, 500 ng / mL FGF4, 2 μmol / L CHIR 99021 and 10 μmol / L SB431542; The third-stage induction medium was: DMEM / F12 medium supplemented with recombinant protein 1. 100 ng / ml BMP4, 0.5 μmol / L all-trans retinoic acid, 3.5 μmol / L CHIR, 1% Glutamax and 2% B27; The fourth-stage induction medium was: Advanced DMEM / F12 medium supplemented with 3 μmol / L CHIR, 10 ng / mL FGF10, 10 ng / mL KGF, 20 μmol / L DAPT, 1% B27, and 1% N2. The fifth-stage induction medium consisted of Ham's F12 medium supplemented with 50 nmol / L dexamethasone, 100 nmol / L 8-Br-cAMP, 100 nmol / L 3-isobutyl-1-methylxanthine, 10 ng / mL KGF, 3 μmol / L CHIR, 10 μmol / L SB431542, 1% ITS supplement, 1% B-27, and 0.25% BSA.

[0016] The technical solution provided in this disclosure has the following technical contributions: (1) This disclosure provides a soft scaffold suspension self-assembly system for alveolar organoid culture. By using a low-adsorption culture plate, a suspension culture environment with a physical barrier is created, eliminating hard contact between cells and substrates. Then, a low concentration of matrix gel provides key polar signals and growth factors, while solving the problems of loose structure, hypoxia, and lack of extracellular matrix proteins caused by low adsorption. This not only solves the dependence on traditional high-concentration matrix gel (added at a volume ratio of matrix gel:(matrix gel + differentiation medium) (50-75):100) embedding to a certain extent, realizing high throughput, standardization, and clear composition of the culture system, but also strongly promotes cell self-aggregation and self-organization behavior through its physical properties. This three-dimensional construction process driven by the cells themselves is closer to the natural principle of organ development in vivo, which can effectively guide intercellular interactions, thereby significantly enhancing the differentiation potential of cell lineages and generating alveolar organoids with more mature functions and more complex structures.

[0017] (2) This disclosure also provides a method for culturing alveolar organoids induced from human embryonic stem cells. Based on a soft scaffold suspension self-assembly system, the extremely low gel concentration avoids the formation of a dense gel shell while maintaining a suspended state, solving the problems of difficult drug penetration, large passage damage, and abnormal mechanical signals. It successfully induces human pluripotent stem cells to differentiate into multiple key cell types, including proximal airway epithelium (such as basal cells and goblet cells) and distal alveolar epithelium (including functional type 1 and type 2 alveolar cells), after 5 weeks. The alveolar organoid model formed by this soft scaffold suspension self-assembly system provides a more reliable and standardized in vitro platform for studying human lung development, lung disease mechanisms, and innovative drug screening.

[0018] (3) This disclosure breaks through the technical limitations of traditional high-concentration matrix gel embedding. In a 100 mL differentiation culture system (matrix gel + differentiation culture medium), the amount of matrix gel used is greatly reduced from the traditional 50-75 mL to 1-2 mL, which significantly reduces the cost and improves the stability of experimental batches. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the induction of alveolar organoids from human embryonic stem cells according to an embodiment of this disclosure.

[0020] Figure 2 This is a bright-field microscope image of alveolar organoids cultured using a soft scaffold suspension self-assembly system composed of extremely high concentration of matrix gel provided in Comparative Example 1 of this disclosure. Figure 2 The results showed that in a differentiation culture system composed of a matrix gel and differentiation culture medium at a volume ratio of 75:100, the gel layer was dense and hard during the culture process, and a concentration gradient was easily formed inside the differentiation culture system. The cells in the core area were in a state of malnutrition for a long time, and a hypoxic microenvironment was formed inside the gel, which led to the experimental results deviating from the physiological state.

[0021] Figure 3 This is a bright-field microscope image of alveolar organoids cultured using a soft scaffold suspension self-assembly system composed of extremely low concentration matrix gel provided in Comparative Example 2 of this disclosure. Figure 3 The results showed that the differentiation culture system composed of matrix gel (matrix gel + differentiation culture medium) at a volume ratio of 0.25:100 had extremely poor gel mechanical strength during the culture process, which could not effectively support the three-dimensional morphology of organoids, and the 3D structure of alveolar organoids dissociated and collapsed.

