Chemically defined airway basal stem cell culture medium

Abstract

Disclosed is a chemically defined airway basal stem cell culture medium. The provided culture medium involves a simple preparation method and high safety, and does not require a feeder layer or a matrigel-coated culture plate. By means of the culture medium, primary cultured airway basal stem cells achieve good stability; and after multiple passages, the normal morphology of the basal stem cells can be maintained and good differentiation potential can be maintained. In addition, the culture medium has clear components, and can meet both scientific research-grade and clinical-grade applications of airway basal stem cells.

Description

A culture medium for airway basal stem cells with clearly defined chemical composition Technical Field

[0001] This invention relates to the field of stem cell technology, and more specifically to a culture medium for airway basal stem cells with a clearly defined chemical composition. Background Technology

[0002] Basal cells (BC) are commonly referred to as human bronchial epithelial cells (HBEC). 1 Located in the basal layer of the airway epithelium, airway basal stem cells have the ability to self-renew and differentiate into various airway epithelial cells such as ciliated cells, goblet cells, and rod cells. Airway basal stem cells play an important role in maintaining airway epithelial homeostasis and repairing damage, and are a very promising functional cell for the treatment of respiratory diseases. In addition, airway basal stem cells can also serve as a very useful tool for airway development research and disease model construction. Whether for clinical treatment or scientific research, it is necessary to achieve stable culture and expansion of airway basal stem cells in vitro. However, the currently commonly used culture systems have many defects, such as: (1) requiring animal cells (such as mouse embryonic fibroblasts) as a feeder layer to inoculate airway basal stem cells onto feeder layer cells for co-culture. 2,3 This method has the potential for exogenous contamination and the culture process is complex, which greatly increases the culture time and cost of airway basal stem cells. (2) Using components with unclear chemical composition, such as serum, pituitary extract or platelet lysate, for culture can lead to batch-to-batch quality variations and poor reproducibility of experimental results, which is not only detrimental to scientific research but also limits the future clinical application of cell-based therapy. (3) A few commercially available serum-free and pituitary extract-free culture media with clearly defined components have not disclosed their formulations and cannot stably culture airway basal stem cells for a long time. As the number of culture passages increases, the cell division ability will decrease significantly, the morphological changes will be abnormally obvious, and the cell heterogeneity will be very high.

[0003] Therefore, developing a culture medium with clearly defined chemical components, such as serum-free, pituitary extract-free, and platelet lysate-free, that can stably culture human airway basal stem cells and achieve culture without feeder layers and matrix gel assisted culture, has significant research value and good commercial prospects. Summary of the Invention

[0004] The first aspect of the present invention is to provide a composition for culturing airway basal stem cells.

[0005] The second aspect of the present invention aims to provide the application of the composition of the first aspect of the present invention.

[0006] A third aspect of the present invention is to provide a culture medium for culturing airway basal stem cells.

[0007] The fourth aspect of this invention is to provide an application of the culture medium of the third aspect of this invention.

[0008] The fifth aspect of this invention aims to provide a method for culturing airway basal stem cells.

[0009] The sixth aspect of this invention is to provide an airway basal stem cell preparation.

[0010] The seventh aspect of this invention aims to provide a reagent kit.

[0011] In a first aspect, the present invention provides a composition for culturing airway basal stem cells, comprising: EGF, FGF10 and a γ-secretase inhibitor.

[0012] In some embodiments, the composition does not contain serum, pituitary extract (preferably bovine pituitary extract), and / or platelet lysate.

[0013] In some embodiments, the working concentration of the EGF is 0.1-50 ng / mL; further, 0.1-1.2 ng / mL; even further, 0.1-1.1 ng / mL; and still further, 0.9-1.1 ng / mL.

[0014] In some embodiments, the EGF content in the composition is sufficient to make its working concentration in the range of 0.1-50 ng / mL, for example: 0.1-1.2 ng / mL, 0.1-1.1 ng / mL, or 0.9-1.1 ng / mL.

[0015] In some embodiments, the working concentration of FGF10 is 0.04-100 ng / mL; further, 0.04-2.5 ng / mL; and even further, 0.3-0.5 ng / mL.

[0016] In some embodiments, the content of FGF10 in the composition is sufficient to make its working concentration in the range of 0.04-100 ng / mL, for example: 0.04-2.5 ng / mL, or 0.3-0.5 ng / mL.

[0017] In some embodiments, the working concentration of the γ-secretase inhibitor is 0.5-5 μM; further, 0.5-1.5 μM; even further, 0.8-1.2 μM; and still further, 0.9-1.1 μM.

[0018] In some embodiments, the composition contains an amount of γ-secretase inhibitor sufficient to make its working concentration in the range of 0.5-5 μM, for example: 0.5-1.5 μM, 0.8-1.2 μM, or 0.9-1.1 μM.

[0019] In some embodiments, the γ-secretase inhibitor comprises at least one of Dibenzazepine (DBZ), DAPT, LY-411575, CompoundE, RO4929097 (RG-4733), PF03084014, Crenigacestat (LY3039478), BMS906024, Nirogacestat, and MK-0752; further comprising DAPT.

[0020] In some embodiments, the composition further comprises a ROCK inhibitor.

[0021] In some embodiments, the working concentration of the ROCK inhibitor is 5-15 μM; further, 8-12 μM; even further, 9-11 μM; and still further, 9-10 μM.

[0022] In some embodiments, the composition contains a sufficient amount of ROCK inhibitor to achieve a working concentration in the range of 5-15 μM, for example: 8-12 μM, 9-11 μM, or 9-10 μM.

[0023] In some embodiments, the ROCK inhibitor comprises at least one of Y-27632, ZINC00881524, Thiazovivin, GSK429286A, hydroxyfasudil (HA-1100), HA100, H-1152, Blebbistatin, HA-1077, KD-025, Y-33075, and Narciclasine; more particularly Y-27632.

[0024] In some embodiments, the composition further comprises a BMP inhibitor.

[0025] In some embodiments, the working concentration of the BMP inhibitor is 0.5-5 μM; further, 0.8-1.2 μM; even further, 0.9-1.1 μM; and still further, 0.9-1 μM.

[0026] In some embodiments, the composition contains a sufficient amount of BMP inhibitor to achieve a working concentration in the range of 0.5-5 μM, for example: 0.8-1.2 μM, 0.9-1.1 μM, or 0.9-1 μM.

[0027] In some embodiments, the BMP inhibitor comprises at least one of DMH-1, Noggin, LDN193189, LDN212854, Dorsomorphin, K02288, follistatin, chordin, and gremlin; further comprising DMH-1.

[0028] In some embodiments, the composition further comprises: a SMAD inhibitor.

[0029] In some embodiments, the working concentration of the SMAD inhibitor is 0.5-10 μM; further, 0.8-1.2 μM; even further, 0.9-1.1 μM; and still further, 0.9-1 μM.

[0030] In some embodiments, the composition contains an amount of SMAD inhibitor sufficient to achieve a working concentration in the range of 0.5-10 μM, for example: 0.8-1.2 μM, 0.9-1.1 μM, or 0.9-1 μM.

[0031] In some embodiments, the SMAD inhibitor comprises at least one of A-83-01, SB-431542, LY364947, and SB505124; more specifically, A-83-01.

