Compositions and methods for improved generation of organoids

Abstract

The present disclosure relates to cell culture applications using organoids, and in specific embodiments to cell culture applications related to generating respiratory system organoids. Methods include differentiating a population of cells, such as of the respiratory system, through exposure / culture to an incubation temperature above 25°C and lower than 37°C. Methods for obtaining apical-out organoids, such as comprising cells of the respiratory system, at an incubation temperature above 25°C and lower than 37°C are also described. Reliable and robust organoid formation / differentiation are reported by practicing the disclosed methods and / or through use of disclosed culture media formulations.

Description

COMPOSITIONS AND METHODS FOR IMPROVED GENERATION OF ORGANOIDSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 586,478, filed September 29, 2023, the entire contents of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to cell culture applications, and more specifically to cell culture applications using organoids, and still more specifically to cell culture applications related to generating respiratory system organoids.BACKGROUND

[0003] The nasal cavity present in the upper respiratory tract is lined with the nasal epithelium that warms and humidifies inhaled air before it travels down the respiratory tract. The nasal epithelium acts as a physical barrier between the human host and external environmental agents such as viruses, bacteria, allergens and air pollutants. It also has important functions in mucociliary clearance and mediating innate and acquired immune responses. Therefore, it is important to gain insights into the mechanism of action of human nasal epithelial cells for effective management of respiratory diseases and respiratory tract infections (RTI).

[0004] Recent advances in cell culture have paved the way for organoid-based in vitro models that better recapitulate the complex structure and function of in vivo tissues. Such organoid models have emerged as promising alternatives to in vitro cell lines and primary cell culture for better assessment of disease mechanisms and sensitivity to drugs and therapeutic compounds.

[0005] In the field of pulmonary research, there is a need to develop better in vitro experimental models that recapitulate the highly differentiated nasal epithelium for high-throughput screening and drug response assessment. Although organoid models exist for airway epithelial cells, there is a need for improved methods for generating organoids with high efficiency to obtain high-quality nasal epithelial cell models.

[0006] The present disclosure provides a method to reliably and robustly differentiate and / or generate organoids from a population of cells of the respiratory system. The methods disclosed herein represent a significant advance in the development of physiologically relevant models to study the human respiratory tract or the airway epithelium.SUMMARY

[0007] In one aspect of this disclosure are provided methods of culturing a population of cells that in their native context are exposed to air. Such cells may correspond to cells of the respiratory system, skin, or otherwise. If corresponding to the respiratory system, the cells may be cells of the nasal epithelium and / or nasal mucosa. In related aspects, such methods of culturing may more specifically relate to one or more of a method of expanding cells that in their native context are exposed to air, a method of differentiating cells that in their native context are exposed to air, and / or a method of generating organoids from the population of cells that in their native context are exposed to air.

[0008] In such aspect, methods of this disclosure may comprise providing the population of cells in a culture medium, and exposing the population of cells to an unconventional environmental condition, such as to an incubation temperature above 25°C and lower than 37°C. In specific embodiments, the incubation temperature may be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C, or any integer within this range, or may fall within the range 32°C ± 3°C.

[0009] In such aspect, methods of this disclosure may comprise providing the population of cells in a culture medium under non-adherent conditions. Thus, in some embodiments, the population of cells do not come into contact with an added or exogenous extracellular matrix or added or exogenous extracellular matrix protein, whether via culture media or a culture vessel.

[0010] Culture media, whether used in methods of this disclosure or described in a separate aspect, may comprise a basal medium and one or both of an inhibitor of notch signaling and / or an inhibitor of signaling through a TGF. Culture media may further be free of an added extracellular matrix or extracellular matrix protein. Culture media may still further be serum-free.

[0011] Methods of this disclosure may further comprise culturing the cells (exposed to the culture environment) for between 1 to 90 days.

[0012] Methods of this disclosure may further comprise aggregating the population of cells, whether prior to, during, or after providing the population of cells in the culture medium. Aggregating the population of cells may further comprise depositing between lO and 2000 cells per well of a microwell patterned plate or device in the culture medium. In one embodiment, distinct aggregates of cells of the respiratory system are formed in each well of a microwell patterned plate or device.

[0013] In another aspect of this disclosure are provided methods for generating / obtaining organoids comprising cells that in their native context are exposed to air. Such cells may correspond to cells of the respiratory system, skin, or otherwise. If corresponding to the respiratory system, the cells may be cells of the nasal epithelium and / or nasal mucosa. The organoids, or at least some of thegenerated / obtained organoids, may be apical-out organoids (wherein an apical surface of at least a portion of the organoids is faced away from a core thereof).

[0014] In such aspect, methods of this disclosure may comprise providing the population of cells in a culture medium, and exposing the population of cells to an unconventional environmental condition, such as to an incubation temperature above 25°C and lower than 37°C. In specific embodiments, the incubation temperature may be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C, or any integer within this range, or may fall within the range 32°C ± 3°C.

[0015] In such aspect, methods of this disclosure may comprise providing the population of cells in a culture medium under non-adherent conditions. Thus, in some embodiments, the population of cells do not come into contact with an added or exogenous extracellular matrix or added or exogenous extracellular matrix protein, whether via culture media or a culture vessel.

[0016] Culture media, whether used in methods of this disclosure or described in a separate aspect, may comprise a basal medium and one or both of an inhibitor of notch signaling and / or an inhibitor of signaling through a TGF. Culture media may further be free of an added extracellular matrix or extracellular matrix protein. Culture media may still further be serum-free.

[0017] Methods of this disclosure may further comprise culturing the cells (exposed to the culture environment) for between 1 to 90 days.

[0018] Methods of this disclosure may further comprise aggregating the population of cells, whether prior to, during, or after providing the population of cells in the culture medium. Aggregating the population of cells may further comprise depositing between lO and 2000 cells per well of a microwell patterned plate or device in the culture medium. In one embodiment, distinct aggregates of cells of the respiratory system are formed in each well of a microwell patterned plate or device.

[0019] In one embodiment, the number of organoids generated at an incubation temperature of 32°C ± 3°C is at least 30% higher than the number of organoids generated at an incubation temperature of 37°C.

[0020] In one embodiment, at least 25% of cells of the apical-out organoids are ciliated.

[0021] In one embodiment, the organoids support infection by viruses.

[0022] In one embodiment, methods of this disclosure may further comprise assessing organoid responses to one or more therapeutic agents, or one or more pathogens.

[0023] In another aspect of this disclosure are provided systems or kits for culturing (e.g. expanding, differentiating, and / or obtaining organoids) cells that in their native context are exposed to air. Thesystem(s) or kit(s) may comprise one or more of: culture media, whether provided as whole media or as a basal medium + supplement(s); cells that in their native context are exposed to air; incubation temperature conditions; and instructions. In such an aspect the nature of the media, cells, and incubation conditions may be as described within this disclosure.

[0024] In another aspect of this disclosure are provided methods for expanding a population of cells that in their native context are exposed to air (e.g. cells of the respiratory system), the method comprising providing the population of cells in a culture medium (as described in this disclosure), and exposing the population of cells to an incubation temperature above 25°C and lower than 37°C. In specific embodiments, the incubation temperature may be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C, or any integer within this range, or may fall within the range 32°C ± 3°C.

