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WO2025129376A1 - Procédé pour améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire - Google Patents

Procédé pour améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire Download PDF

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Publication number
WO2025129376A1
WO2025129376A1 PCT/CN2023/139398 CN2023139398W WO2025129376A1 WO 2025129376 A1 WO2025129376 A1 WO 2025129376A1 CN 2023139398 W CN2023139398 W CN 2023139398W WO 2025129376 A1 WO2025129376 A1 WO 2025129376A1
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WIPO (PCT)
Prior art keywords
dental pulp
cells
mesenchymal stem
enhancing
stem cells
Prior art date
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Pending
Application number
PCT/CN2023/139398
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English (en)
Chinese (zh)
Inventor
陈俊莉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guanhao Biotech Co Ltd
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Guanhao Biotech Co Ltd
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Filing date
Publication date
Application filed by Guanhao Biotech Co Ltd filed Critical Guanhao Biotech Co Ltd
Priority to PCT/CN2023/139398 priority Critical patent/WO2025129376A1/fr
Publication of WO2025129376A1 publication Critical patent/WO2025129376A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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

Definitions

  • the invention relates to a method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells, and belongs to the fields of stem cell biology and regenerative medicine.
  • the methods used in the industry to enhance the anti-inflammatory ability of DPSC in vitro mainly include adding pro-inflammatory cytokines to the culture medium for co-culture (such as IFN- ⁇ , TNF- ⁇ , IL-1, IL-6, etc.) and gene editing to overexpress anti-inflammatory related factors, etc., to enhance the secretion of anti-inflammatory factors to improve the anti-inflammatory function.
  • pro-inflammatory cytokines such as IFN- ⁇ , TNF- ⁇ , IL-1, IL-6, etc.
  • gene editing to overexpress anti-inflammatory related factors, etc.
  • the purpose of the present invention is to provide a method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells, by combining the addition of interleukin factors and cell hypoxia culture to continuously and stably improve the anti-inflammatory ability of dental pulp mesenchymal stem cells without affecting cell proliferation, immunogenicity, and the safety of subsequent clinical applications.
  • the present invention provides the following technical solutions:
  • a method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells comprising the following steps:
  • step (1) the teeth are cleaned and disinfected using a physiological saline buffer solution containing penicillin-streptomycin.
  • step (2) the digestion is performed using 0.3-0.5% collagenase for 50-70 min, the centrifugation speed is 800-1000 g, and the time is 5-8 min.
  • the density of the P0 generation cells in step (3) is 4500-6500 cells/cm 2 .
  • the concentration of the lipopolysaccharide additive in step (3) is 1.0 ⁇ 2.0ng/ml.
  • the interleukin factor added in step (3) is at a concentration of 2-6 ng/ml.
  • the interleukin factor added in step (3) is at a concentration of 4 ng/ml.
  • the seeding density of the cell passage in step (3) is 5000-8000 cells/cm 2 .
  • the cells in step (3) are cultured under hypoxic conditions.
  • the method of enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells described in the present invention can more stably enhance the anti-inflammatory immunoregulatory ability of DPSC, and has stronger anti-inflammatory function than adding interleukin alone, better sustainability, and more stable batch processing.
  • the use of a low oxygen culture environment during MSC cell culture can better exert the immunoregulatory effect of MSC.
  • a method for enhancing the immunoregulatory ability of dental pulp mesenchymal stem cells comprising:
  • (1) Primary cell culture Take the extracted deciduous teeth, disinfect them, take the dental pulp, rinse them 2-3 times with physiological saline buffer containing penicillin-streptomycin, add 8 ml of 2 mg/ml type I collagenase, 3 mg/ml protease and complete human mesenchymal culture medium, and digest them in a 37°C, 5% CO2 incubator for 1 hour to form a dental pulp suspension; after digestion, collect the dental pulp suspension in a 15 mL centrifuge tube, place it in a centrifuge and centrifuge it at 800 g/min at room temperature for 5 minutes, and remove the supernatant.
  • the cells were seeded at 6000/ cm2 in a culture flask, IL-6 at a concentration of 2-4 ng/ml was added to the culture medium and mixed evenly, and then cultured in a three-gas incubator under hypoxic culture conditions of 5% O2 , 5% CO2 , and 37°C.
  • PBMC peripheral blood mononuclear cells
  • DPSC dental pulp mesenchymal stem cells
  • CD73, CD105 and CD90 which are surface markers of mesenchymal stem cells, were detected.
  • the positive expression rate of CD73, CD105, and CD90 is greater than 95%.
  • the results show that the method used in the present invention enhances anti-inflammatory and immune activities without affecting the changes of key markers on the surface of MSC cells, thereby ensuring cell quality while reducing the risks of the application process.
  • the present invention adopts a combination of adding interleukin factors and cell hypoxia culture during the cell culture process, which can continuously and stably improve the anti-inflammatory ability of dental pulp mesenchymal stem cells without affecting cell proliferation and immunogenicity, thereby improving the safety of subsequent clinical applications.
  • This method can be widely used in various types of cell culture processes.