[0032] In some embodiments, the composition comprises the following components: EGF, FGF10, γ-secretase inhibitor, ROCK inhibitor, BMP inhibitor, and SMAD inhibitor.

[0033] A second aspect of the invention provides the use of the composition of the first aspect of the invention in a1) or a2);

[0034] a1) Culture airway basal stem cells;

[0035] a2) Prepare a culture medium for culturing airway basal stem cells.

[0036] A third aspect of the invention provides a culture medium for culturing airway basal stem cells, comprising the composition of the first aspect of the invention.

[0037] In some embodiments, the culture medium is a basal culture medium containing the composition of the first aspect of the present invention.

[0038] In some embodiments, the concentration of EGF in the culture medium (i.e., the culture medium for culturing or expanding airway basal stem cells) is 0.1-50 ng / mL; further, 0.1-1.2 ng / mL; even further, 0.1-1.1 ng / mL; and still further, 0.9-1.1 ng / mL.

[0039] In some embodiments, the concentration of FGF10 in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.04-100 ng / mL; further, 0.04-2.5 ng / mL; and even further, 0.3-0.5 ng / mL.

[0040] In some embodiments, the concentration of the γ-secretase inhibitor in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.5-5 μM; further, 0.5-1.5 μM; even further, 0.8-1.2 μM; and still further, 0.9-1.1 μM.

[0041] In some embodiments, the concentration of the ROCK inhibitor in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 5-15 μM; further, 8-12 μM; even further, 9-11 μM; and still further, 9-10 μM.

[0042] In some embodiments, the concentration of the BMP inhibitor in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.5-5 μM; further, 0.8-1.2 μM; even further, 0.9-1.1 μM; and still further, 0.9-1 μM.

[0043] In some embodiments, the concentration of the SMAD inhibitor in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.5-10 μM; further, 0.8-1.2 μM; even further, 0.9-1.1 μM; and still further, 0.9-1 μM.

[0044] In some embodiments, the basal culture medium is a basal culture medium containing supplementary components, which include: B27, serum albumin, glutamine, and L-ascorbic acid.

[0045] In some embodiments, the concentration of B27 in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.5-1.5×; further, 0.8-1.2×; further, 0.9-1.1×.

[0046] In some embodiments, the serum albumin is human serum albumin.

[0047] In some embodiments, the concentration of serum albumin in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.05%-1% by weight / volume ratio (w / v, g / mL); further, 0.05%-0.15%; even further, 0.08%-0.12%; and still further, 0.09%-0.11%.

[0048] In some embodiments, the glutamine is GlutaMAX.

[0049] In some embodiments, the concentration of glutamine in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 1-6 mM; further, 1-3 mM; even further, 1.5-2.5 mM; and still further, 1.8-2.2 mM.

[0050] In some embodiments, the concentration of L-ascorbic acid in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 20-300 μg / mL; further, 40-60 μg / mL; and even further, 45-55 μg / mL.

[0051] In some embodiments, the supplemental ingredient further comprises: thioglycerol (i.e., 1-thioglycerol) and hydrocortisone.

[0052] In some embodiments, the concentration of the thioglycerol in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.1-1 mM; further, 0.2-0.6 mM; and even further, 0.3-0.5 mM.

[0053] In some embodiments, the concentration of hydrocortisone in the culture medium (i.e., the culture medium for culturing airway basal stem cells) is 0.5-15 μg / mL; further, 0.5-5 μg / mL; even further, 0.8-1.2 μg / mL; and even further, 0.9-1.1 μg / mL.

[0054] In some embodiments, the supplemental ingredient comprises the following components: B27, serum albumin, glutamine, L-ascorbic acid, thioglycerol, and hydrocortisone.

[0055] In some embodiments, the basal culture medium comprises at least one of DMEM / F12 medium, Basal Medium Eagle (BME) medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, Advanced RPMI 1640 medium, and Advanced DF-12 medium; further comprises IMDM medium and Ham's F12 medium; and even further comprises IMDM medium and Ham's F12 medium.

[0056] In some embodiments, the volume ratio of the IMDM medium to Ham's F12 medium is 1:(0.5-1.5); further, 1:(0.8-1.2); and even further, 1:(0.9-1.1).

[0057] In some embodiments, the culture medium for culturing airway basal stem cells consists of the following components: the composition of the first aspect of the invention, and a basal culture medium containing supplementary components.

[0058] In some embodiments, the culture medium for culturing airway basal stem cells consists of the following components: the composition of the first aspect of the invention, supplementary ingredients, and basal culture medium (i.e., basal culture medium without supplementary ingredients).

[0059] A fourth aspect of the present invention provides the use of the culture medium of the third aspect of the present invention in culturing airway basal stem cells.

[0060] A fifth aspect of the present invention provides a method for culturing airway basal stem cells, comprising the step of using a composition of the first aspect of the present invention or a culture medium of the third aspect of the present invention.

[0061] In some embodiments, the method includes the step of culturing airway basal stem cells using the culture medium of the third aspect of the present invention.

[0062] In some embodiments, the culture conditions are 33–40°C and 3–7% CO2; more specifically, 36–38°C and 4–6% CO2.

[0063] A sixth aspect of the present invention provides an airway basal stem cell preparation comprising: airway basal stem cells; and

[0064] The composition of the first aspect of the present invention or the culture medium of the third aspect of the present invention.

[0065] In some embodiments, the airway basal stem cells are obtained by the method of the fifth aspect of the present invention.

[0066] A seventh aspect of the invention provides a kit comprising: a composition using the first aspect of the invention, a culture medium using the third aspect of the invention, or an airway basal stem cell preparation using the sixth aspect of the invention.

[0067] In this invention:

[0068] In some embodiments, the airway basal stem cells are derived from animals.

[0069] In some embodiments, the animal includes mammals; further includes at least one of humans and non-human mammals; further includes humans.

[0070] In some embodiments, the non-human mammal includes at least one of non-human primates, rodents, cattle, pigs, sheep, dogs, rabbits, cats, and horses; more specifically, rodents.

[0071] In some embodiments, the non-human primates include at least one of orangutans, apes, and monkeys.

[0072] In some embodiments, the rodent includes at least one of mice, rats, hamsters, and guinea pigs.

[0073] In some embodiments, the airway basal stem cells are primary airway basal stem cells.

[0074] In some embodiments, the airway basal stem cells comprise at least one of bronchial epithelial cells and small respiratory tract epithelial cells; further comprising at least one of human bronchial epithelial cells and human small respiratory tract epithelial cells.

[0075] In some embodiments, the culture includes amplification or proliferation.

[0076] In some embodiments, the culture of airway basal stem cells does not require a feeder layer and / or matrix gel-assisted culture.

[0077] This invention provides a composition for culturing airway basal stem cells, which exhibits excellent cell stability. After multiple passages, the cells maintain uniform morphology and minimal change in average diameter, thus preserving the normal morphology and good differentiation potential of basal stem cells. The composition is simple to prepare, free of pathogenic microorganisms and allergens, and has high safety. It does not require a feeder layer or substrate-coated culture plate.

[0078] Furthermore, the composition has a clearly defined composition and does not contain components with unclear chemical composition such as serum, pituitary extract (preferably bovine pituitary extract), and / or platelet lysate. It can simultaneously meet the research-grade and clinical-grade applications of airway basal stem cells and can be used to culture airway basal stem cells or to prepare culture media for culturing airway basal stem cells.

