Human airway basal stem cell culture medium with definite chemical components

Abstract

The invention discloses a human airway basal stem cell culture medium as well as a preparation method and application thereof. The culture medium provided by the invention is simple in preparation method and high in safety, a culture plate does not need to be coated with a feeding layer or matrigel, and the airway basal stem cells subjected to primary culture are good in stability; after multiple passage, the normal form of the basal stem cells can be maintained, and good differentiation potential can be maintained; meanwhile, components of the culture medium are clear, and scientific research-grade and clinical-grade application of the airway basal stem cells can be met at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell technology, in particular to a chemically defined culture medium for stable culture and expansion of human airway basal stem cells, a preparation method and application thereof. BACKGROUND

[0002] Airway basal stem cells (Basal Cell, BC) are usually referred to as human bronchial epithelial cells (Human Bronchial Epithelial Cell, HBEC) 1 , which are located in the basal layer of the airway epithelium and have the ability to self-renew and differentiate into various airway epithelial cells such as ciliated cells, goblet cells and club cells. Airway basal stem cells play an important role in the homeostasis maintenance and injury repair of airway epithelium, and are a very promising functional cell for the treatment of respiratory diseases. In addition, airway basal stem cells can also be used as a very useful tool for airway development research and disease model construction. Whether for clinical treatment or scientific research, it is necessary to realize the stable culture and expansion of airway basal stem cells in vitro. However, the current commonly used culture system has many defects, for example: (1) animal cells (such as mouse embryonic fibroblasts) are needed as feeder layers, and airway basal stem cells are inoculated onto the feeder layer cells for co-culture 2,3 . This method has the risk of exogenous contamination, and the culture process is complex, which greatly increases the culture time and cost of airway basal stem cells. (2) Serum, pituitary extract or platelet lysate and other chemical components with unknown components are used for culture. Such culture medium with unknown components will cause batch-to-batch quality variation, leading to poor reproducibility of experimental results, which is not conducive to scientific research and also limits future clinical applications based on cell therapy. (3) A few commercial serum-free, pituitary extract-free, and component-defined culture media do not disclose the formula, and cannot stably culture airway basal stem cells for a long time. With the increase of culture passage number, the cell division ability will be significantly reduced, the cell morphology will change abnormally obviously, and the cell heterogeneity will be very high.

[0003] Therefore, it is of great research value and good commercial prospect to develop a serum-free, pituitary extract-free, and chemical component-defined culture medium for stable culture of human airway basal stem cells, and to realize culture without feeder layer and matrix glue assistance. SUMMARY

[0004] The human lower respiratory tract is composed of trachea, bronchi, bronchioles of different 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 closely attached to the basal layer of the airway, which specifically express transformation related protein 63 (TRP63 or P63), keratin 5 (KRT5) and nerve growth factor receptor (NGFR). Basal stem cells also exist in the trachea and bronchi of mice, and a large number of literatures show that they play similar functions as human airway basal stem cells 4,5 The cells used in the present patent are primary human airway basal stem cells, which are obtained from the bronchial / tracheal epithelium of large airways (diameter > 2 mm) and small airway epithelium (located after the 8th bronchus, diameter < 2 mm), preferably human bronchial epithelial cells (HBEC) from Lifeline Cell Technology, product number FC-0035; preferably human small airway epithelial cells (HSAEC) from ATCC, product number PCS-301-010.

[0005] The present application provides a chemically defined human airway basal stem cell culture medium for maintaining the culture or expansion of human airway basal stem cells.

[0006] In the present application, the chemically defined culture medium is characterized in that the components and properties of the culture medium are clear, which is conducive to cell culture, easy to separate and purify the product, helps to regulate the physiological response of cells, and is conducive to more accurate evaluation of the influence of drugs on cell function.

[0007] In the present application, the basal medium is a medium that does not require the addition of specific components for the growth of microorganisms with no specific nutritional requirements, for example: DMEM / F12, Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow MEM, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham's medium, RPMI 1640, Fischer's medium or a mixed medium thereof.

[0008] In some embodiments, the application described herein includes:

[0009] 1. An airway basal stem cell medium comprising a basal medium, a supplement, growth factors comprising EGF and FGF10, and inhibitors comprising a gamma-secretase inhibitor, a Smad inhibitor, a BMP inhibitor, and a ROCK inhibitor.

[0010] 2. The airway basal stem cell medium of item 1, wherein the supplement comprises HSA, B27, GlutaMAX, and L-ascorbic acid.

