Methods and compositions for culturing alveolar cells

Abstract

The present invention provides a method and composition for use in growing alveolar epithelial cells.The method can include the use of a three-dimensional substrate and an improved technique for growing cells.The improved composition for culturing alveolar epithelial cells can include at least one or more of the following: a TGF-β pathway inhibitor, a Wnt pathway activator, a ROCK inhibitor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), and fetal bovine serum.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Application No. 62 / 892,206, filed on 27 August 2019, which is incorporated herein by reference in its entirety under Section 119(e) of the U.S. Patent Act.

[0002] This application relates, as a whole, to cell culture, more particularly to methods and compositions for culturing alveolar cells, but not limited to them, and to cells produced by such methods. [Background technology]

[0003] In regenerative medicine, the proliferation of alveolar epithelial cells is a significant challenge and a common obstacle. Alveolar epithelium, which accounts for 95% of the lung epithelial surface area, is composed of type 1 alveolar (AT1) cells, responsible for gas exchange, and type 2 alveolar (AT2) cells, which constitute the remaining distal epithelium and produce surfactant. AT1 cells are thin and fragile, making them difficult to isolate and proliferate. In contrast, AT2 cells proliferate in vivo in response to lung injury and transdifferentiate into AT1 cells to reconstruct the damaged lung. Consequently, AT2 cell proliferation is more readily achievable. However, under standard in vitro 2D culture conditions, human AT2 cells transdifferentiate into non-proliferating AT1-like cells, resulting in minimal proliferation and rapid loss of the AT2 cell phenotype. In addition, often, a small starting population of contaminating airway basal cells or stromal cells grows excessively compared to AT2 cultures. Furthermore, while studies have shown that AT2 cells can proliferate in organoid cultures (e.g., Matrigel disks), such cultures are not scalable. Therefore, there is an unmet need for cell cultures and methods that can aid in promoting the successful proliferation of alveolar epithelial cells. [Overview of the project]

[0004] This disclosure addresses the shortcomings of known methods by providing methods and compositions for culturing alveolar epithelial cells on a three-dimensional substrate. Three-dimensional culture offers certain advantages over standard two-dimensional culture of alveolar epithelial cells, as evidenced by improved maintenance of alveolar epithelial cell function and reduced overgrowth of airway basal cells, which are a cause of contamination.

[0005] A method for producing alveolar epithelial cells preferably includes the steps of preparing a plurality of three-dimensional substrates in a cell culture vessel, seeding the plurality of alveolar epithelial cells by combining the three-dimensional substrates and alveolar epithelial cells in the cell culture vessel and creating conditions suitable for enabling cell adhesion to the three-dimensional substrates and realizing suspension culture, promoting the growth of alveolar epithelial cells on or within the three-dimensional substrates, monitoring the culture with respect to cell proliferation, and harvesting the plurality of alveolar epithelial cells from the three-dimensional substrates. In some embodiments, the alveolar epithelial cells may be AT2 cells. In certain other embodiments, the AT2 cells may be human type II alveolar epithelial cells.

[0006] The three-dimensional substrate may be at least one of non-porous, microporous, or macroporous three-dimensional substrates. In certain embodiments, the three-dimensional substrate may contain multiple microcarriers. In certain embodiments, the substrate may be a macroporous gelatin microcarrier. In some embodiments, the culture vessel may be a bioreactor or any vessel of similar volume dimensions. In some embodiments, the cell culture vessel may be a spinner flask. The three-dimensional substrate may also be present at concentrations of about 1 to 10 mg per 1 mL of culture medium, for example, 1 to 8, 1 to 6, 1 to 4, or 1 to 2 milligrams of the substrate per 1 mL of culture medium.

[0007] Seeding may also include adding alveolar epithelial cells (e.g., fresh isolated or cryopreserved) to the culture medium in a cell culture vessel. In some embodiments, seeding may include agitating the culture. In certain embodiments, the three-dimensional substrate culture is agitated at about 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) on a stirring plate in an incubator or agitated in a bioreactor. In other embodiments, bioreactor culture is carried out on a tabletop, where an external motor controls an impeller inside the vessel to induce mixing. In such embodiments, gas regulation is maintained using a control device, and the vessel is heated using a heat jacket. In some embodiments, agitation is performed intermittently. In some embodiments, agitation includes cycles. In some embodiments, stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period without stirring. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, or 50 minutes or more. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the ratio of stirring time to non-stirring time may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4.These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. The cycle can be repeated at least 12 times, at least 24 times, at least 36 times, or at least 64 times. In some embodiments, the cycle can be repeated 24 to 64 times or 36 to 64 times. In some cases, stirring is performed for about 5 minutes at about 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a period of about 30 minutes without stirring. This cycle can be repeated about 32 to 64 times (optionally, about 32, 35, 40, 45, 50, 55, 60, or 64 times). In certain embodiments of the method, after approximately 18–36 hours of intermittent stirring (optionally 18, 24, 28, 30, or 36 hours), the culture is then continuously stirred at approximately 20 RPM or higher (optionally 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) for the remainder of the culture period. Seeding may be carried out at a volume of approximately one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0008] In some embodiments, the culture can be monitored by supplying nutrients to the culture, performing a LIVE / DEAD® assay on the culture at least once, and / or evaluating the cell coverage on a three-dimensional substrate. The culture can be supplied with nutrients at intervals of approximately 2 to 4 days, and metabolic samples can be collected daily and after nutrient supply. In some embodiments, the culture can be supplied with nutrients more frequently or even continuously, and metabolic samples can be collected hourly, daily, or approximately every 12 hours. In some cases, monitoring and / or sample measurement is performed daily for the duration of the culture process. In other cases, monitoring and / or sample measurement is performed once a day, twice a day, or as required to ensure accurate readings of the measurements. In additional cases, monitoring and / or sample measurement is performed every other day, every two days, every three days, or every four days. In further cases, monitoring and / or sample measurement is performed continuously for the duration of the culture process. In some embodiments of the method, the sample is monitored and maintained to determine at least one of the following: pH, glucose, lactate, glutamine, ammonium, dissolved oxygen level, or biocapacitance. In some embodiments of the method, cell counting is performed daily to assess growth. In some cases where cells are cultured in a bioreactor, the measurement is performed via the use of a probe.

[0009] The steps for harvesting the culture may also include at least one of the following steps and any combination thereof: harvesting multiple alveolar epithelial cells from the three-dimensional substrate by precipitating the three-dimensional substrate; removing a certain amount of culture medium from the cell culture vessel; washing the cell culture vessel; adding a certain amount of agent to detach the cells from the three-dimensional substrate or dissolve the three-dimensional substrate; stirring the cell culture vessel; collecting the cell solution into a sterile bioprocess container; rinsing the cell culture vessel; collecting a certain amount of rinse solution from the cell culture vessel; neutralizing the detachment enzyme; centrifuging a certain amount of the cell solution; aspirating the supernatant of the pelletized cell solution; and resuspending any sample in phosphate-buffered saline, cell culture medium, or cryopreservation medium. In some embodiments, harvesting is performed between approximately 10 and 18 days of culture.

[0010] In some embodiments, the recovered cells are further processed. In some cases, the recovered cells are seeded onto a new three-dimensional substrate and culture is continued. The agent used to detach the cells from the three-dimensional substrate or to dissolve the three-dimensional substrate may be at least one detaching enzyme, optionally trypsin or tryp-LE. In other cases, the recovered cells are resuspended in cryopreservation medium and then frozen for storage.

[0011] In some embodiments of the method, multiple recovered alveolar epithelial cells express surfactant protein C precursor (pSP-C), indicating that the alveolar cells remain functional even after proliferation. In some preferred embodiments, multiple recovered alveolar epithelial cells lose less than 30% of pSP-C expression during the first 14, 15, 18, 20, 25, 30, 35, or 40 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose less than 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of pSP-C expression during the first 14, 15, 18, 20, 25, 30, 35, or 40 days of culture. In some embodiments, multiple recovered alveolar epithelial cells include a population with over 30% pSP-C expression after approximately 14, 15, 18, 20, 25, 30, 35, or 40 days. In some cases, multiple recovered alveolar epithelial cells include a population with over 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pSP-C expression after approximately 14, 15, 18, 20, 25, 30, 35, or 40 days. Multiple recovered alveolar epithelial cells may also express HT2-280, a type 2 alveolar cell marker. In certain embodiments, the recovered alveolar epithelial cells do not express excessive CK5 or contain overgrown airway basal cells.

[0012] Microcarriers used in some embodiments can have a hardness between approximately 1 kPa and approximately 100 kPa. In a particular embodiment, the microcarriers have a hardness of approximately 4 kPa.

[0013] Cell culture medium compositions for culturing alveolar epithelial cells are also provided herein. In some embodiments, the composition may include a transforming growth factor β (TGF-β) pathway inhibitor, a Wnt pathway activator, a Rho kinase (ROCK) inhibitor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), and / or fetal bovine serum (FBS).

[0014] In some embodiments of the composition, the TGF-β pathway inhibitor may be present in the culture medium at a level of approximately 1 μM to approximately 10 μM. In some embodiments, the Wnt pathway activator may be present in the culture medium at a level of approximately 1 μM to approximately 10 μM. In some embodiments, the ROCK inhibitor may be present in the culture medium at a level of approximately 1 μM to approximately 10 μM. In some embodiments, EGF accounts for approximately 25 ng to approximately 200 ng per mL of composition. In some embodiments, KGF accounts for approximately 25 ng to approximately 200 ng per mL of composition. In some embodiments, fetal bovine serum accounts for approximately 1% to approximately 10% by volume. In certain embodiments, the TGF-β inhibitor comprises at least one of A-83-01 or DMH1. In certain embodiments, the Wnt pathway activator comprises CHIR99021. In other embodiments, the ROCK inhibitor comprises Y27632. In some cases, the cell culture medium is O-WREKT medium.

[0015] In further embodiments of the composition, the composition may further comprise a plurality of three-dimensional substrates. In a particular preferred embodiment, the three-dimensional substrates may be microcarriers.