[0079] The human lower respiratory tract consists of the trachea, bronchi, bronchioles of varying sizes, and alveoli. Human lung basal stem cells are distributed throughout the trachea, bronchi, and terminal bronchioles. Basal stem cells are a group of cells tightly attached to the basal layer of the airway, specifically expressing transformation-related protein 63 (TRP63 or P63), keratin 5 (KRT5), and nerve growth factor receptor (NGFR). Basal stem cells are also present in the trachea and bronchi of rats (and mice), and numerous studies have shown that their functions are similar to those of human airway basal stem cells. 4,5 The cells used in this invention are primary human airway basal stem cells, which are derived from the bronchial / tracheal epithelial and small airway epithelial (located behind the 8th bronchus, with a diameter <2mm) of the large airways (diameter >2mm). Preferably, they are human bronchial / tracheal epithelial cells (HBEC) from Lifeline Cell Technology, catalog number FC-0035; and preferably human small airway epithelial cells (HSAEC) from ATCC, catalog number PCS-301-010.

[0080] This invention provides a culture medium for human airway basal stem cells with clearly defined chemical composition, for maintaining or expanding human airway basal stem cells.

[0081] In this invention, the characteristic of the culture medium with a clearly defined chemical composition is that the composition and properties of the culture medium are clearly defined, which is conducive to cell culture, facilitates the separation and purification of products, helps to regulate cell physiological responses, and facilitates more accurate assessment of the effects of drugs on cell function.

[0082] In this invention, the basal culture medium is a culture medium that can support the growth of microorganisms without specific nutritional requirements without the addition of specific components, such as: DMEM / F12, Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow MEM, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham's medium, RPMI 1640, Fischer's medium, or a mixture thereof.

[0083] In some implementations, the invention described herein includes the following:

[0084] 1. A culture medium for airway basal stem cells, comprising a basal culture medium, supplementary components, growth factors and inhibitors, wherein the growth factors include EGF and FGF10, and the inhibitors include γ-secretase inhibitors, Smad inhibitors, BMP inhibitors and ROCK inhibitors.

[0085] 2. The airway basal stem cell culture medium described in Project 1, wherein the supplementary components include HSA, B27, GlutaMAX and L-ascorbic acid.

[0086] 3. The airway basal stem cell culture medium described in Project 1 or 2, wherein the supplementary ingredients further comprise 1-thioglycerol and hydrocortisone.

[0087] 4. The airway basal stem cell culture medium according to any one of items 1-3, wherein the growth factors comprise 0.1-1 ng / mL EGF and 0.04-2.5 ng / mL FGF10.

[0088] 5. The airway basal stem cell culture medium according to any one of items 1-4, wherein the γ-secretase inhibitor is selected from DAPT, Dibenzazepine (DBZ), LY-411575, Compound E, preferably DAPT; the Smad inhibitor is selected from A-83-01, SB-431542, LY364947, preferably A-83-01; the BMP inhibitor is selected from DMH-1, Noggin, LDN193189, LDN212854, Dorsomorphin, K02288, preferably DMH-1; the ROCK inhibitor is selected from Y-27632, fasudil (HA1077), Thiazovivin, hydroxyfasudil (HA-1100), preferably Y-27632.

[0089] 6. The airway basal stem cell culture medium according to any one of items 1-5, wherein the inhibitor comprises 1-5 μM DAPT, 1-10 μM MA-83-01, 1-5 μM DMH-1 and 5-10 μM Y-27632.

[0090] 7. The airway basal stem cell culture medium described in any one of items 1-6, wherein the basal culture medium is selected from DMEM / F12, Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow MEM, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham's medium, RPMI 1640, Fischer's medium, or a mixture thereof;

[0091] Preferably, the basal culture medium is selected from a mixture of IMDM and Ham's F12, and more preferably, the mixing ratio of IMDM and Ham's F12 is 1:1.

[0092] 8. A method for culturing airway basal stem cells, characterized in that airway basal stem cells are cultured using the airway basal stem cell culture medium described in any one of items 1-7.

[0093] 9. The airway basal stem cell culture medium of any one of items 1-7 or the method of item 8, wherein the airway basal stem cells are derived from mammals, preferably human, mouse or rat.

[0094] 10. The airway basal stem cell culture medium of any one of items 1-7 or the method of item 8, wherein the airway basal stem cells are human bronchial epithelial cells (HBEC) or human small respiratory tract epithelial cells (HSAEC).

[0095] 11. A kit comprising the airway basal stem cell culture medium described in any one of items 1-7.

[0096] In this invention, IMDM medium (Iscove's modified dulbecco's medium) is Iscove's modified DMEM medium, which is commonly used for high-density cell culture for rapid proliferation, and is preferably derived from Gibco, catalog number 12440061.

[0097] Ham's F12 nutrient medium is a serum-free medium for animal cell culture; preferably sourced from Gibco, catalog number 11765054.

[0098] B27 (50X) is a serum-free additive commonly used in neural cell culture. It can effectively maintain cell growth, and the working concentration is 1X. Preferably, B27 is sourced from Gibco, catalog number 17504044.

[0099] HSA (Human serum albumin) is the most abundant protein in blood plasma. It provides nitrogen and essential amino acids that are important for cell growth and helps maintain osmotic pressure balance in the cell culture environment. The working concentration is 0.05% to 1%. Preferably, HSA is from Sigma, catalog number A9731.

[0100] GlutaMAX additive is an alternative to L-glutamine with better stability. L-glutamine is an essential amino acid, an important component of culture medium, and the main energy source for cultured cells. The working concentration is 2-6 mM. Preferably, GlutaMAX is from Gibco, catalog number 35050061.

[0101] L-Ascorbic acid, also known as vitamin C, is a water-soluble vitamin and a highly effective antioxidant used to reduce oxidative stress in cells. It also participates in important intracellular biosynthetic processes, with a working concentration of 50–300 μg / mL. Preferably, L-Ascorbic acid is sourced from Sigma, catalog number A4544.

[0102] Monothioglycerol (MTG) has the same effect as β-mercaptoethanol in cell culture medium and is used to stimulate proliferation at a working concentration of 0.1-1 mM. Preferably, monothioglycerol is derived from Sigma, catalog number M6145.

[0103] Hydrocortisone can enhance the cell's ability to respond to harmful stimuli, promote gluconeogenesis, slow down glucose breakdown, and increase protein catabolism. The working concentration is 1-15 μg / mL. Preferably, hydrocortisone is derived from STEMCELL, catalog number 07926.

[0104] EGF (Epidermal Growth Factor) is an important endocrine cell growth factor in the human body with strong physiological activity. It exerts its effects by binding with a high affinity to the cell surface receptor—epidermal growth factor receptor (EGFR / ErbB). EGF plays a crucial role in the survival and growth of airway basal stem cells, with a working concentration of 0.1-1.0 ng / mL; preferably, the EGF is derived from Sigma, catalog number SRP3027.

[0105] FGF10 (fibroblast growth factor) is a heparin-binding growth factor belonging to the FGF family. FGF10 plays a central role in promoting the proliferation of basal stem cells, with a working concentration of 0.04-2.5 ng / mL; preferably, FGF10 is sourced from R&D Systems, catalog number 345-FG-250.