[0011] 3. The airway basal stem cell medium of item 1 or 2, wherein the supplement further comprises 1-thioglycerol and hydrocortisone.

[0012] 4. The airway basal stem cell medium of 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.

[0013] 5. The airway basal stem cell medium of any one of items 1-4, wherein the gamma- 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; and the ROCK inhibitor is selected from Y-27632, Fasudil (HA1077), Thiazovivin, Hydroxyfasudil (HA-1100), preferably Y-27632.

[0014] 6. The airway basal stem cell medium of any one of items 1-5, wherein the inhibitors comprise 1-5 μΜ DAPT, 1-10 μΜ A-83-01, 1-5 μΜ DMH-1, and 5-10 μΜ Y-27632.

[0015] 7. The airway basal stem cell culture medium of any one of items 1 to 6, wherein the basal medium is selected from the group consisting of DMEM / F12, Basal Medium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow MEM, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham's medium, RPMI 1640, Fischer's medium or a mixture thereof.

[0016] Preferably, the basal medium is selected from a mixture of IMDM and Ham's F12, more preferably, the IMDM and Ham's F12 medium are mixed in a ratio of 1 : 1.

[0017] 8. A method of culturing airway basal stem cells, characterized in that the airway basal stem cells are cultured using the airway basal stem cell culture medium of any one of items 1 to 7.

[0018] 9. The airway basal stem cell culture medium of any one of items 1 to 7 or the method of item 8, wherein the airway basal stem cells are from a mammal, preferably a human, a mouse or a rat.

[0019] 10. The airway basal stem cell culture medium of any one of items 1 to 7 or the method of item 8, wherein the airway basal stem cells are human bronchial epithelial cells (HBEC) or human small airway epithelial cells (HSAEC).

[0020] 11. A kit comprising the airway basal stem cell culture medium of any one of items 1 to 7.

[0021] In the present invention, IMDM medium (Iscove's modified dulbecco's medium) is a commonly used medium for rapid propagation of high-density cell culture, preferably from Gibco, item number 12440061.

[0022] Ham's F12 medium (Ham's F12 nutrient medium) is a serum-free medium for animal cell culture; preferably from Gibco, item number 11765054.

[0023] B27 (50X) is a serum-free additive commonly used for neural cell culture, which can better maintain cell growth, and the working concentration is 1X; preferably, B27 is from Gibco, item number 17504044.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Gamma-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. Gamma-secretase inhibitors are substances that can reduce gamma-secretase expression and / or gamma-secretase activity, including but not limited to Dibenzazepine (DBZ), DAPT, LY-411575, Compound E, and RO4929097 (RG-4733). In the embodiments of this patent, DAPT is preferred, with a working concentration of 1-5 μM. The preferred DAPT is sourced from Sigma, catalog number D5942.

[0032] 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 the embodiments of this patent, Y27632 is preferred, with a working concentration of 5-10 μM. Preferably, the Y27632 is derived from Selleck, catalog number S1049.

[0033] 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 this patent, the preferred SMAD inhibitor is A-83-01, with a working concentration of 1-10 μM; preferably, A-83-01 is from Sigma, catalog number SML0788; the preferred BMP inhibitor is DMH1, with a working concentration of 1-5 μM, preferably, DMH1 is from Sigma, catalog number D8946.

[0034] The commercial culture medium compared in this patented experiment was 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.

[0035] In a specific implementation, the concentration of HSA in the airway basal stem cell culture medium is 0.05% to 1%, preferably 0.1%.

[0036] In a specific implementation plan, the concentration of GlutaMAX in the airway basal stem cell culture medium is 2-6 mM, preferably 2 mM.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] In a specific implementation scheme, the concentration of DAPT in the airway basal stem cell culture medium is 1-5 μM, preferably 1 μM.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Compared with the prior art, the beneficial effects of the present invention are one or more of the following:

[0047] 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.

[0048] 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

[0049] Figure 1 The results show the morphological observation of HBEC cells from generation P3 to P8 cultured in Ex-Plus medium; where A represents generation P3 cells, B represents generation P4 cells, C represents generation P5 cells, D represents generation P6 cells, E represents generation P7 cells, and F represents generation P8 cells.

[0050] Figure 2 The results show the morphological observation of HBECs from generation P3 to P15 cultured using the culture medium formulated according to the present invention; where A represents generation P3 cells, B represents generation P4 cells, C represents generation P5 cells, D represents generation P6 cells, E represents generation P7 cells, F represents generation P8 cells, G represents generation P9 cells, H represents generation P10 cells, I represents generation P11 cells, J represents generation P12 cells, K represents generation P13 cells, L represents generation P14 cells, and M represents generation P15 cells.

