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WO2016168950A1 - Procédé de construction in vitro d'organoïdes de glande salivaire et d'analogues d'acinus - Google Patents

Procédé de construction in vitro d'organoïdes de glande salivaire et d'analogues d'acinus Download PDF

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Publication number
WO2016168950A1
WO2016168950A1 PCT/CN2015/000295 CN2015000295W WO2016168950A1 WO 2016168950 A1 WO2016168950 A1 WO 2016168950A1 CN 2015000295 W CN2015000295 W CN 2015000295W WO 2016168950 A1 WO2016168950 A1 WO 2016168950A1
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salivary gland
cells
matrigel
medium
vitro
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WO2016168950A9 (fr
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赵振民
吕璘
张辰
刘磊
韩婷璐
张翔宇
杜水果
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

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  • the invention belongs to the technical field of tissue and organ reconstruction, and particularly relates to a method for constructing a bioengineered salivary gland organ and an acinoid based on human small salivary gland stem cells.
  • Human salivary glands mainly consist of three large salivary glands and many small parotid glands distributed under the oral mucosa. They are composed of parenchyma (acinus, duct) and stroma (connective tissue, blood vessels, lymphatic vessels, nerves, etc.). Studies on stem cells in salivary glands have focused on large salivary glands. It has been experimentally confirmed that mesenchymal stem cells exist in human parotid glands and can be induced to differentiate into osteogenic, adipogenic and chondrocytes in vitro. This is another new source of mesenchymal stem cells.
  • pluripotent progenitor cells isolated from the submandibular gland have stem cell characteristics and can differentiate into endoderm cells. They are implanted into the liver through the portal vein and found to be integrated into the trabecular bone and secrete albumin.
  • salivary gland cells were isolated from the submandibular glands of murine animals and cultured in vitro by a floating sphere culture system containing cells expressing stem cell markers Sca-1, c-Kit and Musashi-1, and confirmed that these stem cells were derived from the ductal epithelium. In vitro, these cells differentiate into salivary gland duct cells and acinar cells that secrete mucin and amylase.
  • the stem cells were enriched by labeling flow cytometry with c-Kit, and these cells were differentiated into acinar cells secreting amylase in vitro. Intra-gieririal injection of small amounts of c-Kit+ cells in the body results in long-term recovery of salivary gland morphology and function. This indicates that the salivary gland contains two kinds of stem cells, mesenchymal stem cells and epithelial stem/progenitor cells. Although these studies confirmed the presence of these two types of stem cells in large salivary gland tissue, it is clinically difficult to obtain an appropriate amount of tissue from the large salivary glands to isolate and culture these stem cells.
  • the submucosal parotid gland has homology with large salivary gland tissue: it began to be cut in the 1980s. A biopsy of the mucous glands of the lips was performed to diagnose some of the diseases of the large salivary glands, indicating the homology of the small parotid glands with the large salivary glands.
  • the cell components of the small parotid gland under the mucosa are the same as those of the large salivary gland. They are composed of acinar cells, ductal epithelial cells, myoepithelial cells and mesenchymal cells. In theory, the small parotid gland should also contain the above. Two kinds of stem cells.
  • the small parotid glands under the mucosa are widely distributed, the source is rich, the material is easy to obtain, the surgical trauma is small, the donor area is concealed, and the function of the donor area is basically not affected after cutting.
  • Organ-like units are structures formed by three-dimensional culture in vitro in an extracellular matrix with various cell types in tissues. This method is widely used in the study of small intestinal stem cells and tissue engineering construction. It has been reported in the literature that small intestinal stem cells isolated from Lgr5+ can differentiate into organ-like units in Matrigel. After detecting this type of organ unit, it was found to have different cell types, including intestinal epithelial cells, mucus secreting cells and endocrine cells, and a intestinal-like structure was observed. Based on this, the researchers conducted research on tissue engineering small intestine and various stem cell proliferation and differentiation. Recently, some scholars obtained epithelial cells and mesenchymal cells from the submandibular glandular tissue of mouse embryos from 13.5 days to 14 days.
  • the two cells were put together for three-dimensional culture in vitro, and three days later, bioengineered salivary gland buds were formed, and then Transplanted orthotopically into the submandibular gland or parotid gland, a mature functional salivary gland is formed in the body 30 days after transplantation.