[0106] γ-secretase is an intramembrane proteolytic enzyme composed of four subunits, primarily involved in the cleavage and hydrolysis of important transmembrane proteins such as β-amyloid precursor (APP) and Notch. γ-secretase inhibitors are substances that can reduce γ-secretase expression and / or γ-secretase activity, including but not limited to Dibenzazepine (DBZ), DAPT, LY-411575, CompoundE, and RO4929097 (RG-4733). In embodiments of this invention, DAPT is preferred, with a working concentration of 1-5 μM. Preferably, the DAPT is sourced from Sigma, catalog number D5942.

[0107] Rho-associated kinase (ROCK) can activate numerous downstream targets, including actin and intermediate filament proteins, through phosphorylation, thereby regulating the function of these proteins. Therefore, ROCK can regulate a variety of key cellular functions, including cell proliferation, migration, and activity. Rho inhibitors are substances that can reduce Rho expression and / or Rho activity, including but not limited to ZINC00881524, Thiazovivin, GSK429286A, and Y-27632. In embodiments of this invention, Y27632 is preferred, with a working concentration of 5-10 μM. Preferably, the Y27632 is derived from Selleck, catalog number S1049.

[0108] The TGF-β pathway coordinates many cellular processes, including cell growth, differentiation, cell migration, invasion, and extracellular matrix remodeling. The TGF-β family is broadly divided into two subfamilies: TGF-β ligands and bone morphogenetic protein (BMP) ligands. TGF-β signaling is induced by ligands, serine / threonine protein kinases, and their binding to homologous cell membrane receptors. Cell membrane receptors are classified as type I or type II receptors. Type II receptors are constitutively activated. After ligand binding, TGFβ approaches and phosphorylates and activates the type I receptor. Receptor activation induces C-terminal phosphorylation of SMAD. The phosphorylated SMAD then forms a complex with the co-mediators SMAD and SMAD4; SMAD4 translocates to the nucleus, where it binds to gene promoters. TGFβ receptor inhibitors are selected from, but are not limited to, ALK5 inhibitor II, SB431542, LY364947, DMH1, and A83-01. In the embodiments of the present invention, the preferred SMAD inhibitor is A-83-01, with a working concentration of 1-10 μM; preferably, A-83-01 is derived from Sigma, catalog number SML0788; the preferred BMP inhibitor is DMH1, with a working concentration of 1-5 μM, preferably, DMH1 is derived from Sigma, catalog number D8946.

[0109] The commercial culture medium used for comparison in the experiments of this invention is PneumaCult. TM -Ex Plus Medium, sourced from STEMCELL, catalog number 05040, is formulated without serum or bovine pituitary extract (BPE). When used in cell culture, it must be composed of or substantially composed of at least one SMAD inhibitor, at least one transforming growth factor β (TGF-β) inhibitor, at least one bone morphogenetic protein (BMP) inhibitor, and at least one ROCK inhibitor. Specifically, the medium must contain three inhibitors: Y-27632, A-83-01, and DMH-1. This medium will be abbreviated as Ex-Plus below.

[0110] In a specific implementation, the concentration of HSA in the airway basal stem cell culture medium is 0.05% to 1%, preferably 0.1%.

[0111] In a specific implementation plan, the concentration of GlutaMAX in the airway basal stem cell culture medium is 2-6 mM, preferably 2 mM.

[0112] In a specific implementation plan, the concentration of L-ascorbic acid in the airway basal stem cell culture medium is 50–300 μg / mL, preferably 50 μg / mL.

[0113] In a specific implementation scheme, the concentration of 1-thioglycerol in the airway basal stem cell culture medium is 0.1-1 mM, preferably 0.4 mM.

[0114] In a specific implementation scheme, the concentration of hydrocortisone in the airway basal stem cell culture medium is 1-15 μg / mL, preferably 1 μg / mL.

[0115] In a specific implementation scheme, the concentration of EGF in the airway basal stem cell culture medium is 0.1-1.0 ng / mL, preferably 1.0 ng / mL.

[0116] In a specific implementation plan, the concentration of FGF10 in the airway basal stem cell culture medium is 0.04-2.5 ng / mL, preferably 0.4 ng / mL.

[0117] In a specific implementation scheme, the concentration of DAPT in the airway basal stem cell culture medium is 1-5 μM, preferably 1 μM.

[0118] In a specific implementation plan, the concentration of Y-27632 in the airway basal stem cell culture medium is 5-10 μM, preferably 10 μM.

[0119] In a specific implementation scheme, the concentration of DMH-1 in the airway basal stem cell culture medium is 1-5 μM, preferably 1 μM.

[0120] In a specific implementation scheme, the concentration of A-83-01 in the airway basal stem cell culture medium is 1-10 μM, preferably 1 μM.

[0121] Compared with the prior art, the beneficial effects of the present invention are one or more of the following:

[0122] 1. The culture medium provided by this invention has the advantages of simple preparation method, no pathogenic microorganisms, no allergens, and high safety. It does not require a feeder layer or substrate coating treatment for the culture plate, and the primary cultured cells have good stability. After multiple passages, the cell morphology is uniform and the average diameter changes little, which can maintain the normal morphology of basal stem cells and maintain good differentiation potential.

[0123] 2. The culture medium system has clearly defined components and does not contain components with unclear chemical composition such as serum, bovine pituitary extract (BPE), or platelet lysate, which can simultaneously meet the research-grade and clinical-grade applications of airway basal stem cells. Attached Figure Description

[0124] Figure 1 shows the morphological observation results of HBEC cells from generation P3 to P8, which were continuously cultured in Ex-Plus medium; where A is generation P3 cells, B is generation P4 cells, C is generation P5 cells, D is generation P6 cells, E is generation P7 cells, and F is generation P8 cells, with a scale bar of 200 μm.

[0125] Figure 2 shows the morphological observation results of HBEC cells from generation P3 to P15 continuously cultured using the culture medium of formulation 1 of this invention; where A is generation P3 cells, B is generation P4 cells, C is generation P5 cells, D is generation P6 cells, E is generation P7 cells, F is generation P8 cells, G is generation P9 cells, H is generation P10 cells, I is generation P11 cells, J is generation P12 cells, K is generation P13 cells, L is generation P14 cells, and M is generation P15 cells, with a scale bar of 200 μm.

[0126] Figure 3 shows the morphological observation results of HBECs continuously cultured in Ex-Plus medium after passage to P7, and cultured in Ex-Plus medium and the medium of formulation 1 of the present invention for two days respectively; A is Ex-Plus medium, B is medium of formulation 1 of the present invention, scale bar 200 μm.

[0127] Figure 4 is a bright field diagram of P3 generation HBEC cultured for 4 days using culture media of formulations 1, 2-6 of the present invention; where A is culture medium of formulation 2 of the present invention, B is culture medium of formulation 3 of the present invention, C is culture medium of formulation 1 of the present invention, D is culture medium of formulation 4 of the present invention, E is culture medium of formulation 5 of the present invention, and F is culture medium of formulation 6 of the present invention, with a scale bar of 200 μm.

[0128] Figure 5 is a bright field diagram of P3 generation HBEC cultured for 4 days using the culture media of formulation 1 and formulations 7-12 of the present invention; where A is the culture medium of formulation 7 of the present invention, B is the culture medium of formulation 8 of the present invention, C is the culture medium of formulation 1 of the present invention, D is the culture medium of formulation 9 of the present invention, E is the culture medium of formulation 10 of the present invention, F is the culture medium of formulation 11 of the present invention, and G is the culture medium of formulation 12 of the present invention, with a scale bar of 200 μm.

