Serum-free culture medium and application thereof

Abstract

The invention discloses a serum-free culture medium and application thereof. The serum-free culture medium comprises an MCDB153 basal culture medium, a high amino acid composition and a small molecule signal channel regulator composition. According to the serum-free culture medium for fine lung slice culture of the human airway epithelial stem cells and the in-vitro lung tissue, the MCDB153 basic culture medium, the high-amino-acid composition with specific components and concentrations and the small-molecule signal channel regulator composition are adopted, the chemical components of a culture system are definite, and the stability among batches is good; the long-term subculture (gt; 15 generations), maintaining stemness and differentiation potential; the establishment of a complete three-dimensional culture and directional differentiation scheme is facilitated, and technical support is provided for disease model construction and drug screening.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, in particular to a serum-free culture medium for culturing human airway epithelial stem cells and isolated lung tissue fine lung slices. Background Art

[0002] Airway epithelial tissue is an important component of the respiratory system, with complex regional-specific cell composition and functional characteristics. From the proximal airways to the distal alveoli, epithelial stem cells in different anatomical regions exhibit unique morphological characteristics and functional properties. Traditional cell culture methods are difficult to simulate this complex regional specificity and cellular heterogeneity. In contrast, the in vitro culture technology of fine lung slices has unique advantages: 1) maintaining the complete tissue structure; 2) allowing for regional studies; 3) closer to the in vivo microenvironment. However, this technology currently still faces the following challenges: 1) lack of a standardized culture system; 2) limited maintenance time of tissue activity; 3) imperfect evaluation index system; 4) the need to optimize regional-specific research methods.

[0003] Patent CN201711223340.0 discloses a method for rapidly isolating and culturing human airway epithelial stem cells. However, this method dissociates tissue into single cells, losing the original tissue structure and regional characteristics. However, maintaining tissue integrity is important for understanding disease progression and drug response. Tissues in different anatomical regions exhibit significant differences in their response to treatment, making it crucial to study the regional specificity of lung tissue in different disease states.

[0004] Therefore, establishing a standardized culture system that can maintain tissue integrity and support regional specificity research is of great significance for respiratory disease research and personalized treatment. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art and provide a serum-free culture medium for culturing human airway epithelial stem cells and isolated lung tissue fine lung slices.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect is to provide a serum-free culture medium, comprising MCDB153 basal culture medium, a high amino acid composition, and a small molecule signaling pathway regulator composition;

[0008] The high amino acid composition comprises: 50-800 μM L-histidine, 0.1-5 mM L-isoleucine, 10-500 μM L-methionine, 10-500 μM L-phenylalanine, 5-500 μM L-tryptophan, and 10-500 μM L-tyrosine;

[0009] The small molecule signaling pathway regulator composition includes: 5-15 μM Y-27632; 0.5-2 μM DMH-1; 0.5-2 μM A83-01; 10-25 μM UK5099;

[0010] The concentration of each added component is based on the total volume of the serum-free medium;

[0011] Based on the total volume of the serum-free medium, the liquid volume of the MCDB153 basal medium is 700-850 mL / L.

[0012] Furthermore, the serum-free culture medium also includes a growth factor composition.

[0013] Furthermore, the growth factor composition comprises: 1-100 ng / mL of recombinant fibroblast growth factor 1, 0.1-50 ng / mL of recombinant epidermal growth factor, and 1-100 ng / mL of recombinant insulin-like growth factor 1.

[0014] The second aspect is to provide the use of the above serum-free culture medium in the culture of human airway epithelial stem cells.

[0015] Furthermore, a three-dimensional cell scaffold system is used for culture, and the three-dimensional cell scaffold system provides an ideal three-dimensional support structure for cell culture.

[0016] The third aspect is to provide the use of the serum-free culture medium in the culture of fine lung slices of ex vivo lung tissue.

[0017] Furthermore, a three-dimensional cell scaffold system is used for culture, and the three-dimensional cell scaffold system provides an ideal three-dimensional support structure for cell culture.