[0022] Figure 4 This is an immunofluorescence staining image of cells cultured on day 4 in this embodiment of the present disclosure, where green represents SOX17 protein, red represents FOXA2 protein, and blue represents DAPI. Figure 4 The results showed that on day 4 of induced differentiation, the nuclei of the vast majority of cells simultaneously expressed the marker proteins SOX17 and FOXA2 of the morphological endoderm. The two proteins showed obvious yellow colocalization signals in the combined image, indicating that the induction system disclosed in this paper can efficiently induce human pluripotent stem cells to differentiate into the morphological endoderm.

[0023] Figure 5 This is a bright-field microscope image of foregut endoderm cells and spheres cultured on day 8 in this embodiment of the present disclosure. Figure 5 The results showed that on day 8 of culture, foregut endoderm cells aggregated and bulged outward, forming a unique "semi-adherent-suspended" spherical structure. This structure formed foregut endoderm spheres with one end adhering to the wall and the other end floating, exhibiting obvious budding phenomena, similar to lung bud-like structures in vivo, which is the structural basis for the subsequent formation of airway branches.

[0024] Figure 6 These are bright-field micrographs and immunofluorescence staining images of foregut endoderm cells cultured on day 8 in this embodiment of the present disclosure, where red represents TBX3 protein and blue represents DAPI. Figure 6 As shown on day 8 of differentiation, the foregut endoderm marker protein TBX3 was mainly concentrated in the cell nucleus, overlapping with the blue DAPI signal and appearing purplish-red in the merged image. This indicates that the cells had successfully differentiated into the foregut endoderm.

[0025] Figure 7 This is an immunofluorescence detection image of lung bud organoids cultured on day 21 in this embodiment of the present disclosure, where green represents NKX2.1 protein, blue represents DAPI, and the scale bar is 50 μm. Figure 7 The cells, cultured for 21 days, had self-assembled into distinct organoid spherical structures. The lung progenitor cell marker protein NKX2.1 was clearly visible and specifically located within the cell nucleus. The cells had successfully differentiated from the early endoderm into lung progenitor cells through a soft scaffold suspension self-assembly system.

[0026] Figure 8 This is an immunofluorescence image of alveolar organoids cultured for 35 days in this embodiment, expressing multiple proximal airway cell types. The green cells represent SOX2, MUC5AC, P63, and E-cadherin, respectively, and the blue cells represent DAPI. The scale bar is 50 μm. Figure 8 By day 35 of culture, the organoids had successfully differentiated into various mature proximal airway epithelial cell types, including lung airway progenitor cells (marker protein SOX2), goblet cells (marker protein MUC5AC), and basal cells (marker protein P63), and formed polar epithelial tissue structures (marker protein E-cadherin). In the soft scaffold suspension self-assembly system disclosed in this paper, the organoids can mature and contain a variety of functionally defined airway epithelial cell types.

[0027] Figure 9 This is an immunofluorescence image of mature alveolar organoid AT1 and AT2 cells cultured for 35 days in this embodiment of the present disclosure. In the image, red represents SOX9 and PDPN proteins, green represents SFTPC protein, and blue represents DAPI. The scale bar is 50 μm. Figure 9 The results showed that by day 35 of culture, the organoids had successfully differentiated and matured into functional alveolar AT2 cells (marker protein SFTPC), alveolar progenitor cells (marker protein SOX9), and alveolar AT1 cells (marker protein PDPN). The AT1 cells were flattened and distributed on the surface of the alveolar organoid chambers, confirming the ability of this soft scaffold suspension self-assembly system to induce the maturation of the distal lung lineage.

[0028] Figure 10This is a bright-field microscope image of alveolar organoids cultured on day 35 in this embodiment of the present disclosure. Figure 10 On day 35 of culture, a distinct cavity was visible in the center of the organoid, which was tightly wrapped by a dense layer of epithelial cells, forming a typical sac-like structure similar to lung development in vivo. In the soft scaffold suspension self-assembly system disclosed in this paper, the organoid exhibited a typical spherical structure with a clear outline, smooth edges, no deformation or spreading into a single layer, and no structural collapse or dissolution. Detailed Implementation

[0029] The technical solutions in the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all of them. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0030] Unless otherwise indicated, the practice of this disclosure will employ conventional techniques within the scope of the art, including molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology.

[0031] In the following embodiments, unless otherwise specified, the terms used are explained as follows: In this disclosure, unless otherwise specified, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0032] In this disclosure, unless otherwise specified, the term "human embryonic stem cells" (hESCs) is the "seed" of current regenerative medicine and organoid research. They originate from the inner cell mass (ICM) of early human embryos (blastocyst stage) and possess two core characteristics: unlimited self-renewal capacity and multi-directional differentiation potential.