[0129] Figure 6 shows the morphological observation results of P3 generation HBEC cells continuously cultured in culture medium formulated with the present invention, where A represents P3 generation cells, B represents P4 generation cells, C represents P5 generation cells, and D represents P6 generation cells. The scale bar is 200 μm.

[0130] Figure 7 shows the morphological observation results of HSAEC cells from generation P3 to generation P9 after continuous culture in Ex-Plus medium; where A is generation P3 cells, B is generation P4 cells, C is generation P5 cells, D is generation P6 cells, E is generation P7 cells, F is generation P8 cells, and G is generation P9 cells, with a scale bar of 200 μm.

[0131] Figure 8 shows the morphological observation results of HSAEC cells from generation P3 to generation P9 continuously cultured in the culture medium of formulation 1 of the present invention; where A is generation P3 cells, B is generation P4 cells, C is generation P5 cells, D is generation P6 cells, E is generation P7 cells, F is generation P8 cells, G is generation P9 cells, and the scale bar is 200 μm.

[0132] Figure 9 shows the doubling curve of the airway basal stem cell population cultured using the culture medium of Formula 1 of the present invention; A is HBEC, and B is HSAEC.

[0133] Figure 10 shows the average diameter values ​​and statistical graphs of airway basal stem cells cultured using Ex-Plus medium and the medium of formulation 1 of this invention; where A is the average diameter value of HBEC cells, B is the average diameter value of HSAEC cells, C is the statistical graph of the average diameter of HBEC cells, and D is the statistical graph of the average diameter of HSAEC cells.

[0134] Figure 11 shows the immunofluorescence results of molecular markers of P4 generation HBEC cultured in culture medium formulated according to the present invention; A is the staining image of P63 and KRT5 under 5X field of view, B is the staining image of P63 and KRT5 under 20X field of view, C is the staining image of NGFR and KI67 under 5X field of view, and D is the staining image of NGFR and KI67 under 20X field of view.

[0135] Figure 12 shows the immunofluorescence results of molecular markers of P7 generation HSAEC cultured in culture medium formulated according to the present invention; A is the staining image of P63 and KRT5 under 5X field of view, B is the staining image of P63 and KRT5 under 40X field of view, C is the staining image of NGFR and KI67 under 5X field of view, and D is the staining image of NGFR and KI67 under 40X field of view.

[0136] Figure 13 shows the immunofluorescence staining results of HBEC cultured in the culture medium of Formula 1 of this invention on day 21 of induced differentiation; A and B are bright field images of P7 generation HBECs on day 21 of differentiation under different fields of view; C is the staining image of MUC5AC and Acetylated-Tubulin under 40X field of view; and D is the staining image of MUC5AC and Acetylated-Tubulin under 100X field of view.

[0137] Figure 14 shows the immunofluorescence staining results of HSAECs cultured in the culture medium of Formula 1 of this invention on day 21 of induced differentiation; A and B are bright field images of P7 generation HSAECs on day 21 of differentiation under different fields of view; C is the staining image of MUC5AC and Acetylated-Tubulin under 40X field of view; and D is the staining image of MUC5AC and Acetylated-Tubulin under 100X field of view.

[0138] Figure 15 shows the bright field images of P3 generation HBEC cells cultured in culture media using formulations ①-④ on the third day, where A, B, C, and D correspond to culture media using formulations ①, ②, ③, and ④, respectively.

[0139] Figure 16 shows a bright field image of P4 generation HBEC cells after 24 hours of culture, where A, B, C, and D correspond to culture media using formulations ①, ②, ③, and ④, respectively. Detailed Implementation

[0140] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0141] Example 1. Preparation method of airway basal stem cell culture medium

[0142] The formulation of the airway basal stem cell culture medium is shown in Table 1.

[0143] In Table 1, IMDM medium was sourced from Gibco (catalog number 12440061); Ham's F12 medium was sourced from Gibco (catalog number 11765054); B27 medium was sourced from Gibco (catalog number 17504044); HSA medium was sourced from Sigma (catalog number A9731); GlutaMAX medium was sourced from Gibco (catalog number 35050061); L-ascorbic acid was sourced from Sigma (catalog number A4544); 1-thioglycerol was sourced from Sigma (catalog number M6145); hydrocortisone was sourced from STEMCELL (catalog number 07926); EGF was sourced from Sigma (catalog number SRP3027); and FGF10 was sourced from R&D. Systems, catalog number 345-FG-250; DAPT is from Sigma, catalog number D5942; Y27632 is from Selleck, catalog number S1049; A-83-01 is from Sigma, catalog number SML0788; DMH1 is from Sigma, catalog number D8946; In Table 1, the final concentrations of IMDM medium and Ham's F12 medium are their final concentrations in the basal medium, and the final concentrations of other components are their final concentrations in the airway basal stem cell medium.

[0144] The preparation methods for different formulas are the same, and the specific steps are as follows:

[0145] (1) Dilute EGF and FGF10 with 5% trehalose buffer (PBS buffer containing 5% (w / v, g / mL));

[0146] (2) Dissolve DATP, Y-27632, DMH-1 and A-83-01 respectively using dimethyl sulfoxide;

[0147] (3) Mix IMDM and Ham's F12 medium in a 1:1 ratio (volume ratio) to obtain the basic medium;

[0148] (4) Add (1), (2) and supplementary ingredients to (3) and mix well to obtain airway basal stem cell culture medium. The culture medium needs to be protected from light during preparation and can be stored at 4°C for 7 days.

[0149] Example 2. Effects of airway basal stem cell culture medium on the proliferation capacity and cell morphology of basal stem cells in large and small airways.

[0150] 1. Experimental Methods

[0151] (1) Culture of airway basal stem cells

[0152] The culture medium formulated in Example 1 and the commercially available Ex-Plus (PneumaCult) culture medium were used respectively. TM -Ex Plus Medium, sourced from STEMCELL (catalog number 05040), was used in 12-well plates to culture HBEC (Lifeline Cell Technology, catalog number FC-0035) and HSAEC (ATCC, catalog number PCS-301-010). (In this invention, commercially available HBEC and HSAEC were revived (i.e., P1), expanded and passaged using commercially available cell culture media to obtain P2 generation cells, which were used for the experiments described below). The cells were cultured at 37°C in a 5% CO2 cell culture incubator without a feeder layer. The medium was changed every two days. Once the cell density reached approximately 80%, the cells were digested and passaged using ACCUTASE digestive enzyme (#A6964, Sigma). After digestion, the cells were counted, and the cell diameter was recorded. Subculturing was continued in 12-well plates or seeded into cell culture dishes at appropriate cell densities for subsequent assays.

[0153] (2) Morphological observation

[0154] The growth morphology and state of basal stem cells in the large and small airways were observed under a microscope.

[0155] 2. Experimental Results

[0156] (1) Effects on cell morphology

[0157] As shown in Figure 1, HBEC cells continuously cultured in Ex-Plus medium showed good adherence and growth in passages P3-P6, with tight intercellular connections, clear edges, and most cells exhibiting a cuboidal morphology, demonstrating good overall homogeneity. Passages P7-P8 showed poor adherence, moderate growth, slower growth rate, abnormal cell morphology, including vesicle formation and filamentation, and generally poor cell homogeneity. HBEC passages were discontinued at passage 8.

