WO2023274043A1 - Cellule souche de glande lacrymale humaine, son procédé de culture par différenciation et son application - Google Patents

Cellule souche de glande lacrymale humaine, son procédé de culture par différenciation et son application Download PDF

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WO2023274043A1
WO2023274043A1 PCT/CN2022/100948 CN2022100948W WO2023274043A1 WO 2023274043 A1 WO2023274043 A1 WO 2023274043A1 CN 2022100948 W CN2022100948 W CN 2022100948W WO 2023274043 A1 WO2023274043 A1 WO 2023274043A1
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lacrimal gland
human
stem cells
culture
gland stem
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PCT/CN2022/100948
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English (en)
Chinese (zh)
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叶琳
王玲
韩春霞
张咏鑫
殷国干
王媛
杨美娜
郭慧
詹文珠
廖子芳
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深圳市眼科医院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0633—Cells of secretory glands, e.g. parotid gland, salivary glands, sweat glands, lacrymal glands
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of cell engineering, and relates to a human lacrimal gland stem cell and its differentiation and cultivation method and application.
  • the lacrimal gland is an important organ of the eye. Its main function is to secrete tears and keep the eyes moist. Dysfunction can lead to dry eye syndrome. If the lacrimal gland function is damaged or becomes diseased, it usually leads to diseases such as dry eye syndrome in the human eye. Patients with dry eye syndrome are usually accompanied by corneal inflammation, which can even cause damage to corneal cells and ulcers, resulting in decreased vision and affecting their quality of life. For patients with dry eye, although the current drug treatment can relieve the symptoms, it still cannot fundamentally solve the problem of lacrimal gland dysfunction.
  • the lacrimal gland can obtain human lacrimal gland stem cells that can be stably cultured and passaged in vitro, and construct an artificial lacrimal gland, it will be possible to use the patient's own lacrimal gland cells to reconstruct the function of the lacrimal gland, which is of great significance to the research and application of regenerative medicine.
  • 3D air-liquid interface culture can grow organoids similar to in vivo epithelial tissues. Due to the ability to cultivate living cells in an in vitro model similar to the in vivo state, the air-liquid interface is often used to study respiratory diseases, digestive system diseases, including intercellular signal transmission and disease modeling. But there is no report about its use in lacrimal gland stem cell culture.
  • the primary purpose of the present invention is to provide a human lacrimal gland stem cell for the deficiencies of the prior art.
  • the cells are isolated and cultured from normal Chinese lacrimal gland tissue. The cells do not introduce any foreign genes, can proliferate indefinitely, and can be induced to exhibit acinar characteristics.
  • Another object of the present invention is to provide a method for primary isolation and culture of the above-mentioned human lacrimal gland stem cells.
  • Another object of the present invention is to provide a method for infinite subculture of the above-mentioned human lacrimal gland stem cells.
  • the object of the present invention is also to provide the application of the human lacrimal gland stem cells.
  • the present invention provides a method for primary isolation and culture of lacrimal gland stem cells, comprising the following steps:
  • the PBS buffer is 0.01M, pH 7.4.
  • the step of removing residual fat includes placing the lacrimal gland tissue sample into a sterile petri dish containing pre-cooled PBS, and removing the residual fat in the lacrimal gland tissue sample under a dissecting microscope; In some embodiments, the pre-cooling is on ice.
  • the digestion solution in step (3) is a DMEM medium containing collagenase and dispase; in some embodiments, based on the total volume of the digestion solution, the collagenase and dispase The concentrations are 0.2 ⁇ 0.5mg/mL.
  • the amount of the digestive solution in step (3) is preferably 10-12 times the volume of the tissue sample.
  • the digestion condition in step (3) is 37°C for 1-3 hours.
  • the digestion in step (4) is digestion on ice for 1-3 hours or digestion at 37° C. for 10-15 minutes.
  • the centrifugation in steps (4), (5) and (6) is at 1000-1500 rpm for 3-5 minutes.