[0016] In certain embodiments, a kit comprising a plurality of alveolar epithelial cells obtained by the method described herein or using the cell culture medium composition described herein is further disclosed herein.

[0017] The above summary, drawings, and detailed descriptions below are for illustrative and illustrative purposes only. They are intended to provide further details of this disclosure and should not be construed as limiting. Other purposes, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of this disclosure. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows images of the results of a biostaining assay of AT2 cells on a three-dimensional substrate across three generations of passages. [Figure 2] Graph showing the phenotypic stability of alveolar epithelial cell culture on a three-dimensional substrate over three generations of subculture. [Figure 3] Graph showing a graphical comparison of three-dimensional culture versus two-dimensional culture over multiple tests regarding the maintenance of functional type II alveolar cells. [Figure 4-1] Graph exemplifying the overview of all spinner flask AT2 microcarrier growths performed separately for each passage (Figure 4A). [Figure 4-2] Graph exemplifying the AT2 fold change (Figure 4B) and in-process cell count (Figure 4C) of a 250 mL spinner flask passage 0 growth trial performed using GE Cultispher GL microcarriers in O-WREKT medium. [Figure 4-3] Graph exemplifying the AT2 fold change (Figure 4B) and in-process cell count (Figure 4C) of a 250 mL spinner flask passage 0 growth trial performed using GE Cultispher GL microcarriers in O-WREKT medium. [Figure 4-4] Figure exemplifying AT2 cell growth at various passages in a spinner flask by cell growth measurement metrics including in-process cell count, as well as AT2 fold change, population doubling level, and population doubling time (hours) (Figure 4D). [Figure 5] Figure exemplifying an exemplary bioreactor growth process. [Figure 6-1] Graph exemplifying cell growth at various scales. [[ID=*]] [Figure 6-2] Graph exemplifying cell growth at various passages in a bioreactor. [Figure 7] Graph exemplifying the bioreactor growth and measurement metrics from passage 0 to passage 2. [Figure 8] Graph exemplifying bioreactor growth phenotypic analysis. [Figure 9] Figure exemplifying an exemplary process scale-up / out for use in one or more of the methods described herein. [Modes for carrying out the invention]

[0019] Embodiments of the present disclosure are described below in more detail. However, embodiments of the present disclosure may be embodied in different ways and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to make the present disclosure detailed and complete and to fully convey the scope of the invention to those skilled in the art. The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit them.

[0020] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art to which this invention pertains. Terms as defined in commonly used dictionaries should be interpreted as having the meaning consistent with the meaning in the context of this application and in the relevant art, and should not be interpreted in an ideal or overly formal sense unless such a definition is explicitly stated herein. Although not explicitly defined below, such terms should be interpreted according to their general meaning.

[0021] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including its definitions, shall prevail. Other embodiments are described in the subsequent claims.

[0022] Unless otherwise instructed, the implementation of this technology shall utilize conventional techniques within the scope of the art, including tissue culture, immunology, molecular biology, microbiology, chemical engineering, and cell biology.

[0023] Unless otherwise indicated by the context, the various features described herein are expressly intended to be usable in any combination. Furthermore, this disclosure also intends that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For example, where this specification indicates that a complex comprises components A, B, and C (or A, B, and / or C), it is expressly intended that any one of A, B, or C, or any combination thereof, may be omitted and discarded individually or in any combination.

[0024] Unless otherwise explicitly indicated, all embodiments, features, and terms described herein are intended to include both the embodiments, features, or terms described herein and their biological equivalents.

[0025] All numerical values, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that may vary (+) or (-) by units of 1.0 or 0.1, or by variations of + / - 15%, 10%, 5%, or 2%, as appropriate. While not always explicitly indicated, it should be understood that all numerical values ​​are preceded by the term "approximately."

[0026] definition When used in the description of the present invention and the claims, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise indicated in the context.

[0027] When referring to measurable values, such as quantity or concentration, the term “approximately” as used herein means encompassing variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified quantity.

[0028] When used to describe any selection of ingredients, ranges, dosage forms, etc. disclosed herein, the terms “acceptable,” “effective,” or “sufficient” mean that such ingredients, ranges, dosage forms, etc. are suitable for the purposes of this disclosure.

[0029] Furthermore, as used herein, “and / or” encompasses all possible combinations of one or more of the items listed with this term, and the absence of any combination when interpreted disjunctly ("or").

[0030] As used herein, “optional” or “optional” means that the event or situation described thereafter may or may not occur, and that the description includes both instances in which the event or situation occurs and instances in which it does not occur.

[0031] As used herein, “microcarrier” refers to a support matrix that enables cell growth in a bioreactor. Microcarrier beads, containers, or vessels may consist of any material suitable for tissue culture, including but not limited to glass, polystyrene, poly(caprolactone), nylon, poly(ethylene terephthalate) (PET), poly(glycolic acid) (PGA), gelatin, and / or dextran. Microcarriers may also include materials that are magnetic or can become magnetic, such as Fe3O4. Microcarriers may be of any size and / or shape suitable for culturing cells, typically having a diameter in the range of about 25 μm to about 500 μm, but may be larger or smaller. Microcarriers may be porous (e.g., microporous or macroporous) or nonporous.

[0032] As used herein, the terms “complete media” and “complete medium” refer to cell culture media optimized for alveolar epithelial cell growth (e.g., type II alveolar epithelial cells, optionally human AT2 cells). In some cases, complete media contain inorganic salts, trace elements, vitamins, amino acids, lipids, carbohydrates, cytokines, growth factors, small molecules, and / or additional proteins, with the ratios of each component optimized for cell growth. Exemplary additional proteins include albumin, transferrin, fibronectin, and insulin. Exemplary carbohydrates include glucose. Exemplary inorganic salts include sodium, potassium, and calcium ions. Exemplary trace elements include zinc, copper, selenium, and tricarboxylic acids. Examples of amino acids include essential amino acids such as L-glutamine (e.g., alanyl-l-glutamine or glycyl-l-glutamine), or non-essential amino acids (NEAAs) such as glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and / or L-serine. In some embodiments, the complete medium also contains one or more of the following: sodium bicarbonate (NaHCO3), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phenol red, antibiotics, and / or β-mercaptoethanol. In some cases, the complete medium is serum-free. In some cases, the complete medium is xeno-free.

[0033] As used herein, the term “synthetic medium” refers to a cell culture medium in which the composition and concentration of all components are known. Synthetic media differ from complete media in that complete media may contain components whose composition and / or concentration are not known, such as animal-derived components. Complete media may also be synthetic media if the composition and concentration of all components are known.

[0034] In some cases, "Xenofree" media contain no animal-derived (non-human) components. In other cases, Xenofree media contain one or more human-derived components, such as human serum, growth factors, and insulin.

[0035] In some embodiments, the "serum-free" medium does not contain serum or plasma, but may contain components derived from serum or plasma. In some cases, the "serum-free" medium contains animal-derived components such as bovine serum albumin (BSA).

[0036] In some embodiments, the “minimal” medium contains the minimum necessary substances for the growth of target cells. In some cases, the minimal medium contains inorganic salts, a carbon source, and water. In some cases, supplements such as cytokines and / or proteins such as albumin (e.g., HSA) are added to the minimal medium. As used herein, supplements include trace elements, vitamins, amino acids, lipids, carbohydrates, cytokines, growth factors, or combinations thereof.

[0037] Methods for the proliferation of alveolar epithelial cells In certain embodiments, methods for producing alveolar epithelial cells, optionally type II alveolar epithelial cells, are disclosed herein. In some embodiments, the method may include the steps of preparing a plurality of three-dimensional substrates in a cell culture vessel, seeding the plurality of alveolar epithelial cells by combining the three-dimensional substrates with the alveolar epithelial cells in the cell culture vessel and creating conditions suitable for enabling cell adhesion to the three-dimensional substrates and achieving suspension culture, promoting the growth of the alveolar epithelial cells on or within the three-dimensional substrates, monitoring the culture with respect to cell proliferation, and harvesting the plurality of alveolar epithelial cells from the three-dimensional substrates. In some embodiments, the method may optionally include the step of supplying a new three-dimensional substrate after the harvesting step to continue the growth and proliferation of the alveolar epithelial cells. The alveolar epithelial cells may be type II alveolar epithelial cells, such as human AT2 cells.

[0038] In some embodiments, the three-dimensional substrate may be at least one of non-porous, microporous, or macroporous three-dimensional substrates. Alveolar epithelial cells can be cultured on, inside, or both on and inside the three-dimensional substrate. In certain preferred embodiments, the three-dimensional substrate may consist of multiple microcarriers.

[0039] In some embodiments, the substrate is a microporous substrate. In such cases, alveolar epithelial cells (e.g., type II alveolar epithelial cells) are cultured on or on the surface of the three-dimensional microporous substrate. In some cases, the microporous substrate is used in stirred-tank partial medium exchange culture. In some embodiments, the microporous substrate is used in stirred-tank perfusion culture. In some embodiments, the microporous substrate may be a high-density microporous substrate for use in fluidized-bed perfusion culture. In other embodiments, the microporous substrate may be a high-density macroporous substrate for use in packed-bed perfusion culture.

[0040] In some embodiments, the substrate is a macroporous substrate. In such cases, alveolar epithelial cells (e.g., type II alveolar epithelial cells) are cultured on, inside, or both on and inside the three-dimensional macroporous substrate. In some embodiments, the macroporous substrate is used in stirred-tank partial medium exchange culture. In some embodiments, the macroporous substrate is used in stirred-tank perfusion culture. In some embodiments, the macroporous substrate may be a high-density macroporous substrate for use in fluidized-bed perfusion culture. In other embodiments, the macroporous substrate may be a high-density macroporous substrate for use in packed-bed perfusion culture.