[0158] As shown in Figure 2, HBEC cells were continuously cultured using Formula 1 medium. Cells from passages P3 to P10 showed good adherence and growth, with tight intercellular connections, clear edges, a cuboidal cell morphology, and excellent overall homogeneity. From passages P10 to P15, cells adhered well and grew well, but the growth rate was relatively slow. Intercellular connections remained tight, the cell morphology was still cuboidal, and overall homogeneity remained good.

[0159] As shown in Figure 3A, HBECs continuously cultured in Ex-Plus medium showed vesicular cell formation two days after passage, with inconsistent cell morphology and small clones. Figure 3B shows HBECs continuously cultured in Ex-Plus medium, which was replaced with Formula 1 medium on the day of passage to P7. Two days later, the P7 cells showed clear clones, more uniform cell morphology, and were mostly cuboidal. This demonstrates that Formula 1 medium can salvage the poor cell morphology and proliferation observed in the later stages of HBEC culture in Ex-Plus medium.

[0160] As shown in Figure 4, P3 generation HBECs were cultured in formulations 1 and 2–6, with the same number of cells seeded and cultured for 4 days. In formulation 2 (Figure 4A) (without EGF), cells did not adhere to the culture medium. In formulations 4–6 (Figures 4D–F), cells became elongated and exhibited significant deformation. In formulations 1 (Figure 4C) and 3 (Figure 4B), cells maintained good morphology and growth. Therefore, EGF is the key factor in this formulation, and a concentration in the range of 0.1–1 ng / mL is suitable.

[0161] As shown in Figure 5, P3 generation HBECs were cultured in media containing formulations 1 and 7–12, with the same number of cells seeded and cultured for 4 days. In media containing formulations 10–12 (Figures 5E–G), cells showed significant morphological changes. In media containing formulation 7 (Figure 5A), cell proliferation was extremely slow, failing to achieve normal expansion. In media containing formulations 1 (Figure 5C), 8 (Figure 5B), and 9 (Figure 5D), cells maintained good morphology and growth, with good overall homogeneity. Therefore, FGF10 promotes cell proliferation, and a concentration in the range of 0.04–2.5 ng / mL is suitable.

[0162] As shown in Figure 6, when HBECs were continuously cultured in Formula 13 medium (without DAPT), the cells could only maintain a passage of 4, exhibiting phenomena such as filamentation, abnormal growth, and heterogeneous morphology. Complete medium with a final DAPT concentration of 5 μM could also be used for continuous HBEC culture, maintaining a passage of 7 (P3-P9), but with a significant number of dead cells during culture (data not provided in the image). Compared to Formula 1 (with DAPT) (Figure 2), the added inhibitor DAPT in the formula helped maintain the morphology of airway basal stem cells and promote long-term passage growth.

[0163] In summary, the results show that HBECs cultured in Formula 1 medium exhibited good overall morphology and condition. Even after continuous subculturing up to 15 generations, the cells maintained good morphology and growth status, and showed good overall uniformity.

[0164] As shown in Figure 7, HSAEC cells continuously cultured in Ex-Plus medium showed good adherence and growth in passages P3 to P6, with tight intercellular connections, clear edges, and most cells exhibiting a cuboidal morphology, demonstrating good overall uniformity. Passages P7 to P9 showed generally poor growth, with slower expansion and abnormal cell morphology, including vesicle formation and filamentation.

[0165] As shown in Figure 8, HSAEC cells were cultured to the 9th generation using the same culture medium (Formula 1). Cells from P3 to P9 showed good adherence and growth, with tight intercellular connections, clear edges, a cuboidal cell morphology, and excellent overall uniformity.

[0166] The results in summary indicate that the use of Formula 1 medium can also maintain the normal passage growth of HSAECs, and compared with Ex-Plus medium, the cultured cells have better overall morphology and uniformity.

[0167] (2) Effects on cell proliferation capacity and diameter

[0168] As shown in Figure 9A, HBECs were continuously cultured using Formula 1 medium, and the population doubling curve reflected the sustained good proliferation level of cells during the P3 to P15 generation culture process.

[0169] As shown in Figure 9B, HSAECs were continuously cultured using Formula 1 medium, and the population doubling curve reflected the sustained good proliferation level of cells during the P3 to P9 generation culture.

[0170] As shown in Figure 10A, HBEC cells continuously cultured in Ex-Plus medium were passaged up to P7 with an average cell diameter of 19.19 μm (15.43 μm–23.94 μm). In contrast, HBEC cells continuously cultured in Formula 1 medium had an average cell diameter of 16.65 μm (14.57 μm–18.98 μm) and could be passaged up to P15 and beyond. Furthermore, the statistical graph of average cell diameter shown in Figure 10C clearly demonstrates that the diameter of cells continuously cultured in Formula 1 medium did not change significantly.

[0171] As shown in Figure 10B, HSAECs were continuously cultured in Ex-Plus medium and passaged to the P9 generation, with an average cell diameter of 18.88 μm (15.58 μm–20.95 μm). HSAECs were also continuously cultured in Formula 1 medium and passaged to the P9 generation, with an average cell diameter of 15.96 μm (13.31 μm–17.33 μm). As shown in Figure 10D, the statistical graph of average cell diameter clearly shows that the diameter of cells continuously cultured using Formula 1 medium did not change significantly.

[0172] The results in summary indicate that airway basal stem cells cultured in Formula 1 can be passaged multiple times (≥P10; since EXplus can only be passaged up to 9 generations, Formula 1 only shows up to P9 generation, but it can be passaged up to P24 generation). After multiple passages, the cells can still maintain good proliferative capacity and the average cell diameter changes little.

[0173] Example 3. Immunofluorescence detection of airway basal stem cells

[0174] 1. Experimental Methods

[0175] (1) Culture of airway basal stem cells

[0176] Airway basal stem cells were cultured in the culture medium of Formula 1 from Example 1.

[0177] Airway basal stem cells were cultured in 12-well plates using the culture medium formulated as described in Example 1, without the addition of feeder cells. The medium was changed every two days. When the cells reached a density of approximately 80%, they were digested and passaged using ACCUTASE digestive enzyme. After digestion, the cells were counted and the cell number was determined. Finally, the cells were divided into 5 × 10⁻⁶ wells. 4 Seeds were placed in confocal dishes at a density of 1 cell per cell.

[0178] (2) Immunofluorescence detection

[0179] After 3–5 days of adherent culture, cells were fixed with 4% paraformaldehyde and immunofluorescence was performed using molecular marker antibodies as shown in Table 2.

[0180] Immunofluorescence assay: Fixative was removed, the slide was permeabilized with permeabilizing buffer for 10 minutes, washed three times with PBS, and blocked with blocking buffer for 10 minutes. Primary antibody (as shown in Table 2) was then added, and the slide was incubated overnight at 4°C. The next day, the primary antibody was recovered, the slide was washed three times with PBS, and the secondary antibody was added and incubated at room temperature for 60 minutes. The secondary antibody was removed, the slide was washed three times with PBS, and DAPI was added for staining for 15 minutes. DAPI was removed, the slide was washed with PBS, and the slide was mounted with anti-quenching mounting medium. Images were taken using an LSM 900 laser confocal microscope (ZEISS, Germany).