[0018] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0019] The serum-free culture medium of the present invention, for culturing human airway epithelial stem cells and isolated lung tissue fine lung slices, utilizes MCDB153 basal culture medium as well as a high amino acid composition and a small molecule signaling pathway regulator composition of specific components and concentrations. The culture system has a clear chemical composition and good batch-to-batch stability. It can support long-term cell subculture (>15 generations) and maintain stemness and differentiation potential. It also facilitates the establishment of a complete three-dimensional culture and directed differentiation protocol, providing technical support for disease model construction and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Microscopic images (A) and statistical graphs (B) show the expression results of KRT5, Ki67 and SA-β-gal (characterized by immunofluorescence staining) after epithelial cells of different anatomical origins (proximal, terminal, and cellular areas balf) were cultured in the serum-free culture medium of the present invention under two-dimensional culture conditions.

[0021] Figure 2 The present invention demonstrates the ability of the serum-free culture medium to support cell therapy research while maintaining the three-dimensional structural integrity of lung tissue. (A) shows the expression of fibrosis-related markers (Collagen / α-SMA), stem cell differentiation-related markers (SCGB1A1 / SCGB3A2 / RAGE / SFTPC), and stemness-related marker (KRT5) 14 days after treatment with fluorescent (GFP)-labeled stem cells, as well as a comparison with an untreated control group. (B) Statistical analysis of changes in fibrosis indicators reflects disease progression and treatment efficacy. (C) is a statistical graph showing the differentiation ratios of various lung cell types. (D) shows MASSON staining of cultured lung sections (indicating the degree of fibrosis) to demonstrate changes in the degree of fibrosis in the treatment and disease groups. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present invention may be combined with each other.

[0023] Example 1

[0024] This embodiment provides a method for preparing a serum-free culture medium, and the specific steps are as follows:

[0025] 1. Preparation of high amino acid composition

[0026] 1) Preparation of amino acid stock solutions:

[0027] L-histidine stock solution: 200 mM, dissolved in H2O;

[0028] L-isoleucine stock solution: 500 mM, dissolved in 1 M HCl;

[0029] L-methionine stock solution: 200 mM, dissolved in H2O;

[0030] L-phenylalanine stock solution: 200 mM, dissolved in 1 M HCl;

[0031] L-tryptophan stock solution: 100 mM, dissolved in 1 M HCl;

[0032] L-Tyrosine stock solution: 200 mM in 1 M HCl.

[0033] 2) Prepare 100× high amino acid mixture:

[0034] The amino acid stock solutions were mixed in proportion, sterilized by filtration through a 0.22 μm filter membrane, and stored at 4°C.

[0035] 2. Preparation of small molecule signaling pathway modulator compositions

[0036] 1) Preparation of each regulator stock solution:

[0037] Y-27632 stock solution: 10 mM, dissolved in DMSO;

[0038] DMH-1 stock solution: 5 mM, dissolved in DMSO;

[0039] A83-01 stock solution: 5 mM, dissolved in DMSO;

[0040] UK5099 stock solution: 20 mM, dissolved in DMSO.

[0041] 2) Prepare 100× small molecule regulator mixture:

[0042] The stock solutions of each regulator were mixed in proportion, sterilized by filtration with a 0.22 μm filter membrane, and stored in aliquots at -80°C.

[0043] 3. Preparation of Growth Factor Composition

[0044] 1) Preparation of growth factor stock solutions:

[0045] Recombinant FGF1 stock solution: 100 μg / ml, dissolved in PBS containing 0.1% HSA;

[0046] Recombinant EGF stock solution: 50 μg / ml, dissolved in PBS containing 0.1% HSA;

[0047] Recombinant IGF1 stock solution: 100 μg / ml, dissolved in PBS containing 0.1% HSA.

[0048] 2) Prepare 100× growth factor mixture:

[0049] The growth factor stock solutions were mixed in proportion, sterilized by filtration through a 0.22 μm filter membrane, and stored in aliquots at -80°C.