[0041] In some embodiments, the substrate is a non-porous substrate. In such cases, alveolar epithelial cells (e.g., type II alveolar epithelial cells) are cultured on or on the surface of the three-dimensional non-porous substrate. In some cases, the non-porous substrate is used in stirred-tank partial medium exchange culture. In some embodiments, the non-porous substrate is used in stirred-tank perfusion culture. In some embodiments, the non-porous substrate may be a high-density non-porous substrate for use in fluidized-bed perfusion culture. In other embodiments, the non-porous substrate may be a high-density non-porous substrate for use in packed-bed perfusion culture.

[0042] The cell culture vessel may be a spinner flask or a bioreactor, and the three-dimensional substrate occupies approximately 1 to 10 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1.2 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1.4 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1.5 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1.6 mg / mL during culture. In some cases, the three-dimensional substrate occupies approximately 1.8 mg / mL during culture. In some cases, the three-dimensional substrate accounts for approximately 2 mg / mL of the culture.

[0043] In some embodiments, the cell culture vessel is a spinner flask, and the culture can be at least 125 mL, 250 mL, 500 mL, 1 L, 3 L, or 10 L.

[0044] In some embodiments, the cell culture vessel is a bioreactor, and the culture may be at least 1 L, 3 L, 3.75 L, 5 L, 7 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, or 60 L. In some cases, the culture may be at least 3 L, 3.75 L, 10 L, or 40 L.

[0045] Cell seeding may further include adding alveolar epithelial cells (e.g., fresh isolated or cryopreserved) from a cell culture medium to a cell culture vessel. Seeding may also include agitating the culture. For example, in some embodiments of the methods of the present disclosure, a three-dimensional substrate culture may be agitated at about 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) on a stirring plate in an incubator. In certain embodiments of the methods, agitation is performed intermittently and includes a cycle in which agitation is performed for a first period at about 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) followed by a second period in which no agitation is performed. In some cases, this cycle repeats approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or more. In some cases, the second period is approximately 30 minutes or more. In some cases, the cycle repeats approximately 32 to approximately 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the ratio of stirring time to non-stirring time may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can last for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. The cycle can be repeated at least 12 times, at least 24 times, at least 36 times, or at least 64 times. In some embodiments, the cycle can be repeated 24 to 64 times or 36 to 64 times.In some cases, stirring is performed intermittently, including a cycle in which stirring is performed for about 5 minutes at a rate of approximately 20 RPM or higher (optionally 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher), followed by a period of approximately 30 minutes without stirring. This cycle is then repeated approximately 32 to 64 times (optionally 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, after approximately 18–36 hours (optionally 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at approximately 20 RPM or higher (optionally 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) until the cells are harvested. In some cases, seeding is performed at a volume of approximately one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0046] In some embodiments, the three-dimensional substrate culture may be stirred at approximately 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) in a 250 mL spinner flask on a stirring plate in an incubator. In such cases, the three-dimensional substrate culture may be stirred at approximately 25 RPM or higher, 30 RPM or higher, 35 RPM or higher, or 40 RPM or higher on a stirring plate in an incubator. In some cases, the three-dimensional substrate culture may be stirred at approximately 35 RPM or higher on a stirring plate in an incubator. In some embodiments, stirring is performed intermittently and includes a cycle in which stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period in which no stirring occurs. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or longer. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the time ratio of stirring to non-stirring may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. The cycle can be repeated at least 12 times, at least 24 times, at least 36 times, or at least 64 times.In some embodiments, the cycle may be repeated 24 to 64 times or 36 to 64 times. In some cases, stirring is performed intermittently, optionally including a cycle in which stirring is performed at approximately 35 RPM or higher for approximately 5 minutes, followed by a period of no stirring for approximately 30 minutes, and this cycle is then repeated approximately 32 to 64 times. In some embodiments, after approximately 18 to 36 hours (optionally, 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at approximately 35 RPM or higher until the cells are harvested. In some cases, seeding is performed at a volume of approximately one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0047] In some embodiments, the three-dimensional substrate culture may be stirred at approximately 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 40 RPM or higher, 50 RPM or higher, or 60 RPM or higher) in a 1 L spinner flask on a stirring plate in an incubator. In such cases, the three-dimensional substrate culture may be stirred at approximately 20 RPM or higher, 25 RPM or higher, 30 RPM or higher, or 35 RPM or higher on a stirring plate in an incubator. In some cases, the three-dimensional substrate culture may be stirred at approximately 20 RPM or higher on a stirring plate in an incubator. In some embodiments, stirring is performed intermittently and includes a cycle in which stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period in which no stirring occurs. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or longer. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the time ratio of stirring to non-stirring may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. The cycle can be repeated at least 12 times, at least 24 times, at least 36 times, or at least 64 times.In some embodiments, the cycle may be repeated 24 to 64 times or 36 to 64 times. In some cases, stirring is performed intermittently, optionally including a cycle in which stirring is performed at or above 20 RPM for about 5 minutes, followed by a period of no stirring for about 30 minutes, and this cycle is then repeated approximately 32 to 64 times. In some embodiments, after about 18 to 36 hours (optionally, 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at or above 20 RPM until the cells are harvested. In some cases, seeding is performed at a volume of about one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0048] In some embodiments, the three-dimensional substrate culture may be stirred in a bioreactor at approximately 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 39 RPM or higher, 40 RPM or higher, 42 RPM or higher, 45 RPM or higher, 48 RPM or higher, 50 RPM or higher, 52 RPM or higher, 55 RPM or higher, or 60 RPM or higher). In some cases, the bioreactor culture is performed on a tabletop, where an external motor controls an impeller inside the container to induce mixing. In such embodiments, gas regulation is maintained using a control device, and the container is heated using a heat jacket. In certain embodiments of the method, stirring may be performed intermittently and may include a cycle in which stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period in which no stirring occurs. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or longer. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the time ratio of stirring to non-stirring may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours.The cycle can be repeated at least 12, at least 24, at least 36, or at least 64 times. In some embodiments, the cycle can be repeated 24 to 64 times or 36 to 64 times. In some embodiments, stirring is performed intermittently and includes a cycle in which stirring is performed for about 5 minutes at about 20 RPM or more (optionally 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 39 RPM or more, 40 RPM or more, 42 RPM or more, 45 RPM or more, 48 RPM or more, 50 RPM or more, 52 RPM or more, 55 RPM or more, or 60 RPM or more), followed by a period of about 30 minutes without stirring, after which this cycle is repeated about 32 to 64 times (optionally about 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, after approximately 18–36 hours (optionally 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at approximately 20 RPM or higher (optionally 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 39 RPM or higher, 40 RPM or higher, 42 RPM or higher, 45 RPM or higher, 48 RPM or higher, 50 RPM or higher, 52 RPM or higher, 55 RPM or higher, or 60 RPM or higher) until the cells are harvested. After seeding, the culture medium may be added until the total culture volume is reached.

[0049] In some embodiments, the three-dimensional substrate culture may be stirred in a 1 L bioreactor at approximately 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 39 RPM or higher, 40 RPM or higher, 42 RPM or higher, 45 RPM or higher, 48 RPM or higher, 50 RPM or higher, 52 RPM or higher, 55 RPM or higher, or 60 RPM or higher). In such cases, the three-dimensional substrate culture may be stirred in a 1 L bioreactor at approximately 45 RPM or higher, 48 RPM or higher, 50 RPM or higher, or 52 RPM or higher. In some cases, the three-dimensional substrate culture may be stirred in a 1 L bioreactor at approximately 48 RPM or higher or 52 RPM or higher. In some embodiments, stirring is performed intermittently and includes a cycle in which stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period in which no stirring occurs. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or longer. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the time ratio of stirring to non-stirring may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours.The cycle can be repeated at least 12, 24, 36, or 64 times. In some embodiments, the cycle may be repeated 24–64 times or 36–64 times. In some cases, stirring is performed intermittently, optionally including a cycle in which stirring is performed at about 48 RPM or higher for about 5 minutes, followed by a period of no stirring for about 30 minutes, and this cycle is then repeated about 32–64 times. In some embodiments, after about 18–36 hours (optionally, 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at about 48 RPM or higher until the cells are harvested. In some cases, seeding is performed at a volume of about one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0050] In some embodiments, the three-dimensional substrate culture may be stirred in a 3.75 L bioreactor at approximately 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 39 RPM or higher, 40 RPM or higher, 42 RPM or higher, 45 RPM or higher, 48 RPM or higher, 50 RPM or higher, 52 RPM or higher, 55 RPM or higher, or 60 RPM or higher). In such cases, the three-dimensional substrate culture may be stirred in a 3.75 L bioreactor at approximately 45 RPM or higher, 50 RPM or higher, 55 RPM or higher, or 60 RPM or higher. In some cases, the three-dimensional substrate culture may be stirred in a 3.75 L bioreactor at approximately 50 RPM or higher or 55 RPM or higher. In some embodiments, stirring is performed intermittently and includes a cycle in which stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period in which no stirring occurs. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or longer. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the time ratio of stirring to non-stirring may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours.The cycle can be repeated at least 12, 24, 36, or 64 times. In some embodiments, the cycle may be repeated 24–64 times or 36–64 times. In some cases, stirring is performed intermittently, optionally including a cycle in which stirring is performed at about 50 RPM or higher for about 5 minutes, followed by a period of no stirring for about 30 minutes, and this cycle is then repeated about 32–64 times. In some embodiments, after about 18–36 hours (optionally, 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at about 50 RPM or higher until the cells are harvested. In some cases, seeding is performed at a volume of about one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0051] In some embodiments, the three-dimensional substrate culture may be stirred in a 10L bioreactor at approximately 20 RPM or higher (optionally, 25 RPM or higher, 28 RPM or higher, 30 RPM or higher, 32 RPM or higher, 35 RPM or higher, 38 RPM or higher, 39 RPM or higher, 40 RPM or higher, 42 RPM or higher, 45 RPM or higher, 48 RPM or higher, 50 RPM or higher, 52 RPM or higher, 55 RPM or higher, or 60 RPM or higher). In such cases, the three-dimensional substrate culture may be stirred in a 10L bioreactor at approximately 35 RPM or higher, 39 RPM or higher, 40 RPM or higher, or 42 RPM or higher. In some cases, the three-dimensional substrate culture may be stirred in a 10L bioreactor at approximately 39 RPM or higher or 42 RPM or higher. In some embodiments, stirring is performed intermittently and includes a cycle in which stirring is performed for a first period at approximately 20 RPM or more (optionally, 25 RPM or more, 28 RPM or more, 30 RPM or more, 32 RPM or more, 35 RPM or more, 38 RPM or more, 40 RPM or more, 50 RPM or more, or 60 RPM or more), followed by a second period in which no stirring occurs. In some cases, this cycle is repeated approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 times or more. In some cases, the first period is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes or more. In some cases, the first period is approximately 5 minutes or more. In some cases, the second period is approximately 10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes or longer. In some cases, the second period is approximately 30 minutes or longer. In some cases, the cycle is repeated approximately 32 to 64 times (optionally, approximately 32, 35, 40, 45, 50, 55, 60, or 64 times). In some embodiments, the cycle of stirring and non-stirring is repeated with a time ratio of approximately 1:10 (e.g., 1 minute of stirring and 10 minutes of non-stirring), 1:8, 1:6, 1:5, 1:4, 1:3, or 1:2. For example, the time ratio of stirring to non-stirring may be between 1:10 and 1:2, 1:8 and 1:3, or 1:7 and 1:4. These cycles can continue for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours.The cycle can be repeated at least 12, 24, 36, or 64 times. In some embodiments, the cycle may be repeated 24–64 times or 36–64 times. In some cases, stirring is performed intermittently, optionally including a cycle in which stirring is performed at about 39 RPM or higher for about 5 minutes, followed by a period of no stirring for about 30 minutes, and this cycle is then repeated about 32–64 times. In some embodiments, after about 18–36 hours (optionally, 18, 24, 28, 30, or 36 hours), the culture may be continuously stirred at about 39 RPM or higher until the cells are harvested. In some cases, seeding is performed at a volume of about one-quarter, one-third, or one-half of the final culture volume. After seeding, culture medium may be added until the total culture volume is reached.