[0025] In such aspect, methods of this disclosure may comprise providing the population of cells in a culture medium under non-adherent conditions. Thus, in some embodiments, the population of cells do not come into contact with an added or exogenous extracellular matrix or added or exogenous extracellular matrix protein, whether via culture media or a culture vessel.

[0026] In such aspect, methods of this disclosure may further comprise culturing the cells (exposed to the culture environment) for between 1 to 90 days, or for at least 1 week in the culture medium.

[0027] In such aspect, the culture media may comprise a basal medium and one or both of an inhibitor of notch signaling and / or an inhibitor of signaling through a TGF. Culture media may be free of an added extracellular matrix or extracellular matrix protein. Culture may further be serum-free.

[0028] In such aspect, methods of this disclosure may comprise expanding the population of cells a higher number of passages at an incubation temperature above 25°C and lower than 37°C compared to a population of cells expanded at 37°C.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0030] Figure 1 shows the results of organoid generation from cells of two donors at different temperatures. Representative images of day 25 organoids formed at 25.5°C, 29°C, 32.5°C or 37°C (A). Scale bars represent 50 pm. Bar graphs quantify organoid number and organoid generation efficiencypercentage (B) and ciliated cell percentage (C) among organoids formed from human nasal epithelial cells (hNECs) of 2 donors at 25.5°C, 29°C, 32.5°C or 37°C. Points in each bar represent independent technical replicates. Organoids formed at 37°C were collected after 15 days in culture while organoids formed at other temperature conditions (25.5°C, 29°C & 32.5°C) were collected after 28 days in culture. Ciliated cell % and organoid numbers were assessed at these timepoints respectively. 2D expansion of hNECs in a medium of this disclosure at 37°C (D) and 32°C (E) respectively. Data for population doublings versus days in culture is shown for hNECs derived from two commercially available donors (Donor 857 & Donor 869).

[0031] Figure 2 shows the effects of incubation temperatures during organoid formation. Representative images of day 15 and day 28 apical-out organoids formed at either 32°C, 37°C, or initiated at 32°C and after 5 days transitioned to 37°C (32°C to 37°C) (A). Scale bars represent 50 pm. Representative images at two different magnifications (20X and 40X) of day 15 organoids formed at either 37°C or 32°C (B). Scale bars represent 50 pm. Bar graph quantifying organoid number and formation efficiency after forming organoids from hNECs at 37°C, 32°C, or 32°C to 37°C (C). Points in each bar represent independent technical replicates.

[0032] Figure 3 shows the characterization of apical-out organoids generated from hNECs at 32.5°C. Bar graphs quantifying organoid number and organoid generation efficiency percentage (A), and ciliated cell percentage (B) for organoids formed from 3 donors of either passage 3 (p3) or passage 4 (p4). Histogram of organoid size generated from each donor (C). Size assessment was based on calculation of the Feret diameter of at least 200 p4 organoids per hNEC donor.

[0033] Figure 4 shows the cellular composition of apical-out organoids generated from hNECs at 32.5°C. Representative immunocytochemistry images of organoids from a donor stained with markers for acetylated a-tubulin, KRT5, DAPI and MUC5AC (A & B, as denoted by arrows). Scale bars represent 50 pm. Bar graph quantifying the percentage of organoids comprising goblet cells (C). The results of qRT-PCR experiments for various markers of cells of the respiratory system (KRT5, ITGA6, F0XJ1, TUBB4B, SGCB1A1, MUC5AC, ASCL1, TSLP & FOXI1) in apical-out organoids generated at 32.5°C (D). Gene expression among the cells of apical-out organoids formed from different donors at 32.5°C was compared to donor cells (p3 hNECs) expanded in PneumaCult™ ExPlus at 37°C (STEMCELL Technologies).DETAILED DESCRIPTION

[0034] This disclosure relates to media and methods of culturing cells that in their native context are exposed to (external) air. Culturing such cells may relate to expanding such cells, differentiating suchcells, and / or obtaining / generating / forming organoids from such cells. More specifically, this disclosure relates to culturing using cells of the respiratory system. In some embodiments, the disclosed methods relate to differentiating cells of the respiratory system, such as pulmonary lineage cells. More specifically, this disclosure relates to cells that in their native context are exposed to (external) air in a culture environment in which at least one parameter deviates from conventional culture conditions.

[0035] Where used in this disclosure, the term "cells that in their native context are exposed to air" relates to cells that are not embedded deep inside the body such as within an internal organ or tissue. Thus, cells that in their native context are exposed to air refers to cells in a reasonable proximity to external air, or directly exposed to air external the tissue or organ. Non-limiting examples of such cells include those cells of the respiratory system, cells of the skin, cells of the mouth, cells of the ears, hair follicles, cells that give rise to nails, and the like. In some cases, cells that in their native context are exposed to air may relate to epithelial cells.

[0036] Where used in this disclosure, the term "cells of the respiratory system" refers to one of various types of cells that may be isolated from the airway or respiratory tract or differentiated to resemble cells of the airway or respiratory tract. The cells of the respiratory system may comprise cells from the upper or lower respiratory tract. The cells of the respiratory system may comprise cells from the nasal cavity, mouth, pharynx, larynx, trachea, bronchi, bronchiole or alveoli. In a specific aspect, the cells of the respiratory system are from the nasal cavity or nasal mucosa (e.g. nasal epithelial cells). The cells of the respiratory system may be from any species, but are preferably mammalian, and more preferably are human. In one embodiment, the cells of the respiratory system are human pulmonary epithelial cells. In one embodiment, the cells of the respiratory system are human airway epithelial cells. In one embodiment, the cells of the respiratory system are human nasal epithelial cells. In one embodiment, the human nasal epithelial cells may be cells that stain positive for cytokeratin.

[0037] Where used in this disclosure, the term "apical-out" refers to an organoid, and in some embodiments an epithelial organoid, wherein the apical surface is in direct contact with the external environment (i.e. a cell culture medium). Among apical-out organoids, some or all of such organoids may comprise a lumen, in which case a basolateral side thereof is in direct contact with the lumen. However, some apical-out organoids may not comprise a lumen, but may rather exhibit a compact / dense conformation, in which case the basolateral side thereof may be comprised in or adjacent a central core of such apical-out organoids.Media

[0038] In one aspect of this disclosure are provided media for culturing cells that in their native context are exposed to air. Where culturing such cells corresponds to differentiation culture, this disclosure may relate to culture media for differentiating cells that in their native context are exposed to air, such as cells of the respiratory system (e.g. nasal epithelial cells or pulmonary lineage cells). Where culturing such cells corresponds to organoid formation culture, this disclosure may relate to culture media for forming organoids from cells that in their native context are exposed to air, such as organoids initiated from cells of the respiratory system. Where culturing such cells corresponds to expansion culture, this disclosure may relate to culture media for expanding cells that in their native context are exposed to air, such as cells of the respiratory system (e.g. nasal epithelial cells or pulmonary lineage cells). Thus, culture media of this disclosure may be any media that support one or more of forming organoids, such as from cells of the respiratory system, differentiating of cells, such as cells of the respiratory system, or expanding cells, such as cells of the respiratory system. Media for differentiating cells may also be media for forming organoids, such as when differentiation occurs as or after organoids form.