[0050] 4. Preparation of complete culture medium

[0051] The above components were added to MCDB153 basal medium in the following proportions:

[0052] 100× high amino acid mixture: 1%;

[0053] 100× small molecule modulator mixture: 1%;

[0054] 100× growth factor mixture: 1%;

[0055] Sterilize by filtration with a 0.22 μm filter membrane, store at 4°C, and use within 2 weeks.

[0056] This example also provides a method for preparing a three-dimensional cell scaffold that combines Matrigel matrix with air-liquid interface (ALI) culture technology. The scaffold can effectively simulate the in vivo cell growth microenvironment and provide an ideal three-dimensional support structure for cell culture:

[0057] 1) Mix Matrigel and rat tail collagen in a volume ratio of 2:1 and pre-cool on ice to 4°C;

[0058] 2) Take an appropriate amount of the pre-cooled mixture (200 μL per well) and smear it on the air-liquid interface culture plate using a sterile smear tool (such as a cell scraper or sterile pipette tip);

[0059] 3) Apply the mixture in a spiral motion from the inside out, ensuring that the mixture is evenly distributed on the surface of the culture plate, forming a thin layer approximately 0.5-1.0 mm thick;

[0060] 4) Place the coated culture plate in a clean bench and let it stand at room temperature (25°C) for 15 minutes to allow the mixture to fully gel;

[0061] 5) Place the gelled culture plate under ultraviolet light for 30 minutes for sterilization;

[0062] 6) After sterilization, cells can be inoculated on the scaffold for air-liquid interface culture.

[0063] Example 2

[0064] Using the preparation method of Example 1, this example provides a serum-free culture medium for culturing human airway epithelial stem cells and isolated lung tissue fine lung slices, comprising MCDB153 basal culture medium, a high amino acid composition, a small molecule signaling pathway regulator composition, and a growth factor composition;

[0065] The high amino acid composition includes: 400 μM L-histidine, 2 mM L-isoleucine, 300 μM L-methionine, 400 μM L-phenylalanine, 400 μM L-tryptophan, and 300 μM L-tyrosine;

[0066] The small molecule signaling pathway modulator composition includes: 10 μM Y-27632; 1 μM DMH-1; 1.5 μM A83-01; 20 μM UK5099;

[0067] The growth factor composition includes: 80 ng / mL of recombinant fibroblast growth factor 1, 30 ng / mL of recombinant epidermal growth factor, and 80 ng / mL of recombinant insulin-like growth factor 1;

[0068] The concentration of each added component was based on the total volume of serum-free medium;

[0069] Based on the total volume of serum-free medium, the liquid volume of MCDB153 basal medium is 850 ml / L.

[0070] Example 3

[0071] Using the preparation method of Example 1, this example provides a serum-free culture medium for culturing human airway epithelial stem cells and isolated lung tissue fine lung slices, comprising MCDB153 basal culture medium, a high amino acid composition, a small molecule signaling pathway regulator composition, and a growth factor composition;

[0072] The high amino acid composition includes: 300 μM L-histidine, 1 mM L-isoleucine, 200 μM L-methionine, 300 μM L-phenylalanine, 250 μM L-tryptophan, and 200 μM L-tyrosine;

[0073] The small molecule signaling pathway modulator composition includes: 5 μM Y-27632; 0.5 μM DMH-1; 1 μM A83-01; 15 μM UK5099;

[0074] The growth factor composition includes: 50 ng / mL of recombinant fibroblast growth factor 1, 20 ng / mL of recombinant epidermal growth factor, and 50 ng / mL of recombinant insulin-like growth factor 1;

[0075] The concentration of each added component was based on the total volume of serum-free medium;

[0076] Based on the total volume of serum-free medium, the liquid volume of MCDB153 basal medium is 850 ml / L.

