Compositions and methods for biasing the polarity of organoids

Abstract

The present disclosure relates to the medium composition and / or supplements added to organoid medium, and the method of generating apical-outer organoid.Apical-outer organoid shows its apical surface facing the external environment and its basolateral surface facing inward.Apical-outer organoid can be formed in the organoid medium comprising a basal medium and one or more of Notch signaling inhibitor and TGF-mediated signaling inhibitor.The apical-outer organoid formed according to the disclosed method or in the disclosed medium can be used to assay host response to pathogens.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 210,248, filed June 14, 2021, and U.S. Provisional Patent Application No. 63 / 286,173, filed December 6, 2021, the contents of both of which are incorporated by reference in their entireties herein.

[0002] The present disclosure relates to cell culture applications, more particularly to cell culture applications that use organoids, and even more particularly to cell culture applications in which the polarity of the organoids may be biased. [Background technology]

[0003] Cell-based research, diagnosis and therapy often rely on tissue and / or organ models, but often lack the existence of relevant physiological models. In recent years, organoid technology has shown promise as a relevant physiological model for studying developmental biology, disease, susceptibility to viral infection, toxicology, susceptibility to drugs and compounds, personalized medicine, etc.

[0004] A challenge with culturing organoids, particularly epithelial organoids, and using them in downstream assays is that organoids tend to form and grow with the apical surface facing the organoid's core (and often the central lumen), while the basolateral surface facing the external environment. However, access to the apical side via the direct external environment is often useful, and in some cases, necessary, to perform certain studies.

[0005] Therefore, there is a need for improved media formulations and methods for generating so-called "apical-lateral" organoids. Summary of the Invention

[0006] The present disclosure relates to the medium composition and / or the supplements added to the medium, and the method for generating organoid, particularly including "apical-outer" organoid.The medium and method disclosed herein produce such "apical-outer" organoid with high efficiency.

[0007] In one broad aspect of the present disclosure, the organoid medium for generating apical-lateral organoid is provided, the medium comprises a basal medium and one or more of Notch signal transduction inhibitor, transforming growth factor ("TGF") signal transduction inhibitor and cytoskeleton structure disrupting agent.In one embodiment, the organoid medium is a lung organoid medium.

[0008] In one embodiment, the organoid medium is serum-free.

[0009] In one embodiment, organoid medium does not contain or contact with added extracellular matrix or extracellular matrix protein.In one embodiment, organoid does not form in the presence of added extracellular matrix or extracellular matrix protein.In one embodiment, a population of cells, such as a population of lung lineage cells, does not contact with added extracellular matrix or extracellular matrix protein.In one embodiment, i) organoid medium does not contain or contact with added extracellular matrix or extracellular matrix protein, ii) organoid does not form in the presence of added extracellular matrix or extracellular matrix protein, and iii) cells (e.g., lung lineage cells) do not contact with added extracellular matrix or extracellular matrix protein.

[0010] In embodiments in which the organoid medium comprises a Notch signaling inhibitor, the Notch signaling inhibitor is a gamma secretase inhibitor.

[0011] In embodiments in which the organoid medium comprises a TGF-mediated signaling inhibitor, the TGF-mediated signaling inhibitor is a TGF-beta signaling inhibitor.

[0012] In embodiments in which the organoid medium comprises a factor that modifies the cytoskeleton, the factor that modifies the cytoskeleton is an inhibitor of Rho-associated protein kinase.

[0013] In one embodiment, the organoid medium does not contain the factor that modifies cytoskeleton.In one embodiment, the organoid medium does not contain the inhibitor of Rho-associated protein kinase.

[0014] In one broad aspect of the present disclosure, a method for forming organoids in culture is provided, the method comprises contacting a population of cells, such as a population of lung lineage cells, with organoid medium, and culturing the cells in the organoid medium.In one embodiment, the population of cells is cultured in the absence of added extracellular matrix or extracellular matrix protein.

[0015] In one embodiment, the method may further include forming an organoid, wherein at least a portion of the apical surface of the organoid is in contact with an organoid medium.

[0016] In one embodiment, the population of lung lineage cells is bronchial epithelial cells or nasal epithelial cells. In one embodiment, the population of lung lineage cells is human or murine.

[0017] In one embodiment, the method further comprises aggregating the population of cells. In one embodiment, aggregating the population of cells is in organoid medium. In one embodiment, aggregating the population of cells is for 1-7 days. In one embodiment, the aggregation comprises depositing 10-2000 cells into the microwell device. In one embodiment, the deposited cells are single cells or are included in clumps.

[0018] In one embodiment, the organoid medium is serum-free.

[0019] In one embodiment, the organoid medium comprises a basal medium.

[0020] In one embodiment, organoid medium does not contain or contact with added extracellular matrix or extracellular matrix protein.In one embodiment, organoid does not form in the presence of added extracellular matrix or extracellular matrix protein.In one embodiment, cells such as a group of lung lineage cells do not contact with added extracellular matrix or extracellular matrix protein.In one embodiment, i) organoid medium does not contain or contact with added extracellular matrix or extracellular matrix protein, ii) organoid does not form in the presence of added extracellular matrix or extracellular matrix protein, and iii) cells (e.g., lung lineage cells) do not contact with added extracellular matrix or extracellular matrix protein.

[0021] In one embodiment, the organoid medium comprises a basal medium supplemented with one or more of a Notch signaling inhibitor, a TGF-mediated signaling inhibitor, and an agent that disrupts the cytoskeleton structure.

[0022] In one embodiment, the organoid medium comprises a Notch signal transduction inhibitor.In one embodiment, the Notch signal transduction inhibitor is a gamma secretase inhibitor.