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé permettant d'améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire, appartenant au domaine de la biologie des cellules souches et de la médecine regénérative. Le procédé comporte les étapes de prétraitement de l'échantillon de dent, de culture primaire et de sous-culture des cellules souches mésenchymateuses de pulpe dentaire, etc. Dans le processus de culture cellulaire, un mode de combinaison d'ajout de facteurs d'interleukine et de culture en hypoxie cellulaire est utilisé, ce qui permet d'améliorer de manière continue et stable les propriétés anti-inflammatoires des cellules souches mésenchymateuses de pulpe dentaire, de ne pas affecter la prolifération cellulaire et l'immunogénicité, et donc d'améliorer la sécurité des applications cliniques subséquentes.
PCT/CN2023/139398 2023-12-18 2023-12-18 Procédé pour améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire Pending WO2025129376A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/139398 WO2025129376A1 (fr) 2023-12-18 2023-12-18 Procédé pour améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/139398 WO2025129376A1 (fr) 2023-12-18 2023-12-18 Procédé pour améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire

Publications (1)

Publication Number Publication Date
WO2025129376A1 true WO2025129376A1 (fr) 2025-06-26

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Application Number Title Priority Date Filing Date
PCT/CN2023/139398 Pending WO2025129376A1 (fr) 2023-12-18 2023-12-18 Procédé pour améliorer les propriétés immunomodulatrices des cellules souches mésenchymateuses de pulpe dentaire

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WO (1) WO2025129376A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090035376A1 (en) * 2004-07-28 2009-02-05 Francesco Carinci Stem cells obtained from pulp of deciduous or permanent teeth and of dental germ, able to produce human bone tissue
CN104694465A (zh) * 2015-02-13 2015-06-10 王劲茗 一种低氧后侧群分选人牙髓干细胞的实验方法
CN106978394A (zh) * 2017-02-23 2017-07-25 北京恒峰铭成生物科技有限公司 一种抗慢性炎症的间充质干细胞的制备方法及其应用
CN109161520A (zh) * 2018-08-17 2019-01-08 北昊干细胞与再生医学研究院有限公司 间充质干细胞的分离培养方法及相应的保存方法和无血清培养基
CN114621919A (zh) * 2022-05-17 2022-06-14 天九再生医学(天津)科技有限公司 一种增强间充质干细胞免疫调节能力的方法
CN116875542A (zh) * 2023-09-08 2023-10-13 苏州依科赛生物科技股份有限公司 一种间充质干细胞培养增强剂及其应用
WO2023227972A1 (fr) * 2022-05-24 2023-11-30 Iceta - Instituto De Ciências, Tecnologias E Agroambiente Da Universidade Do Porto Procédé de préparation et de cryoconservation de pulpe dentaire provenant de dents définitives et produits associés à base de cellules souches mésenchymateuses isolées

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090035376A1 (en) * 2004-07-28 2009-02-05 Francesco Carinci Stem cells obtained from pulp of deciduous or permanent teeth and of dental germ, able to produce human bone tissue
CN104694465A (zh) * 2015-02-13 2015-06-10 王劲茗 一种低氧后侧群分选人牙髓干细胞的实验方法
CN106978394A (zh) * 2017-02-23 2017-07-25 北京恒峰铭成生物科技有限公司 一种抗慢性炎症的间充质干细胞的制备方法及其应用
CN109161520A (zh) * 2018-08-17 2019-01-08 北昊干细胞与再生医学研究院有限公司 间充质干细胞的分离培养方法及相应的保存方法和无血清培养基
CN114621919A (zh) * 2022-05-17 2022-06-14 天九再生医学(天津)科技有限公司 一种增强间充质干细胞免疫调节能力的方法
WO2023227972A1 (fr) * 2022-05-24 2023-11-30 Iceta - Instituto De Ciências, Tecnologias E Agroambiente Da Universidade Do Porto Procédé de préparation et de cryoconservation de pulpe dentaire provenant de dents définitives et produits associés à base de cellules souches mésenchymateuses isolées
CN116875542A (zh) * 2023-09-08 2023-10-13 苏州依科赛生物科技股份有限公司 一种间充质干细胞培养增强剂及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI JUAN : "Biological characteristics and clinical application potential of dental pulp-derived mesenchymal stem cells", ORAL BIOMEDICINE, vol. 14, no. 2, 16 June 2023 (2023-06-16), pages 134 - 140, XP093326676 *

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