[0033] In this disclosure, unless otherwise specified, the term "organoid" refers to an organ-specific cell aggregate formed through three-dimensional culture, aggregation, or recombination of stem cells, possessing self-renewal capabilities, and is also known as a "miniature organ" or "pseudo-organ." Organoids have a morphology similar to real organs and can be used to conduct studies in vitro that are difficult to perform in animal models, such as molecular signal regulation. Therefore, organoids are extremely useful in basic research and can be widely applied in various fields such as human development processes, disease model establishment, drug efficacy evaluation and screening, drug toxicity assessment platform development, and cell therapy development, but this disclosure is not limited thereto.

[0034] In this disclosure, unless otherwise specified, the term "Matrigel" refers to a soluble basement membrane extract obtained from mouse EHS sarcoma (Engelbreth-Holm-Swarm tumor). It has an extremely complex composition, containing a variety of extracellular matrix proteins and growth factors. It rapidly polymerizes at room temperature (>15°C) to form a gel, mimicking the basement membrane in vivo and providing a 3D growth environment for cells.

[0035] In this disclosure, unless otherwise specified, the term "SOX17 (SRY-box transcription factor 17)" refers to a key transcription factor of the Sry-associated HMG-box family. In alveolar organoid culture, the core plays a role in endoderm specialization and lineage fate regulation, and is an early key marker and functional regulator of differentiation from pluripotent stem cells (PSCs / iPSCs) into the lung / alveolar lineage.

[0036] In this disclosure, unless otherwise specified, the term "FOXA2 (Forkhead Box A2)," also known as HNF-3β, is a core member of the forkhead box transcription factor family. Like SOX17, it is a key marker of the definitive endoderm (DE) in alveolar organoid culture and lung developmental biology, but its role extends throughout the entire process from endoderm specialization to alveolar maturation.

[0037] In this disclosure, unless otherwise specified, the term "TBX3" refers to a member of the T-box transcription factor family. In alveolar organoid culture and lung developmental biology, it is a core marker of early distal lung epithelial progenitors and a key factor in maintaining progenitor characteristics and regulating alveolar lineage specialization, and is known as the "commander" of the alveolar lineage.

[0038] In this disclosure, unless otherwise specified, the term "NKX2.1" (also known as TTF-1 or TITF1) is the most important and central "master switch" in alveolar organoid culture. It is a homeobox transcription factor whose expression marks the differentiation of cells from pan-endoderm to the lung / thyroid lineage of foregut endoderm. Without NKX2.1, there is no lung tissue.

[0039] In this disclosure, unless otherwise specified, the term "lung progenitor cells" refers to immature, proliferating lung stem cells that serve as a crucial bridge connecting the "defined endoderm (DE)" and the "mature alveolar epithelium (AT1 / AT2)." Lung progenitor cell spheroids typically appear as smooth-edged, densely structured spheroids under a microscope. If the spheroids are loose or the cells migrate outwards in a fibroblast-like manner, it indicates a loss of progenitor cell characteristics.

[0040] In this disclosure, unless otherwise specified, the term "P63" refers to a member of the p53 transcription factor family, which is a core marker of basal cells in alveolar organoids and lung developmental biology, and a key regulator of the proximal airway and lung stem cell niche.

[0041] In this disclosure, unless otherwise specified, the term "MUC5AC" is a gel-forming mucin that is a specific marker of goblet cells in the respiratory system.

[0042] In this disclosure, unless otherwise specified, the term "E-cadherin" is a transmembrane glycoprotein and a core component of adherens junctions. In alveolar organoid culture, it acts as a "gatekeeper of epithelial properties."

[0043] In this disclosure, unless otherwise specified, the term "SOX2 (SRY-box transcription factor 2)" refers to a key transcription factor of the Sry-associated HMG-box family. In alveolar organoid (organoid) culture, at the initial stage: it is one of the "three giants" maintaining stem cell (hESC / iPSC) stemness (alongside OCT4 and NANOG); at the differentiation stage: it is a core marker of the proximal airways (trachea / bronchus).

[0044] In this disclosure, unless otherwise specified, the term "SOX9 (SRY-box transcription factor 9)" is a key transcription factor of the Sry-related HMG-box family. In alveolar organoid culture, it maintains the proliferative capacity of distal progenitor cells and guides the branching morphology of alveolar structures. It is the "commander of proliferation and shaping" and a key bridge connecting "lung progenitor cells" and "mature cells" in alveolar organoid culture.

[0045] In this disclosure, unless otherwise specified, the term "PDPN" (also known as T1α or Aggrus) is a type I transmembrane glycoprotein that is primarily expressed on the apical surface of AT1 cells and is a core marker of type I alveolar epithelial cells (AT1) in alveolar organoid culture.