[0023] In one embodiment, organoid medium comprises TGF-mediated signal transduction inhibitor.In one embodiment, TGF-mediated signal transduction inhibitor is TGF beta signal transduction inhibitor.

[0024] In one embodiment, the organoid medium comprises a factor that modifies the cytoskeleton.In one embodiment, the organoid medium does not comprise a factor that modifies the cytoskeleton.

[0025] In one embodiment, the agent that modifies the cytoskeleton is an inhibitor of Rho-associated protein kinase.

[0026] In one embodiment, the culture of the population of lung lineage cells in organoid medium is under non-adherent conditions. In one embodiment, the culture is for 5 to 25 days.

[0027] In one embodiment, at least 25% of the cells in the organoid portion are ciliated.In one embodiment, at least 50% of the cells in the organoid portion are ciliated.

[0028] In one embodiment, at least 60% of the organoids have their apical surface in contact with organoid medium.In one embodiment, at least 80% of the organoids have their apical surface in contact with organoid medium.

[0029] In one embodiment, the organoid expresses one or more markers of ciliogenesis. In one embodiment, the one or more markers of ciliogenesis include FOXJ1 and / or TUBB4B.

[0030] In one embodiment, the organoid expresses one or more of TJP1 (i.e., ZO-1) and ACE2.

[0031] In one embodiment, the organoid does not express MUC5AC.

[0032] In one embodiment, the organoids support viral replication.

[0033] In one embodiment, the method can further comprise evaluating the organoid response to one or more therapeutic agents, such as antiviral agents.In one embodiment, the method can further comprise evaluating the organoid response to one or more pathogens, such as viruses or bacteria.

[0034] Thus, advantages of the methods and media disclosed herein include, but are not limited to, i) ECM-free workflows may be superior to ECM-removed workflows in terms of efficiency in generating apical-lateral organoids; ii) easy efficient removal of dead, dying or other sloughed cells from the culture since they do not accumulate in the lumen of "apical-lateral" organoids; iii) rapid readout of ciliary beating, potentially of use in studying cilia-related diseases; iv) elimination of spatial constraints imposed by forming organoids in extracellular matrix and / or potential for efficient scale-up; and v) studying viral infection and infectivity titers, and / or screening of antiviral drugs.

[0035] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, and that various changes and modifications which will become apparent to those skilled in the art from this detailed description are within the spirit and scope of the present invention.

[0036] For a better understanding of the various embodiments described herein, and to more clearly show how they may be effectively practiced, reference is made to the accompanying drawings, which illustrate at least one exemplary embodiment, and which will now be described, the drawings being not intended to limit the scope of the teachings described herein. [Brief description of the drawings]

[0037] [Figure 1]Figure 1 shows the results of initial characterization of apical-lateral organoids generated using the disclosed method or medium. Organoid movement was measured by visually assessing ciliated organoids and scoring the movement of such ciliated organoids (Panel A). Organoids from three different donors were evaluated for motility over six consecutive passages starting from passage 3. Histogram showing the distribution of mean organoid diameters measured at passage 3 from three different donors (Panel B). 750 organoids were equally counted from three wells for each donor. Box plot of organoid diameter distribution when organoids are formed from different starting cell numbers. Unshaded values ​​correspond to measurements taken at a relatively early time point, while shaded values ​​correspond to measurements taken after organoids have been in suspension culture for at least another week (Panel C). The center line represents the median value, and the whisker boundaries represent the maximum and minimum values. 75 organoids were measured for each condition (n=1). [Diagram 2] Figure 1 shows a comparison of the efficiency of apical-lateral airway organoid formation in various conditions. Bar graph showing the efficiency of forming apical-lateral organoids using either an ECM-free workflow (ECM-free Ap-O AO) or a workflow where the organoids are first formed in a Matrigel™ dome and then removed from the Matrigel to attempt organoid inversion (ECM Ap-O AO) for three different donors. Individual points represent technical replicates. Data are presented as the mean ± SD of five independent experiments. P values ​​are calculated by unpaired Student's t-test (**=p<0.01, ***=p<0.001). Representative images of organoid fusion typically observed between organoids that can be formed with the ECM Ap-O AO workflow (scale bar = 200 μm). White arrows indicate organoids that are fused but not completely fused together (Panel B). [Diagram 3]Figure 1 shows a graph quantifying the percentage of cells in ciliated organoids.The epithelial cells of the first donor (panels A) and B)) and the second donor (panels C) and D) are formed into apical-lateral organoids according to the present disclosure.In panels A) and C), the concentration of Notch signaling inhibitor is titrated, while the concentration of TGF beta signaling inhibitor is roughly constant.In panels B) and D), the concentration of TGF beta signaling inhibitor is titrated, while the concentration of Notch signaling inhibitor is roughly constant. [Figure 4] Graphs are shown quantifying the number of apical-lateral organoids and the percentage of cells in ciliated organoids. The epithelial cells of the first donor (panels A) and B)) and the second donor (panels C) and D)) are formed into apical-lateral organoids according to the present disclosure. The effect of removing Notch signaling inhibitor on the total number of organoids is shown in panels A) and C). The effect of removing Notch signaling inhibitor on the percentage of ciliated cells in apical-lateral organoids is shown in panels B) and D). [Diagram 5] Figure 1 shows the graphs quantifying the number of apical-lateral organoids and the percentage of ciliated organoid cells.Donor epithelial cells are formed into apical-lateral organoids according to the present disclosure.The effect of combining Notch signaling inhibitor with TGF beta signaling inhibitor on the total number of organoids (panel A) and the percentage of ciliated cells in apical-lateral organoids (panel B) is shown. [Figure 6] Graphs are shown quantifying the efficiency of apical-lateral organoid formation across multiple donors and multiple passages. Following the formation of apical-lateral organoids according to the present disclosure, the number of motile organoids is quantified over successive passages (Panel A). During each passage, dissociated cells are analyzed for the presence of cilia (Panel B). Each point represents a different well of a 24-well plate (technical replicate). n=2 replicates per donor (up to p5). [Figure 7]Representative images of apical-lateral organoids formed under different conditions are shown. P6 aggregates cultured in organoid medium containing TGF beta signaling inhibitor and Y-27632 show patchy ciliation (Panel A). P6 aggregates cultured in organoid medium containing Notch signaling inhibitor show extensive ciliation (Panel B). P6 aggregates cultured in organoid medium containing Notch signaling inhibitor and TGF beta signaling inhibitor show extensive ciliation (Panel C). [Figure 8] Representative images of apical-lateral organoids stained for various markers are shown. Apical-lateral organoids are positive for acetylated alpha tubulin (arrow) and ZO-1 (arrowhead) but negative for MUC5AC (Panel A). Apical-lateral organoids are positive for acetylated alpha tubulin (arrow) and ACE2 (arrowhead) (Panel B). [Figure 9] Illustrates the results of qRT-PCR experiments for various markers of lung lineage cells.The gene expression between the cells of the apical-lateral organoid (AOAO) formed according to the present disclosure is compared with various controls, namely the donor cells cultured under air-liquid interface conditions (ALI) in PneumaCult ALI (STEMCELL Technologies), the donor cells formed into organoids using PneumaCult Airway Organoid Kit (AOK) (STEMCELL Technologies), and the donor cells expanded in PneumaCult ExPlus (Ex+) (STEMCELL Technologies). [Figure 10]Figure 1 shows a graph quantifying the number of apical-outer organoids over multiple passages and the percentage of cells in ciliated organoids.Apical-outer organoids were formed according to the present disclosure from four donors (panels A) and B), panels C) and D), panels E) and F), and panels G) and H) in either organoid medium containing Notch signaling and TGF beta signaling inhibitor (AOAO), or control organoid medium containing TGF beta signaling inhibitor and Y-27632 (control).The number of apical-outer organoids formed from four donor samples in two media is shown in panels A), C), E), and G).The percentage of ciliated cells in apical-outer organoids formed from four donor samples in two media is shown in panels B), D), F), and H). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present disclosure relates to medium compositions and / or supplements added to medium, and methods for generating "apical-outer" organoids.The medium and methods disclosed herein produce such "apical-outer" organoids with high efficiency.