[0052] In some embodiments, the culture is monitored for growth and proliferation throughout the entire duration of the culture process. In some embodiments, monitoring may include supplying nutrients to the culture, performing at least one LIVE / DEAD® assay on the culture, and evaluating cell coverage on a three-dimensional substrate. The culture can be supplied with nutrients at intervals of approximately 2 to 4 days, and metabolic samples can be collected, for example, daily and / or after nutrient supply. In some cases, monitoring and / or sample measurement is performed daily for the duration of the culture process. In other cases, monitoring and / or sample measurement is performed once a day, twice a day, or as required to ensure accurate readings of the measurements. In additional cases, monitoring and / or sample measurement is performed every other day, every two days, every three days, or every four days. In further cases, monitoring and / or sample measurement is performed continuously for the duration of the culture process. Monitoring may monitor at least one of the following, and any combination thereof: pH, glucose, lactate, glutamine, ammonium, and / or dissolved oxygen levels, and / or biocapacitance. Table 1 illustrates exemplary low and high levels of pH, glucose, lactate, glutamine, ammonium, and / or dissolved oxygen in some cases. In some embodiments of the method, cell counting is performed daily to assess growth. In some cases where cells are cultured in a bioreactor, the measurement is performed via the use of a probe.

[0053] [Table 1]

[0054] In some embodiments of the method of this disclosure, the step of recovering a plurality of alveolar epithelial cells from a three-dimensional substrate may include: precipitating the three-dimensional substrate and removing a certain amount of culture medium from the cell culture vessel; washing the cell culture vessel; adding a certain amount of agent to detach the cells from the three-dimensional substrate; stirring the cell culture vessel; collecting the cell solution into a sterile bioprocess container; rinsing the cell culture vessel; collecting a certain amount of rinse liquid from the cell culture vessel; neutralizing the detachment enzyme; centrifuging a certain amount of cell solution; aspirating the supernatant of the pelletized cell solution; and resuspending any sample in phosphate-buffered saline, cell culture medium, or cryopreservation medium. In some embodiments, recovery is performed about 10 to 18 days of culture, about 12 to 16 days of culture, or about 12 to 14 days of culture. After recovery, the recovered cells can be seeded on a new three-dimensional substrate and culture can be continued.

[0055] Cells can be detached from a three-dimensional substrate, or the three-dimensional substrate can be dissolved. In some embodiments of the methods of this disclosure, the agent for detaching cells from the three-dimensional substrate or dissolving the three-dimensional substrate may be at least one detachment enzyme, optionally trypsin or tryp-LE.

[0056] Multiple recovered alveolar epithelial cells may also express certain biological markers as measures of other indicators of cell health, adhesion, or successful proliferation. Multiple recovered alveolar epithelial cells may further express HT2-280, a biomarker specific to the apical plasma with biochemical characteristics of an endogenous membrane protein. HT2-280 is an AT2 cell identification marker that demonstrates the presence of multiple AT2 cells in culture. In some embodiments of the method, multiple recovered epithelial cells may express pSP-C. In some preferred embodiments, multiple recovered alveolar epithelial cells lose less than 30% pSP-C expression during the first 14, 15, 18, 20, 25, 30, 35, or 40 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 25%, 30, 35, or 40% of pSP-C expression at the first 14, 15, 18, 20, 25, 30, 35, or 40 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 15 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 18 days of culture.In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 20 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 25 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 30 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 35 days of culture. In some embodiments, multiple recovered alveolar epithelial cells lose 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 18%, 16%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1% pSP-C expression during the first 40 days of culture.

[0057] In some embodiments, multiple recovered alveolar epithelial cells include a population with more than 30% pSP-C expression after approximately 14, 15, 18, 20, 25, 30, 35, or 40 days. In some cases, multiple recovered alveolar epithelial cells include a population with more than 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pSP-C expression after approximately 14, 15, 18, 20, 25, 30, 35, or 40 days. In some cases, multiple recovered alveolar epithelial cells contain populations with approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% pSP-C expression after approximately 14 days. In some cases, multiple recovered alveolar epithelial cells contain populations with approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% pSP-C expression after approximately 15 days. In some cases, multiple recovered alveolar epithelial cells contain populations with approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% pSP-C expression after approximately 18 days. In some cases, multiple recovered alveolar epithelial cells contain populations with approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% pSP-C expression after approximately 20 days. In some cases, multiple recovered alveolar epithelial cells contain populations with pSP-C expression of approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% after about 25 days.In some cases, multiple recovered alveolar epithelial cells contain populations with approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% pSP-C expression after approximately 30 days. In some cases, multiple recovered alveolar epithelial cells contain populations with approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% pSP-C expression after approximately 35 days. In some cases, multiple recovered alveolar epithelial cells contain populations with pSP-C expression of approximately 30%, 40%, 41%, 50%, 60%, 70%, 72%, 74%, 75%, 79%, 80%, 85%, 86%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% after about 40 days.

[0058] pSP-C is a functional marker that can indicate that more pSP-C expression is retained than in two-dimensional culture. In some embodiments of the method, multiple recovered alveolar epithelial cells may not express excessive CK5 or contain overgrowth of airway basal cells.

[0059] Microcarriers used in some embodiments of the methods of this disclosure may have a stiffness between approximately 1 kPa and approximately 100 kPa. In certain embodiments, the microcarriers have a stiffness of approximately 4 kPa (e.g., within 10% or 20% of 4 kPa), which is within a range configured to mimic the stiffness of the lung, particularly the human lung. In some cases, the microcarriers are nonporous, microporous, or macroporous. In some cases, approximately 1 to 10 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1.2 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1.4 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1.5 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1.6 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 1.8 mg / mL of microcarriers are added to the cell culture vessel. In some cases, approximately 2 mg / mL of microcarriers are added to the cell culture vessel.

[0060] In some embodiments, cultured alveolar epithelial cells make up about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more of the cells in culture. In some cases, cultured alveolar epithelial cells make up about 70% of the cells in culture. In some cases, cultured alveolar epithelial cells make up about 75% of the cells in culture. In some cases, cultured alveolar epithelial cells make up about 80% of the cells in culture. In some cases, cultured alveolar epithelial cells make up about 85% of the cells in culture. In some cases, cultured alveolar epithelial cells make up about 90% of the cells in culture.

[0061] In some embodiments, the cultures described herein contain contaminants at concentrations of approximately 30%, 28%, 25%, 24%, 22%, 20%, 18%, 16%, 15%, 13%, 12%, 10%, 8%, or less than 5%. In some cases, the contaminants include undesirable cells, such as cells that are not alveolar epithelial cells, cells that are not type II alveolar epithelial (AT2) cells (optionally), and cells that are not human AT2 cells (optionally).

[0062] In some embodiments, alveolar epithelial cells are cultured and grown for one or more passages, two or more passages, three or more passages, four or more passages, five or more passages, or six or more passages. In some cases, alveolar epithelial cells are cultured and grown for one, two, three, four, five, or six or more passages. In some cases, the number of cells in a passage increases by 1x, 2x, 3x, 4x, 5x, 6x, 6.5x, 6.6x, 7x, 8x, 9x, 10x, 11x, 12x, 15x, 20x, 50x, 100x, or more than 1000x. In some cases, the number of cells collected from one passage increases by 1x. In some cases, the number of cells collected from one passage increases by 2x. In some cases, the number of cells collected from one passage increases by 5x. In some cases, the number of cells collected from a single passage increases by 6 times, 6.5 times, or 6.6 times. In some cases, the number of cells collected from a single passage increases by 8 times. In some cases, the number of cells collected from a passage increases by 10 times. In some cases, the number of cells collected from passage 0 (P0) increases by 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 6.5 times, 6.6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 15 times, 20 times, 50 times, 100 times, or more than 1000 times. In some cases, the number of cells collected from passage 1 (P1) increases by 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 6.5 times, 6.6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 15 times, 20 times, 50 times, 100 times, or more than 1000 times. In some cases, the number of cells collected from passage 2 (P2) increases by 1x, 2x, 3x, 4x, 5x, 6x, 6.5x, 6.6x, 7x, 8x, 9x, 10x, 11x, 12x, 15x, 20x, 50x, 100x, or more than 1000x.