[0039] Culture media comprise a basal medium. Basal media typically include one or more of: amino acids, vitamin(s), organic and / or inorganic salt(s), buffer(s), antioxidant(s), protein(s), energy (e.g., carbon) source(s), and the like for supporting the growth of cells. If basal media do not include one or more of the foregoing components, and if such missing components are essential or beneficial, they may added to a basal media. Numerous basal media are known and commercially available, including Dulbecco's Modified Eagle's Medium (DMEM), F12, Roswell Park Memorial Institute Medium (RPMI) 1640, Iscove's Modified Dulbecco's Medium (IMDM), Advanced DMEM, Advanced DMEM / F-12, PneumaCult™-branded basal media, and various others marketed specifically for the culture of epithelial cells. In one embodiment, the basal medium is a PneumaCult™-branded basal medium.

[0040] In the context of an organoid formation and / or cell differentiation medium, a basal medium will typically be appropriately supplemented or formulated, such as by inclusion of, for example, cytokines, growth factors, small molecules, serum / albumin / serum replacement, proteins, lipids, etc. Where a medium is an organoid forming medium (e.g. for forming apical-in or -out organoids comprising or initiated from epithelial cells), it may include one or more of: a mitogen; a fibroblast growth factor; an activator and / or an inhibitor of signaling through a bone morphogenic protein; an activator and / or an inhibitor of signaling through a wnt; and an activator and / or an inhibitor of signaling through transformation growth factor (e.g. transformation growth factor beta).

[0041] Culture media of this disclosure may comprise an inhibitor of notch signaling. Examples of inhibitors of notch signaling include DBZ, DAPT, Compound E, Compound W, SAHM1, and FLI 06. Inone embodiment, the inhibitor of notch signaling is a gamma secretase inhibitor. In one embodiment, the inhibitor of notch signaling is DAPT.

[0042] If present in a culture medium of this disclosure, an inhibitor of notch signaling will be present at an effective concentration while also being present at a concentration that does not result in any or in significant levels of cell toxicity or death. In some embodiments, a concentration of the inhibitor of notch signaling may range between about 1 nM and 1 mM, between about 5 nM and 500 pM, between about 10 nM and 200 pM, between about 50 nM and 100 pM, between about 100 nM and 50 pM, or between about 0.5 pM and 20 pM.

[0043] Culture media of this disclosure may further comprise an inhibitor of signaling through a TGF. An inhibitor of signaling through a TGF may be a natural inhibitor, such as an endogenous protein, or may be a synthetic inhibitor, such as a small molecule. Examples of inhibitors of signaling through a TGF include A83-01, A77-01, SB 431542, LY364947, and LDN 214117. In one embodiment, the inhibitor of signaling through a TGF is A83-01. In one embodiment, the inhibitor of signaling through a TGF is A77-01. In one embodiment, the inhibitor of signaling through a TGF is SB 431542. In one embodiment, the TGF is TGFbeta.

[0044] If present in a culture medium of this disclosure, an inhibitor of signaling through a TGF will be present at an effective concentration while also being present at a concentration that does not result in significant levels of cell toxicity or death. In some embodiments, a concentration of the inhibitor of signaling through a TGF may range between about 1 nM and 1 mM, between about 5 nM and 500 pM, between about 10 nM and 200 pM, between about 50 nM and 100 pM, between about 100 nM and 50 pM, or between about 0.5 pM and 20 pM.

[0045] In one embodiment, culture media of this disclosure comprise both i) one or more inhibitor of notch signaling (e.g. a gamma secretase inhibitor) and ii) one or more inhibitor of signaling through a TGF. In one embodiment, culture media of this disclosure comprise one of i) one or more inhibitor of notch signaling (e.g. a gamma secretase inhibitor) and ii) one or more inhibitor of signaling through a TGF.

[0046] Culture media of this disclosure may be free of an added extracellular matrix or extracellular matrix protein. In addition, culture media of this disclosure may neither contain nor come into contact with an added extracellular matrix or extracellular matrix protein. Accordingly, organoid media and / or differentiation media of this disclosure may not require contact with Matrigel™ or any other extracellular matrix to form the organoids. The absence of an added extracellular matrix or extracellular matrix protein, or the absence of contact with an exogenous extracellular matrix orextracellular matrix protein may be particularly pertinent when forming (and / or differentiating) apical-out organoids.

[0047] In some cases, culture media of this disclosure for forming organoids and / or differentiating cells, even in the context of apical-out organoids, may be inhibitory if an extracellular matrix or extracellular matrix protein is above a threshold level. Thus, culture media of this disclosure may comprise an extracellular matrix or an extracellular matrix protein, but at a low and / or a sub-gelation concentration.

[0048] Culture media of this disclosure may be serum-free, but may nevertheless comprise a different albumin source, such as BSA, HSA, recombinant albumin, a serum replacement, or the like.

[0049] Culture media of this disclosure may comprise a factor that modifies the cytoskeleton, or culture media of this disclosure may be free of an exogenously added factor that modifies the cytoskeleton. Examples of factors that modify the cytoskeleton (included or specifically excluded from the culture medium) include a Rho-associated protein kinase inhibitor, a p21-activated kinase (PAK) inhibitor, or a myosin II inhibitor. Examples of a Rho-associated protein kinase inhibitor include Y- 27632, SR 3677, 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, culture media of this disclosure are free of an exogenously added factor that modifies the cytoskeleton.

[0050] Culture media of this disclosure specifically for forming epithelial organoids may comprise one of, one or more of, two of, or each of an inhibitor of notch signaling (e.g. a gamma secretase inhibitor), an inhibitor of signaling through a TGF, and a factor that modifies / disrupts cytoskeletal structure. If forming apical-out organoids, the foregoing media may either be free of an added extracellular matrix or extracellular matrix protein or not having come into contact with an exogenous extracellular matrix or extracellular matrix protein.

[0051] Culture media of this disclosure specifically for differentiating cells (such as while forming epithelial organoids or after they have formed) may comprise one of, one or more of, two of, or each of an inhibitor of notch signaling (e.g. a gamma secretase inhibitor), an inhibitor of signaling through a TGF, and a factor that modifies / disrupts cytoskeletal structure. If differentiating apical-out organoids, the foregoing media may either be free of an added extracellular matrix or extracellular matrix protein or not having come into contact with an exogenous extracellular matrix or extracellular matrix protein.Methods

[0052] In another aspect of this disclosure are provided methods for culturing cells that in their native context are exposed to air. Where culturing such cells corresponds to differentiation culture, this disclosure may relate to methods for differentiating cells that in their native context are exposed to air, such as cells of the respiratory system (e.g. nasal epithelial cells or pulmonary lineage cells). Where culturing such cells corresponds to organoid formation culture, this disclosure may relate to methods for forming organoids from cells that in their native context are exposed to air, such as organoids initiated from cells of the respiratory system. Where culturing such cells corresponds to expansion culture, this disclosure may relate to methods for expanding cells that in their native context are exposed to air, such as cells of the respiratory system (e.g. nasal epithelial cells or pulmonary lineage cells). Methods for differentiating cells may also relate to methods of forming organoids, such as when differentiation occurs as or after organoids form.