[0039] As used in this disclosure, the term "organoid" refers to a multicellular structure that can be generated ex vivo and shows a higher level of organization similar to that observed in the corresponding tissue. Organoids corresponding to various tissue types can be formed using kits and protocols commercially available, for example, by STEMCELL Technologies. This disclosure focuses primarily on epithelial organoids, but is not necessarily limited to epithelial organoids only. Examples of epithelial tissues that can be formed into the organoids of this disclosure (according to the medium and method disclosed herein) include lung organoids, whether they correspond to proximal airway, distal airway, or both.

[0040] As used in this disclosure, the term "lung lineage cells" refers to one of various types of cells that can be isolated from lung tissue, including the nasal cavity, trachea, or bronchi, bronchioles, or alveoli. The term "lung lineage cells" may also refer to any of various types of cells differentiated from pluripotent stem cells (PSCs), including embryonic stem cells or induced pluripotent stem cells. When differentiated from PSCs, lung lineage cells may share one or more common characteristics with the corresponding or closely corresponding primary (i.e., patient-derived) cell type. The lung lineage cells of the present disclosure may be from any species, but are preferably mammalian, more preferably human or mouse. In one embodiment, the lung lineage cells are human lung epithelial cells. In one embodiment, the lung lineage cells are human bronchial epithelial cells. In one embodiment, the lung lineage cells are basal cells.

[0041] As used in this disclosure, the term "organoid medium" or "organoid media" refers to a cell culture medium, including a basal medium, appropriately supplemented to form the organoids of the present disclosure. In fact, basal media are well known in the art and are routinely formulated to include one or more of salt(s), amino acid(s), carbohydrate(s), buffer(s), trace elements, etc. Examples of commercially available basal media include DMEM, Adv-DMEM, DMEM / F-12, RPMI, Iscoves, and various other media that are specifically marketed for the culture of epithelial cells. The specific supplementation of the basal medium, including, for example, cytokines, growth factors, small molecules, serum / albumin / serum replacement, lipids, etc., will depend on the application to which the organoid medium is put. In the embodiment where the organoid is a pulmonary organoid, it generally comprises one or more of mitogens, fibroblast growth factors, activators and / or inhibitors of signal transduction through bone morphogenetic protein, activators and / or inhibitors of signal transduction through wnt, vitamin A analogs, precursors or metabolites / derivatives, and activators and / or inhibitors of signal transduction through transforming growth factor (e.g., transforming growth factor beta).In one embodiment, the organoid medium comprises basal medium and notch signal transduction inhibitor and / or TGF signal transduction inhibitor.

[0042] As used in the present disclosure, the term "apical-outer" refers to organoid, and in some embodiments, epithelial organoid, where the apical surface is in direct contact with the external environment (i.e., cell culture medium).Among the apical-outer organoids, some or all of such organoids may contain a lumen, where the basolateral side is in direct contact with the lumen.However, some apical-outer organoids may not contain a lumen, but rather show a compact / dense conformation, where the basolateral side may be included in or adjacent to the central core of such apical-outer organoid.