[0181] Table 2. Molecular marker antibodies used for immunofluorescence identification of airway basal stem cells.

[0182] 2. Experimental Results

[0183] As shown in Figure 11, A shows the staining images of P63 and KRT5 under 5X field of view for HBEC cultured from P3 to P4 generations using Formula 1 medium; B shows the staining images of P63 and KRT5 under 20X field of view; C shows the staining images of NGFR and KI67 under 5X field of view; and D shows the staining images of NGFR and KI67 under 20X field of view. The basal cell-specific markers P63, KRT5, and NGFR were all expressed with a high positive rate; the cell proliferation marker KI67 also showed strong positive expression.

[0184] As shown in Figure 12, A shows the staining images of P63 and KRT5 under 5X field of view for HSAECs cultured from P3 generation HBEC to P7 generation using Formula 1 medium; B shows the staining images of P63 and KRT5 under 40X field of view; C shows the staining images of NGFR and KI67 under 5X field of view; and D shows the staining images of NGFR and KI67 under 40X field of view. The basal cell-specific markers P63, KRT5, and NGFR were all expressed with a high positive rate; the cell proliferation marker KI67 also showed strong positive expression.

[0185] The results in summary indicate that airway basal stem cells cultured in Formula 1 medium showed high positive rates for the expression of specific markers P63, KRT5, and NGFR, indicating high cell purity, and also showed high KI67 expression, thus demonstrating strong proliferative capacity.

[0186] Example 4: Detection of the ability of airway basal stem cells to induce differentiation

[0187] 1. Experimental Methods

[0188] (1) Culture of airway basal stem cells

[0189] Airway basal stem cells were cultured in 6-well plates using the culture medium formulation 1 of Example 1, without adding feeder cells. The medium was changed every two days. Once the cells reached approximately 80% density, they were passaged using ACCUTASE digestive enzyme. After digestion, the cells were counted to determine the cell number. P7 HBEC / HSAEC were cultured at a ratio of 3 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of 1,000 cells into the upper chamber of a transwell (#3460, Corning) coated with type IV collagen (#C5533, Sigma) and cultured using formulation 1 medium.

[0190] (2) Differentiation induction by gas-liquid interface culture method

[0191] After 3 days of proliferation, when cell confluence is close to 100%, remove the culture medium from the upper chamber of the transwell and keep the cell in a dry state. Replace the lower chamber with differentiation induction medium (PneumaCult for HBEC). TM -ALI Medium, #05001, STEM CELL; HSAEC uses PneumaCult TM -ALI-S Medium, #05050, STEM CELL); change the medium in the lower chamber of the transwell every 2 days, and wash the mucus in the upper chamber with PBS every 7 days, and maintain the gas-liquid interface for 21 days.

[0192] (3) Immunofluorescence detection

[0193] On day 21 of differentiation, the upper chamber was fixed with 4% paraformaldehyde, and immunofluorescence detection was performed using molecular marker antibodies as shown in Table 3.

[0194] Immunofluorescence assay: Fixative was removed, the membrane was permeabilized with permeabilizing buffer for 10 minutes, washed three times with PBS, and blocked with blocking buffer for 10 minutes. The primary antibody (as shown in Table 3) was then added, and the membrane was incubated overnight at 4°C. The primary antibody was recovered the next day, the membrane was washed three times with PBS, and the secondary antibody was added and incubated at room temperature for 60 minutes. The secondary antibody was removed, the membrane was washed three times with PBS, and DAPI was added for staining for 15 minutes. The DAPI was removed, the membrane was washed with PBS, and the membrane was cut with a blade and mounted with anti-quenching mounting medium. Images were taken using an FV3000 laser confocal microscope (Olympus, Japan).

[0195] Table 3. Molecular marker antibodies used for immunofluorescence identification of airway basal stem cell differentiation capacity.

[0196] 2. Experimental Results

[0197] As shown in Figure 13, 13A-13B are bright-field images under different fields of view under a microscope on the 21st day after the differentiation of P7 generation HBECs, 13C is a staining image of MUC5AC and Acetylated-Tubulin under a 40X field of view, and 13D is a staining image of MUC5AC and Acetylated-Tubulin under a 100X field of view.

[0198] As shown in Figure 14, 14A-14B are bright-field images under a microscope at different fields of view on day 21 of differentiation of P7 generation HSAECs, 14C is a staining image of MUC5AC and Acetylated-Tubulin under a 40X field of view, and 14D is a staining image of MUC5AC and Acetylated-Tubulin under a 100X field of view.

[0199] The results in summary indicate that airway basal stem cells cultured in Formula 1 medium can differentiate into well-functioning airway epithelial cells and have good differentiation potential.

[0200] Comparative Example 1 verifies whether the culture medium for differentiating airway progenitor cells can be used to culture airway basal stem cells.

[0201] The formulations disclosed in the patents (US2024052319A1; US2021254016A1) have similar components to those in this invention. Both patents describe methods for differentiating pluripotent stem cells or lung progenitor cells into epithelial cells or basal cells.

[0202] In patent (US2024052319A1), during the stage of pluripotent stem cells generating immature airway progenitor cells, a completely serum-free differentiation medium (cSFDM) + airway differentiation medium (ADM) was used. The cSFDM formulation is as follows: 375 mL IMDM, 125 mL Ham's F-12 medium, 5 mL GlutaMAX, 5 mL B27 supplement, 3.3 mL 7.5% BSA, 2.5 mL N2 supplement, 500 μL 50 mg / mL ascorbic acid, 1.5 mL 13 μL / mL MTG (thioglycerol), and 500 μL Primocin are mixed and sterilely filtered. Patent (US2024052319A1) has similar components to this invention: basal medium IMDM and Ham's F-12, with supplements of GlutaMAX, B27, ascorbic acid, and thioglycerol. According to calculations, the final concentration of some supplementary components in the completely serum-free differentiation medium (cSFDM) is the same as the final concentration of the medium formulation 1 of this invention, which is 2 mM GlutaMAX, 50 μg / mL L-ascorbic acid, and 0.4 μM 1-thioglycerol. However, the basal medium IMDM and Ham's F-12 in the aforementioned patent (US2024052319A1) are configured in a 3:1 ratio, and B27 is 0.5X, which differs from this invention. Specifically, the airway differentiation medium (ADM) is prepared by mixing 45 mL of cSFDM medium, 5 mL of 10X cAMP / IBMX, 50 μL of 250 pg / mL rhFGF2, 500 μL of 10 pg / mL rhFGF10, 25 μL of 100 pM dexamethasone, and 50 μL of 10 mM Y-27632 and aseptically filtering. The components similar to those in the present invention in patent (US2024052319A1) include FGF10 and Y-27632. Calculations show that the final concentration of FGF10 in airway differentiation medium (ADM) is 100 ng / mL, while the final concentration of FGF10 in formulation 1 of the present invention is 0.4 ng / mL. The suitable range for FGF10 in the culture medium of the present invention is 0.04-2.5 ng / mL. The final concentration of Y-27632 is the same as that in formulation 1 of the present invention, i.e., 10 μM.

[0203] In patent (US2021254016A1), during the stage of airway progenitor cells / immature basal cells maturing into basal stem cells, the third culture medium used is PneumacultExPlus. TM And add Y-27632, DMH-1, A-83-01.