[0051] Figure 3 The images show P7 generation HBECs cultured two days after subculturing using Ex-Plus medium and the patented formulation 1 medium; A is Ex-Plus medium, and B is the patented formulation 1 medium.

[0052] Figure 4 Bright field diagrams showing the culture of P3 generation HBECs using culture media of this patent formulation 1, formulations 2-6, after 4 days of culture; where A is culture medium of this patent formulation 2, B is culture medium of this patent formulation 3, C is culture medium of this patent formulation 1, D is culture medium of this patent formulation 4, E is culture medium of this patent formulation 5, and F is culture medium of this patent formulation 6.

[0053] Figure 5 Bright field diagrams of P3 generation HBEC cultured using culture media of this patent formulation 1 and formulations 7-12 after 4 days of culture; where A is culture media of this patent formulation 7, B is culture media of this patent formulation 8, C is culture media of this patent formulation 1, D is culture media of this patent formulation 9, E is culture media of this patent formulation 10, F is culture media of this patent formulation 11, and G is culture media of this patent formulation 12.

[0054] Figure 6 To culture P3 to P6 generation HBECs using the patented formula 13 culture medium, where A is P3 generation cells, B is P4 generation cells, C is P5 generation cells, and D is P6 generation cells.

[0055] Figure 7 The results show the morphological observation of P3-P9 generation HSAECs cultured in Ex-Plus medium; where A is P3 generation cells, B is P4 generation cells, C is P5 generation cells, D is P6 generation cells, E is P7 generation cells, F is P8 generation cells, and G is P9 generation cells.

[0056] Figure 8 The results show the morphological observation of P3-P9 generation HSAECs cultured using the culture medium of this patented formula 1; where A is P3 generation cells, B is P4 generation cells, C is P5 generation cells, D is P6 generation cells, E is P7 generation cells, F is P8 generation cells, and G is P9 generation cells.

[0057] Figure 9The graph shows the doubling curve of the airway basal stem cell population cultured using the culture medium of this patented formula 1; A represents HBEC, and B represents HSAEC.

[0058] Figure 10 The figures show the average diameter values ​​and statistical graphs of airway basal stem cells cultured using Ex-Plus medium and the medium of this patented formulation 1; 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.

[0059] Figure 11 The results of immunofluorescence of molecular markers for P4 generation HBEC cultured using the culture medium of this patented formulation 1 are shown; A and C are staining images under 5X field of view, and B and D are staining images under 20X field of view.

[0060] Figure 12 The results of immunofluorescence of molecular markers in P7 generation HSAEC cultured using the culture medium of this patented formulation 1 are shown; A and C are staining images under 5X field of view, and B and D are staining images under 40X field of view.

[0061] Figure 13 The results of immunofluorescence staining on day 21 of induced differentiation of HBEC cultured using the culture medium of this patented formula 1 are shown. A is a bright field image of P7 generation HBEC on day 21 of differentiation under different fields of view, B is a staining image under 40X field of view, and C is a staining image under 100X field of view.

[0062] Figure 14 The results of immunofluorescence staining on day 21 of induced differentiation of HSAEC cultured using the culture medium of this patented formula 1 are shown. A is a bright field image of P7 generation HSAECs on day 21 of differentiation under different fields of view, B is a staining image under 40X field of view, and C is a staining image under 100X field of view.

[0063] Figure 15 Bright field images of P3 generation HBEC cells cultured on day 3 using formulations ①-④, where A, B, C, and D correspond to formulations ①, ②, ③, and ④, respectively.

[0064] Figure 16 Bright field images 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

[0065] 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.

[0066] Example 1. Preparation method of airway basal stem cell culture medium

[0067] The culture medium formulation, consisting of basal medium, supplementary components, growth factors, and inhibitors, is shown in Table 1.

[0068]

[0069] The preparation methods for different formulas are the same, and the specific steps are as follows:

[0070] (1) Dilute EGF and FGF10 with 5% trehalose buffer;

[0071] (2) Dissolve DATP, Y-27632, DMH-1 and A-83-01 respectively using dimethyl sulfoxide;

[0072] (3) Mix IMDM and Ham's F12 medium in a 1:1 ratio to obtain the basic medium;

[0073] (4) Prepare the basic culture medium according to the formula shown in Table 1. Add (1), (2) and supplementary ingredients to (3) and mix well to obtain the complete culture medium for airway basal stem cells. The culture medium needs to be protected from light during preparation and can be stored at 4°C for 7 days.