[0053] Methods of this disclosure may involve providing a population of cells that in their native context are exposed to air, such as cells of the respiratory system, in a culture medium (as described above). Following, methods of this disclosure may involve exposing the cells to an incubation environment that supports expansion and / or differentiation and / or organoid formation of the cells.

[0054] The incubation environment is not particularly limited, provided that it is supportive of expansion and / or differentiation and / or organoid formation of cells that in their native context are exposed to (external) air. Preferably, the incubation environment recapitulates (as closely as possible) at least one parameter of the typical environment which cells in their native context are exposed to. Thus, it has been surprisingly shown that for cells which in their native context are exposed to air, one parameter of the incubation environment that may impact upon culture performance relates to temperature, which will be described in more detail further below.

[0055] Cells of this disclosure are not particularly limited, provided that such cells in their native context are exposed to air, or more particularly to external air. Non-limiting examples of such cells include cells of the respiratory tract, and more particularly cells of the airway epithelium, and still more particularly nasal epithelial cells. Other non-limiting examples of such cells include skin cells, such as epithelial or other cells of the skin, hair follicles, cells that give rise to nails, cells of the ear, mouth, eyes, etc.

[0056] Cells of this disclosure may preferably be epithelial cells, however, non-epithelial cells may likewise be cultured in accordance with the methods disclosed herein.

[0057] In the context of cells of the respiratory system, a population comprising such cells may comprise cells of the upper or lower respiratory tract, such as cells of the nasal cavity, nasal mucosa,mouth, pharynx, larynx, trachea, bronchi, bronchiole or alveoli. Such cells may be obtained / isolated by brushing, scraping, or lavage, and may be obtained / isolated from any species, but are preferably mammalian, and more preferably are human. Preferably, cells of this disclosure (that in their native context are exposed to (external) air) that are obtained / isolated correspond to a low passage number (e.g. PO, Pl or P2).

[0058] If cells of this disclosure (that in their native context are exposed to (external) air) are cells of the respiratory system, such cells may be pulmonary lineage cells (e.g pulmonary epithelial cells, airway epithelial cells, nasal epithelial cells, etc.). Where the cells (that in their native context are exposed to (external) air) are nasal epithelial cells, such cells may be characterized as cells that stain positive for cytokeratin.

[0059] Providing a population of cells (that in their native context are exposed to (external) air) in a culture medium may support expansion / proliferation or a limited expansion / proliferation of the cells and / or differentiation or a limited differentiation of the cells of the respiratory system and / or selforganization of the cells into organoids. Culture media that may be used in the methods of this disclosure may be as described in detail hereinabove, and such description is fully incorporated by reference into these methods.

[0060] As described above, culture media (which may be used in methods of this disclosure) comprise a basal medium and may further comprise one or more factors that support the desired culture of the cells (that in their native context are exposed to (external) air), whether it is expansion and / or differentiation and / or organoid formation.

[0061] Culture media (which may be used in methods of this disclosure) may comprise one or both of an inhibitor of notch signaling and / or an inhibitor of signaling through a TGF. In embodiments where a culture medium comprises an inhibitor of notch signaling, the inhibitor of notch signaling may be a gamma secretase inhibitor. In embodiments where a culture medium comprises an inhibitor of signaling through a TGF, the inhibitor of signaling through a TGF may be an inhibitor of TGFbeta signaling.

[0062] Culture media (which may be used in methods of this disclosure) may comprise a factor that modifies the cytoskeleton. In embodiments where a culture medium comprises a factor that modifies the cytoskeleton, such factor may be an inhibitor of a Rho-associated protein kinase. As described above, culture media for use in methods of this disclosure may comprise a basal medium supplemented with one or more of an inhibitor of notch signaling, an inhibitor of signaling through a TGF, and an agent that disrupts cytoskeletal structure. In some embodiments, culture medium do notcomprise a factor that modifies the cytoskeleton, such as an inhibitor of a Rho-associated protein kinase.

[0063] Culture media used in the methods of this disclosure may be free of an added extracellular matrix or extracellular matrix protein.

[0064] Exposing cells (that in their native context are exposed to (external) air) to an incubation environment that supports expansion and / or differentiation and / or organoid formation of the cells may comprise incubation parameters that are standard in the art. Standard incubation parameters, such as in terms of oxygen levels, carbon dioxide levels, atmospheric pressure, and temperature, are well known. However, it has been surprisingly shown that an incubation environment for cells that in their native context are exposed to (external) air may deviate from such standard cell culture conditions in one or more ways.

[0065] A deviation from standard culture conditions may comprise exposing the cells to an incubation temperature that is not 37°C, such as to an incubation temperature above 25°C and lower than 37°C. Thus, the incubation temperature may be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C, or any increment of any of the foregoing temperatures within the range between 25°C and 37°C. In some embodiments, an incubation temperature may fall within the range 27°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 29°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 31°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 33°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 32°C ± 3°C.

[0066] Where methods of this disclosure specifically relate to forming organoids, such as apical-out organoids, from cells that in their native context are exposed to (external) air (e.g. cells of the respiratory system), it may be important to avoid contact with or exposure to an (added) extracellular matrix or an extracellular matrix protein. Thus, methods of this disclosure may be performed in environments (including vessels and culture media) free of an added extracellular matrix or extracellular matrix protein and / or free from contact with an exogenous extracellular matrix or exogenous extracellular matrix protein. Accordingly, culture media may not comprise an added extracellular matrix or extracellular matrix protein and / or have come into contact with an exogenous extracellular matrix or exogenous extracellular matrix protein. And, vessels / substrates may not comprise an added extracellular matrix or extracellular matrix protein and / or have come into contact with an exogenous extracellular matrix or exogenous extracellular matrix protein. In some embodiments, organoids (whether apical-out or apical-in organoids) may form when cultured in or incontact with low / limited percentages or quantities (e.g. at <10% or <5% concentration, or a subgelation threshold) of an extracellular matrix or an extracellular matrix protein.

[0067] Methods of this disclosure may be performed under one or more of animal component-free, serum-free, feeder-free, or bovine pituitary extract-free conditions. Thus, with regard to media (as may be used in methods of this disclosure), they may be serum-free and / or animal component-free. Conversely, media (as may be used in methods of this disclosure) may be serum-containing and / or animal component-containing.

[0068] Methods of this disclosure may comprise providing a population of cells that in their native context are exposed to (external) air (e.g. cells of the respiratory system) in a culture medium under non-adherent or suspension conditions. Methods of this disclosure may further comprise culturing cells that in their native context are exposed to (external) air (e.g. cells of the respiratory system) in a culture medium under non-adherent or suspension conditions. In such methods, they may comprise aggregating the cells before, during or after the providing step, or before, during after the exposing step, or before, during or after the contacting step.

[0069] Methods of this disclosure that comprise aggregating the population of cells, may be performed in round bottom microwell plates (e.g. 96-well plates) or in a microwell device (e.g. AggreWell™ plates) under non-adherent conditions. In some cases, coating aggregation devices with an anti-adherence solution, as may be provided with AggreWell™ plates, may be desirable.

[0070] Aggregating cells may comprise depositing between 10 and 2000 cells per well of a microwell or patterned plate or device in a culture medium to form distinct aggregates of cells. In one embodiment, aggregating the cells comprises depositing about 300 cells per well of a microwell or patterned plate or device. In one embodiment, aggregating the cells comprises depositing about 200 cells per well of a microwell or patterned plate or device. The deposited cells may be single cells or comprised in clumps, or a mixture thereof.