  • the mesenchymal stem cell culture medium for MSCM The mesenchymal stem cell culture medium for MSCM.
  • the keratinocyte medium is KM medium.
  • a method for constructing a salivary gland organ and an acinoid-like body in vitro is carried out as follows:
  • the basement membrane matrigel is the extracellular matrix Matrigel.
  • the specific operation steps of mixing the three-dimensional culture of the small meibomian mesenchymal stem cells and the small parotid epithelial stem/progenitor cells in the basement membrane matrigel are: culturing the small parotid glands to three or more generations The mesenchymal stem cells and epithelial stem/progenitor cells are digested into single cells, mixed with 1:1 cells, seeded in Matrigel, and cultured in equal proportions of mesenchymal stem cell culture medium and keratinocyte culture medium.
  • the enzymatically digested mesenchymal stem cells and epithelial stem/progenitor cells were single cell suspensions, counted, resuspended in medium, mixed with an equal volume of 4 ° C thawed Matrigel, seeded in 24-well plates, and placed in a 37 ° C cell culture incubator. After 20 minutes, the cells were coagulated with Matrigel, and the medium was added. Each well was inoculated with 2.5 ⁇ 10 5 small mesenchymal stem cells and 2.5 ⁇ 10 5 small parotid epithelial stem/progenitor cells, mixed with 50 ⁇ L of Matrigel, and inoculated successfully.
  • the liquid was changed every other day, and after one day of in vitro culture, the cells aggregated into a branch shape, and after 3 days, organoid formation of the alveolar and tubular branch structures was observed. It can grow to the maximum in 6-10 days, and can be switched to salivary gland medium after 6 days to promote its differentiation and maturation.
  • the specific operation step of three-dimensionally culturing the human parotid epithelial stem/progenitor cells in the basement membrane matrigel is: digesting the small salivary gland epithelial stem/progenitor cells cultured to three or more generations into single cells Inoculated in Matrigel, cultured with keratinocyte culture medium, the specific operation is trypsin digestion of epithelial stem/progenitor cells as single cell suspension, counted, resuspended in medium, and mixed with an equal volume of 4 ° C thawed Matrigel, Inoculate in a 24-well plate and place in a 37 °C cell culture incubator.
  • the method of the present invention is carried out by inoculation of human small salivary gland adult mesenchymal stem cells and human small salivary gland epithelial stem/progenitor cells in a three-dimensional culture on an extracellular matrix Matrigel to obtain a human salivary gland organ, or simply Human salivary glandular acinar tissue was obtained by three-dimensional culture of human small salivary gland epithelial stem cells/progenitor on extracellular matrix Matrigel.
  • the invention has a wide source of materials, is easy to cut, has small trauma, and is concealed in the oral donor area. The material is taken from the adult tissue and can be used for autologous transplantation by in vitro expansion.
  • the invention has broad application prospects for growth and development of salivary glands in vitro or in vivo, research on salivary gland disease model, study on physiological functions and pathological changes of saliva, and treatment of salivary gland diseases.
  • Fig. 1 is a photomicrograph of a mixed culture of human salivary gland epithelial stem/progenitor cells and mesenchymal stem cells on Matrigel.
  • Figure 2 is a photomicrograph of the human small parotid epithelial stem cell culture on Matrigel.
  • DMEM/F12 is supplemented with 10% fetal bovine serum, 1% double antibody and 1% glutamine;
  • Human parotid mesenchymal stem cell culture Mesenchymal stem cell culture medium: DMEM/F12 supplemented with 5% fetal bovine serum, 1% double antibody, and BSA 10 ⁇ g/mL, apo-transferr in 10 ⁇ g/mL, insulin 5 mg/ mL, EGF 2ng/mL, FGF-2 2ng/mL and hydrocortisone 1mg/mL.
  • Mesenchymal stem cell culture medium DMEM/F12 supplemented with 5% fetal bovine serum, 1% double antibody, and BSA 10 ⁇ g/mL, apo-transferr in 10 ⁇ g/mL, insulin 5 mg/ mL, EGF 2ng/mL, FGF-2 2ng/mL and hydrocortisone 1mg/mL.
