WO2014107784A1 - Procédé de collecte pour la congélation et le stockage de cellules souches à partir de pulpe de dents temporaires - Google Patents

Procédé de collecte pour la congélation et le stockage de cellules souches à partir de pulpe de dents temporaires Download PDF

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Publication number
WO2014107784A1
WO2014107784A1 PCT/BR2014/000015 BR2014000015W WO2014107784A1 WO 2014107784 A1 WO2014107784 A1 WO 2014107784A1 BR 2014000015 W BR2014000015 W BR 2014000015W WO 2014107784 A1 WO2014107784 A1 WO 2014107784A1
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WIPO (PCT)
Prior art keywords
freezing
collection
tooth
storage
stem cells
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Ceased
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PCT/BR2014/000015
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English (en)
Portuguese (pt)
Inventor
Jose Ricardo MUNIZ FERREIRA
Roberto DALTO FANGANIELLO
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Publication of WO2014107784A1 publication Critical patent/WO2014107784A1/fr
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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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0664—Dental pulp stem cells, Dental follicle stem cells

Definitions

  • Patent Descriptive Report "COLLECTION PROCESS FOR FREEZING AND STORAGE OF TRUNK CELLS FROM DECIDENT Teeth PULP".
  • the present invention relates to a collection process for freezing and storing stem cells from primary tooth pulp, more precisely opening another possibility of collecting stem cells.
  • PI 0513773-0 A Stem cells obtained from deciduous pulp or permanent teeth and dental germs, capable of producing Human bone tissue. This technology provides for the isolation, growth, application for production of autologous bone "in vitro", with the preservation of this living bone and its application in bone tissue reconstruction in patients.
  • the collection process for freezing and storing stem cells from primary teeth pulp deals with the sequence of procedures from primary teeth collection, stem cell isolation, expansion, to storage of stem cells under favorable conditions. and when necessary, and for the operation of the process a Collection Kit was developed to allow the Dentist to perform the collection in his own office according to pre-established technical protocols and deadlines. which was called the collection kit.
  • COLLECTION (Phase 1): The collection of primary teeth will be done by accredited dentists and pediatric dentists, after signature, by the child's parents or legal guardian and the responsible physician, of the service contract. It will be recommended that before extraction the child is healthy and free of oral infectious diseases. It will also be recommended that before the time of extraction the tooth be inspected for possible cavities or infections. The service can only be hired by parents or legal guardians of children free of infectious diseases at the time of tooth extraction.
  • the tooth be washed with 10 mL saline solution (solution included in the Collection Kit) inspected by accredited clinicians (A) and immediately immersed in collection media, packed in a collection tube (included in the Collection Kit), placed in the Collection Kit box and stored in a refrigerator (average temperature 4 ° C) until time of shipment.
  • PROCESSING (Phase 3): Upon arrival of the material, the tooth will be visually inspected for caries or infectious conditions that may increase the risk of cell culture contamination or decrease the efficiency of dental pulp stem cell isolation. The tooth will undergo three washes in PBS IX (phosphatebuffered saline) solution: 137 mMNaCl; 2.7 mMKCl; 4.3 mM Na 2 HP0 4 ; 1.47 mM KH 2 P0 4 , pH adjusted to 7.4) for 1 minute, 1 solution wash 1% povidoneiodine for 2 minutes, 1 wash in 0.1% sodium thiosulfate solution for 1 minute and 1 wash in PBS IX solution for 30 seconds.
  • PBS IX phosphatebuffered saline
  • the root will be separated from the dental crown using a sterile disposable scalpel.
  • the dental pulp will be accessed and isolated with the aid of Gracey's lime or curette, washed twice in antibiotic-supplemented PBS IX solution (100U / mL penicillin and 100 ⁇ g / mL streptomycin).
  • the pulps will be placed in type I and II collagenase solution and incubated at 37 ° C for 40 minutes.
  • the culture flasks will be monitored daily and bottles with contamination or evidence of onset of contamination will be discarded immediately. Flasks with healthy cell populations will be monitored for cell growth, with culture medium changed every 3 days, and also quality control (B).
  • FREEZING AND STORAGE phase 4: After reaching semi-confluence, the culture medium will be removed and the cells will be washed once with PBS IX. We will add PBS IX and the cells will be released from the bottom of the bottles using the CellLifter (Corning) instrument.
  • Cells will be counted using CellCountess R AutomatedCellCounter (Life Technologies) and aliquots of 0.5x10 5 cells will be diluted in cell freezing medium (20% basal medium, 70% AB-HumanSerum and 10% dimethylsulfoxide) in cryotubes and frozen at a reduction of 1 ° C per minute (with the aid of the 5100 Cryo 1 ° C Freezing Container - ThermoScientific apparatus) to -85 ° C and then transferred to gallons of liquid nitrogen.
  • cell freezing medium (20% basal medium, 70% AB-HumanSerum and 10% dimethylsulfoxide
  • cryotubes frozen at a reduction of 1 ° C per minute (with the aid of the 5100 Cryo 1 ° C Freezing Container - ThermoScientific apparatus) to -85 ° C and then transferred to gallons of liquid nitrogen.
  • the Collection Kit in which the collected material will be transported is stored in a Styrofoam container, disposable tube with 10 ml saline, collection tube (2 ml volume) with a 5 ml collection medium (Stern Pro MSC SFM XenoFreelOOU / mL penicillin and 100 ⁇ g / mL streptomycin), 1 packet of liquid ice and 1 instruction sheet for storing the tooth and shipping the kit.
  • a Styrofoam container disposable tube with 10 ml saline, collection tube (2 ml volume) with a 5 ml collection medium (Stern Pro MSC SFM XenoFreelOOU / mL penicillin and 100 ⁇ g / mL streptomycin), 1 packet of liquid ice and 1 instruction sheet for storing the tooth and shipping the kit.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Rheumatology (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