[0046] In this disclosure, unless otherwise specified, the term "SFTPC (surfactant protein C)" refers to an extremely hydrophobic protein that binds to phospholipids to form pulmonary surfactant. SFTPC is the core and most specific "ultimate goal" in alveolar organoid culture. The expression of SFTPC signifies that the organoid has successfully differentiated from "lung progenitor cells" into functional "mature alveolar cells".

[0047] In this disclosure, unless otherwise specified, the term "AT1 cell," also known as Type I alveolar epithelial cells, refers to cells that are flattened and paved-like, with a slight bulge around the nucleus and an extremely thin cytoplasm (only 0.1-0.2 μm thick). AT1 cells act as the "membranes and windows" of the alveoli, constituting over 95% of the alveolar surface area and serving as the physical channels for oxygen entry into the blood and carbon dioxide expulsion. The presence of AT1 cells signifies the formation of a functional gas exchange interface.

[0048] In this disclosure, unless otherwise specified, the term "AT2 cells," also known as Type II alveolar epithelial cells, refers to the "stem cell bank" of the alveoli. AT2 cells are responsible not only for secreting the "lubricant" (surfactant) that maintains alveolar opening but also for being a crucial source of repair after alveolar injury. AT2 cells are the ultimate goal of alveolar organoid culture. The purity and maturity of AT2 cells are the gold standard for measuring the success of organoid culture.

[0049] Unless otherwise specified in this disclosure, the term "StemFlex™ medium" refers to a feeder-free, chemically defined, xeno-free culture medium specifically for human pluripotent stem cells (hPSC / hESC / iPSC). It is optimized for high-pressure applications such as single-cell passage, gene editing, and reprogramming, and can be changed every other day (no need to change it on weekends). Long-term culture shows no karyotype abnormalities and can be seamlessly integrated with the CTS Essential 8® clinical-grade system.

[0050] In this disclosure, unless otherwise specified, the term "RPMI 1640 medium" is a commonly used cell culture medium, originally developed by Moore et al. in 1966 at the Roswell Park Memorial Institute in the United States, hence its name. RPMI 1640 medium was initially designed specifically for lymphocyte culture, but has now been found suitable for a variety of mammalian cells, including HeLa cells, Jurkat cells, MCF-7 cells, PC12 cells, PBMC cells, astrocytes, and cancer cells. RPMI 1640 contains reduced glutathione and high concentrations of vitamins, making it rich in nutrients and offering unique benefits, thus it is widely used for the in vitro culture of various mammalian cells.

[0051] In this disclosure, unless otherwise specified, the term "DMEM / F12 medium" is a widely used basal medium that can be used to support the growth of many different types of mammalian cells. Cells that have been successfully cultured in DMEM / F-12 include MDCK, glial cells, fibroblasts, human endothelial cells, and rat fibroblasts.

[0052] In this disclosure, unless otherwise specified, the term "Advanced DMEM / F12 medium" refers to a widely used basal medium for culturing mammalian cells with reduced fetal bovine serum (FBS) addition. Compared to classic DMEM / F-12, serum addition can be reduced by 50–90% without alteration of cell growth rate or morphology. Cells that can be successfully cultured in Advanced DMEM / F-12 without adaptation include MRC-5, SP2, Vero, WI-38, and Jurkat.

[0053] In this disclosure, unless otherwise specified, the term "Ham's F12 medium" is designed for low-serum / serum-free culture, primary epithelial cells (especially lung epithelial cells / lung progenitor cells), CHO cells, etc., containing L-glutamine and phenol red, HEPES-free, sodium bicarbonate buffered, and requiring 5–10% CO2 for culture.

[0054] The following description is based on specific embodiments. Example 1 like Figure 1As shown, this disclosure provides a flexible scaffold suspension self-assembly system for alveolar organoid culture, comprising: a low-adsorption culture plate for containing differentiation culture medium, and a matrix gel added to the differentiation culture medium to provide polar signals and growth factors and maintain a suspension culture environment. The matrix gel and the differentiation culture medium constitute a differentiation culture system, and the volume ratio of matrix gel to the differentiation culture system is (1-2):100. This flexible scaffold suspension self-assembly system is applied in the culture stages of ventral foregut endoderm, lung bud organoids, and alveolar organoids during the induction of alveolar organoids from human embryonic stem cells. Using this system, the culture time can be shortened to 35 days to detect PDPN+ AT1 cells and SFTPC+ AT2 cells.