[0074] Example 2. Effects of culture medium on the proliferation and cell morphology of basal stem cells in large and small airways

[0075] 1. Experimental Methods

[0076] (1) Culture of airway basal stem cells

[0077] HBECs and HSAECs were cultured in 12-well plates using the formulation medium from Example 1 and the commercial Ex-Plus medium, respectively, without adding a feeder layer. 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 (#A6964, Sigma). After digestion, the cells were counted, and the cell diameter was recorded. Subcultures were continued in 12-well plates or seeded into cell culture dishes at appropriate cell densities for subsequent assays.

[0078] (2) Morphological observation

[0079] The growth morphology and state of basal stem cells in the large and small airways were observed under a microscope.

[0080] 2. Experimental Results

[0081] (1) Effects on cell morphology

[0082] like Figure 1 As shown, HBEC cells continuously cultured in Ex-Plus medium exhibited good adherence and growth in passages P3-P6, with tight intercellular connections, clear edges, and the majority of cells being cuboidal in shape, 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 after the 8th passage.

[0083] like Figure 2 As shown, HBECs were continuously cultured in Formula 1 medium. From P3 to P10, the cells adhered well and grew well, with tight intercellular connections, clear edges, and a cuboidal cell morphology, exhibiting excellent overall homogeneity. From P10 to P15, the cells adhered well and grew well, but the growth rate was relatively slow. The intercellular connections remained tight, and the cell morphology was still cuboidal, with good overall homogeneity.

[0084] like Figure 3 As shown in Figure A, HBEC cells continuously cultured in Ex-Plus medium showed vesicular formation two days after passage, with inconsistent cell morphology and small clones. Figure 3 As shown in Figure B, HBECs cultured continuously in Ex-Plus medium were replaced with Formula 1 medium on the day of passage to P7. Two days later, the P7 cell clones were more pronounced, and the cell morphology was more uniform, mostly cuboidal. This indicates that Formula 1 medium can salvage the poor cell morphology and poor proliferation of HBECs in the later stages of Ex-Plus medium culture.

[0085] like Figure 4 As shown, 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 medium ( Figure 4 A) (without EGF), cells do not adhere to the cell wall and grow; in formulations 4-6 ( Figure 4 (D~F) The cells become elongated in appearance, exhibiting obvious deformation. In formula 1 ( Figure 4 C) and Formula 3 Figure 4 In culture medium B), cells maintain good morphology and growth. Therefore, EGF is a key factor in this formulation, and a concentration in the range of 0.1–1 ng / mL is suitable.

[0086] like Figure 5As shown, P3 generation HBECs were cultured in formulations 1 and 7-12, with the same number of cells seeded and cultured for 4 days, then in formulations 10-12. Figure 5 E~G), the cells exhibited significant deformation in the culture medium formulated with formula 3 (E~G), Figure 5 A) Cell proliferation is extremely slow, failing to achieve normal expansion. In formula 1 ( Figure 5 C), Formula 4 Figure 5 B) and Formula 5 Figure 5 In the D) culture medium, cells maintain good morphology and growth status, and exhibit good overall homogeneity. Therefore, FGF10 promotes cell proliferation, and a concentration in the range of 0.04–2.5 ng / mL is suitable.

[0087] like Figure 6 As shown, HBECs were continuously cultured in Formula 13 medium (without DAPT), but the cells only lasted for 4 passages, 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 cell passages for 7 generations (i.e., P3-P9), but a significant number of cells died during the culture process (data not provided in the image). Compared to Formula 1 (with DAPT)... Figure 2 In contrast, the inhibitor DAPT added to the formula helps maintain the morphology of airway basal stem cells and promote long-term passage growth.

[0088] The results in summary indicate 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.

[0089] like Figure 7 As shown, HSAEC cells continuously cultured in Ex-Plus medium exhibited good adherence and growth in passages P3-P6, with tight intercellular connections, clear edges, and the majority of cells being cuboidal in shape, demonstrating good overall uniformity. Passages P7-P9 showed generally poor growth, with slower expansion and abnormal cell morphology, including vesicle formation and filamentation.

[0090] like Figure 8 As shown, HSAEC cells were cultured to the 9th generation using Formula 1 medium. Cells from P3 to P9 showed good adherence and growth, tight intercellular connections, clear edges, a cuboidal cell morphology, and excellent overall homogeneity.

