Dental pulp marrow similar cells (DPMSC) and methods of isolating and using
a technology of dental pulp and similar cells, which is applied in the field of compositions and methods for isolating and using dental pulp marrow similar cells (dpmscs), can solve the problems of significant cost, both financially and physically, for the patient to receive anti-rejection medications at a significant cost, and achieve the effect of easing cardiac failur
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example 1a
Isolation of DPMSC
[0223]Cell Culture
[0224]Dental pulp was extracted from normal exfoliated human deciduous teeth of 5- to 7-year old children (10 patients) with informed consent of the donors. Dental pulp was extracted using needle and was transferred into 35-mm Petri dishes F12 / Medium 199 / CMRL 1066 supplemented with 1.25% Human serum and supplemented with 1-50 ng / ml (preferably about 5-15 ng / ml) platelet derived growth factor-BB (PDGF-BB), 1-50 ng / ml (preferably about 1-15 ng / ml) epidermal growth factor (EGF), 1-50 ng / ml (preferably about 1-15 ng / ml) insulin-like growth factor (IGF), 1-50 ng / ml (preferably about 1-15 ng / ml) fibroblast growth factor-b (FGF-b), 10−10 to 10−8 M dexamethasone or other appropriate steroid, 0-1 μg / mL linoleic acid, and 10-50 mg / L ascorbic acid. Tissue explant of dental pulp was used to isolate immature DPMSC. The growing culture of DPMSC was maintained under these conditions for 1 day and then the cells were placed, optionally dissociated, in a collagena...
example 1b
Method of Isolating and / or Culturing DPMSC
[0229]Dental pulp was extracted from normal exfoliated human deciduous teeth of 5- to 7-year-old children (12 patients) with informed consent of the donors. Dental pulp was extracted using needle and was transferred into 35-mm Petri dishes (Falcon, BD-Biosciences, Italy) with a proliferation medium composed of F-12 Coon's modified / Ambesi's modified (Gibco) / Medium 199 (Sigma Aldrich, Germany) / CMRL 1066 (Sigma Aldrich, Germany) supplemented with 1.25% of Human serum 1-50 ng / ml platelet-derived growth factor-BB (PDGF-BB, Immunotools, Germany), 1-50 ng / ml epidermal growth factor (EGF, Immunotools, Germany), 1-50 ng / ml insulin-like growth factor-I (IGF-I, Immunotools, Germany), 1-50 ng / ml fibroblast growth factor-I (FGF-b, Immunotools, Germany), 10−10 to 10−8 M dexamethasone (MP), 20-100 μg / L linoleic acid (Sigma, Germany), 10-50 mg / L ascorbic acid (Sigma) and 0.5 ml / L gentamycin (Gibco). The growing culture of DPMSC was maintained under these co...
example 2a
Methods of Phenotype Analysis of DPMSC Cell Population
[0231]Immunofluorescence, Flow-Cytometry, FISH and Immunoblot
[0232]Immunofluorescence and FISH analyses were performed on 4% buffered paraformaldehyde or methanol / acetone fixed cells, while FACS analysis was performed on P3-P5 cells detached from the culture substrate through a short incubation in CTC. Staining was performed either using properly conjugated primary antibodies or with un-conjugated primary antibodies followed by an incubation with conjugated secondary antibodies. Intracellular staining was performed after a permeabilization step utilizing the Intrastain Fixation and Permeabilization kit (Dako, Danmark), following the manufacturer's instructions.
[0233]Image acquisition was carried out by a Confocal Laser Microscope (Leica TCS-SP2, Leica Microsystems, Italy), utilizing either a 63× oil immersion objective (numerical aperture: 1.40) or a 40× oil immersion objective (numerical aperture: 1.25). Epifluorescence and phas...
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