[0077] Example 4

[0078] Using the preparation method of Example 1, this example provides a serum-free culture medium for culturing human airway epithelial stem cells and isolated lung tissue fine lung slices, comprising MCDB153 basal culture medium, a high amino acid composition, a small molecule signaling pathway regulator composition, and a growth factor composition;

[0079] The high amino acid composition includes: 700 μM L-histidine, 4 mM L-isoleucine, 450 μM L-methionine, 450 μM L-phenylalanine, 500 μM L-tryptophan, and 400 μM L-tyrosine;

[0080] The small molecule signaling pathway modulator composition includes: 15 μM Y-27632; 1.5 μM DMH-1; 2 μM A83-01; 25 μM UK5099;

[0081] The growth factor composition includes: 100 ng / mL of recombinant fibroblast growth factor 1, 50 ng / mL of recombinant epidermal growth factor, and 100 ng / mL of recombinant insulin-like growth factor 1;

[0082] The concentration of each added component was based on the total volume of serum-free medium;

[0083] Based on the total volume of serum-free medium, the liquid volume of MCDB153 basal medium is 850 ml / L.

[0084] Example 5 Isolation and expansion of human airway epithelial stem cells

[0085] 3.1 Primary cell isolation

[0086] 1) Sample collection and processing: Human airway tissue samples were obtained after surgical resection and rinsed three times in pre-cooled sterile phosphate-buffered saline (PBS) to completely remove residual blood.

[0087] 2) Preparation of the enzymatic digestion system: Prepare the complex enzyme digestion solution according to the following final concentrations:

[0088] Collagenase IV: 2 mg / mL;

[0089] Elastase: 0.5 mg / mL;

[0090] DNase I: 0.1 mg / mL;

[0091] Rho kinase inhibitor Y-27632: 10 μM;

[0092] 3) Tissue Digestion: Immerse the minced tissue in digestion solution and digest in a 37°C water bath with shaking for 30-45 minutes until the tissue is completely dissociated into a single-cell suspension.

[0093] 4) Cell purification: Filter through a 70 μm pore size cell sieve to remove undigested debris, centrifuge at 300 × g for 5 minutes to collect the cell pellet, resuspend in PBS, and count.

[0094] 3.2 Primary cell culture

[0095] 1) Inoculation conditions: 2×10 5 pieces / cm 2 The cells were seeded in ordinary culture plates at an initial density of 1.

[0096] 2) Culture using the complete medium of Example 2.

[0097] 3) Culture parameters: Adherent culture was performed at 37°C, 7.5% CO2 and saturated humidity, and fresh culture medium was replaced every 48-72 hours.

[0098] 4) Growth monitoring: Observe cell morphology and colony formation status daily using an inverted phase contrast microscope. Start subculturing when the cell confluence reaches 80-90%.

[0099] 3.3 Subculture and amplification

[0100] 1) Cell dissociation: Discard the old culture medium, add TrypLE Select enzymatic solution, incubate at 37°C for 5 minutes to terminate the digestion, and collect the cells by centrifugation.

[0101] 2) Subculture ratio: Divide the cells into new culture vessels at a ratio of 1:3 to 1:4. Supplement the subculture medium with 5 μM Y-27632 to improve cell attachment and survival rate.

[0102] 3) Record cell morphological changes and passage time.

[0103] Example 6 Molecular phenotype identification and functional analysis of airway epithelial stem cells

[0104] Multi-parameter characterization was performed using immunofluorescence staining. The specific process is as follows:

[0105] 1) Fixation: Fix with 4% paraformaldehyde at room temperature for 15 minutes, then rinse three times with PBS;

[0106] 2) Permeabilization: 0.1% Triton X-100 treatment for 5 minutes, followed by PBS washing;

[0107] 3) Blocking: Block with 5% bovine serum albumin (BSA) at room temperature for 1 hour;

[0108] 4) Primary antibody incubation: Add the following primary antibodies and incubate at 4°C overnight:

[0109] anti-keratin KRT5 monoclonal antibody (1:200);

[0110] Anti-proliferation marker Ki67 monoclonal antibody (1:100)

[0111] Anti-aging marker sa-β-gal antibody (1:100)

[0112] 5) Incubate with fluorescent secondary antibody at room temperature for 1 hour;

[0113] 6) Nuclear staining: DAPI solution (1 μg / mL) was used to stain the nucleus for 5 minutes;

[0114] 7) Observe and take photos using a fluorescence microscope.