  • the lacrimal gland stem cell culture medium in step (7) is: DMEM medium and F12 medium are mixed at a volume ratio of 3:1, and 5% L-glutamine, 10% fetal bovine serum, 0.1% hydrocortisone, 8.4 ng/mL cholera toxin, 10 ng/mL epidermal growth factor (epithelial growth factor (EGF), 1/50 B27, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 0.25 ⁇ g/mL Amphotericin B (Fungizone), 30 ⁇ M Fasudil.
  • DMEM medium and F12 medium are mixed at a volume ratio of 3:1, and 5% L-glutamine, 10% fetal bovine serum, 0.1% hydrocortisone, 8.4 ng/mL cholera toxin, 10 ng/mL epidermal growth factor (epithelial growth factor (EGF), 1/50 B27, 100 U/mL penicillin, 100 ⁇ g/mL strepto
  • the culture medium is filtered through a filter membrane with a pore size of 0.22 ⁇ m.
  • the culture conditions in step (7) are 37°C, 5% CO 2 .
  • the present invention further provides the human lacrimal gland stem cells obtained by the above primary isolation and culture method.
  • the human lacrimal gland stem cells are derived from human normal lacrimal gland stem cells, the chromosomes are diploid, and the STR (short tandem repeat sequence) genotype is represented by 22 "STR locus/allelic length": AMEL /X/X, D3S1358/15/16, D13S317/8/12, D7S820/11/12, D16S539/10/11, Penta D/9/12, D2S441/10/11, TPOX/11/12, TH01/ 7/7, D2S1338/17/17, CSF1PO/11/12, Penta E/14/14, D10S1248/15, D19S433/13/15.2, vWA/14/16, D21S11/29/33.2, D18S51/12/20 , D6S1043/10/18, D8S1179/14/15, D5S818/11/11, D12S391/18/21, FGA/19/24.
  • the human lacrimal gland stem cells are preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202076.
  • the culture condition of the human lacrimal gland stem cells is cultured with lacrimal gland stem cells based on 37°C and 5% CO 2 culture;
  • the lacrimal gland stem cell culture medium is: DMEM and serum-free medium F12 in a volume ratio of 3:1 Mix and add 5% (v/v) FBS (fetal bovine serum), and 0.4 ⁇ g/mL cortisol (hydrocortisone), 5 ⁇ g/mL insulin (insulin), 8.4 ng/mL cholera toxin (cholera toxin), 10 ng/mL epidermal growth factor (EGF), 24 ⁇ g/mL adenine, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 0.25 ⁇ g/mL amphoteric Mycin B (Fungizone), 30 ⁇ M Fasudil (Fasudil), the above culture medium needs to be filtered through a 0.22 ⁇ m pore size filter.
  • FBS fetal bovine serum
  • the present invention also provides a subculture method for the above-mentioned human lacrimal gland stem cells, comprising the following steps:
  • the PBS buffer is 0.01M, pH 7.4, and the mass volume fraction of trypsin in the trypsin-EDTA solution is 0.05%-0.1%.
  • the digestion time in step (1) is 2-5 minutes.
  • the centrifugation in step (2) is at 1000-1500 rpm for 3-5 minutes.
  • the culture conditions in step (2) are preferably 37°C, 5% CO 2 .
  • the present invention further provides the application of the above-mentioned human lacrimal gland stem cells, including performing differentiation culture of the human lacrimal gland stem cells through a 3D air-liquid culture interface differentiation culture method to obtain a human lacrimal gland acinar organ model.
  • the 3D air-liquid culture interface differentiation culture method includes the following steps:
  • the human lacrimal gland stem cells are spread into the cell culture insert (insert), and the liquid level of the lacrimal gland stem cell culture medium inside and outside the insert is kept consistent;
  • Air-liquid culture was continuously cultured for 7 days, and the differentiation medium outside the insert was replaced every day;
  • the insert described in step (1) is placed in a multi-well cell culture plate, such as a 24-well plate, a 48-well plate or a 96-well plate.