[0055] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that a soft scaffold suspension self-assembly system is constructed by using ultra-high concentration matrix gel (added at a volume ratio of matrix gel:(matrix gel + differentiation culture medium) 75:100) and low adsorption culture plate.

[0056] like Figure 2 As shown, Comparative Example 1 uses an ultra-high concentration matrix gel. The gel layer is dense and rigid, easily forming a concentration gradient within the differentiation culture system. Cells in the core area are chronically undernourished, creating a hypoxic microenvironment within the gel, leading to experimental results deviating from physiological conditions. The high rigidity of the gel layer after solidification requires stronger lysis and digestion conditions for subsequent cell digestion, gel dissociation, and RNA / protein extraction, potentially reducing the accuracy of downstream detections. Furthermore, excessively high concentrations mean that even small errors in gel preparation and plating thickness can lead to significant differences in mechanical and diffusion properties, making experimental reproducibility difficult to guarantee.

[0057] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that a soft scaffold suspension self-assembly system is constructed using ultra-low concentration matrix gel (added at a volume ratio of matrix gel:(matrix gel + differentiation culture medium) 0.25:100) and low-adsorption culture plate.

[0058] like Figure 3 As shown, Comparative Example 2 uses an extremely low concentration of matrix gel, resulting in a gel with extremely poor mechanical strength, which cannot effectively support the three-dimensional morphology of organoids. During the culture process, it is very easy to collapse and dissociate, reattaching to the wall to form a two-dimensional monolayer, thus losing the core advantage of three-dimensional culture.

[0059] Example 2 Based on the soft scaffold suspension self-assembly system provided in Example 1, this disclosure also provides a method for culturing alveolar organoids induced from human embryonic stem cells, the specific steps of which are as follows: S0: Culture of human embryonic stem cells This disclosure utilizes a serum-free and feeder-free culture system to expand and maintain human pluripotent stem cells. After resuscitation of human embryonic stem cells (hESC H9), 4 mL of StemFlex™ medium (ThermoFisher Scientific, A3349401) is added. After centrifugation at 1000 rpm for 5 min, the supernatant is discarded, and H9 cells are transferred to cell culture flasks or dishes pre-coated with stem cell culture-specific matrix gel (Corning, 354277). The medium needs to be changed daily for 6-7 consecutive days. During this stage, the volume ratio of matrix gel to the differentiation culture system (Matrix gel + StemFlex™ medium) is 0.2:100, i.e., 1 mL of matrix gel is added to 499 mL of StemFlex™ medium. Coating with matrix gel promotes stem cell adherence and growth.

[0060] S1: Culture of definitive endoderm (DE), Day 1-Day 4 hESC H9 cells were digested from the culture dish using H9 cell digestion solution (catalog number: H9Med-iCell-CA003, brand: iCell) and then transferred to 24-well cell culture plates (Corning, 3524) lined with Matrigel (Corning, 356231). Approximately 5000 cells were seeded per well, and 400 μL of first-stage induction medium was added to each well to begin the first stage of differentiation induction. During this stage, the volume ratio of Matrigel to the differentiation culture system (Matrigel + first-stage induction medium) was 0.2:100, i.e., 1 mL of Matrigel was added to 499 mL of first-stage induction medium.

[0061] The induction medium for the first stage was as follows: On the first day, 100 ng / mL Activin A (Peprotech, 120-14E-50) recombinant protein was added to RPMI1640 medium (Gibco, C11875500BT); on the second day, the old medium from the first day was discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 0.2% fetal bovine serum (EcoSy, FSD500); on the third day, the old medium from the second day was discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 2% fetal bovine serum.

[0062] Initially, typical hESC clones gradually depolymerize, cells spread and elongate, becoming loosely connected, irregular spindle-shaped or fusiform cells, losing clonal boundaries, and forming a homogeneous monolayer. Immunofluorescence staining results are as follows: Figure 4As shown, on day 4, more than 80% of the cells highly expressed SOX17+ and FOXA2+ endoderm, indicating that the cells had been successfully induced into a defined endoderm.

[0063] In the initial stage of hESC differentiation into alveolar organoids (endoderm differentiation), co-expression of SOX17 and FOXA2 is the gold standard marker for defining the endoderm, indicating that cells have successfully exited pluripotency and entered the endoderm lineage, laying the foundation for subsequent lung / alveolar differentiation.