[0063] Figure 1 shows images of the results of a biostaining assay of AT2 cells on a three-dimensional substrate over three passages using the method disclosed herein. In all three passages, the images of cells after seeding (upper panel) and before harvesting (lower panel) show an increase in cell coverage within the microcarrier (white: living cells).

[0064] Figure 2 illustrates a graph of the phenotypic stability of alveolar epithelial cell cultures on a three-dimensional substrate over three passages. HT2-280 is an identification marker for AT2 cells. The upper panel of Figure 2 shows that HT2-280 expression was better maintained in three-dimensional culture compared to two-dimensional culture for two culture tests. Overgrowth of airway basal cells (indicated by CK5 expression) is a significant challenge in AT2 proliferation under standard culture conditions. The data in the lower panel demonstrate that CK5 expression increases more in two-dimensional culture or when cells are switched from three-dimensional to two-dimensional culture. Therefore, 3D culture conditions can reduce the amount of airway basal cells in the culture medium compared to 2D culture conditions. The notation 2D / 3D in the graph captions represents the culture platform in chronological order over three passages, separated by a hyphen.

[0065] Figure 3 shows a graphical comparison of three-dimensional culture versus two-dimensional culture over multiple tests regarding the maintenance of functional AT2 cells. Since AT2 cells mainly act to produce surfactant in the lung to reduce surface tension, surfactant protein C precursor is an important functional marker of AT2 cells. The upper panel of Figure 3 shows the pSP-C expression of three generations of passages in the case of a sample set with n = 2, demonstrating the maintenance of long-term pSP-C expression in three-dimensional culture compared to two-dimensional culture. In the lower panel of Figure 3, the loss of functional AT2 cells at passage 0 was calculated as the difference in the percentage of pSP-C positive cells (pSP-C+) divided by the percentage of HT2-280 positive (HT2-280+) cells from the initial culture to passage 0, and this calculation was performed assuming that all pSP-C+ cells are HT2-280+. This data demonstrates that there is a loss of approximately 11% in three-dimensional culture compared to approximately 44% loss in two-dimensional culture with respect to the percentage of functional AT2 cells, indicating that AT2 cells maintain their function better on a three-dimensional culture substrate compared to a two-dimensional culture substrate. 3D culture conditions can increase the amount of pSP-C+ cells in the culture medium compared to 2D culture conditions.

[0066] The culture yield depends on various factors including culture time, conditions, and volume. The methods described herein can result in a yield of at least 1×10 6 cells / culture, 1×10 7 cells / culture, 1×10 8 cells / culture, 1×10 9 cells / culture, or 5×10 9 cells / culture. In some embodiments, the cell yield can be at least 1×10 5 cells / mL, 2×10 5 cells / mL, 3×10 5 cells / mL, 4×10 5 cells / mL, or 5×10 5 cells / mL. These resulting cells can be AT2 cells or cells having one or more functional characteristics of AT2 cells.

[0067] In some embodiments, alveolar epithelial cells are cultured and grown using one or more of the methods described herein and / or the culture medium compositions described herein for use in regenerative medicine, for example, for use in tissue or organ engineering. In some embodiments, alveolar epithelial cells cultured using one or more of the methods described herein and / or the culture medium compositions described herein are also used for cell therapy, for example, for the treatment of one or more diseases or conditions such as cancer.

[0068] Composition for culturing alveolar epithelial cells Cell culture medium compositions for culturing alveolar epithelial cells are also provided herein. In some embodiments, the cell culture medium composition for culturing alveolar epithelial cells may comprise a TGF-β pathway inhibitor, a Wnt pathway activator, a ROCK inhibitor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), and fetal bovine serum (FBS). In some cases, the cell culture medium is a complete cell medium supplemented with one or more of the TGF-β pathway inhibitor, Wnt pathway activator, ROCK inhibitor, EGF, or KGF at the discretion of the user. In some cases, the cell culture medium is an FBS-based medium supplemented with one or more of the TGF-β pathway inhibitor, Wnt pathway activator, ROCK inhibitor, EGF, or KGF at the discretion of the user. In some cases, the cell culture medium is a serum-free medium supplemented with one or more of the TGF-β pathway inhibitor, Wnt pathway activator, ROCK inhibitor, EGF, or KGF at the discretion of the user. In some cases, the cell medium is a synthetic medium supplemented with one or more of the following: TGF-β pathway inhibitors, Wnt pathway activators, ROCK inhibitors, EGF, KGF, or FBS. In some cases, the cell medium is a minimal medium supplemented with one or more of the following: TGF-β pathway inhibitors, Wnt pathway activators, ROCK inhibitors, EGF, KGF, or FBS. In some cases, the cell medium further comprises one or more amino acid supplements (e.g., L-glutamine) and / or antibiotics. In some cases, the cell culture medium composition is used in the methods described above for culturing alveolar epithelial cells, optionally type II alveolar epithelial cells, and optionally human AT2 cells.

[0069] In some embodiments of the culture medium composition, the TGF-β pathway inhibitor may be present in molar concentrations of approximately 1 μM to approximately 10 μM or any value or partial range in between. Further formulation concentrations of the TGF-β pathway inhibitor include approximately 1.25 μM, 1.5 μM, 1.75 μM, 2.0 μM, 2.25 μM, 2.5 μM, 2.75 μM, 3.0 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4.0 μM, 4.25 μM, 4.5 μM, 4.75 μM, 5.0 μM, 5.25 μM, and 5 They may be included in molar concentrations of 0.5 μM, 5.75 μM, 6.0 μM, 6.25 μM, 6.5 μM, 6.75 μM, 7.0 μM, 7.25 μM, 7.5 μM, 7.75 μM, 8.0 μM, 8.25 μM, 8.5 μM, 8.75 μM, 9.0 μM, 9.25 μM, 9.5 μM, 9.75 μM, or 10.0 μM. In some cases, TGF-β pathway inhibitors are included in molar concentrations of approximately 1 μM or approximately 2 μM. In some embodiments, TGF-β pathway inhibitors include any inhibitors that modulate or interfere with the interaction between TGF-β and its respective receptors, TGF-β receptor kinase function, or TGF-β signaling. In some cases, one or more TGF-β pathway inhibitors are included in the culture medium composition. In some embodiments, the TGF-β pathway inhibitor may be at least one of A-83-01 or DMH1.

[0070] In some embodiments of the culture medium composition, the Wnt pathway activator may be present in molar concentrations of approximately 1 μM to approximately 10 μM or any value or partial range in between. The formulation concentrations of the Wnt pathway activator are approximately 1.25 μM, 1.5 μM, 1.75 μM, 2.0 μM, 2.25 μM, 2.5 μM, 2.75 μM, 3.0 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4.0 μM, 4.25 μM, 4.5 μM, 4.75 μM, 5.0 μM, 5.25 μM, 5.5 It may be included in molar concentrations of μM, 5.75μM, 6.0μM, 6.25μM, 6.5μM, 6.75μM, 7.0μM, 7.25μM, 7.5μM, 7.75μM, 8.0μM, 8.25μM, 8.5μM, 8.75μM, 9.0μM, 9.25μM, 9.5μM, 9.75μM, or 10.0μM. In some cases, the Wnt pathway activator is included at a molar concentration of approximately 2μM. In some embodiments, the Wnt pathway activator encompasses any Wnt signaling activator or Wnt / β-catenin pathway activator. In certain embodiments, the Wnt pathway activator may be CHIR99021.

[0071] In some embodiments of the culture medium composition, the ROCK inhibitor may be present in molar concentrations of approximately 1 μM to approximately 10 μM or any value or partial range in between. Further formulation concentrations of the ROCK inhibitor include approximately 1.25 μM, 1.5 μM, 1.75 μM, 2.0 μM, 2.25 μM, 2.5 μM, 2.75 μM, 3.0 μM, 3.25 μM, 3.5 μM, 3.75 μM, 4.0 μM, 4.25 μM, 4.5 μM, 4.75 μM, 5.0 μM, 5.25 μM, and 5. It may be included in molar concentrations of 5 μM, 5.75 μM, 6.0 μM, 6.25 μM, 6.5 μM, 6.75 μM, 7.0 μM, 7.25 μM, 7.5 μM, 7.75 μM, 8.0 μM, 8.25 μM, 8.5 μM, 8.75 μM, 9.0 μM, 9.25 μM, 9.5 μM, 9.75 μM, or 10.0 μM. In some cases, the ROCK inhibitor is included at a molar concentration of approximately 10 μM. In some cases, the ROCK inhibitor is a ROCK1 inhibitor. In other cases, the ROCK inhibitor is a ROCK2 inhibitor. In some cases, the ROCK inhibitor may be Y27632. In some cases, the ROCK inhibitor may be fasudil.

[0072] In some embodiments of the culture medium composition, the EGF may be in the range of about 25 ng / mL to about 200 ng / mL or any value or partial range in that range. In some cases, EGF is included at a concentration of about 50 ng / mL.

[0073] In some embodiments of the culture medium composition, fetal bovine serum (FBS) may be present at a volume concentration of about 1% to about 10% (v / v) or any value or partial range in between. Certain formulation concentrations may include fetal bovine serum at volume concentrations (v / v) of approximately 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%, 7.25%, 7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10.0%. In some cases, FBS is included at a concentration of approximately 5%.

[0074] In some embodiments of the culture medium composition, keratinocyte growth factor (KGF) may be present in any value or partial range between approximately 25 ng / mL and approximately 200 ng / mL. Further formulation concentrations of KGF may include concentrations of approximately 25 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, or 200 ng / mL. In some cases, KGF is present in concentrations of approximately 50 ng / mL to approximately 100 ng / mL.