  • the differentiation medium described in step (2) is: FGF7 and FGF10 are added to the lacrimal gland stem cell medium, preferably at a working concentration of 100 ng/ml.
  • the upper layer does not add medium and is in direct contact with air; the lower layer adds differentiation medium to provide nutrients for cell growth and differentiation.
  • the present invention also provides the application of the human lacrimal gland stem cells in the preparation of medicines for treating diseases related to lacrimal gland dysfunction.
  • the lacrimal gland dysfunction-related disease is dry eye.
  • a normal person refers to a human individual whose lacrimal gland function is normal, for example, its lacrimal gland can secrete tears normally, and the lacrimal gland function has not been damaged nor has any pathological changes occurred; Damaged and undisturbed lacrimal gland tissue.
  • the human lacrimal gland stem cells provided by the present invention are primary isolated and cultured from human normal lacrimal gland tissue.
  • the cells have not introduced any foreign genes.
  • the stem cells are identified by STR genotyping and are a kind of human that has never been registered at home and abroad. It can be used for physiological research of human normal cells, lacrimal gland and lacrimal gland-related diseases including dry eye, pathogenesis research of lacrimal secretion, toxicological research of environmental pollutants on lacrimal gland, pharmacological research of drugs, etc., also for human Cell therapy provides the basis for related diseases such as lacrimal gland dysfunction.
  • FIG. 1 is a cell morphology diagram of human lacrimal gland stem cells in Example 1.
  • FIG. 1 is a cell morphology diagram of human lacrimal gland stem cells in Example 1.
  • Fig. 2 is the growth curve of the human lacrimal gland stem cells in Example 2.
  • Fig. 3 is a graph of STR genotyping of human lacrimal stem cells in Example 3.
  • Fig. 4 is the H&E staining diagram of normal human lacrimal gland tissue (Fig. 4-A) and the 3D air-liquid culture interface of human lacrimal gland stem cells in Example 5 (Fig. 4-B).
  • Figure 5 is the expression of AQP5 molecules in the 3D air-liquid culture interface of human lacrimal gland stem cells and human normal lacrimal gland tissue in Example 6, wherein Figures A, B, and C are sequentially the AQP5 in the 3D air-liquid culture interface of human lacrimal gland stem cells in the embodiment of the present invention Photos of antibody labeling, DAPI fluorescent staining, and merged fluorescent staining (Merge); Figures D, E, and F are photos of AQP5 antibody labeling, DAPI fluorescent staining, and merged fluorescent staining (Merge) in human normal lacrimal gland tissue in sequence.
  • Figure 6 shows the 3D air-liquid culture interface of human lacrimal gland stem cells in Example 7 and the expression of CK15 molecules in normal human lacrimal gland tissue, where Figures A, B, and C are sequentially CK15 in the 3D air-liquid culture interface of human lacrimal gland stem cells according to the embodiment of the present invention Photos of antibody labeling, DAPI fluorescent staining, and merged fluorescent staining (Merge); Figures D, E, and F are photos of CK15 antibody labeling, DAPI fluorescent staining, and merged fluorescent staining (Merge) in human normal lacrimal gland tissue in sequence.
  • Figure 7 is the 3D air-liquid culture interface of human lacrimal gland stem cells in Example 8 and the expression of DSG-1 molecules in normal human lacrimal gland tissue
  • Figures A, B, and C are the 3D air-liquid culture interface of human lacrimal gland stem cells in accordance with the embodiment of the present invention
  • Figures D, E, and F are photos of DSG-1 antibody labeling, DAPI fluorescent staining, and merged fluorescent staining (Merge) in human normal lacrimal gland tissue.
  • Fig. 8 is the 3D air-liquid culture interface of human lacrimal gland stem cells in Example 9 (Fig. 8-A) and the acinar structure of normal human lacrimal gland tissue (Fig. 8-B).