[0204] The HSA, hydrocortisone, EGF, and DAPT involved in the formulation of this invention are not present in the formulations disclosed in the patents (US2024052319A1; US2021254016A1).

[0205] Based on the present invention and the formulations in the published patents (US2024052319A1; US2021254016A1), four formulations as shown in Table 4 below were prepared for testing (the reagent dosage is based on the preparation of 50 mL of culture medium).

[0206] Table 4 shows four culture medium formulations used to verify their suitability for culturing airway basal stem cells.

[0207] HBEC cells of generation P3 were cultured in media formulated ①-④, as shown in Figure 15. After 3 days of culture, the cells exhibited significant deformation. Passaged at this morphology and density, the bright-field plot after 24 hours is shown in Figure 16, indicating that the cells could not survive normally.

[0208] In conclusion, the airway differentiation medium (ADM) used to generate immature airway progenitor cells from pluripotent stem cells cannot be used for the culture and uncoated expansion of airway basal stem cells; and the addition of FGF10, Y-27632, A83-01, and DMH-1 to completely serum-free differentiation medium (cSFDM) also cannot be used for the culture and uncoated expansion of airway basal stem cells.

[0209] References

[0210] 1. Hawkins FJ, Suzuki S, et al. Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells. Cell Stem Cell. 2021 Jan 7;28(1):79-95.

[0211] 2. ZhangY, Lin T, et al. STAT3 mutation-associated airway epithelial defects in Job syndrome. J Allergy Clin Immunol. 2023 Aug; 152(2):538-550.

[0212] 3.Wagner R, Amonkar GM, et al. A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia. Am J Respir Crit Care Med. 2023 May 1; 207(9):1214-1226.

[0213] 4.Wu H,Tang N.Stem cells in pulmonary alveolar regeneration.Development.2021 Jan 18;148(2):dev193458.

[0214] 5. Usmani OS, Dhand R, et al. Why We Should Target Small Airways Disease in Our Management ofChronic Obstructive Pulmonary Disease. Mayo Clin Proc. 2021 Sep; 96(9):2448-2463.

[0215] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A composition for culturing airway basal stem cells, comprising: EGF, FGF10 and a γ-secretase inhibitor.

2. The composition according to claim 1, characterized in that, The composition further comprises: a ROCK inhibitor; and / or The composition further comprises: a BMP inhibitor; and / or The composition also contains: an SMAD inhibitor.

3. The composition according to claim 1 or 2, characterized in that, The working concentration of the EGF is 0.1-50 ng / mL; and / or The working concentration of the FGF10 is 0.04-100 ng / mL; and / or The working concentration of the γ-secretase inhibitor is 0.5-5 μM; and / or The γ-secretase inhibitor comprises at least one of Dibenzazepine (DBZ), DAPT, LY-411575, Compound E, RO4929097 (RG-4733), PF03084014, Crenigacestat (LY3039478), BMS906024, Nirogacestat, and MK-0752; and / or The working concentration of the ROCK inhibitor is 5-15 μM; and / or The ROCK inhibitor comprises at least one of Y-27632, ZINC00881524, Thiazovivin, GSK429286A, hydroxyfasudil (HA-1100), HA100, H-1152, Blebbistatin, HA-1077, KD-025, Y-33075, and Narciclasine; and / or The working concentration of the BMP inhibitor is 0.5-5 μM; and / or The BMP inhibitor comprises at least one of DMH-1, Noggin, LDN193189, LDN212854, Dorsomorphin, K02288, follistatin, chordin, and gremlin; and / or The working concentration of the SMAD inhibitor is 0.5-10 μM; and / or The SMAD inhibitor comprises at least one of A-83-01, SB-431542, LY364947, and SB505124.

4. The use of the composition according to any one of claims 1-3 in a1) or a2); a1) Culture airway basal stem cells; a2) Prepare a culture medium for culturing airway basal stem cells.

5. A culture medium comprising the composition according to any one of claims 1-3.

6. The culture medium according to claim 5, characterized in that, The culture medium is a basal culture medium containing the composition according to any one of claims 1-3.

7. The culture medium according to any one of claims 5-6, characterized in that, The basal culture medium is a basal culture medium containing supplementary components, which include: B27, serum albumin, glutamine, and L-ascorbic acid; Preferably, the supplementary ingredients further comprise: thioglycerin and hydrocortisone.

8. The culture medium according to any one of claims 6-7, characterized in that, The basal culture medium comprises at least one of DMEM / F12 medium, Basal Medium Eagle medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, Advanced RPMI 1640 medium, and Advanced DF-12 medium; and further comprises IMDM medium and Ham's F12 medium. Preferably, the volume ratio of the IMDM medium to the Ham's F12 medium is 1:(0.5-1.5); more preferably, it is 1:(0.8-1.2).

9. The culture medium according to any one of claims 5-8, characterized in that, The concentration of EGF in the culture medium is 0.1-50 ng / mL; more preferably 0.1-1.2 ng / mL; even more preferably 0.1-1.1 ng / mL; and / or The concentration of FGF10 in the culture medium is 0.04-100 ng / mL; more preferably 0.04-2.5 ng / mL; and / or The concentration of the γ-secretase inhibitor in the culture medium is 0.5-5 μM; more preferably 0.5-1.5 μM; even more preferably 0.8-1.2 μM; and / or The concentration of the ROCK inhibitor in the culture medium is 5-15 μM; more preferably 8-12 μM; even more preferably 9-11 μM; and / or The concentration of the BMP inhibitor in the culture medium is 0.5-5 μM; more preferably 0.8-1.2 μM; even more preferably 0.9-1.1 μM; and / or The concentration of the SMAD inhibitor in the culture medium is 0.5-10 μM; further, 0.8-1.2 μM; and even further, 0.9-1.1 μM.

10. The culture medium according to any one of claims 7-9, characterized in that, The concentration of B27 in the culture medium is 0.5-1.5×; and / or The concentration of serum albumin in the culture medium is 0.05%-1% by mass-volume ratio; more preferably 0.05%-0.15%; and / or The concentration of glutamine in the culture medium is 1-6 mM; more preferably 1-3 mM; and / or The concentration of L-ascorbic acid in the culture medium is 20-300 μg / mL; more preferably 40-60 μg / mL; and / or The concentration of the thioglycerol in the culture medium is 0.1-1 mM; more preferably 0.2-0.6 mM; and / or The concentration of hydrocortisone in the culture medium is 0.5-15 μg / mL; further, 0.5-5 μg / mL; and even further, 0.8-1.2 μg / mL.

11. The use of the culture medium according to any one of claims 5-10 in culturing airway basal stem cells.

12. A method for culturing airway basal stem cells, comprising the step of using the composition of any one of claims 1-3 or the culture medium of any one of claims 5-10.

13. An airway basal stem cell preparation comprising: airway basal stem cells; and The composition according to any one of claims 1-3 or the culture medium according to any one of claims 5-10.

14. A kit comprising: the composition of any one of claims 1-3, the culture medium of any one of claims 5-10, or the airway basal stem cell preparation of claim 13.

15. The composition, application, culture medium, method, airway basal stem cell preparation, or kit according to any one of claims 1-14, characterized in that, The airway basal stem cells are derived from animals; Preferably, the animal comprises a mammal; further comprises at least one of a human or a non-human mammal; and further comprises a human.

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