[0115] The results are as follows Figure 1 As shown:

[0116] 1) Clone formation analysis: Bright field microscopy was used to compare the clonal morphological differences of stem cells from different anatomical sources (proximal, terminal, and cellular areas). Figure 1 A) The control and proximal groups maintained a clonal morphology with clear boundaries and regular internal morphology, while the pathological cells in the terminal and cellular regions (balf) showed obvious clonal aging (keratinized bead formation and some cells did not proliferate) in two-dimensional culture, reflecting the maintenance of cellular characteristics at the two-dimensional culture level.

[0117] 2) Evaluation of maintenance of stemness, proliferation, and senescence: Quantitative analysis of KRT5 staining showed that the expression of stemness markers in proximal airway-derived cells was significantly higher than that in other groups; Comparison of proliferation capacity: Ki67 positive rate statistics showed that the proliferation activity of primary cells decreased by 40% after passage three; Senescence phenotype detection: sa-β-gal staining showed that the positive rate of long-term passaged cells (P5) reached 25%, suggesting that the number of passages should be controlled ( Figure 1 B) demonstrates that the culture system of the present invention can maintain the original biological characteristics of cells from different sources.

[0118] Example 7 Preparation and in vitro culture of fine lung slices

[0119] 5.1 Lung tissue acquisition and pretreatment

[0120] 1) Sampling: Fresh lung tissue was obtained from surgical specimens.

[0121] 2) Fast processing:

[0122] ① Immediately place the tissue in 4°C pre-cooled PBS containing antibiotics (formula: penicillin 100 U / ml, streptomycin 100 μg / ml, amphotericin B 2.5 μg / ml);

[0123] ② Gently rinse 3 times to remove surface blood and impurities.

[0124] 5.2 Anatomical Positioning and Precise Sampling

[0125] 1) Anatomical landmark identification:

[0126] ① Using the pre-terminal bronchioles (pre-TBs) as landmarks, locate the junction between the terminal bronchioles and respiratory bronchioles;

[0127] ② Verify the accuracy of the sampling area through H&E staining and distinguish normal areas from pathological areas (such as fibrosis areas).

[0128] 2) Regional-specific section preparation: Based on H&E staining results, samples were collected from the following airway regions:

[0129] ① proximal airway;

[0130] ②Intermediate airway;

[0131] ③Terminal bronchioles;

[0132] ④ respiratory bronchioles;

[0133] ⑤ Honeycomb lesion area.

[0134] 5.3 Preparation of fine sections

[0135] 1) Prepare 300 μm thick biopsies using a tissue slicer.

[0136] 2) Storage and quality inspection:

[0137] ① Transfer the slices to a six-well plate containing pre-cooled complete culture medium (Example 2);

[0138] ② Check the integrity of the slices under a microscope (no tears, clear cell structure).

[0139] 5.4 In vitro gas-liquid interface culture system

[0140] 1) Culture device:

[0141] ①Use a porous membrane culture insert (pore size 0.4 μm) and place the slice on the membrane to form an air-liquid interface;

[0142] ②The culture medium only covers the bottom of the membrane to avoid immersing the slices.

[0143] 2) Optimize the culture medium formula (based on the improvement of Example 2):

[0144] Add the following to the basal medium:

[0145] ① Vitamin A (100nM), retinol (50nM);

[0146] ②N-acetylcysteine (1mM);

[0147] ③Bovine transferrin (5μg / ml), hydrolyzed bovine placenta extract (15μg / ml).

[0148] 3) Culture condition control:

[0149] ① Constant temperature 37℃, CO2 concentration 7.5%;

[0150] ②Replace the culture medium every 24 hours to maintain the gas-liquid interface.

[0151] Example 8 Disease Model Construction and Stem Cell Therapy Verification Based on Fine Lung Slices

[0152] 6.1 Establishment of stem cell co-culture model

[0153] 1) Stem cell acquisition and expansion

[0154] ① Isolation of basal stem cells from the airways of healthy donors by in vitro brushing;

[0155] ② Expand to the third generation in a culture medium containing growth factors.

[0156] 2) Co-culture system: The expanded stem cells were co-cultured with the IPF patient lung slices prepared in Example 7 to simulate the stem cell treatment microenvironment.