[0091] 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.

[0092] (2) Effects on cell proliferation capacity and diameter

[0093] like Figure 9 As shown in Figure A, 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.

[0094] like Figure 9 As shown in B, 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 process.

[0095] like Figure 10 As shown in Figure A, HBECs cultured continuously in Ex-Plus medium could be passaged up to the P8 generation, with an average cell diameter of 19.19 μm (15.43 μm–23.94 μm); while HBECs cultured continuously 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 the P15 generation and beyond. Figure 10 The statistical chart of average cell diameter shown in Figure C visually demonstrates that cells continuously cultured using Formula 1 medium do not show significant changes in diameter.

[0096] like Figure 10 As shown in B, HSAECs were continuously cultured in Ex-Plus medium, and the cells were continuously 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 the cells were continuously passaged to the P9 generation, with an average cell diameter of 15.96 μm (13.31 μm–17.33 μm), and as... Figure 10 The statistical chart of average cell diameter shown in D visually demonstrates that cells continuously cultured using Formula 1 medium do not show significant changes in diameter.

[0097] The results in summary indicate that airway basal stem cells cultured in Formula 1 medium can be passaged multiple times (≥P10), and after multiple passages, the cells can still maintain good proliferative capacity with little change in average cell diameter.

[0098] Example 3. Immunofluorescence detection of airway basal stem cells

[0099] 1. Experimental Methods

[0100] (1) Culture of airway basal stem cells

[0101] Airway basal stem cells were cultured in the culture medium of Formula 1 from Example 1.

[0102] 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.

[0103] (2) Immunofluorescence detection

[0104] 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.

[0105] 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).

[0106] Table 2. Molecular marker antibodies used for immunofluorescence identification of airway basal stem cells.

[0107]

[0108]

[0109] 2. Experimental Results

[0110] like Figure 11 As shown, A is the staining image of P63 and KRT5 in P4 generation HBEC cultured in Formula 1 medium, viewed at 5X field; B is the staining image of P63 and KRT5 in 20X field; C is the staining image of NGFR and KI67 in 5X field; and D is the staining image of NGFR and KI67 in 20X field. 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.

[0111] like Figure 12 As shown, A is the staining image of P63 and KRT5 in P7 generation HSAECs cultured in the formulated medium at 5X field of view; B is the staining image of P63 and KRT5 at 40X field of view; C is the staining image of NGFR and KI67 at 5X field of view; and D is the staining image of NGFR and KI67 at 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.

[0112] 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.

[0113] Example 4: Detection of the ability of airway basal stem cells to induce differentiation

[0114] 1. Experimental Methods

[0115] (1) Culture of airway basal stem cells

[0116] Airway basal stem cells were cultured in 6-well plates using the culture medium formulated as described in Example 1, without adding feeder cells. The medium was changed every 2 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. P7HBEC / 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.

[0117] (2) Differentiation induction by gas-liquid interface culture method

[0118] 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, maintaining the gas-liquid interface for 21 days.

[0119] (3) Immunofluorescence detection

[0120] 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.

[0121] 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).

[0122] Table 3. Molecular marker antibodies used for immunofluorescence identification of airway basal stem cell differentiation capacity.

[0123]

[0124] 2. Experimental Results

[0125] like Figure 13 As shown, A to B are bright-field images under a microscope on day 21 of P7 generation HBEC differentiation under different fields of view; C is an image of MUC5AC stained with Acetylated-Tubulin under a 40X field of view; and D is an image of MUC5AC stained with Acetylated-Tubulin under a 100X field of view.

[0126] like Figure 14 As shown, A to B are bright-field images under a microscope on day 21 of differentiation of P7 generation HSAECs under different fields of view; C is an image of MUC5AC stained with Acetylated-Tubulin under a 40X field of view; and D is an image of MUC5AC stained with Acetylated-Tubulin under a 100X field of view.

[0127] 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.

[0128] Comparative Example 1 verifies whether the culture medium for differentiating airway progenitor cells can be used to culture airway basal stem cells.

[0129] The formulations disclosed in the patents (US2024052319A1; US2021254016A1) have similar components to those in this application. Both patents describe methods for differentiating pluripotent stem cells or lung progenitor cells into epithelial cells or basal cells.