[0075] In some embodiments of the culture medium composition, the composition comprises a basal medium further supplemented with one or more additional components, such as a TGF-β pathway inhibitor, a Wnt pathway activator, a ROCK inhibitor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), fetal bovine serum (FBS), and optionally an amino acid such as L-glutamine and / or an antibiotic. In some cases, the basal medium is DMEM / F-12 medium. In some cases, the composition contains L-glutamine (e.g., GlutaMAX®). In some cases, the culture medium composition contains an antibiotic in an amount of about 50 μg / mL to about 200 μg / mL, optionally about 100 μg / mL to about 200 μg / mL, or about 100 μg / mL to about 150 μg / mL. In some cases, the culture medium composition contains an antibiotic in an amount of about 100 μg / mL. In some cases, the antibiotic is Primocin®.

[0076] In some embodiments, the culture medium composition includes a basal medium selected from DMEM / F-12 medium. In some cases, the culture medium composition further includes about 2.5 mM L-glutamine, about 5% FBS, about 2 μM of a first TGF-β pathway inhibitor, about 1 μM of a second TGF-β pathway inhibitor, about 2 μM of a Wnt pathway activator, about 50 ng / mL of EGF, about 50-100 ng / mL of KGF, about 10 μM of a ROCK inhibitor, and about 100 μg / mL of an antibiotic.

[0077] In some embodiments, the culture medium composition includes a basal medium selected from DMEM / F-12 medium. In some cases, the culture medium composition further includes GlutaMAX® at a concentration of 2.5 mM, about 5% FBS, about 2 μM A-83-01, about 1 μM DHM1, about 2 μM CHIR99021, about 50 ng / mL EGF, about 50-100 ng / mL KGF, about 10 μM Y27632, and about 100 μg / mL Primocin®. In some cases, this culture medium composition is also referred to herein as O-WREKT medium.

[0078] A composition for culturing alveolar epithelial cells may further comprise a plurality of three-dimensional substrates. In some embodiments, the three-dimensional substrate may be at least one of a plurality of non-porous, microporous, or macroporous three-dimensional substrates. In certain embodiments, the microporous three-dimensional substrate further comprises microcarriers.

[0079] Kits and manufactured articles In some embodiments, the kit or manufactured article described herein includes one or more alveolar epithelial cell populations obtained by the method described above, or one or more alveolar epithelial cell populations cultured in the cell culture medium composition described above. In some cases, the kit or manufactured article described herein further includes one or more containers, such as a transport container, packaging container, or container partitioned to accommodate vials, tubes, etc., each of the one or more containers includes one of the distinct elements used in the method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from a variety of materials such as glass or plastic.

[0080] The manufactured articles provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging materials suitable for the selected formulation and the intended method of administration and treatment.

[0081] The kit typically includes labels and / or instructions for use listing the contents, as well as accompanying documentation containing instructions for use. A set of instructions is also typically included. [Examples]

[0082] These embodiments are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

[0083] [Example 1] Parameter optimization Microcarrier Information: The following microcarriers were tested for AT2 cell adhesion: 1) Percell CultiSpher S microcarrier, 2) Percell CultiSpher GL microcarrier, 3) Corning collagen soluble microcarrier, 4) Corning SyntheMax II soluble microcarrier, 5) Percell CultiSpher G microcarrier, and 6) GE Cytodex3 microcarrier. AT2 cells adhered to all microcarriers, but adhesion was most pronounced with CultiSpher GL and Corning collagen soluble microcarriers, based on LIVE / DEAD® imaging of AT2 cells after 24 hours.

[0084] Culture medium optimization: Various cell culture media were tested to determine the optimal growth conditions. In one assay, three distinct cell culture media were tested: 1) Gibco DMEM F-12 medium containing a TGF-β pathway inhibitor, a Wnt pathway activator, a ROCK inhibitor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), and fetal bovine serum (FBS); 2) Lonza SAGM medium; and 3) Stemcell Technologies Pneumacult ALI medium. The medium described in 1) performed better than the other growth media tested.

[0085] Seeding optimization: Intermittent and continuous agitation seeding were tested to improve the adhesion efficiency of AT2 cells to microcarriers. Intermittent agitation was found to improve AT2 adhesion, as determined by viability / death staining at 18 hours.

[0086] protocol Microcarrier Preparation: First, the microcarriers were prepared, followed by seeding. Microcarrier preparation involved adding 0.2 grams of Percell CultiSpher GL microcarriers to a 250 mL spinner flask. The microcarriers were then hydrated in 100–150 mL of Dulbecco's phosphate-buffered saline (DPBS) for at least 1 hour (ranging from 1 hour to overnight), and sterilized by autoclaving in the spinner flask. The DPBS was aspirated, and then the microcarriers were washed with DPBS. The DPBS was then replaced with 100 mL of cell culture medium. The spinner flask was transferred to 37°C and 5% CO2 and incubated. Equilibrium was achieved by stirring on a stirring plate in the incubator at 32 RPM for approximately 2 hours. After seeding, culture medium could be added until the total culture volume was reached.

[0087] AT2 cell seeding: In one example, after equilibration, 5,000-10,000 cells / cm³ were seeded in the cell culture medium. 2 AT2 cells were added to each spinner flask and seeded for 18 hours using intermittent stirring. Stirring was performed at the following intervals: ON time: 5 minutes, stirring: 32 RPM, OFF time: 30 minutes. In this exemplary embodiment of the method, approximately 31 cycles were completed. After approximately 18 hours, continuous stirring was started at approximately 32 RPM for the remainder of the culture period.

[0088] Nutrient supply to the culture: The culture was supplied with nutrients approximately every 2 to 4 days, and metabolic samples were collected daily and after nutrient supply. Nutrient supply was performed in a biological safety cabinet after sufficient time had elapsed to allow the microcarriers to precipitate.

[0089] Culture monitoring: Approximately 2 mL of culture medium sample was taken using a syringe and passed through a NOVA Flex2 bioanalyzer. The sample was analyzed for at least pH, glucose, lactate, glutamine, and ammonium levels. The spinner flask was then returned to the stirring plate.

[0090] Next, samples were collected, and the LIVE / DEAD® assay was performed approximately every 2-3 days throughout the culture. Microcarriers were suspended in a spinner flask in a biological safety cabinet. Approximately 0.5 mL of sample was taken from the microcarriers and culture medium and added to a microcentrifuge tube. The microcarriers were then stained according to the manufacturer's protocol (ThermoFisher LIVE / DEAD® Cell Imaging Kit, product number R37601), and imaging was performed to evaluate cell density and coverage.

[0091] Alveolar epithelial cell recovery: In exemplary embodiments of the claimed method, AT2 cells were recovered from microcarriers. Recovery date was based on the density assessment of the accompanying 2D cell culture and live / death imaging assay (typically around 13–16 days of culture). Microcarriers were allowed to settle, and almost all of the medium was aspirated from each spinner. The spinner flasks were washed twice with 200 mL of DPBS, with almost all of the DPBS aspirated each time. Approximately 150 mL of approximately 0.25% trypsin was then added to each spinner. The spinners were then returned to the incubator and stirred at approximately 32 RPM for approximately 15–20 minutes. The cell solution was then collected in approximately 50 mL of centrifuge tubes, and each spinner was rinsed with approximately 50 mL of DPBS containing 2% fetal bovine serum (FBS). The rinse was then collected in centrifuge tubes. The cells were centrifuged at approximately 300 × g for approximately 15 minutes. Next, the supernatant was aspirated, and each sample was resuspended in 20 mL of DPBS, and the cells were counted.

[0092] If the culture is continued over subsequent subculturings after harvesting, each step of this protocol can be repeated in the subsequent subculturings.

[0093] result The protocol described herein demonstrates the feasibility of culturing AT2 cells on microcarriers in a glass spinner flask of approximately 250 mL by culturing AT2 cells over three passages. As shown in Figure 1, the increase in the number of AT2 cells on the microcarriers was clearly evident in all three passages, indicating successful adhesion and growth of AT2 cells on the microcarriers.

[0094] As illustrated by the graph in the upper panel of Figure 2, HT2-280 expression, a marker used to identify AT2 cells, was better maintained in three-dimensional culture on microcarriers compared to standard two-dimensional culture in two trials. Figure 2 also shows that overgrowth of contaminating airway basal cells (CK5+), which has been one of the biggest obstacles to AT2 proliferation to date, was slower in AT2 cell culture on microcarriers in two trials. Overgrowth of airway basal cells was observed within 1-2 passages compared to AT2 cells cultured in two-dimensional culture.

[0095] Figure 3 shows that AT2 function was better maintained on the microcarrier than in standard two-dimensional culture, as evidenced by the better maintenance of SP-C expression in the first passage of the culture. These results are compared to standard two-dimensional culture, where SP-C expression is typically lost within the first few days of culture.

[0096] [Example 2] AT2 cell proliferation was performed more than 50 times in 250 mL spinner flasks. Cells from 11 different lung donors were tested. Figure 4A illustrates an overview of all spinner flask AT2 microcarrier proliferations performed in 250 mL spinner flasks, separated by passage. The same number of trials were performed from fresh isolated cells and pre-frozen cells. Figures 4B-4C illustrate the AT2 magnification change (average 6.6) and average in-process cell number for 250 mL spinner flask passage 0 proliferation trials performed using Percell Cultispher GL microcarriers in O-WREKT medium. Figure 4D shows the in-process cell number and proliferation characteristics (AT2 magnification change, population doubling level, and population doubling time) from 250 mL spinner flask proliferations performed for 5 passages (passages 0-4) on Percell Cultispher GL microcarriers in O-WREKT medium. This data demonstrates substantial growth over three passages from passage 0 to passage 3. As described above, O-WREKT medium contains DMEM / F-12 medium, GlutaMAX® at a concentration of 2.5 mM, approximately 5% FBS, approximately 2 μM A-83-01, approximately 1 μM DHM1, approximately 2 μM CHIR99021, approximately 50 ng / mL EGF, approximately 50-100 ng / mL KGF, approximately 10 μM Y27632, and approximately 100 μg / mL Primocin®.