[0071] Methods of this disclosure may comprise aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium if such cells will be cultured at an incubation temperature above 25°C and lower than 37°C, as compared to the number of cells (per well of a microwell patterned plate or device) in a culture medium if such cells will be cultured at an incubation temperature of about 37°C. Aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium at an incubation temperature above 25°C and lower than 37°C may yield a comparable number of organoids (e.g. ±10% or ±5%) compared to the organoids generated at an incubation temperature of 37°C. Aggregating a lower number of cells (per well of amicrowell patterned plate or device) in a culture medium at an incubation temperature above 25°C and lower than 37°C may yield a comparable percentage efficiency of organoid generation (e.g. ±10% or ±5%) compared to the organoids generated at an incubation temperature of 37°C. Aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium at an incubation temperature above 25°C and lower than 37°C may yield organoids with a similar ciliated cell percentage (e.g. ±10% or ±5%) compared to the organoids generated at an incubation temperature of 37°C.

[0072] Aggregating cells may be for between 1 and 7 days and may take place in the same culture medium which the cells will be contacted with. Aggregating cells may be for between 1 and 6 days and may take place in the same culture medium which the cells will be contacted with. Aggregating cells may be for between 1 and 5 days and may take place in the same culture medium which the cells will be contacted with.

[0073] Following exposing the cells (that in their native context are exposed to (external) air) to an incubation environment, as described above, the cells may be cultured in a culture medium, as described above, for between 1 to 90 days. In one embodiment, culturing is for between 10 to 35 days. In one embodiment, culturing is for 15 days. In one embodiment, culturing is for 25 days. In one embodiment, culturing is for 28 days.

[0074] In aspects of this disclosure relating to forming or generating organoids, at least a proportion of which may be apical-out organoids. Methods of forming or generating organoids (e.g. apical-out organoids) of this disclosure preferably involve a population of cells that in their native context are exposed to (external) air (e.g. cells of the respiratory system), as described above.

[0075] Methods of forming apical-out organoids may comprise providing a population of cells in a culture medium (as described above) and exposing the cells of the respiratory system to a culture environment (as described above). As described above, a culture environment of this disclosure may comprise an incubation temperature that is not 37°C, such as an incubation temperature above 25°C and lower than 37°C. Thus, the incubation temperature may be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C, or any increment of any of the foregoing temperatures within the range between 25°C and 37°C. In some embodiments, an incubation temperature may fall within the range 27°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 29°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 31°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 33°C ± 2°C. In some embodiments, an incubation temperature may fall within the range 32°C ± 3°C.

[0076] Methods of this disclosure may yield apical-out organoids wherein an apical surface of at least a portion of the apical-out organoids faces away from a core (e.g. a lumen) thereof, but in any event the apical surface of apical-out organoids will be in direct contact with the external environment. In one embodiment, a majority of the formed organoids (i.e the portion) exhibit an apical-out morphology / organization. In one embodiment, among at least 50% of the organoids the apical surface faces away from the core (or, lumen). In one embodiment, among at least 60% of the organoids the apical surface faces away from the core (or, lumen). In one embodiment, among at least 80% of the organoids the apical surface faces away from the core (or, lumen). In one embodiment, about 90% or more of the organoids exhibit an apical surface that faces away from the core (or, lumen) thereof. In one embodiment, the converse of the foregoing applies to methods of forming (apical-in) organoids.

[0077] Methods for generating / obtaining apical-out organoids may comprise providing a population of cells that in their native context are exposed to (external) air (e.g. cells of the respiratory system) in the culture medium, essentially as described above. Also essentially as described above, the providing step may be under non-adherent or suspension conditions. Thus, the population of cells may be free from contact with an exogenous or added extracellular matrix or exogenous extracellular matrix protein, whether via culture media or culture vessel, as described above. The aforementioned extracellular matrix may be Matrigel®, and the aforementioned extracellular matrix protein may be one which is comprised in Matrigel®, such as vitronectin, fibronectin, entactin, etc.

[0078] The same non-adherent or suspension conditions as described above may also be carried forward in an exposing step.

[0079] The same non-adherent or suspension conditions as described above may also be carried forward in a culturing step. Culturing the cells (exposed to the incubation temperature) in a culture medium may be for between 1 to 90 days. In one embodiment, culturing is for between 10 to 35 days. In one embodiment, culturing is for 15 days. In one embodiment, culturing is for 25 days. In one embodiment, culturing is for 28 days.

[0080] Thus, organoids do not form / generate in the presence of an exogenously added extracellular matrix or extracellular matrix protein. In one embodiment, the cells (e.g. the cells of the respiratory system) do not come into contact with an exogenously added extracellular matrix or extracellular matrix protein(s). In one embodiment, neither the cells of the respiratory system, the aggregated cells of the respiratory system, nor the organoids come into contact with an exogenously added extracellular matrix protein.

[0081] Culture media used in this aspect may be as described above.

[0082] Prior to the methods of this disclosure (e.g. before the providing step) it may be desirable to increase the number of starting cells. Various commercial culture media are available forthis purpose. In the specific context of expanding cells of the respiratory system, an appropriate PneumaCult™- branded medium (STEMCELL Technologies) may be used. In the specific context of expanding cells of the skin, an appropriate DermaCult™-branded medium (STEMCELL Technologies) may be used.

[0083] Methods of obtaining / generating organoids (e.g. apical-out organoids) may comprise aggregating a population of cells, as may be performed in round bottom microwell plates (e.g. 96-well plates) or in a microwell device (e.g. AggreWell™ plates) under non-adherent conditions. In some cases, coating aggregation devices with an anti-adherence solution, as may be provided with AggreWell™ plates, may be desirable.

[0084] Aggregating cells may comprise depositing between 10 and 2000 cells per well of a microwell or patterned plate or device in a culture medium to form distinct aggregates of cells. In one embodiment, aggregating the cells comprises depositing about 300 cells per well of a microwell or patterned plate or device. In one embodiment, aggregating the cells comprises depositing about 200 cells per well of a microwell or patterned plate or device. The deposited cells may be single cells or comprised in clumps, or a mixture thereof.

[0085] Methods of this aspect may comprise aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium if such cells will be cultured at an incubation temperature above 25°C and lower than 37°C, as compared to the number of cells (per well of a microwell patterned plate or device) in a culture medium if such cells will be cultured at an incubation temperature of about 37°C. Aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium at an incubation temperature above 25°C and lower than 37°C may yield a comparable number of organoids (e.g. ±10% or ±5%) compared to the organoids generated at an incubation temperature of 37°C. Aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium at an incubation temperature above 25°C and lower than 37°C may yield a comparable percentage efficiency of organoid generation (e.g. ±10% or ±5%) compared to the organoids generated at an incubation temperature of 37°C. Aggregating a lower number of cells (per well of a microwell patterned plate or device) in a culture medium at an incubation temperature above 25°C and lower than 37°C may yield organoids with a similar ciliated cell percentage (e.g. ±10% or ±5%) compared to the organoids generated at an incubation temperature of 37°C.