[0043] Organoid Media In one aspect of the present disclosure, the organoid medium is provided for generating apical-outer organoid.In a preferred embodiment, the organoid medium is for generating apical-outer lung organoid, such as starting from lung epithelial cells.Therefore, when the organoid medium is used for generating apical-outer lung organoid, it can be characterized as lung organoid medium.

[0044] Organoid medium can contain Notch signal transduction inhibitor.In one embodiment, Notch signal transduction inhibitor is gamma secretase inhibitor.Examples of Notch signal transduction inhibitor include DBZ, DAPT, compound E, compound W, SAHM1 and FLI06.In one embodiment, Notch signal transduction inhibitor is DAPT.

[0045] When present in organoid medium, Notch signal transduction inhibitor will be present at effective concentration, but also at a concentration that does not cause any level or significant level of cytotoxicity.In one embodiment, the concentration of Notch signal transduction inhibitor is in the range of about 1nM to 1mM, about 5nM to 500μM, about 10nM to 200μM, about 50nM to 100μM, about 100nM to 50μM, or about 0.5μM to 20μM.

[0046] In one embodiment, the organoid medium comprises a TGF-mediated signaling inhibitor. In one embodiment, the TGF is TGF beta. In one embodiment, the TGF-mediated signaling inhibitor is a natural inhibitor, such as an endogenous protein. In one embodiment, the TGF-mediated signaling inhibitor is a synthetic inhibitor, such as a small molecule. Examples of TGF-mediated signaling inhibitors include A83-01, A77-01, SB431542, LY364947, and LDN214117. In one embodiment, the TGF-mediated signaling inhibitor is A83-01. In one embodiment, the TGF-mediated signaling inhibitor is A77-01. In one embodiment, the TGF-mediated signaling inhibitor is SB431542.

[0047] When included in organoid medium, TGF-mediated signal transduction inhibitor will be present at an effective concentration, but also at a concentration that does not cause significant levels of cytotoxicity.In one embodiment, the concentration of TGF-mediated signal transduction inhibitor is in the range of about 1nM to 1mM, about 5nM to 500μM, about 10nM to 200μM, about 50nM to 100μM, about 100nM to 50μM, or about 0.5μM to 20μM.

[0048] In one embodiment, organoid medium comprises Notch signal transduction inhibitor (e.g., gamma secretase inhibitor) and TGF-mediated signal transduction inhibitor or both.In one embodiment, organoid medium comprises only Notch signal transduction inhibitor (e.g., gamma secretase inhibitor) or TGF-mediated signal transduction inhibitor.

[0049] In one embodiment, organoid medium does not contain extracellular matrix or extracellular matrix protein.Therefore, in one embodiment, organoid medium does not contain (or does not contain or contact with) added extracellular matrix or extracellular matrix protein.Therefore, organoid medium does not require Matrigel™ or any other extracellular matrix to form apical-lateral organoid.

[0050] In one embodiment, organoid medium does not contain or contact with extracellular matrix or extracellular matrix protein (unless naturally produced by cells in culture).In one embodiment, organoid is not formed in the presence of exogenously added extracellular matrix or extracellular matrix protein.In one embodiment, cells (e.g., lung lineage cells) do not contact with exogenously added extracellular matrix or extracellular matrix protein(s).In one embodiment, neither lung lineage cells, aggregated lung lineage cells, nor organoids contact with exogenously added extracellular matrix protein.

[0051] In one embodiment, organoid medium does not contain serum.In some embodiments of serum-free medium, medium can contain albumin or different serum substitutes.When organoid medium contains albumin, it can be isolated from serum, more specifically from animal serum.Or, albumin can be recombinant and expressed in cell system, such as bacteria, fungi, plant or animal cell system.

[0052] In one embodiment, the organoid medium comprises a factor that modifies the cytoskeleton. In one embodiment, the organoid medium does not comprise a factor that modifies the cytoskeleton. Examples of factors that modify the cytoskeleton (included or specifically excluded in the organoid medium) include Rho-associated protein kinase inhibitors, p21-activated kinase (PAK) inhibitors, or myosin II inhibitors. Examples of Rho-associated protein kinase inhibitors include Y-27632, SR3677, thiazovivin, HA1100 hydrochloride, HA1077, and GSK-429286. An example of a PAK inhibitor is IPA3. An example of a myosin II inhibitor is blebbistatin. In a preferred embodiment, the organoid medium does not comprise a factor that modifies the cytoskeleton.

[0053] In one embodiment, the organoid medium comprises one, more than one, more than two, or each of a Notch signaling inhibitor (e.g., a gamma secretase inhibitor), a TGF-mediated signaling inhibitor, and a factor that modifies / disrupts the cytoskeletal structure.

[0054] method In one aspect of the present disclosure, the method for generating organoid is provided, and some of them are apical-lateral organoid.In a preferred embodiment, the method is for generating apical-lateral lung organoid, such as starting from lung epithelial cell.

[0055] The method of forming organoids (e.g., apical-lateral organoids) in culture will include contacting a population of cells (e.g., lung lineage cells) with organoid medium. In one embodiment, the organoid medium of the present disclosure comprises a basal medium and one or both of a Notch signaling inhibitor and / or a TGF-mediated signaling inhibitor. In one embodiment, the organoid medium of the present disclosure comprises a basal medium and both of a Notch signaling inhibitor and a TGF-mediated signaling inhibitor. In one embodiment, the organoid medium of the present disclosure comprises only a basal medium and one or both of a Notch signaling inhibitor or a TGF-mediated signaling inhibitor. In one embodiment, the organoid medium of the present disclosure comprises one or more of a basal medium and a Notch signaling inhibitor, a TGF-mediated signaling inhibitor including both, and an agent that disrupts cytoskeleton structure. In any case, the organoid medium used in the method of the present disclosure, for example, a lung organoid medium, can be formulated as described above.