[0097] [Example 3] Figure 5 illustrates an exemplary bioreactor growth process. In some embodiments, AT2 cells were isolated and purified from human donor lung tissue. Next, microcarriers were prepared for cell seeding. Microcarrier preparation involved adding 10 grams of Percell CultiSpher GL microcarriers to a bottle. The microcarriers were then hydrated in 2–3 L of Dulbecco's phosphate-buffered saline (DPBS) for at least 1 hour (ranging from 1 hour to overnight) and autoclaved for sterilization. The DPBS was then removed and replaced with 4.5 L of cell culture medium. The microcarriers were transferred to a bioreactor. Equilibration was achieved by stirring at approximately 40 RPM for approximately 2 hours.

[0098] AT2 cell seeding: In one example, after equilibration, 550e in cell culture medium. 6 Individual AT2 cells were added to a bioreactor and seeded for 18 hours using intermittent stirring. Stirring was performed at the following intervals: ON time: 5 minutes, stirring: 42 RPM, OFF time: 30 minutes. In this exemplary embodiment of the method, approximately 32 cycles were completed. After approximately 18 hours, continuous stirring was started at approximately 39 RPM for the remainder of the culture period. After seeding, culture medium can be added until the total culture volume is reached.

[0099] Nutrient supply to the culture: The culture was supplied with nutrients approximately every two days, and metabolic samples were collected daily and after nutrient supply. Nutrient supply was performed after sufficient time had elapsed to allow the microcarriers to precipitate.

[0100] Culture monitoring: In-process counting and probes were used to measure one or more of the following: pH, glucose, lactate, glutamine, ammonium, dissolved oxygen level, or biocapacitance.

[0101] Alveolar epithelial cell recovery: In an exemplary embodiment of the claimed method, AT2 cells were recovered from microcarriers. The microcarriers were allowed to settle, and almost all of the culture medium was removed from the bioreactor. The microcarriers were washed once with 5 L of DPBS to remove almost all of the DPBS. Approximately 3–4 L of approximately 0.25% trypsin was then added to the bioreactor and stirred at approximately 39 RPM for approximately 15–45 minutes. The cell solution was then collected in a sterile bioprocess container. The bioreactor was rinsed with approximately 1 L of DPBS containing 5% fetal bovine serum (FBS). The rinse was then collected in the same bioprocess container and subsequently transferred to a centrifuge tube. The cells were centrifuged at approximately 300 × g for approximately 15 minutes. The supernatant was then aspirated, and each sample was resuspended in approximately 1 L of DPBS, and the cells were counted.

[0102] If the culture is continued over subsequent subculturings after harvesting, each step of this protocol can be repeated in the subsequent subculturings.

[0103] Figures 6A and 6B illustrate growth profiles at various scales (spinner flask and bioreactor) and at various subculturing stages in the bioreactor.

[0104] Figure 7 illustrates the bioreactor growth measurement indicators from subgeneration 0 to subgeneration 2.

[0105] Figure 8 illustrates the bioreactor proliferation phenotypic analysis. HT2-280 and SP-C were maintained across three passages. No CK5+ basal cell or CD90+ stromal cell overgrowth was observed.

[0106] Figure 9 illustrates an exemplary process scale-up / out for use in one or more of the methods described herein.

[0107] While certain embodiments have been illustrated and described, it should be understood that changes and modifications thereto are possible in accordance with the ordinary art in the art without departing from the broader embodiments of the art as defined in the following claims.

[0108] Embodiments described herein as illustrative examples can be suitably implemented in the absence of any one or more elements or limitations, even if they are not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are descriptive rather than restrictive, and in using such terms and expressions, there is no intention to exclude any equivalents of the exhibited or described features or parts thereof, and it should be recognized that various modifications are possible within the scope of the claimed technology. In addition, the phrase “essentially consisting of” should be understood to include the specifically described elements and any further elements that do not substantially affect the fundamental and novel features of the claimed technology. The phrase “consisting of” excludes any elements not explicitly stated.

[0109] This disclosure should not be limited in terms of the specific embodiments described in this application. As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope of this disclosure. In addition to those enumerated herein, functionally equivalent methods and compositions within the scope of this disclosure will also be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the appended claims. This disclosure should be limited only by the terminology of the appended claims and the entire scope of equivalents granted by such claims. It should be understood that this disclosure is not limited to any particular methods, reagents, compounds, or compositions, and that they are naturally modifiable. It should also be understood that the terminology used herein is intended solely to describe and not to limit specific embodiments.

[0110] In addition, if any feature or aspect of the present disclosure is described in terms of a Markush group, a person skilled in the art will recognize that the present disclosure is also described in terms of any individual element or subgroup of elements of the Markush group.

[0111] As will be understood by those skilled in the art, for all purposes, and especially in terms of providing written explanations, all scopes disclosed herein also encompass all possible sub-scopes and combinations thereof, including endpoints. Any scope enumerated can be readily recognized as sufficiently describing and acknowledging that the same scope can be divided into at least two, three, four, five, ten, etc. As a non-limiting example, each scope discussed herein can readily be divided into a lower third, a middle third, an upper third, etc. As will be further understood by those skilled in the art, all terms such as “maximum,” “at least,” “greater than,” and “less than” refer to scopes that include the stated number and can subsequently be divided into the sub-scopes discussed above. Finally, as will be understood by those skilled in the art, a scope includes each individual element.

[0112] All publications, patent applications, granted patents, and other documents referenced herein are incorporated by reference in such a way that it is specifically and individually indicated that each individual publication, patent application, granted patent, or other document in its entirety is incorporated by reference. Any definitions contained in the text incorporated by reference that conflict with the definitions in this disclosure are excluded.

[0113] Other embodiments are described in the following claims. This application also relates to the following aspects. (1) A method for producing alveolar epithelial cells, A step of preparing multiple three-dimensional substrates in a cell culture vessel, A step of seeding multiple alveolar epithelial cells, wherein the seeding includes combining the three-dimensional substrate and the alveolar epithelial cells in the cell culture vessel and creating conditions suitable for enabling the cells to adhere to the three-dimensional substrate and realizing suspension culture. A step of promoting the growth of alveolar epithelial cells on or within the three-dimensional substrate, The steps include monitoring the culture with respect to cell proliferation, and A step of collecting multiple alveolar epithelial cells from the three-dimensional substrate. Methods that include... (2) The method according to (1) above, wherein the alveolar epithelial cells include type II alveolar epithelial (AT2) cells. (3) The method according to (1) or (2) above, wherein the alveolar epithelial cells include human type II alveolar epithelial cells (AT2). (4) The method according to (1) above, wherein the three-dimensional substrate comprises at least one of non-porous, microporous, or macroporous three-dimensional substrates. (5) The method according to (4) above, wherein the alveolar epithelial cells are cultured on or inside the three-dimensional substrate, or both on and inside. (6) The method according to (4) or (5) above, wherein the three-dimensional substrate includes a plurality of microcarriers. (7) The method according to (1) above, wherein the cell culture vessel includes a spinner flask or a bioreactor. (8) The method according to any one of (1) to (7) above, wherein the three-dimensional substrate accounts for approximately 1 to 2 mg / mL. (9) The method according to (1) above, further comprising adding alveolar epithelial cells in a cell culture medium to the cell culture vessel for seeding. (10) The method according to (9) above, further comprising sowing and stirring the culture. (11) The method according to (10) above, wherein the three-dimensional substrate culture is stirred at approximately 20 RPM or more in the cell culture vessel. (12) The method according to (10) above, wherein the stirring includes a cycle in which stirring is performed at a rate of 20 RPM or higher for about 5 minutes, followed by a period of no stirring for about 30 minutes, and the cycle is repeated about 31 times. (13) The method according to (12) above, wherein after approximately 18 hours, the culture is continuously stirred at approximately 20 RPM or more for the remainder of the culture period. (14) The method according to (1) above, wherein monitoring includes supplying nutrients to the culture, performing at least one viability / death assay on the culture, evaluating the results of measurements of pH, glucose, lactate, glutamine, ammonium, and / or dissolved oxygen levels, and / or biocapacitance, evaluating the cell coverage on the three-dimensional substrate, or a combination thereof. (15) The method according to (14) above, wherein the culture is supplied with nutrients at intervals of approximately 2 to 4 days, and metabolic samples are collected daily and / or after nutrient supply. (16) The method according to (15) above, wherein the sample is monitored and maintained to determine at least one of the following: pH, glucose, lactic acid, glutamine, ammonium, and / or dissolved oxygen level, and / or biocapacitance. (17) The method according to (14) above, wherein a live / death assay is performed to monitor and maintain the culture. (18) The method according to (10), wherein the step of recovering a plurality of alveolar epithelial cells from the three-dimensional substrate further comprises: precipitating the three-dimensional substrate and removing a certain amount of culture medium from the cell culture vessel; washing the cell culture vessel; adding a certain amount of agent to detach the cells from the three-dimensional substrate; stirring the cell culture vessel; collecting the cell solution into a centrifuge tube; rinsing the cell culture vessel; collecting a certain amount of rinse solution from the cell culture vessel; centrifuging the certain amount of rinse solution; aspirating the supernatant of the rinse solution; and resuspending any sample in phosphate-buffered saline, and further, the recovery is carried out during about 10 to about 18 days of culture. (19) The method according to any one of (10) to (18) above, wherein the recovered cells are seeded onto a new three-dimensional substrate and cultured further, or cryopreserved. (20) The method according to (18) above, wherein the agent for detaching the cells from the three-dimensional substrate comprises at least one detachment enzyme, optionally trypsin or tryp-LE. (21) The method according to (1) above, wherein the plurality of recovered alveolar epithelial cells express surfactant protein C precursor (pSP-C). (22) The method according to (1) above, wherein the plurality of recovered alveolar epithelial cells lose 25% or less of pSP-C expression after culturing for up to approximately 40 days. (23) The method according to (1) above, wherein the plurality of recovered alveolar epithelial cells include a population having more than 30% pSP-C expression after a maximum of approximately 40 days and more than 0% pSP-C expression after a total of approximately 28 days of culture. (24) The method according to (1) above, wherein the plurality of recovered alveolar epithelial cells express HT2-280. (25) The method according to (1) above, wherein the plurality of recovered alveolar epithelial cells do not express excessive CK5 or contain overgrowth of airway basal cells. (26) The method according to any one of (1) to (25) above, wherein the alveolar epithelial cells are cultured for one or more passages, two or more passages at the discretion of the person, three or more passages, four or more passages, five or more passages, or six or more passages. (27) The method according to any one of (1) to (26) above, wherein the alveolar epithelial cells collected from a single passage increase by 1x or more, and optionally by 2x, 3x, 4x, 5x, 6x, 6.5x, 6.6x, 7x, 8x, 9x, 10x, 11x, 12x, 15x, or 20x or more. (28) The method according to (6) above, wherein the microcarriers have a hardness between approximately 1 kPa and approximately 100 kPa. (29) The method according to (6) above, wherein the microcarriers have a hardness of approximately 4 kPa. (30) TGF-β pathway inhibitors, Wnt pathway activators, ROCK inhibitors, epidermal growth factor (EGF), Keratinocyte growth factor (KGF), and Bovine fetal serum A cell culture medium composition for culturing alveolar epithelial cells, comprising: (31) The composition according to (30) above, wherein the TGF-β pathway inhibitor accounts for approximately 1 μM to approximately 10 μM. (32) The composition according to (30) above, wherein the Wnt pathway activator accounts for approximately 1 μM to approximately 10 μM. (33) The composition according to (30) above, wherein the ROCK inhibitor accounts for approximately 1 μM to approximately 10 μM. (34) The composition according to (30) above, wherein the EGF is approximately 25 ng / mL to approximately 200 ng / mL. (35) The composition according to (30) above, wherein the KGF is present in a concentration of approximately 25 ng / mL to approximately 200 ng / mL. (36) The composition described in (30) above, wherein fetal bovine serum accounts for approximately 1% to approximately 10% by volume (v / v). (37) The composition according to (30) above, wherein the TGF-β inhibitor comprises at least one of A-83-01 or DMH1. (38) The composition according to (30) above, wherein the Wnt pathway activator comprises CHIR99021. (39) The composition according to (30) above, wherein the ROCK inhibitor comprises Y27632. (40) The composition according to (30) above, further comprising a plurality of three-dimensional substrates. (41) The composition according to (40) above, wherein the three-dimensional substrate comprises at least one of a plurality of non-porous, microporous, or macroporous three-dimensional substrates. (42) The composition according to (40) above, wherein the microporous three-dimensional substrate further comprises microcarriers. (43) The method according to any one of the above (1 to 29), wherein the alveolar epithelial cells are cultured using the cell culture medium composition described in (30) to (42) above. (44) A kit comprising multiple alveolar epithelial cells obtained by the method described in (1) to (29) above, or using the cell culture medium composition described in (30) to (42) above.