[0086] Aggregating cells may be for between 1 and 7 days, and may take place in the same culture medium which the cells will be contacted with. Aggregating cells may be for between 1 and 6 days,and may take place in the same culture medium which the cells will be contacted with. Aggregating cells may be for between 1 and 5 days, and may take place in the same culture medium which the cells will be contacted with.

[0087] Methods for obtaining organoids, such as apical-out organoids, may comprise exposing cells that in their native context are exposed to (external) air (e.g. cells of the respiratory system) to, and culturing such cells at, an incubation temperature of 32°C ± 3°C in a culture medium.

[0088] The number of organoids generated at an incubation temperature of 32°C ± 3°C may be at least 30% higher than the number of organoids generated at an incubation temperature of 37°C. The number of organoids generated at an incubation temperature of 32°C ± 3°C may be at least 40% higher than the number of organoids generated at an incubation temperature of 37°C. The number of organoids generated at an incubation temperature of 32°C ± 3°C may be at least 50% higher than the number of organoids generated at an incubation temperature of 37°C.

[0089] At least 25% of cells of the organoids (e.g. apical-out organoids) obtained / generated by the methods of this disclosure may be ciliated. In some embodiments, at least 30% of cells of the organoids (e.g. apical-out organoids) obtained / generated by the methods of this disclosure may be ciliated. In some embodiments, at least 35% of cells of the organoids (e.g. apical-out organoids) obtained / generated by the methods of this disclosure may be ciliated. In some embodiments, at least 40% of cells of the organoids (e.g. apical-out organoids) obtained / generated by the methods of this disclosure may be ciliated. In some embodiments, at least 45% of cells of the organoids (e.g. apical- out organoids) obtained / generated by the methods of this disclosure may be ciliated. In some embodiments, at least 50% of cells of the organoids (e.g. apical-out organoids) obtained / generated by the methods of this disclosure may be ciliated. In some embodiments, more than 50% of cells of the organoids (e.g. apical-out organoids) obtained / generated by the methods of this disclosure may be ciliated.

[0090] Organoids (e.g. apical-out organoids) formed in accordance with this disclosure may express one or more markers of ciliogenesis. For example, the one or more markers of ciliogenesis may comprise F0XJ1, TUBB4B, and / or acetylated a-Tubulin.

[0091] Organoids (e.g. apical-out organoids) formed in accordance with this disclosure may comprise goblet cells, which may be identified by the marker, MUC5AC. A majority of organoids formed in accordance with this disclosure may comprise at least one goblet cell, such as ~60%, ~70, ~80, ~90%, or >95%.

[0092] Organoids (e.g. apical-out organoids) formed in accordance with this disclosure may be similar or uniform in size and may exhibit a size range from 50 to 150 pm in diameter.

[0093] Organoids (e.g. apical-out organoids) formed in accordance with this disclosure may support the replication of viruses or infection by viruses, such as respiratory or other viruses. Thus, methods of this disclosure may further comprise assessing organoid responses to one or more therapeutic agents, or one or more pathogens, such as virus or bacteria.

[0094] In embodiments of this disclosure wherein media comprise or methods steps are performed in the presence of an extracellular matrix or extracellular matrix protein, the nature of such extracellular matrix or extracellular matrix protein is not particularly limited.

[0095] In one embodiment, an extracellular matrix comprises or an extracellular matrix protein is one or more of a vitronectin, a laminin, a fibronectin, or a collagen. In one embodiment, the extracellular matrix is any combination or mixture of a vitronectin, a laminin, a fibronectin, or a collagen. In one embodiment, the extracellular matrix may be Matrigel®.

[0096] In one embodiment, extracellular matrix proteins may be recombinant and / or of natural origin. In one embodiment, extracellular matrix proteins may be genetically engineered. In one embodiment, extracellular matrix proteins may be a whole protein or a fragment thereof, such as a peptide fragment. In one embodiment, extracellular matrix proteins may be fusion proteins.

[0097] In one embodiment, the extracellular matrix may be extracellular matrix (ECM)-derived hydrogels or ECM hydrogels. In one embodiment, the extracellular matrix may be ECM derived from Engelbreth-Holm-Swarm (EHS) murine sarcoma.

[0098] If comprised in a culture medium or adopted into methods of this disclosure, an extracellular matrix or extracellular matrix protein will be present at an effective concentration that can range from 0.01 to 100%. In one embodiment, the extracellular matrix or extracellular matrix protein is present at a concentration below its gelation threshold. In one embodiment, a concentration of the extracellular matrix or extracellular matrix protein ranges between 0.01% to 10%. In one embodiment, a concentration of the extracellular matrix or extracellular matrix protein is about 2% ± 1.5%.

[0099] The following non-limiting examples are illustrative of the present disclosure.ExamplesExample 1: Culturing and expanding starting cells

[0100] Primary normal human nasal epithelial cells (HNECs) were commercially sourced, such as fromLONZA or EPITHELIX SARL. In the experiments described herein, all cells were collected from non-smoking, healthy donors. If prior expansion of epithelial cells is required, HNECs may be seeded at passage one into T25 cell culture tissue flasks in PneumaCult™ Ex-Plus (STEMCELL Technologies) medium and incubated at 37°C and 5% CO2in accordance with the manufacturer's protocol. Once cells reach 50-60% confluency, they may be dissociated, such as with the ACF Cell Dissociation kit (STEMCELL Technologies) following the manufacturer's instructions.Example 2: Laboratory ware preparation

[0101] Plates used in downstream assays were coated with Anti-Adherence Rinsing Solution (STEMCELL Technologies). Briefly, 500 pL of Anti-Adherence Rinsing Solution was added to each well to be used and the plate was centrifuged for 10 min at 1300g. Anti-Adherence Rinsing Solution was then removed, and the well was washed once with 1 mL DMEM. The wells were used directly, or they were stored for up to a week at 37°C after adding 500 pL DMEM.Example 3: Forming aggregates

[0102] Cells were cultured and dissociated in accordance with Example 1 and seeded into Aggrewell™ 400 plates (STEMCELL Technologies) that were prepared in accordance with Example 2. Aggregates were generated by seeding roughly 300 cells per microwell of an Aggrewell™ 400 plate. The cells were seeded in 1 ml of culture medium comprising a basal medium, an inhibitor of notch signaling and an inhibitor of signaling through a TGF. Such a culture medium may be referred to as an apical-out culture medium. The AggreWell™ plate was centrifuged for 3 min at 100g in order to sediment the cells to the bottom of the microwell. The plates were then incubated in 5% C02for 24 h to 168h at 37°C or 32.5 °C. Instead of an AggreWell™ plate, it may be possible to aggregate cells in round bottom well of a multi-well plate, such as a 96-well plate, potentially at the expense of uniformity of size.Example 4: Suspension culture of apical-out organoids

[0103] After the aggregates were formed in accordance with Example 3, 500 pL of fresh apical-out culture medium was added to each well to resuspend the aggregates, before distributing the contents of each well of an Aggrewell™ plate into two wells of a 24-well plate (prepared as described in Example 2). The aggregates were incubated under non-adherent conditions in apical-out culture medium for up to 15 or 28 days at different incubation temperature conditions: a) 25.5°C; b) 29°C; c) 32°C; d) 32.5°C; e) 37°C; or f) Incubation at 32°C for 5 days followed by incubation at 37°C (32°C to 37°C)). A partial medium change was performed every second day.Example 5: Dissociating apical-out organoids