[0130] In patent (US2024052319A1), during the stage of generating immature airway progenitor cells from pluripotent stem cells, serum-free differentiation medium (cSFDM) + airway differentiation medium (ADM) were used. The cSFDM formulation was 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 were mixed and aseptically filtered. Similar components to this application in patent (US2024052319A1) include: basal medium IMDM and Ham's F-12, and supplementary components 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 culture medium formulation 1 in the patent application, which is 2 mM GlutaMAX, 50 μg / mL L-ascorbic acid, and 0.4 μM 1-thioglycerol. However, the basal culture medium IMDM and Ham's F-12 in the aforementioned patent (US2024052319A1) are configured in a ratio of 3:1, and B27 is 0.5X, which is different from the present invention. Among them, 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 this application 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 the culture medium formulation 1 of the patent application is 0.4 ng / mL. The suitable range for FGF10 in the patent application's culture medium is 0.04-2.5 ng / mL. The final concentration of Y-27632 is the same as that in formulation 1 of this application, i.e., 10 μM.

[0131] In patent (US2021254016A1), during the stage of airway progenitor cells / immature basal cells maturing into basal stem cells, the third culture medium used is Pneumacult ExPlus. TM And add Y-27632, DMH-1, A-83-01.

[0132] The HSA, hydrocortisone, EGF, and DAPT involved in the formulation of this application are not present in the formulations disclosed in the patents (US2024052319A1; US2021254016A1).

[0133] Based on the similar components of the formulations in this application and the published patents (US2024052319A1; US2021254016A1), the following four formulations were prepared for testing (the reagent dosage is based on preparing 50mL of culture medium).

[0134] Table 4 shows four culture medium formulations used to verify their suitability for culturing airway basal stem cells.

[0135]

[0136] HBECs of generation P3 were cultured in culture media formulated according to formulas ①-④ respectively. Figure 15 As shown, after 3 days of culture, the cells exhibited significant deformation. After passage at this morphology and density, the bright-field plot after 24 hours is shown below. Figure 16 As shown, the cells cannot survive normally.

[0137] 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.

[0138] References

[0139] 1. Hawkins FJ, Suzuki S, et al. Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells. Cell Stem Cell. 2021Jan 7; 28(1):79-95.

[0140] 2. Zhang Y, Lin T, et al. STAT3 mutation-associated airway epithelialdefects in Job syndrome. J Allergy Clin Immunol. 2023Aug; 152(2):538-550.

[0141] 3.Wagner R, Amonkar GM, et al. ATracheal Aspirate-derived Airway BasalCell Model Reveals a Proinflammatory Epithelial Defect in CongenitalDiaphragmatic Hernia. Am J Respir Crit Care Med. 2023May 1; 207(9):1214-1226.

[0142] 4.Wu H,Tang N.Stem cells in pulmonary alveolar regeneration.Development.2021Jan 18;148(2):dev193458.

[0143] 5. Usmani OS, Dhand R, et al. Why We Should Target Small Airways Disease in Our Management of Chronic Obstructive Pulmonary Disease. Mayo ClinProc. 2021Sep; 96(9):2448-2463.

[0144] 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 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.

2. The airway basal stem cell culture medium of claim 1, wherein the supplementary components comprise HSA, B27, GlutaMAX and L-ascorbic acid.

3. The airway basal stem cell culture medium of claim 1 or 2, wherein the supplementary component further comprises 1-thioglycerol and hydrocortisone.

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

5. The airway basal stem cell culture medium according to any one of claims 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.

6. The airway basal stem cell culture medium according to any one of claims 1-5, wherein the inhibitor comprises 1-5 μM MDAPT, 1-10 μM A-83-01, 1-5 μM DMH-1 and 5-10 μM Y-27632.

7. The airway basal stem cell culture medium according to any one of claims 1-6, wherein the basal culture medium is selected from DMEM / F12, BasalMedium Eagle (BME), BGJb medium, CMRL 1066 medium, Glasgow MEM, IMDM, Medium199, Eagle MEM, αMEM, DMEM, Ham's medium, RPMI 1640, Fischer's medium, or a mixture thereof; 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.

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 according to any one of claims 1-7.

9. The airway basal stem cell culture medium according to any one of claims 1-7 or the method according to claim 8, wherein the airway basal stem cells are derived from mammals, preferably humans, mice or rats.

10. The airway basal stem cell culture medium according to any one of claims 1-7 or the method according to claim 8, wherein the airway basal stem cells are human bronchial epithelial cells (HBEC) or human small respiratory tract epithelial cells (HSAEC).

11. A kit comprising the airway basal stem cell culture medium according to any one of claims 1-7.

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