[0056] The method of forming organoid (e.g., apical-lateral organoid) in culture will include culturing a group of cells (e.g., lung lineage cells) in organoid medium.In one embodiment, culturing a group of cells (e.g., lung lineage cells) in organoid medium is performed in the absence of added extracellular matrix or extracellular matrix protein.

[0057] This method produces apical-outer organoids, and at least some of the apical surfaces of the apical-outer organoids face away from their core (e.g., lumen), but in any case, the apical surfaces of the apical-outer organoids will be in direct contact with the external environment. In one embodiment, the majority (i.e., part) of the organoids formed exhibits apical-outer morphology / configuration. In one embodiment, at least 60% of the organoids have apical surfaces facing away from the core (or lumen). In one embodiment, at least 80% of the organoids have apical surfaces facing away from the core (or lumen). In one embodiment, about 90% or more of the organoids exhibit apical surfaces facing away from the core (or lumen).

[0058] In one embodiment, at least 25% of the cells of the apical-lateral organoid are ciliated.In one embodiment, at least 50% of the cells of the apical-lateral organoid are ciliated.

[0059] In one embodiment, the method further comprises aggregating the population of cells (e.g., lung lineage cells). In one embodiment, the method further comprises aggregating the population of cells (e.g., lung lineage cells) prior to or concurrently with the contacting step.

[0060] Cells can be aggregated using any known means.It has been reported that cells can be deposited in the wells of certain types of 96-well plates and form aggregates when they settle, but this approach produces a large variability in aggregate size and the number of aggregates formed per well.A better approach may be to use AggreWell™ microwell device to ensure the formation of a single aggregate per well and obtain aggregates that are roughly uniform in size.

[0061] In one embodiment, aggregating the cells (e.g., lung lineage cells) occurs in the absence of added extracellular matrix or extracellular matrix protein(s). In one embodiment, the aggregating cells and the aggregated cells (e.g., lung lineage cells) are not in contact or come into contact with added extracellular matrix or extracellular matrix protein(s).

[0062] In one embodiment, aggregating a population of cells (e.g., lung lineage cells, such as isolated basal cells) comprises bringing 10-2000 cells into close proximity in a common well. In one embodiment, the number of cells forming the aggregate is 50-150. In one particular embodiment, aggregating a population of lung lineage cells comprises depositing 10-2000 lung lineage cells per well of a microwell device. In one embodiment, the deposited cells are single cells or included in clumps, or a mixture thereof. Interestingly, aggregates / organoids initially formed from different cell numbers may undergo cell "shedding" when cultured in suspension, such that after 5 or more days in suspension culture, they may converge to roughly equal diameters, regardless of the initial organoid diameter.

[0063] In one embodiment, a population of lung lineage cells is aggregated in the organoid medium of the present disclosure.In one embodiment, a population of lung lineage cells is aggregated in organoid medium for 1-7 days.In one embodiment, a population of lung lineage cells is aggregated in organoid medium for 4 days ± 2 days.

[0064] In one embodiment, culturing the population of cells (e.g., lung lineage cells) is under non-adherent conditions. In one embodiment, the aggregation step is performed under non-adherent conditions, for example, by using ultra-low attachment 96-well plates or by coating the laboratory equipment used with an anti-adherent solution provided with the AggreWell™ plates. In one embodiment, the contacting step is performed under non-adherent conditions. In one embodiment, the contacting step and the culturing step, as well as the aggregation step, if performed, are under non-adherent conditions.

[0065] In one embodiment, culturing the population of cells (e.g., lung lineage cells) in organoid medium and in the absence of added extracellular matrix or extracellular matrix protein(s) is for a time sufficient to generate apical-lateral organoids. In one embodiment, the culturing is for 5-25 days. In one embodiment, the culturing is for 9 days ± 3 days. In one embodiment, the culturing is for 15 days. In embodiments where the population of lung lineage cells is aggregated before or during the contacting step, the culturing step can be shortened if it is evident that apical-lateral organoids are emerging.

[0066] As discussed with respect to the organoid medium of the present disclosure, such organoid medium does not contain extracellular matrix or extracellular matrix protein.In one embodiment, organoid medium does not contain or contact with extracellular matrix or extracellular matrix protein (unless naturally produced by cells in culture).Therefore, in one embodiment, organoid medium does not contain (or does not contain or contact with) added extracellular matrix or extracellular matrix protein.Therefore, organoid medium does not require Matrigel™ or any other extracellular matrix to form apical-outer organoid.

[0067] Similarly, the contacting step, the culturing step and, if applicable, the agglomeration step are carried out in the absence of added extracellular matrix or extracellular matrix protein.Therefore, the laboratory equipment used during these steps is not coated with extracellular matrix or extracellular matrix protein.Therefore, the extracellular matrix commonly used for organoid formation, such as Matrigel™ or others, is not necessary to carry out the method of the present disclosure.