[0104] Apical-out organoids formed in accordance with Example 4 were harvested either on day 15 or day 28, transferred to a 15 ml tube, and centrifuged at 150g for 5 min. The supernatant was removed, and the organoids were washed once with DMEM. The washed organoids were resuspended in ACCUTASE™ (STEMCELL Technologies) and were incubated for 15 minutes at room temperature, before being dissociated to single cells by pipetting vigorously with a P1000 pipette. The single-cell suspension was diluted 1:1 with trypan blue and the cells were counted manually. At the time the dissociated cells are counted, the number of ciliated cells can also be determined by visual inspection.Example 6: Characterizing apical-out organoids

[0105] Fully mature organoids, formed in accordance with Example 4, were imaged using a standard microscope camera (e.g Leica DMi8 or EVOS M500). The number of organoids was quantified and the percentage of motile organoids (i.e. % ciliated organoids) was assessed by visual inspection and by manually counting organoids that displayed beating cilia on the outer (apical) side. The organoid formation efficiency was calculated as the percentage of number of organoids obtained divided by the maximum number of organoids that could potentially be generated in the Aggrewell™ plate.

[0106] For immunocytochemistry (ICC) analysis, organoids were stained in accordance with the following generalized protocol. Apical-out organoids were fixed in 4% Paraformaldehyde and subjected to antigen retrieval before being permeabilized 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 the cells at room temperature in a tube with gentle agitation for 4 days. Certain airway epithelial markers were tested including Acetylated a-tubulin (Sigma, cat #T7451, 1:750), MUC5AC (Abeam, cat #ab212636, 1:750) and KRT 5 (Biolegend, cat # 905501, 1:750). Appropriate secondary antibodies were used against the primary antibodies. Cells were washed with PBSTT and the respective secondary antibody was incubated at room temperature in a tube with gentle agitation for 3 days. Cells were washed again with PBSTT before staining with 4', 6-diamidino-2-phenylindole (DAPI) and imaged with LEICA SP8.

[0107] For mRNA expression analysis, total RNA was isolated from the organoids using the Qiagen Easy RNA mini kit (Qiagen) as per the manufacturer's protocol. 500 ng of RNA was DNase treated (Invitrogen) as per the manufacturer's protocol and then reverse-transcribed to 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 found below in Table 1. Samples were amplified as follows: denaturation at 95°C for 20 sec followed by 50 cycles at 95°C for Is and 60°C for20s. The mRNA expression levels of cellular genes were normalized to Tata-binding protein (TBP), as it was found to have the smallest standard deviation amongst a panel of housekeeping genes.Table 1: Accession number of assays used to quantify the expression of various target genes.

[0108] The expression of the markers outlined above was assessed in apical-out organoids formed in accordance with Examples 1-4. The expression of these markers was also assessed in p3 hNECs expanded using PneumaCult™ Ex Plus (STEMCELL Technologies) and the apical-out organoid data was expressed as fold change relative to p3 hNEC expansion data.Example 7: Effect of temperature on expansion and organoid formation

[0109] hNECs were cultured essentially as described in Example 1, except for a few modifications to the protocol outlined below. hNECs derived from two different donors (Donor 857 & Donor 869) were thawed at the second passage (p2) and expanded in Pneumacult™ Ex-Plus at 37°C. After passage, p3 cells were seeded at 32.5°C and 37°C to test expansion potential at these temperatures. Cells at passage number 4 (p4) cultured at 37°C were further used to generate apical-out organoid cultures in accordance with Examples 2, 3, 4 and 5 by seeding 300 cells per microwell of an Aggrewell™ plate and incubating the plates at either: (i) 25.5°C, (ii) 29°C, (iii) 32.5°C and (iv) 37°C.

[0110] The aforementioned apical-out organoid cultures were cultured up to 28 days. However, visual inspection of formed organoids revealed that the organoids formed at 37°C harvested on day 25 displayed a poor morphology with smaller-sized organoids while organoids formed at 32.5°C displayed the best morphology, in terms of smooth edges and a round morphology (Figure 1A). Therefore, in further experiments described below, organoids formed at 37°C were collected after 15 days in culture while organoids generated at other temperature conditions (25.5°C, 29°C & 32.5°C) were collected after 28 days in culture.

[0111] For both donors, organoid generation efficiency and the number of organoids formed was highest at 29°C and 32.5°C and the lowest at 25.5°C (Figure IB). Organoids formed at 37°C were used as a reference for comparison, but the % efficiency of formation and numbers were lower than for the 29°C and 32.5°C culture conditions. The organoids were further evaluated for their differentiation potential by quantifying the number of ciliated cells (Figure 1C). For both donors, organoids formed at 32.5°C had the highest percentage of ciliated cells compared to all other temperature conditions tested. Fewest ciliated cells were observed among organoids formed at 25.5°C and 29°C.

[0112] Population doublings were also assessed as a function of the days in culture at 32.5°C or 37°C (Figure ID & Figure IE). For both donors, cells expanded in 2D at 37°C could be maintained for up to 11 days in culture and underwent ~6 population doublings, while cells expanded in 2D at 32.5°C could be maintained for up to 9 days in culture and underwent ~4 population doublings. Surprisingly, over a period of 9-days in culture cells expanded at either temperature underwent more-or-less the same number of population doublings. Therefore, in addition to improved organoid formation / differentiation at 32.5°C compared to 37°C, hNECs could also be expanded in 2D at 32.5°C.Example 8: Effect of temperature transition on organoid formation

[0113] To further understand the unexpected findings of improved organoid formation efficiencies and numbers at temperatures lower than 37°C, organoids formed in accordance with Examples 1-5 were transitioned after 5-days incubation at 32°C into 37°C for the remaining culture duration. On day 15 or day 28 the formed organoids were harvested and the transition culture (32°C to 37°C) was characterized against organoids formed and maintained at either 32°C or 37°C.

[0114] The apical-out organoids formed from the cells of the respiratory system were visually assessed using brightfield microscopy on day 15 and day 28. Organoids formed at 37°C and at 32°C to 37°C conditions generally displayed a poor morphology at day 15 that further declined in long-term cultures at day 28 (Figure 2A). Observed organoids were small in size, with a rather unclearly defined outer layer characterized by random protrusions, absence of smoothness and heterogeneous shape. However, organoids generated at 32°C generally displayed a much healthier morphology at day 15 that was maintained long term until day 28 (Figure 2A). The outer layer of such organoids was observed to be smooth and clearly defined, with minimal protrusions and irregularities. The organoids were also more uniform in size. Further comparison at higher magnifications showed that day 15 organoids cultured at 37°C displayed a bubbly morphology characterized by multiple vacuoles and protruding cells while day 15 organoids cultured at 32°C displayed smooth edges and a round morphology, with few vacuoles and protruding cells (Figure 2B). Overall, forming / culturing organoids at 32°C showed marked improvements in morphology compared to culturing at other temperatureconditions tested. This was also apparent for longer culture durations. Organoids generated at 32°C displayed high numbers of beating cilia even after 40 days of culture. These organoids were quite robust and did not show any signs of degradation even up to 90 days in culture (data not shown). In contrast, organoids formed at 37°C could not be maintained up to 90 days in culture, and as stated above already displayed poor morphology by day 28 (Figure 2A). Furthermore, the number of organoids formed and the formation efficiency % were found to be higher for dl5 and d28 organoids formed at 32°C compared to those formed at 37°C or 32°C to 37°C conditions (Figure 2C).