[0064] S2: Culture of anterior foregut endoderm (AFE), Day 4-Day 8 On day 4, cell status was observed. The first-stage induction medium for the DE phase was removed by pipette, and the cells were washed once with DPBS (Gibco, C14190000BT). The second-stage induction medium was then added to the 24 wells containing the prepared Matrigel to begin the second-stage differentiation induction. 400 μL of the second-stage induction medium was added to each well, and the medium was changed daily. During this stage, the volume ratio of Matrigel to the differentiation culture system (Matrigel + second-stage induction medium) was 0.2:100, i.e., 1 mL of Matrigel was added to 499 mL of the second-stage induction medium.

[0065] The second-stage induction medium consisted of Advanced DMEM / F12 medium (Gibco, 12634010) supplemented with 200 ng / mL Noggin recombinant protein (nearshore protein, CB89), 500 ng / mL FGF4 (Peprotech, 100-31), 2 μmol / L CHIR 99021 (Sigma, SML1046), and 10 μmol / L SB431542 (sigma, S4317).

[0066] On the 8th day after 4 days of induction culture in this stage, such as Figure 5-6 As shown, the cell morphology changed from cobblestone-like to polygonal or spindle-shaped, and the cells highly expressed TBX3+, indicating differentiation into the foregut endoderm. S3: Culture of ventralized anterior foregut endoderm (VAFE), Day 8-Day 14 On day 8, adherent foregut endoderm spheroids with protrusions were observed in the 24-well culture plates. The foregut endoderm cells and spheroids in the 24-well plates were digested with 0.25% trypsin (Thermo Fisher Scientific, 25200-056), centrifuged at 1000 rpm for 5 minutes, washed with DPBS, and then resuspended in 200 μL of DPBS before being transferred to low-absorption culture plates to promote cell self-aggregation and self-organization. Simultaneously, Matrigel (Corning, 356231) was added to the third-stage induction medium. The volume ratio of Matrigel to the differentiation culture system (Matrigel + third-stage induction medium) in this stage was (1-2):100. This stage was continued for 6 days with 500 μL of third-stage induction medium per well, and the medium was changed every 2-3 days (with Matrigel added in the same proportion to the replacement medium). During medium changes, the organoid spheroids were collected, centrifuged at 1000 rpm for 3 minutes, washed with DPBS, and then re-added to the low-absorption plates for this stage.

[0067] The third-stage induction medium was DMEM / F12 medium (Thermo Fisher Scientific, 11320033) supplemented with recombinant protein 1. 100 ng / ml BMP4 (Proteintech, 120-05ET), 0.5 μmol / L all-trans retinoic acid (Sigma, R2625), 3.5 μmol / L CHIR (Sigma, SML1046), 1% Glutamax (Gibco, 35050061) and 2% B27 (Thermo Fisher Scientific, 17504044).

[0068] Compared to the traditional high-concentration matrix gel (added at a volume ratio of matrix gel:(matrix gel + differentiation medium) (50-75):100) to form a hard gel embedding, this provides a "soft" liquid microenvironment and weak physical support. Combined with the low-absorption plate, it keeps the cells suspended, guiding them to perform gentle self-assembly and establishing a "soft scaffold suspension self-assembly system".

[0069] S4: Culture of lung progenitors (LPs), Day 14-Day 21 Starting from day 14, the fourth stage of induced differentiation begins, maintaining a "soft scaffold suspension self-assembly system." Discard the third stage culture medium, collect the organoids, centrifuge at 1000 rpm for 3 min, wash once with DPBS, resuspend the organoids, and transfer them to a new low-absorption plate. Add Matrigel to the fourth stage culture medium. In this stage, the volume ratio of Matrigel to the differentiation culture system (Matrigel + fourth stage induction medium) is (1-2):100. Continue this stage for 7 days, using 800 μL of fourth stage induction medium per well. Change the medium every 2-3 days (add Matrigel to the replacement medium in the same proportion). Collect the organoids during medium changes, centrifuge at 1000 rpm for 3 min, wash once with DPBS, and re-add to the low-absorption plate for this stage.

[0070] The fourth-stage induction medium was Advanced DMEM / F12 medium (Gibco, 12634010) supplemented with 3 μmol / L CHIR, 10 ng / mL FGF10 (Peprolech, 100-26), 10 ng / mL KGF (novoprotein, CM88), 20 μmol / L DAPT (Sigma, D5942), 1% B27 (Thermo Fisher Scientific, 17504044), and 1% N2 (Thermo Fisher Scientific, 17502048).

[0071] At this point, sac-like folded lung-like organoid structures are clearly visible. For example... Figure 7 As shown, NKX2.1+ lung progenitor cells can be detected in lung bud organoids.