[0157] 6.2 Cell Phenotype Maintenance and Efficacy Evaluation

[0158] 1) Dynamic phenotypic detection

[0159] ① Time points of immunofluorescence detection: 24 hours, 7 days, and 14 days after co-culture;

[0160] ②Test indicators:

[0161] basal cell marker (KRT5);

[0162] Airway secretory cell markers (SCGB3A2 / SCGB1A1);

[0163] alveolar markers (RAGE / SFTPC);

[0164] Fibrosis indicators (type I collagen, α-SMA).

[0165] 2) Histopathological evaluation:

[0166] ① Quantification of collagen deposition area by MASSON trichrome staining;

[0167] ②H&E staining was used to evaluate tissue structure repair.

[0168] The results are as follows Figure 2 As shown, the three-dimensional structure of the lung itself, including small airways, alveoli, respiratory parenchyma and connective tissue, is preserved by the slicing technology, and normal proliferation and differentiation can be maintained in the culture system of the present invention, thereby studying disease-related changes.

[0169] Green fluorescent (GFP)-expressing basal cells were transplanted into lung slices from patients with idiopathic pulmonary fibrosis (IPF) and cultured in vitro. The cells were analyzed on days 1, 7, and 14 ( Figure 2 A). On day 1, GFP+ stem cells were sparsely distributed; on days 7 and 14, they exhibited seamless integration into host tissue. After transplantation, GFP+ stem cells differentiated into distinct cell phenotypes: one subset retained KRT5 positivity, maintaining basal cell identity, while another subset expressed SCGB3A2 alone or both SCGB3A2 and SCGB1A1, hallmarks of secretory cell differentiation ( Figure 2 B). At the same time, compared with the control group, the fibrosis markers (including collagen) in the stem cell transplanted sections were significantly reduced, with a significant decrease on days 7 and 14, and the percentage of fibrosis area was reduced ( Figure 2 B and Figure 2 C; P < 0.05).

[0170] To precisely quantify fibrosis, tissue sections were stained with Masson's trichrome staining ( Figure 2 D). BC transplant sections show a progressive decrease in collagen deposition, reaching a statistically significant reduction on day 14 compared to the control group. The change in fibrosis percentage from day 0 to day 14 further confirms the ability of this culture system to restore the fibrotic pathology and the anti-fibrotic effects of cell therapy.

[0171] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A serum-free culture medium, characterized in that The method comprises an MCDB153 basal culture medium, a high amino acid composition, and a small molecule signaling pathway regulator composition; The high amino acid composition comprises: 50-800 μM L-histidine, 0.1-5 mM L-isoleucine, 10-500 μM L-methionine, 10-500 μM L-phenylalanine, 5-500 μM L-tryptophan, and 10-500 μM L-tyrosine; The small molecule signaling pathway regulator composition includes: 5-15 μM Y-27632; 0.5-2 μM DMH-1; 0.5-2 μM A83-01; 10-25 μM UK5099; The concentration of each added component is based on the total volume of the serum-free medium; Based on the total volume of the serum-free medium, the liquid volume of the MCDB153 basal medium is 700-850 mL / L.

2. The serum-free culture medium according to claim 1, wherein The serum-free culture medium also includes a growth factor composition.

3. The serum-free medium according to claim 2, wherein The growth factor composition comprises: 1-100 ng / mL of recombinant fibroblast growth factor 1, 0.1-50 ng / mL of recombinant epidermal growth factor, and 1-100 ng / mL of recombinant insulin-like growth factor 1.

4. Use of the serum-free medium according to any one of claims 1 to 3 in culturing human airway epithelial stem cells.

5. The use according to claim 4, characterized in that The cells were cultured using a three-dimensional cell scaffold system, which provides an ideal three-dimensional support structure for cell culture.

6. Use of the serum-free culture medium according to any one of claims 1 to 3 in culture of isolated lung tissue fine lung slices.

7. The use according to claim 6, characterized in that The cells were cultured using a three-dimensional cell scaffold system, which provides an ideal three-dimensional support structure for cell culture.

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