[0115] Another notable observation from the experiments was that better results could be obtained with lower seeding densities at 32°C compared to 37°C. While seeding 300 cells per microwell gave the best results for organoids formed / differentiated at 37°C, comparable results could be obtained by seeding 200 cells per microwell for organoids formed / differentiated at 32°C (data not shown).Example 9: Detailed characterization of apical-out organoids generated at 32.5° C

[0116] The apical-out nasal organoids generated at 32.5°C were characterized further in terms of organoid generation, size, composition of cell types and mRNA expression. hNECs derived from three different donors (Donors 1, 2 & 3) were thawed at the third (p3) or fourth passage (p4) and expanded in PneumaCult™ Ex-Plus at 37°C, as described in Example 1. Organoids were formed from these cells in apical-out culture medium as described in Examples 2 to 5. The apical-out organoids formed were harvested on day 28 and characterized further.

[0117] The number of organoids formed, the generation efficiency % and ciliated cell % were calculated as described in Example 6. For all 3 donors, organoid generation efficiency % and the number of organoids formed were higher at p3 compared to p4 (Figure 3A). This was expected as p4 represents older cells. However, organoids could still be generated at considerably good levels from p4 hNECs indicating the robustness of the workflow even with older or late passage starting cells. Apical-out organoids could also differentiate efficiently at 32.5°C and generate beating ciliated cells (Figure 3B). While the ciliated cell % was higher for p3 hNECs compared to p4 hNECs for donor 1, it was comparable across p3 and p4 for donors 2 and 3. Donor 3 showed a considerably lower % of ciliated cells compared to donors 1 and 2. Although donor 3-derived organoids comprised visible cilia based on ICC staining using a cilia-specific marker (Figures 4A, as denoted by arrows), they did not display conspicuous beating cilia, which may explain the discrepancy observed with ciliated cell percentages. The size of the generated organoids was further assessed by calculating the Feret diameter of at least 200 organoids per donor generated using p4 hNECs. The apical-out organoids generated at 32.5°C were substantially homogenous in size across the 3 donors tested with Feret diameters mostly ranging between 50 - 120 pm (Figure 3C).

[0118] The apical-out organoids were further characterized by ICC to assess the cellular composition of the generated organoids. Visible cilia (based on acetylated a-tubulin marker expression) on the outer surface of the organoids and presence of basal cells (by KRT5 marker expression) were observed in the representative donor 3-derived organoids (Figure 4A, as denoted by arrows). Goblet cells identified via MUC5AC expression were also observed in the representative donor 3-derived organoids (Figure 4B, as denoted by arrows). To characterize the percentage of organoids comprising goblet cells, apical-out organoids from all 3 donors were collected on d28 and stained for the goblet cell marker MUC5AC, as described in Example 6. The signal corresponding to goblet cell in each organoid was visually assessed and calculated. When the data was averaged across all donors, it was observed that most (>90%, or ~95%) of the organoids comprised goblet cells (Figure 4C) and were therefore highly homogenous in terms of goblet cell composition. Next, the expression of common RNA markers in apical-out organoids generated at 32.5°C was assessed by qPCR, as described in Example 6. The expression of various constituent cell types such as basal cells (KRT5, ITGA6), ciliated cells (F0XJ1, TUBB4B), club cells (SCGB1A1), goblet cells (MUC5AC), neuroendocrine cells (ASCL1), tuft cells (TSLP) and ionocytes (FOXI1) was assessed. A clear induction of differentiation was observed with the apical- out organoids, with downregulation of basal cells and upregulation of ciliated cells and importantly goblet cells.

[0119] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0120] 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

CLAIMS1) A method of culturing a population of cells that in their native context are exposed to air, the method comprising: a) providing the population of cells in a culture medium; and b) exposing the population of cells to an incubation temperature above 25°C and lower than 37°C.2) The method of claim 1, wherein providing the population of cells in the culture medium is under non-adherent conditions.3) The method of claim 1 or 2, wherein the population of cells is free from contact with an exogenous extracellular matrix or exogenous extracellular matrix protein.4) The method of any one of claims 1 to 3, wherein the culture medium: i) comprises a basal medium and one or both of an inhibitor of notch signaling and / or an inhibitor of signaling through a TGF; and / or ii) is free of an added extracellular matrix or extracellular matrix protein; and / or iii) is serum-free.5) The method of any one of claims 1 to 4, wherein the incubation temperature falls within the range 32°C ± 3°C.6) The method of any one of claims 1 to 5, further comprising aggregating the population of cells.7) The method of claim 6, further comprising depositing between 10 and 2000 cells per well of a microwell patterned plate or device in the culture medium to form distinct aggregates of cells of the respiratory system.8) The method of any one of claims 1 to 7, wherein the cells are cells of the respiratory system.9) The method of any one of claims 1 to 8, wherein the cells are nasal epithelial cells.10) The method of any one of claims 1 to 9, wherein the method of culturing is a method of expanding, a method of differentiating, and / or a method of generating organoids from the population of cells that in their native context are exposed to air.11) A method for generating organoids comprising cells that in their native context are exposed to air, the method comprising: a) providing a population of cells that in their native context are exposed to external air in a culture medium; b) exposing the cells to an incubation temperature above 25°C and lower than 37°C; andc) generating organoids after culture in the culture medium and at the incubation temperature for a sufficient time.12) The method of claim 11, wherein providing the population of cells in the culture medium is under non-adherent conditions.13) The method of claim 11 or 12, wherein the population of cells is free from contact with an exogenous extracellular matrix or exogenous extracellular matrix protein.14) The method of any one of claims 11 to 13, wherein the culture medium: i) comprises a basal medium and one or both of an inhibitor of notch signaling and / or an inhibitor of signaling through a TGF; and / or ii) is free of an added extracellular matrix or extracellular matrix protein; and / or ill) is serum-free.15) The method of any one of claims 11 to 14, wherein the incubation temperature falls within the range 32°C ± 3°C.16) The method of claim 15, wherein the number of organoids generated at the incubation temperature ranging between 32°C ± 3°C is at least 30% higher than the number of organoids generated at an incubation temperature of 37°C.17) The method of any one of claims 11 to 16, further comprising aggregating the population of cells.18) The method of claim 17, further comprising depositing between 10 and 2000 cells per well of a microwell patterned plate or device in the culture medium to form distinct aggregates of cells of the respiratory system.19) The method of any one of claims 11 to 18, wherein the cells are cells of the respiratory system.20) The method of any one of claims 11 to 19, wherein the cells are nasal epithelial cells.21) The method of any one of claims 11 to 20, wherein at least 25% of cells of the organoids are ciliated.22) The method of any one of claims 11 to 21, wherein the organoids support infection by viruses.23) The method of any one of claims 11 to 22, further comprising assessing organoid responses to one or more therapeutic agents, or one or more pathogens.24) The method of any one of claims 11 to 23, wherein the organoids are apical-out organoids and an apical surface of at least a portion of the organoids is faced away from a core thereof.

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