Claims

1. A method for producing alveolar epithelial cells, A step of preparing multiple three-dimensional substrates in a cell culture vessel, A step of seeding a plurality of alveolar epithelial cells, wherein the seeding includes combining the three-dimensional substrate and the alveolar epithelial cells in the cell culture vessel and creating conditions suitable for enabling suspension culture by allowing the cells to adhere to the three-dimensional substrate, wherein the alveolar epithelial cells include type II alveolar epithelial (AT2) cells, and the three-dimensional substrate includes microcarriers having a diameter in the range of 25 μm to 500 μm; a step of promoting the growth of the alveolar epithelial cells on or inside the three-dimensional substrate, wherein the alveolar epithelial cells are cultured in suspension using a cell culture medium composition containing a TGF-β pathway inhibitor, a Wnt pathway activator, a ROCK inhibitor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), and fetal bovine serum; a step of monitoring cell proliferation; and a step of recovering a plurality of alveolar epithelial cells from the three-dimensional substrate. method.

2. The method according to claim 1, wherein the microcarrier is a microcarrier bead.

3. The alveolar epithelial cells include human type II alveolar epithelial cells (AT2), according to claim 1 or 2. The method.

4. The three-dimensional substrate is one of the non-porous, microporous, or macroporous three-dimensional substrates. The method according to claim 1, comprising at least one of the above.

5. The alveolar epithelial cells are cultured on or inside the three-dimensional substrate, or both on and inside. The method according to claim 4.

6. The method according to claim 4 or 5, wherein the three-dimensional substrate includes a plurality of microcarriers.

7. The cell culture vessel comprises a spinner flask or a bioreactor, as described in claim 1. The method.

8. The three-dimensional substrate comprises 1 to 2 mg / mL, as described in any one of claims 1 to 7. method.

9. The seeding further includes adding alveolar epithelial cells in the cell culture medium to the cell culture vessel. The method according to claim 1.

10. The method according to claim 9, further comprising stirring the cell culture medium for seeding.

11. Claim 1, wherein the cell culture medium is stirred at 20 RPM or more in the cell culture vessel. The method described in 0.

12. If the aforementioned stirring is performed at 20 RPM or higher for 5 minutes, and then no stirring continues for 30 minutes... The cycle described in claim 10 is further repeated 31 times, including the said cycle. method.

13. After 18 hours, the cell culture medium is then maintained at 20 RPM or higher for the remainder of the culture period. The method according to claim 12, wherein the mixture is continuously stirred.

14. The aforementioned monitoring step is Perform a viability / death assay on the cell culture medium at least once, and check the pH and glucose level. , lactic acid, glutamine, ammonium, and / or dissolved oxygen levels, and / or This involves monitoring by evaluating the measurement results of biocapacitance. This can be monitored by evaluating the cell coverage rate on the three-dimensional substrate, or The method according to claim 1, which includes monitoring by evaluating the combination. 。

15. The cell culture medium is supplied with nutrients at intervals of 2 to 4 days, and metabolic samples are supplied daily and / or The method according to claim 14, wherein the sample is taken after nutrient supply.

16. The sample contains pH, glucose, lactic acid, glutamine, ammonium, and / or dissolved Determine at least one of the following: oxygen level and / or biocapacitance. The method according to claim 15, which is monitored and maintained for the purpose of monitoring and maintaining.

17. A viability / death assay is performed to monitor and maintain the cell culture medium. The method according to claim 14.

18. The step of recovering multiple alveolar epithelial cells from the three-dimensional substrate is, The three-dimensional substrate is precipitated, and a certain amount of culture medium is removed from the cell culture vessel. Washing the cell culture vessel, and a certain amount of agent to detach the cells from the three-dimensional substrate. Adding Stirring the cell culture vessel, Collect the cell solution in a centrifuge tube. Rinse the cell culture vessel. Collect a certain amount of rinse solution from the cell culture vessel. To centrifuge a certain amount of the rinse solution, The supernatant of the rinse solution is to be aspirated, and The method further includes resuspending the sample in phosphate-buffered saline, and further recovering the sample after 10 to 10 minutes of culture time. The method according to claim 10, which is carried out within 18 days.

19. The recovered cells are either seeded onto a new three-dimensional substrate to continue culturing, or frozen. The method according to any one of claims 10 to 18, which is stored.

20. The agent that detaches the cells from the three-dimensional substrate comprises at least one detachment enzyme, optional The method according to claim 18, comprising, optionally, trypsin or tryp-LE.

21. The aforementioned multiple recovered alveolar epithelial cells are surfactant protein C precursor (pSP) The method according to claim 1, wherein -C) is expressed.

22. The aforementioned multiple recovered alveolar epithelial cells showed a pSP-C development rate of 25% or less after culturing for up to 40 days. The method according to claim 1, which loses its existence.

23. Of the multiple recovered alveolar epithelial cells, more than 30% remained after a maximum of 40 days, and a total of 28 days. The method according to claim 1, comprising a population having more than 0% pSP-C expression after culturing.

24. The method according to claim 1, wherein the plurality of recovered alveolar epithelial cells express HT2-280. Law.

25. The aforementioned multiple recovered alveolar epithelial cells also express excessive CK5, and airway basal cells The method according to claim 1, which does not include overgrowth.

26. The alveolar epithelial cells undergo one or more passages, or two or more passages at the discretion of the user, or three or more passages. Cultured for the above subgeneration, four or more subgenerations, five or more subgenerations, or six or more subgenerations. The method according to any one of claims 1 to 25.

27. The alveolar epithelial cells collected from a single passage increase by more than 1x, or optionally 2x, 3x, or 4x. times, 5 times, 6 times, 6.5 times, 6.6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 1 The method according to any one of claims 1 to 26, which increases by five times or more, or by twenty times or more.

28. The microcarriers have a hardness between 1 kPa and 100 kPa, as described in claim 6. The method.

29. The method according to claim 6, wherein the microcarriers have a hardness of 4 kPa.

30. The method according to claim 1, wherein the TGF-β pathway inhibitor accounts for 1 μM to 10 μM.

31. The method according to claim 1, wherein the Wnt pathway activator accounts for 1 μM to 10 μM.

32. The method according to claim 1, wherein the ROCK inhibitor accounts for 1 μM to 10 μM.

33. The method according to claim 1, wherein the EGF is 25 ng / mL to 200 ng / mL.

34. The method according to claim 1, wherein the KGF is present in a concentration of 25 ng / mL to 200 ng / mL.

35. The method according to claim 1, wherein fetal bovine serum accounts for 1% to 10% by volume (v / v).

36. The method according to claim 1, wherein the TGF-β inhibitor comprises at least one of A-83-01 or DMH1.

37. The method according to claim 1, wherein the Wnt pathway activator comprises CHIR99021.

38. The method according to claim 1, wherein the ROCK inhibitor comprises Y27632.

39. The three-dimensional substrate is a plurality of non-porous, microporous three-dimensional substrates, or macroporous tertiary substrates. The method according to claim 1, comprising at least one of the original substrates.

40. The method according to claim 39, wherein the microporous three-dimensional substrate further comprises microcarriers.

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