[0072] S5: Culture of human alveolar organoids (ALOs), Day 21-Day 35 Starting from day 21, the fifth stage of induced differentiation begins, maintaining the "soft scaffold suspension self-assembly system." The fourth stage induction medium is discarded, organoid spheroids are collected, centrifuged at 1000 rpm for 3 min, washed once with DPBS, resuspended, and transferred to a new low-absorption plate. Add matrix gel to the fifth stage medium. In this stage, the volume ratio of matrix gel to the differentiation culture system (matrix gel + fifth stage induction medium) is (1-2):100. This stage is cultured continuously for 14 days, with 500 μL of fifth stage induction medium per well. The medium is changed every 2-3 days (with matrix gel added to the replacement medium in the same proportion). During medium changes, organoid spheroids are collected, centrifuged at 1000 rpm for 3 min, washed once with DPBS, and re-added to the low-absorption plate for this stage. The low-absorption plate is changed every 7-8 days.

[0073] The fifth-stage induction medium was Ham's F12 medium (Gibco, 21127022) supplemented with 50 nmol / L dexamethasone, 100 nmol / L 8-Br-cAMP, 100 nmol / L 3-isobutyl-1-methylxanthine, 10 ng / mL KGF, 3 μmol / L CHIR, 10 μmol / L SB431542, 1% ITS supplement (Sigma, SCM054), 1% B-27 and 0.25% BSA.

[0074] After continuous culture for 35 days, the organoids were passaged by pipetting, then digested in organoid digestion solution (abs9920, Absin) for 3-5 minutes, centrifuged at 1000 rpm for 5 minutes at 4°C, the culture medium was discarded, the cell pellet was washed twice with DPBS, and then transferred to a new low-absorption plate. Figure 8-10 As shown, the formed alveolar organoids have proximal airway epithelium, and P63+ basal cells, MUC5AC+ goblet cells, E-cadherin+ epithelial cells, SOX2+ airway progenitor cells, SOX9+ alveolar progenitor cells, PDPN+ AT1 cells and SFTPC+ AT2 cells can be detected.

[0075] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this application are indicated by the claims.

[0076] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A flexible scaffold suspension self-assembly system for culturing alveolar organoids, characterized in that, It includes: a low-adsorption culture plate for containing differentiation culture medium, and a matrix gel added to the differentiation culture medium to provide polar signals and growth factors and maintain a suspension culture environment, wherein the matrix gel and the differentiation culture medium constitute a differentiation culture system, and the volume ratio of the matrix gel to the differentiation culture system is (1-2):

100.

2. The flexible scaffold suspension self-assembly system according to claim 1, characterized in that, The alveolar organoids are induced to differentiate from human embryonic stem cells. The human embryonic stem cells are induced to differentiate sequentially into fixed endoderm, foregut endoderm, ventral foregut endoderm, lung bud organoids, and alveolar organoids. The soft scaffold suspension self-assembly system is used in the culture stages of the ventral foregut endoderm, the lung bud organoids, and the alveolar organoids.

3. The flexible scaffold suspension self-assembly system according to claim 2, characterized in that, The differentiation culture medium includes at least one of the following: a culture medium for inducing the differentiation of the foregut endoderm into the ventral foregut endoderm, a culture medium for inducing the differentiation of the ventral foregut endoderm into the lung bud organoids, and a culture medium for inducing the differentiation of the lung bud organoids into the alveolar organoids.

4. A method for culturing alveolar organoids induced from human embryonic stem cells, characterized in that, The cultivation method includes the soft scaffold suspension self-assembly system as described in any one of claims 1-3.

5. The cultivation method according to claim 4, characterized in that, It includes: Culture of the finalized endoderm: From day 1 to day 4, the revived human embryonic stem cells were cultured in multi-well plates using the first-stage induction medium to induce the differentiation of human embryonic stem cells into finalized endoderm cells. The medium was changed daily. Culture of foregut endoderm: From day 4 to day 8, cultured in multi-well plates using stage 2 induction medium to induce the differentiation of shaped endoderm cells into foregut endoderm cells, with the medium changed daily; Culture of ventral foregut endoderm: From day 8 to day 14, foregut endoderm cells were digested and transferred to low-absorption culture plates. Matrix gel was added to the third-stage induction medium at a volume ratio of Matrix gel:(Matrix gel + third-stage induction medium) (1-2):100 to induce differentiation of foregut endoderm cells into ventral foregut endoderm cells. The medium was changed every 2-3 days. Culture of lung bud organoids: From day 14 to day 21, replace with a new low-absorption culture plate and add matrix gel to the fourth-stage induction medium at a volume ratio of matrix gel:(matrix gel + fourth-stage induction medium) (1-2):100 to induce differentiation of ventral foregut endoderm cells into lung bud organoids. Change the medium every 2-3 days. Culture of alveolar organoids: From day 21 to day 35, replace with a new low-adsorption culture plate. Add matrix gel to the fifth-stage induction medium at a volume ratio of matrix gel:(matrix gel + fifth-stage induction medium) (1-2):100 to induce lung bud organoids to differentiate into alveolar organoids. Change the medium every 2-3 days. Replace the low-adsorption plate after 7-8 days of continuous culture.