L'invention concerne un procédé de collecte pour la congélation et le stockage de cellules souches à partir de pulpe de dents temporaires, et notamment un procédé permettant de collecter des cellules souches de dents temporaires à partir de leur extraction, comprenant les étapes suivantes : collecte (phase 1) de cellules souches à partir de dents temporaires appelées à être extraites par des dentistes et des odontopédiatres agréés; envoi (phase 2) de la matière collectée au laboratoire dans les 48 heures après la collecte de la dent, insérée dans une trousse de collecte destinée à son transport; traitement (phase 3) consistant à inspecter visuellement la dent au moment de l'arrivée de la matière, en vue de l'identification de caries ou d'états infectieux susceptibles d'augmenter le risque de contamination de la culture cellulaire ou de réduire l'efficacité de l'isolement des cellules souches de la pulpe dentaire, lesquelles sont ensuite augmentées par congélation et stockage (phase 4).
PCT/BR2014/000015 2013-01-14 2014-01-03 Procédé de collecte pour la congélation et le stockage de cellules souches à partir de pulpe de dents temporaires Ceased WO2014107784A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BR102013000871A BR102013000871A2 (pt) 2013-01-14 2013-01-14 Processo de coleta para congelamento e armazenamento de células tronco a partir da polpa de dentes decíduos
BRBR102013000871-0 2013-01-14

Publications (1)

Publication Number Publication Date
WO2014107784A1 true WO2014107784A1 (fr) 2014-07-17

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PCT/BR2014/000015 Ceased WO2014107784A1 (fr) 2013-01-14 2014-01-03 Procédé de collecte pour la congélation et le stockage de cellules souches à partir de pulpe de dents temporaires

Country Status (2)

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BR (1) BR102013000871A2 (fr)
WO (1) WO2014107784A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106367385A (zh) * 2016-09-30 2017-02-01 广州赛莱拉干细胞科技股份有限公司 一种培养基及其应用与培养牙髓干细胞的方法
CN106479891A (zh) * 2016-12-20 2017-03-08 北京市肿瘤防治研究所 大肠癌原代细胞培养试剂盒及其应用方法
IT201700052399A1 (it) * 2017-06-06 2018-12-06 Costantino Santacroce Nuova metodica di separazione e crioconservazione della polpa dentaria per ottenere cellule staminali mesenchimali multipotenti
CN109468273A (zh) * 2018-12-29 2019-03-15 厦门博瑞思生物科技有限公司 一种从脐带中获得间充质干细胞的方法
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

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017079813A1 (fr) * 2015-11-10 2017-05-18 Muniz Ferreira José Ricardo Procédé de traitement de cellules souches provenant de la pulpe de dents déciduales avec cryoconservation et décongélation postérieures
CN112167246B (zh) * 2020-10-31 2021-07-27 北京泰盛生物科技有限公司 一种牙髓间充质干细胞冻存液及冻存方法

Citations (3)

* Cited by examiner, † Cited by third party
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BRPI0402659A (pt) * 2004-06-23 2006-01-31 Univ Fed Sao Paulo Unifesp Uso de células tronco, método de engenharia tecidual, usos de tecidos dentais e substituto biológico do dente
US20060252151A1 (en) * 2001-10-09 2006-11-09 Sramek Roger A Stem cell and dental pulp harvesting method and apparatus
BRPI0513773A (pt) * 2004-07-28 2008-05-13 Franceso Carinci células tronco obtidas da polpa decìdua ou de dentes permanentes e dos germes dentais, capazes de produzir tecido ósseo humano

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060252151A1 (en) * 2001-10-09 2006-11-09 Sramek Roger A Stem cell and dental pulp harvesting method and apparatus
BRPI0402659A (pt) * 2004-06-23 2006-01-31 Univ Fed Sao Paulo Unifesp Uso de células tronco, método de engenharia tecidual, usos de tecidos dentais e substituto biológico do dente
BRPI0513773A (pt) * 2004-07-28 2008-05-13 Franceso Carinci células tronco obtidas da polpa decìdua ou de dentes permanentes e dos germes dentais, capazes de produzir tecido ósseo humano

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRAGA, P.B. ET AL.: "Projeto ''A Fada do Dente''.", EM INSTITUTO AUTISMO E VIDA, 25 April 2012 (2012-04-25), Retrieved from the Internet <URL:http://www.autismoevida.org.br/2012/04/projeto-fada-do-dente.html> [retrieved on 20140402] *
BRUCATO-THOMAS, D.: "Stem Cells.", REGISTERED DENTAL .HYGIENIST, vol. 29, no. ISSUE, February 2009 (2009-02-01), pages 70 - 95, Retrieved from the Internet <URL:http://www.rdhmag.com/articles/print/volume-29/issue-2/feature/stem-cells.html> *
KERKIS, 1. ET AL.: "Stem Cells in Dental Pulp of Deciduous Teeth.", TISSUE ENGINEERING: PART B, vol. 18, no. 2, 2012 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106367385A (zh) * 2016-09-30 2017-02-01 广州赛莱拉干细胞科技股份有限公司 一种培养基及其应用与培养牙髓干细胞的方法
CN106479891A (zh) * 2016-12-20 2017-03-08 北京市肿瘤防治研究所 大肠癌原代细胞培养试剂盒及其应用方法
IT201700052399A1 (it) * 2017-06-06 2018-12-06 Costantino Santacroce Nuova metodica di separazione e crioconservazione della polpa dentaria per ottenere cellule staminali mesenchimali multipotenti
CN109468273A (zh) * 2018-12-29 2019-03-15 厦门博瑞思生物科技有限公司 一种从脐带中获得间充质干细胞的方法
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

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