  • Example 1 Primary isolation and culture of human lacrimal gland stem cells
  • the lacrimal gland stem cell culture medium is: DMEM medium and F12 medium are mixed at a volume ratio of 3:1, and 5% L-glutamine, 10% fetal bovine serum, 0.1% hydrogenated Cortisone, 8.4 ng/mL cholera toxin, 10 ng/mL epidermal growth factor (epithelial growth factor (EGF), 1/50 B27, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 0.25 ⁇ g/mL Amphotericin B (Fungizone), 30 ⁇ M Fasudil, and the above medium was filtered through a 0.22 ⁇ m pore size filter.
  • the lacrimal gland stem cell culture medium is: DMEM medium and F12 medium are mixed at a volume ratio of 3:1, and 5% L-glutamine, 10% fetal bovine serum, 0.1% hydrogenated Cortisone, 8.4 ng/mL cholera toxin, 10 ng/mL epidermal growth factor (epithelial growth
  • tissue samples in vitro come from the excised lacrimal gland tissue of patients undergoing lacrimal gland ptosis surgery, and the samples are anonymized after informed consent of the patients.
  • step (3) Put 1-2 mm3 tissue samples into the 50 mL centrifuge tube containing 10 mL digestion solution prepared in step (2), and digest at 37°C for 2 hours;
  • Resuspension step (6) The cells were precipitated in lacrimal gland stem cell culture medium, inoculated in T25 culture flasks for culture, and the culture conditions were 37°C, 5% CO 2 .
  • the primary human lacrimal gland stem cells were successfully isolated and cultured according to the above method, and the morphology of the cells observed under a microscope is shown in Figure 1 (tightly arranged, clear cell boundaries, strong three-dimensional, polygonal stem cells).
  • the cells are named human lacrimal gland stem cells, and have been deposited in the China Center for Type Culture Collection (Address: China. Wuhan. Wuhan University) on May 19, 2020, with the preservation number CCTCC NO: C202076.
  • Example 1 When the human lacrimal gland stem cells cultured in the T25 culture flask in Example 1 proliferated to 70-90% abundance, the cells were washed twice with 1 ⁇ PBS (0.01M, pH 7.4), and then washed with 0.05% (mass Volume ratio) Trypsin-EDTA digested monolayer cells for 2-5 minutes.
  • 1 ⁇ 10 6 stem cells can be resuspended in 1-2 mL of cell freezing solution (90% fetal bovine serum and 10% DMSO, v/v), and stored in liquid nitrogen for later use.
  • the human lacrimal gland stem cells were subcultured according to the above method, and the cell growth curve of the cultured line is shown in Figure 2. After continuous subculture for 80 days, the human lacrimal gland stem cells of the present invention can still maintain a proliferative state and grow normally.
  • Human lacrimal gland stem cells (1 ⁇ 10 6 ) grown on the adherent wall were washed twice with 1 ⁇ PBS, the monolayer cells were digested with 0.05% trypsin-EDTA for 5 minutes, and the digestion reaction was neutralized with 10 mL of complete DMEM.
  • PowerPlex® 16 HS system (10) PowerPlex® 16 HS system (DC2101, promega company) was used for DNA multiplex amplification of 21 loci (15 STR loci and 1 sex locus).
  • STR loci are detected, expressed in "STR loci/allele length": AMEL/X/X, D3S1358/15/16, D13S317/8/12, D7S820/11/12, D16S539/10/11, Penta D/9/12, D2S441/10/11, TPOX/11/12, TH01 /7/7, D2S1338/17/17, CSF1PO/11/12, Penta E/14/14, D10S1248/15, D19S433/13/15.2, vWA/14/16, D21S11/29/33.2, D18S51/12/ 20, D6S1043/10/18, D8S1179/14/15, D5S818/11/11, D12S391/18/21, FGA/19/24.