6. The cultivation method according to claim 5, characterized in that, The induction medium for the first stage was as follows: On the first day, 100 ng / mL of recombinant Activin A protein was added to RPMI1640 medium; on the second day, the old medium from the first day was discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 0.2% fetal bovine serum; on the third day, the old medium from the second day was discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 2% fetal bovine serum. The second-stage induction medium was: Advanced DMEM / F12 medium supplemented with 200 ng / mL Noggin recombinant protein, 500 ng / mL FGF4, 2 μmol / L CHIR 99021 and 10 μmol / L SB431542; The third-stage induction medium was: DMEM / F12 medium supplemented with recombinant protein 1. 100 ng / ml BMP4, 0.5 μmol / L all-trans retinoic acid, 3.5 μmol / L CHIR, 1% Glutamax, and 2% B27; The fourth-stage induction medium was: Advanced DMEM / F12 medium supplemented with 3 μmol / L CHIR, 10 ng / mL FGF10, 10 ng / mL KGF, 20 μmol / L DAPT, 1% B27, and 1% N2. The fifth-stage induction medium consisted of Ham's F12 medium supplemented with 50 nmol / L dexamethasone, 100 nmol / L 8-Br-cAMP, 100 nmol / L 3-isobutyl-1-methylxanthine, 10 ng / mL KGF, 3 μmol / L CHIR, 10 μmol / L SB431542, 1% ITS supplement, 1% B-27, and 0.25% BSA.

7. An in vitro lung model, characterized in that, It is constructed by the soft scaffold suspension self-assembly system as described in any one of claims 1-3 or the culture method as described in any one of claims 4-6.

8. The use of the lung in vitro model as described in claim 7, characterized in that, Includes at least one of the following: (1) Drug detection and / or drug screening; further, the drugs include small molecule drugs, antibody drugs, ADC drugs, and immunotherapeutic drugs; (2) Regenerative medicine and in vitro non-therapeutic scientific research; (3) Establish in vitro lung airway or alveolar system disease models to study the corresponding pathogenic mechanisms and drug action mechanisms; (4) Test the toxicity of inhaled substances or the effectiveness of inhaled medications; (5) Detect the effects of pathogen infection on lung tissue; (6) As a screening model for biomarkers and therapeutic targets during acute lung injury and repair.

9. A culture medium composition, characterized in that, The culture medium composition includes: the first-stage induction medium, the second-stage induction medium, the third-stage induction medium, the fourth-stage induction medium, and the fifth-stage induction medium as described in claim 6.

10. The culture medium composition according to claim 9, characterized in that, The induction medium for the first stage was as follows: On the first day, 100 ng / mL of recombinant Activin A protein was added to RPMI1640 medium; on the second day, the old medium from the first day was discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 0.2% fetal bovine serum; on the third day, the old medium from the second day was discarded and replaced with RPMI1640 medium supplemented with 100 ng / mL Activin A and 2% fetal bovine serum. The second-stage induction medium was: Advanced DMEM / F12 medium supplemented with 200 ng / mL Noggin recombinant protein, 500 ng / mL FGF4, 2 μmol / L CHIR 99021 and 10 μmol / L SB431542; The third-stage induction medium was: DMEM / F12 medium supplemented with recombinant protein 1. 100 ng / ml BMP4, 0.5 μmol / L all-trans retinoic acid, 3.5 μmol / L CHIR, 1% Glutamax, and 2% B27; The fourth-stage induction medium was: Advanced DMEM / F12 medium supplemented with 3 μmol / L CHIR, 10 ng / mL FGF10, 10 ng / mL KGF, 20 μmol / L DAPT, 1% B27, and 1% N2. The fifth-stage induction medium consisted of Ham's F12 medium supplemented with 50 nmol / L dexamethasone, 100 nmol / L 8-Br-cAMP, 100 nmol / L 3-isobutyl-1-methylxanthine, 10 ng / mL KGF, 3 μmol / L CHIR, 10 μmol / L SB431542, 1% ITS supplement, 1% B-27, and 0.25% BSA.

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