  • BSA bovine serum albumin
  • transferrin transferrin
  • BPE bovine pituitary extract
  • insulin insulin
  • insulin insulin
  • FGF-2 3ng/mL
  • EGF 1 ng/mL
  • epinephrine adrenalin
  • hydrocortisone hydrocortisone
  • Prostaglandin E2 prostaglandin E2 10 -8 M
  • T3 30 nM T3 30 nM.
  • Salivary gland organ construction and acinar construction extracellular matrix MatrigelTM (BD, US), salivary gland organ (or acinar) medium:
  • DMEM/F-12 (1:1) was added with 10% fetal bovine serum, 100 U ml -1 penicillin, 100 ⁇ g ml -1 streptomycin, 1% glutamine.
  • DMEM/F121 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% insulin-transferrin-selenium supplement, 10 mM nicotinamide, 1 x 10 -7 M dexamethasone, 1 mMb-mercaptoethanol, 20 mg/l epidermal growth factor (EGF) and 20 mg/l hepatocyte growth factor (HGF).
  • FBS fetal bovine serum
  • penicillin/streptomycin 1% insulin-transferrin-selenium supplement
  • 10 mM nicotinamide 10 mM nicotinamide
  • 1 x 10 -7 M dexamethasone 1 mMb-mercaptoethanol
  • HGF hepatocyte growth factor
  • the human parotid gland obtained by biopsy or other means was subjected to tissue block culture by tissue block culture.
  • the specific procedure is: flush the small parotid tissue with cold PBS and carefully remove the residual envelope or other connective tissue.
  • the tissue was fully cut with ophthalmic scissors to a small piece of 0.5 mm 3 size, and attached to the bottom of the T25 flask with tweezers. Each tissue block was separated by 5 mm.
  • the medium was slightly moist and placed in an incubator (37 ° C, 5 The tissue block was sufficiently adhered to the bottom of the culture flask in %CO 2 ).
  • epithelioid cells are surrounded by tissue blocks, and epithelioid cells are surrounded by spindle-like fibroblasts.
  • the two cells were isolated by the cloning loop method. 1
  • the specific operation is to circulate a type of cell with a cloning ring. After applying a small amount of petrolatum at the bottom of the cloning ring, the intracellular cells are digested with 0.25% trypsin and transferred to a six-well plate for culture.
  • the isolated small salivary gland mesenchymal stem cells were cultured with mesenchymal stem cell culture medium MSCM, and when the cells were fused to 80-90%, they were stably passaged 1:3 to 20 passages or more.
  • the isolated epithelial stem/progenitor cells were cultured in keratinocyte culture medium, and when cells were fused to 80-90%, they were also digested with 0.25% trypsin, and passaged according to the cell growth state 1:3 or 1:4. Epithelial stem/progenitor cells can also be stably passaged to 20 passages or more.
  • Small mesenchymal stem cells and epithelial stem/progenitor cells cultured for three or more generations are digested into single cells, mixed in a 1:1 cell number, and then inoculated into Matrigel using an equal proportion of mesenchymal stem cell culture medium.
  • Culture with keratinocyte culture medium The specific operation is that trypsin-digested mesenchymal cells and epithelial stem cells are single cell suspensions, which are counted, resuspended in medium, mixed with an equal volume of 4 ° C thawed Matrigel, inoculated into 24-well plates, and placed in 37 ° C cell culture. The box, after 20 minutes, was allowed to solidify in Matrigel and added to the medium.
  • the above Matrigel may also be replaced by other basement membrane matrigel, such as a basement membrane matrix containing the following parts by weight: 10 parts of laminin, 1 part of scutellaria extract, 20 parts of nestin, 20 parts of heparin glycoprotein, epidermis
  • the growth factor was 0.1
  • the fibroblast growth factor was 0.1
  • the platelet-derived proliferation factor was 0.1
  • the growth hormone release inhibitor was 0.2
  • cortisol was 0.01.
  • the above-mentioned scorpion extract is prepared by pulverizing the scorpion, immersing it in a boiling bag for 10 minutes to 11 hours, transferring it into a boiling pot, boiling at a temperature of 80-120 degrees for 10 minutes to 7 hours, and finally frying.
  • the boiled liquid is sprayed and powdered to obtain a small alfalfa extract.