  • human lacrimal gland stem cells of the present invention are identified by STR genotyping, and are a human normal cell line that has never been registered at home and abroad, and are a new type of human lacrimal gland stem cells.
  • the differentiation medium was: DMEM medium and F12 medium were mixed at a volume ratio of 3:1, and 5% L-glutamine, 10% fetal bovine serum, 0.1% hydrocortisone, and 8.4 ng/mL cholera toxin were added at the same time ( cholera toxin), 10 ng/mL epidermal growth factor (epithelial growth factor (EGF), 1/50 B27, 100ng/ml FGF10, 100ng/ml FGF7, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 0.25 ⁇ g/mL Amphotericin B (Fungizone), 30 ⁇ M Fasudil, and the above medium was filtered through a 0.22 ⁇ m pore size filter.
  • DMEM medium and F12 medium were mixed at a volume ratio of 3:1, and 5% L-glutamine, 10% fetal bovine serum, 0.1% hydrocortisone, and 8.4 ng/mL cholera toxin were added at the
  • the acinar structure is shown in Figure 4, which shows that the human lacrimal gland stem cells of the present invention have a similar acinar structure to the lacrimal gland tissue.
  • the 3D air-liquid culture interface of human lacrimal gland stem cells of the present invention has the same expression of AQP5 molecule as that of human lacrimal gland tissue, wherein AQP5 is one of the specific tissue marker proteins of lacrimal gland tissue.
  • the 3D air-liquid culture interface of human lacrimal gland stem cells of the present invention has the same expression of CK15 molecule as that of human lacrimal gland tissue, wherein CK15 is one of the specific tissue marker proteins of lacrimal gland tissue.
  • the 3D air-liquid culture interface of human lacrimal gland stem cells of the present invention has the same expression of DSG-1 molecule as that of human lacrimal gland tissue, wherein DSG-1 is desmosome junction protein, which means that cells differentiate and form tight junctions.
  • Example 8 Observation of human normal lacrimal gland tissue and acinar structure at the interface of 3D air-liquid culture
  • the 3D air-liquid culture interface of human lacrimal gland stem cells of the present invention has an acinar structure similar to that of human lacrimal gland tissue, and the expression and distribution of AQP5 and Lactoferrin are specific: Lactoferrin (green fluorescence) is mainly distributed in the acinar structure, AQP5 is mainly expressed at ductal sites in the apical surface and in the middle of the acini. It can be seen that the 3D air-liquid interface differentiation method involved in this embodiment can be used to simulate the tissue structure of human lacrimal gland stem cells.

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Abstract

Cellule souche de glande lacrymale humaine, son procédé de culture par différenciation et son application. Le numéro de conservation de la cellule souche de glande lacrymale humaine est CCTCC NO : C202076. La cellule est principalement isolée et cultivée à partir du tissu normal de la glande lacrymale humaine. Aucun gène exogène n'est introduit dans la cellule. La cellule peut être utilisée pour la recherche physiologique sur les cellules normales humaines, la recherche sur la pathogenèse de la glande lacrymale et des maladies liées à la glande lacrymale comprenant la xérophtalmie et la sécrétion de larmes, la recherche toxicologique des polluants environnementaux sur la glande lacrymale, la recherche pharmacologique sur les médicaments et analogues. La cellule sert également de base à la thérapie cellulaire pour des pathologies connexes telles que le dysfonctionnement de la glande lacrymale humaine.
PCT/CN2022/100948 2021-06-27 2022-06-24 Cellule souche de glande lacrymale humaine, son procédé de culture par différenciation et son application WO2023274043A1 (fr)

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XIAO SA, ZHANG YAN: "Establishment of long-term serum-free culture for lacrimal gland stem cells aiming at lacrimal gland repair", STEM CELL RESEARCH & THERAPY, vol. 11, no. 1, 1 December 2020 (2020-12-01), XP093017966, DOI: 10.1186/s13287-019-1541-1 *

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