[0068] The lung lineage cell population can be expanded and passaged using a commercial kit such as PneumaCult™ Ex or PneumaCult™ Ex Plus (STEMCELL Technologies). At each passage, some or all of the expanded cells can be used in the method disclosed herein to obtain apical-lateral organoids. If not all of the expanded cells are used to generate apical-lateral organoids, the remaining cells can be passaged to allow the formation of successive "generations" of apical-lateral organoids. The lung lineage cell population expanded and passaged by PneumaCult™ Ex or PneumaCult™ Ex Plus can be expanded for 5 or more passages or 8 or more passages.

[0069] The apical-lateral organoids formed according to the present disclosure may express one or more markers of ciliogenesis. For example, the one or more markers of ciliogenesis may include FOXJ1 and TUBB4B.

[0070] The apical-lateral organoids formed according to the present disclosure can express one or more markers related to their function or identity.For example, the one or more markers related to their function or identity can include TJP1 (i.e., ZO-1) and ACE2.

[0071] Apical-lateral organoids formed in accordance with the present disclosure may have no MUC5AC expression or may express MUC5AC at below detectable levels.

[0072] The apical-lateral organoid formed according to the present disclosure can support the replication of viruses, such as respiratory or other viruses.In one embodiment, the method can further comprise evaluating the organoid response to one or more therapeutic agents.In one embodiment, the method can further comprise evaluating the organoid response to one or more pathogens, such as viruses or bacteria.

[0073] The following non-limiting examples illustrate the present disclosure. EXAMPLES

[0074] Example 1: Cultivation and expansion of starting cells Primary normal human bronchial epithelial cells (hBECs) were commercially available cells such as LONZA or EPITHELIX SARL. In the experiments described herein, all cells were obtained from non-smoking, healthy donors. Optionally, hBECs were seeded at passage 1 in T25 cell culture flasks in PneumaCult™ EX-PLUS (STEMCELL Technologies, catalogue #05040) and incubated at 37°C and 5% CO according to the manufacturer's protocol. 2 When the cells reached 50-60% confluency, they were dissociated using an animal component-free (ACF) cell dissociation kit (STEMCELL Technologies, catalogue #05426).

[0075] Example 2: Preparation of Laboratory Apparatus Plates used in downstream assays were coated with anti-adhesion rinse solution (STEMCELL Technologies, catalogue #07010). Briefly, 500 μL of anti-adhesion rinse solution was added to each well to be used, and the plates were centrifuged at 1300 g for 10 minutes. The anti-adhesion rinse solution was then removed, and the wells were washed once with 1 mL of DMEM. Wells were either used directly or stored at 37° C. for up to 1 week after adding 500 μL of DMEM.

[0076] Example 3: Aggregate formation Cells were cultured and dissociated according to Example 1 and seeded into Aggrewell™ 400 plates (STEMCELL Technologies) prepared according to Example 2. Aggregates were approximately 1.2×10 per well of the Aggrewell™ 400 plate. 5 The AggreWell™ plates were generated by seeding 100 cells per microwell. The cells were seeded in 500 μL of apical-lateral organoid medium containing basal medium and Notch signaling inhibitor. The AggreWell™ plates were centrifuged at 100 g for 3 minutes to allow the cells to settle to the bottom of the microwells. The plates were then incubated at 37° C. and 5% CO 2 The mixture was incubated at 4°C for 24 to 144 hours.

[0077] Example 4: Suspension culture of apical-lateral organoids After the aggregates are formed and fully matured according to Example 3, 500 μL of fresh apical-lateral airway organoid medium is added to each well.The aggregates are then resuspended using a P1000 pipette, and each well is distributed into two wells of a 24-well plate (prepared as described in Example 2).The aggregates are incubated in apical-lateral airway organoid medium under non-adherent conditions for up to 15 days.Partial medium changes are performed every 2 days.

[0078] At day 15, organoids were evaluated for their motility in suspension as an alternative to disrupting cilia facing the culture environment. Virtually all of the apical-lateral organoids formed from the three different donors exhibited motility when assessed at multiple successive passages (Figure 1A). Thus, the tested conditions for forming apical-lateral organoids appeared to be highly efficient. Day 15 organoids were also evaluated for size (i.e., mean organoid diameter), and apical-lateral organoids formed from the three donor samples showed a narrow size distribution (Figure 1B). Specifically, mean diameters of 72.6 ± 6.7 μm (donor 1), 77.3 ± 4.7 μm (donor 2), and 73.6 ± 0.3 μm (donor 3) were observed in the different populations of organoids. Interestingly, even when more than 100 cells were seeded per microwell (e.g., 200, 300, 400, or 500 cells / well), the average diameter of such organoids measured at day 15 was virtually identical to the average diameter of organoids generated from 100 cells per microwell (Figure 1C).

[0079] Example 5: Dissociation of Apical-Lateral Organoids The efficiency of apical-lateral airway organoid formation was compared between organoids formed according to Example 4 (i.e., ECM-free conditions) and organoids generated by removal from ECM conditions (i.e., ECM-depleted conditions). Briefly, ECM-free conditions involved generating organoids by seeding 2500 human bronchial epithelial cells into Matrigel™ domes and culturing for 7 days using the PneumaCult™ Airway Organoid Kit (STEMCELL Technologies). Matrigel was removed after 7 days by incubating in Gentle Cell Dissociation Reagent (STEMCELL Technologies) for 1 hour at 4° C. on a shaker. The data show that apical-lateral airway organoids either did not form or formed with low efficiency in ECM-free conditions, indicating a marked improvement in efficiency in the ECM-free workflow disclosed herein (FIG. 2A). Furthermore, under ECM-free conditions, multiples of the apical-lateral airway organoids that did form tended to fuse into larger organoids (FIG. 2B). Overall, the ECM-free workflow shows a marked improvement compared to the ECM-free workflow.