  • the cells were aggregated into a branch shape, and after 3 days, obvious organoid formation of acinar and tubular branch structures was observed. After 5 days, these structures will appear more obvious, and the organs will grow significantly. It can grow to the maximum in 6-10 days.
  • Salivary gland culture medium can be used after 6 days.
  • Matrigel may be replaced by type I collagen, hydrogel, or the like.
  • the small salivary gland epithelial stem/progenitor cells cultured for three generations were digested into single cells, seeded in Matrigel, and cultured using KM medium.
  • the specific operation is to trypsinize the epithelial stem/progenitor cells as a single cell suspension, count and resuspend in medium, mix with an equal volume of 4 ° C thawed Matrigel, inoculate in a 24-well plate, and place in a 37 ° C cell culture incubator. After 20 minutes, the Matrigel was allowed to solidify and the medium was added. 5 ⁇ 10 4 salivary gland epithelial stem/progenitor cells were inoculated per well, and mixed with 50 ⁇ L of Matrigel and inoculated.
  • the fluid was changed every other day.
  • the cells were arranged in a circular polarity.
  • a spheroid of acinar-like spheroids was formed.
  • the acinar did not grow up and the number of acinars increased.
  • the volume can grow to a maximum after 6-12 days.
  • the salivary gland medium can be used to promote differentiation and maturation. The result is shown in Figure 2.
  • the above Matrigel may also be replaced by other basement membrane matrix, such as basement membrane matrix containing the following parts by weight: laminin 10, eucalyptus extract 1 part, nestin 20, heparin glycoprotein 20, epidermal growth
  • laminin 10 eucalyptus extract 1 part
  • nestin 20 heparin glycoprotein 20
  • the factor was 0.1
  • the fibroblast growth factor was 0.1
  • the platelet-derived proliferation factor was 0.1
  • the growth hormone release inhibitor was 0.2
  • cortisol was 0.01.
  • the above-mentioned cockroach extract is prepared by pulverizing the cockroach, immersing it in a boiling bag for 10 minutes to 11 hours, transferring it into a boiling pot, boiling at a temperature of 80-120 degrees for 10 minutes to 7 hours, and finally frying.
  • the boiled liquid is sprayed and powdered to obtain an extract of Euphorbia.
  • the above experiment was repeated using the above-mentioned basement membrane matrix.
  • the cells were arranged in a circular polarity.
  • an acinous spheroid was formed, and the acinar was not broken as the culture time was prolonged.
  • Growing up the number of acinars is increasing, and the volume can grow to the maximum by 6-12 days. After 6 days, the salivary gland medium can be used to promote differentiation and maturation.
  • Matrigel may be replaced by type I collagen, hydrogel, or the like.

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Abstract

L'invention concerne un procédé pour construire in vitro, par génie biologique, des organoïdes de glande salivaire et des analogues d'acinus basés sur des cellules souches adultes humaines de glande salivaire mineure. L'invention concerne en particulier un procédé pour l'obtention d'organoïdes avec des structures et des fonctions de glande salivaire par culture mixte de cellules souches mésenchymateuses et de cellules souches/progénitrices épithéliales de glandes salivaires mineures humaines, ou un procédé d'obtention de tissus de type acinus avec des structures et des fonctions d'acinus de glande salivaire par culture individuelle de cellules souches/progénitrices épithéliales de glandes salivaires mineures humaines. Le procédé présente les avantages suivants : les sources d'échantillonnage sont abondantes, elles sont faciles à découper, avec un traumatisme faible ; un site buccal donneur est caché ; le matériel est issu de tissus adultes ; et il est possible d'effectuer une transplantation autoplastique par amplification in vitro. Le procédé offre de vastes perspectives d'application dans la croissance et la recherche-développement sur les glandes salivaires in vitro ou in vivo, la recherche d'un modèle de maladie des glandes salivaires, la recherche sur des fonctions physiologiques et des modifications pathologiques de la salive et le traitement de maladies des glandes salivaires.
PCT/CN2015/000295 2015-04-24 2015-04-29 Procédé de construction in vitro d'organoïdes de glande salivaire et d'analogues d'acinus Ceased WO2016168950A1 (fr)

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US12241090B2 (en) 2014-05-28 2025-03-04 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US10174289B2 (en) 2014-05-28 2019-01-08 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11053477B2 (en) 2014-05-28 2021-07-06 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
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