[0080] Example 6: Dissociation of Apical-Lateral Organoids The apical-lateral airway organoids formed according to Example 4 were harvested on the 15th day, transferred to a 15ml tube, and centrifuged at 150g for 5 minutes. The supernatant was removed, and the organoids were washed once with DMEM. The washed organoids were resuspended in ACCUTASE (STEMCELL Technologies), incubated at room temperature for 15 minutes, and then dissociated into single cells by vigorous pipetting with a P1000 pipette. The single cell suspension was diluted 1:1 with trypan blue, and then loaded into a hemocytometer, where the cells were counted manually. When counting dissociated cells, the number of ciliated cells can also be determined by visual inspection.

[0081] In one experiment, apical-lateral organoids were formed from two donor samples in apical-lateral medium formulated with different concentrations of inhibitors of Notch signaling and TGF-beta signaling. The apical-lateral organoids were dissociated and the cells were examined for the number of ciliated cells (Figure 3). In the first donor (panels A and C), all conditions yielded more than 20% ciliated cells, but in this donor, the highest concentrations of both DAPT and A77-01 revealed a decrease in the percentage of ciliated cells. Based on the results from the first donor, only the best performing conditions were tested in the cells of the second donor (panels B and D). Lower concentrations of A77-01 appeared to be detrimental to the emergence of ciliated cells. Thus, it was found that concentrations of 2.5 μM to 10 μM for the Notch signaling inhibitors and 0.5 μM to 1.5 μM for the TGF-mediated signaling inhibitors worked well enough for the efficient generation of the percentage of ciliated cells.

[0082] Example 7: Characterization of apical-lateral organoids The fully matured organoid formed according to Example 4 is imaged using a standard microscope camera.For example, organoid can be imaged using Leica DMi8 or EVOS M500.The number of organoid is quantified, and the percentage of motile organoid (i.e., cilia organoid%) is evaluated by visual inspection and by manually counting the organoid that shows externally broken cilia.

[0083] Cells from two different donors were formed into apical-lateral organoids using apical-lateral organoid medium formulated with TGF-mediated signaling inhibitors alone or in combination with Notch signaling inhibitors (Figure 4). The presence or absence of Notch signaling inhibitors (combined with TGF-mediated signaling inhibitors) did not appear to significantly affect the number of organoids obtained (Figures 4A and 4C), but the absence of Notch signaling inhibitors significantly affected the percentage of ciliated cells in the organoids (Figures 4B and 4D). Thus, Notch signaling inhibitors appear to be important for efficient differentiation of apical-lateral organoids.

[0084] Conversely, the role of TGF-mediated signaling inhibitors was investigated by adding or omitting them to the apical-lateral organoid medium (containing Notch signaling inhibitors) (Figure 5). Inclusion of TGF-mediated signaling inhibitors was able to increase the number of output organoids (Figure 5A), while its absence seemed to have no effect or a favorable effect on the proportion of ciliated cells in the organoids (Figure 5B).

[0085] The inclusion of TGF-mediated signaling inhibitors was believed to be important for increasing organoid output, and the inclusion of Notch signaling inhibitors was believed to be important for differentiation of donor lung lineage cells, so both were included in the apical-lateral organoid medium used in subsequent studies. After forming apical-lateral organoids from lung lineage cells of four different donors according to Example 4, visual inspection under a microscope revealed that almost all of the formed organoids were motile, indicating sufficient fineness of the organoids (Figure 6A). The lung lineage cells were passaged up to eight times depending on the donor and cell number, and each passage was able to form apical-lateral organoids with high efficiency (Figure 6A). The organoids from each "passage" were dissociated, and the cells were evaluated for ciliated rate (Figure 6B). A significant proportion of the cells showed cilia.

[0086] Figure 7 shows representative images of apical-lateral organoids generated from lung lineage cells from donors at passage 6. Patchy cilia were observed among organoids formed in medium containing TGFbeta signaling inhibitors and Y-27632 inhibitors (Figure 7A), whereas extensive cilia were observed for apical-lateral organoids formed in apical-lateral medium containing Notch signaling inhibitors alone (Figure 7B) or in combination with TGF-mediated signaling inhibitors (Figure 7C).

[0087] For comparison, organoids formed from lung lineage cells of four different donors in medium containing inhibitors of TGF-beta signaling and Y-27632 ("control") were directly compared to the apical-lateral organoids of the present disclosure in terms of output organoid numbers (FIGS. 10A, 10C, 10E, and 10G) and percentage of ciliated cells (FIGS. 10B, 10D, 10F, and 10H). Output organoid numbers were roughly comparable between the control and apical-lateral conditions, but it became clear that the apical-lateral condition was superior to the control condition in differentiating organoids to increase the percentage of ciliated cells.

[0088] Organoids were stained according to the following generalized protocol. Apical-lateral organoids were fixed in 4% paraformaldehyde and subjected to antigen retrieval before permeabilization with 1% Triton X-100 in PBS. Permeabilized organoids were blocked with 0.1% BSA / 5% normal goat serum in PBS+0.1% Tween-20+0.2% Triton X-100 (PBSTT). Primary antibodies were diluted in PBSTT and incubated with cells at room temperature in tubes for 4 days with gentle agitation. Specific airway epithelial markers were tested, including acetylated α-tubulin (Sigma, cat#T7451, 1:500), TMPRSS2 (Sigma, cat#MABF2158), ACE2 (Abcam, cat#ab15348), MUC5AC (Abcam, cat#ab212636). Respective secondary antibodies were used for the primary antibodies. Cells were washed with PBSTT and incubated with the respective secondary antibodies for 3 days in tubes at room temperature with gentle agitation. Cells were washed again with PBSTT before staining with 4',6-diamidino-2-phenylindole (DAPI) and imaged with a LEICA SP8. Representative images are shown in Figure 8.

[0089] Total RNA was isolated from apical-lateral airway organoids using Qiagen Easy RNA mini kit (Qiagen) according to the manufacturer's protocol. 500ng of RNA was DNase treated (Invitrogen) according to the manufacturer's protocol and then reverse transcribed into cDNA using SuperScript III (Invitrogen). TaqMan gene-specific assay primers and probes (Integrated DNA Technologies) were used together with TaqMan™ Fast Universal PCR Master Mix (2X) (Applied Biosystems). Information related to primer / probe sets is shown in Table 1 below. Samples were amplified as follows: denaturation at 95°C for 20 seconds, followed by 50 cycles of 95°C for 1 second and 60°C for 20 seconds. The mRNA expression levels of cellular genes were normalized with the expression levels of TBP, as it was shown to have the least standard deviation among a panel of housekeeping genes. [Table 1]

[0090] The expression of the markers outlined above was evaluated in apical-lateral organoids formed in apical-lateral organoid medium. As a control, the expression of these markers was also evaluated in cells formed using Airway Organoid Kit (STEMCELL Technologies), cells grown at air-liquid interface using PneumaCult™ ALI (STEMCELL Technologies), and cells expanded using PneumaCult™ Ex Plus (STEMCELL Technologies). The analysis results are shown in Figure 9, and the expression was normalized to Tata binding protein (TBP).

[0091] While the present disclosure has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the present disclosure is not limited to the disclosed embodiments, but on the contrary, the present disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0092] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. A method for forming apical-lateral organoids in a culture, comprising: contacting a cell population with an organoid medium containing a Notch signaling inhibitor and / or a signaling inhibitor via TGF; culturing the cell population in the organoid medium and in the absence of added extracellular matrix or extracellular matrix protein to obtain apical-lateral organoids; wherein the apical surface of at least some of the apical-lateral organoids faces away from its core; the method.

2. The method according to claim 1, wherein the cell population is lung lineage cells.

3. The method according to claim 2, wherein the lung lineage cells are bronchial epithelial cells or nasal epithelial cells.

4. The method according to claim 2, wherein the cell population of lung lineage cells is from a human or a mouse.

5. The method according to claim 1, further comprising the step of aggregating the cell population.

6. The method according to claim 5, wherein the step of aggregating the cell population is carried out in the organoid medium.

7. The method according to claim 5, wherein the step of aggregating the cell population is carried out for 1 to 7 days.

8. The method according to claim 5, wherein the step of aggregating comprises depositing 10 to 2000 cells into a microwell device.

9. The method according to claim 8, wherein the cells are single cells or are contained in clumps.

10. The method according to claim 1, wherein the organoid medium does not contain serum.

11. The method according to claim 1, wherein the organoid medium contains a signaling inhibitor via TGF.

12. The method according to claim 11, wherein the TGF is TGF beta.

13. The method according to claim 1, wherein the organoid medium contains a factor that modifies the cytoskeleton.

14. The method according to claim 1, wherein the organoid medium does not contain a factor that modifies the cytoskeleton.

15. The method according to claim 13, wherein the factor that modifies the cytoskeleton is an inhibitor of Rho-associated protein kinase.

16. The method according to claim 1, wherein the step of culturing the cell population is under non-adherent conditions.

17. The method according to claim 1, wherein the culturing step is carried out for 5 to 25 days.

18. The method according to claim 1, wherein at least 25% of the cells in the organoid portion are ciliated.

19. The method according to claim 18, wherein at least 50% of the cells in the organoid portion are ciliated.

20. The method according to claim 1, wherein in at least 60% of the organoids, their apical surfaces are in contact with the organoid medium.

21. The method according to claim 1, wherein in at least 80% of the organoids, their apical surfaces are in contact with the organoid medium.

22. The method according to claim 1, wherein the organoid expresses one or more markers of ciliogenesis.

23. The method according to claim 22, wherein the one or more markers of ciliogenesis include FOXJ1 and / or TUBB4B.

24. The method according to claim 1, wherein the organoid expresses one or more of TJP1 (i.e., ZO-1) and ACE2.

25. The method according to claim 1, wherein the organoid does not express MUC5AC.

26. The method according to claim 1, wherein the organoid supports viral replication.

27. The method according to claim 1, further comprising the step of evaluating the organoid response to one or more therapeutic agents or one or more pathogens.

28. An organoid medium for generating apical-out organoids, the medium comprising a basal medium and a Notch signaling inhibitor and / or a TGF-mediated signaling inhibitor.

29. The organoid medium according to claim 28, wherein the organoid medium does not contain serum.

30. The organoid medium according to claim 28, wherein the organoid medium does not contain or is not in contact with an added extracellular matrix or extracellular matrix protein.

31. The organoid medium according to claim 28, further comprising a TGF-mediated signaling inhibitor.

32. The organoid medium according to claim 31, wherein the TGF is TGF beta.

33. The organoid medium according to claim 28, further comprising a factor that modifies the cytoskeleton.

34. The organoid medium according to claim 28, wherein the organoid medium does not contain a factor that modifies the cytoskeleton. The organoid medium according to claim 33, wherein the factor for modifying the cytoskeleton is an inhibitor of Rho-associated protein kinase.

36. The organoid medium according to claim 28, wherein the organoid medium is a lung organoid medium.

37. Use of the organoid medium according to claim 28 for generating apical-lateral organoids.

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