Preparation method and application of normal stem cells separated and cultured from urine of mitochondria mt3243AG mutation groups

A technology for isolating and culturing mitochondria, which can be used in the medical field to solve problems such as difficult treatment

Pending Publication Date: 2019-04-02
SHANGHAI SIXTH PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology allows us to obtain data on blood samples taken by individuals who have been given certain treatments or drugs based solely upon their genetic makeup rather than just their physical characteristics such as race, weight, height, etc., which may affect how well they respond differently during treatment. By isolating specific types of bone marrow stroma (osteoclast) cells containing different growth factors like BMP2/BMP4, we aimed at developing an improved method called induced pluripotential stemcell(iPSC). These new cells could potentially provide potential sources of tissue regenerative medicine for diseases associated with defective hematopoiesis due to various causes including cancer chemotheraphylaxis.

Problems solved by technology

Technological Problem addressed in this patents relates to finding ways to improve mammals through cell replacement or manipulation techniques like embryonic stem/stroma transplanting (ESFT). While these methods aimed towards improving health outcomes associated with aging, cancer risk, renal failure, brain disorders, spinal cord injuries, etc., current treatments only target specific symptoms but cannot address all issues related to metabolism abnormality. There remains a technical challenge because existing therapeutic agents lack effective targets against certain types of nerve degenerative conditions.

Method used

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  • Preparation method and application of normal stem cells separated and cultured from urine of mitochondria mt3243AG mutation groups
  • Preparation method and application of normal stem cells separated and cultured from urine of mitochondria mt3243AG mutation groups
  • Preparation method and application of normal stem cells separated and cultured from urine of mitochondria mt3243AG mutation groups

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Experimental program
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Embodiment 1

[0029] Detection of urine stem cell mutation rate;

[0030] 1. Isolate and culture stem cells from the urine of mutation patients and monoclonal isolation and culture;

[0031] 2. Extract cell DNA and measure the concentration of sample DNA;

[0032] 3. Perform PCR amplification on the extracted DNA;

[0033] 4. Pyrosequencing, 96-well plate, add 70uL binding buffer mix to each well, each well corresponds to 10uL of PCR product, turn on the shaker, shake at 25°C, 1300rpm for 15Min, add primers and buffer, place the 24-well circular plate on shaker machine, 80°C, 5min, 0rpm, record the results of the mutation rate of A>G as Figure 5 shown;

[0034] M1 and M2 were two mitochondrial 3243A>G mutation carriers, 9 single clones were co-cultured in M1, and 19 single clones were co-cultured in M2, of which 6 and 4 single clones with <2% mutation were respectively. Among them, the mutation rate of M1-1, M1-2 and M2-10 after osteogenic differentiation was still <2% by pyrosequencin...

Embodiment 2

[0036] Osteogenic and adipogenic differentiation of mutation-free urinary stem cells from normal controls and mutation patients

[0037] Osteogenic differentiation: Place the cells in an incubator at 37°C with 5% CO2 for culture. When the cell confluence reaches 60%-70%, carefully suck off the complete medium in the well and add it to the six-well plate 2mL adult bone marrow mesenchymal stem cell osteogenic differentiation complete medium (Cyagen, HUXMA-90021), replaced with fresh adult bone marrow mesenchymal stem cell osteogenic differentiation complete medium every 2 to 3 days (preheated to 37 ℃), after 2-4 weeks of induction, depending on the morphological changes and growth of the cells, stain with Alizarin Red and perform related experiments;

[0038]Adipogenic differentiation: Place the cells in an incubator at 37°C with 5% CO2 for culture. When the cell confluence reaches 100% or overconfluence, carefully suck away the complete medium of mesenchymal stem cells and add ...

Embodiment 3

[0040] Detection of ROS, Membrane Potential and ATP in Urinary Stem Cells Without Mutation in Normal Controls and Mutant Patients

[0041] 1. Isolate and culture stem cells from the urine of mutation patients and normal controls;

[0042] 2. For ATP detection, use a centrifuge tube to centrifuge the cells at 3000rpm / 5min, discard the supernatant, and gently scatter the cells. Add the lysate according to the ratio of 200 microliters of lysate to each well of the 6-well plate, lyse the cells, and lyse the cells. Centrifuge at 12000g at 4°C for 5-10 minutes, take the supernatant for subsequent determinations, dissolve the reagents to be used in an ice bath, dilute the ATP standard solution with the ATP detection lysate to an appropriate concentration gradient, and follow each sample or standard Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 microliters of ATP detection working solution, dissolve the reagent to be used on an ice bath,...

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Abstract

The invention belongs to the technical field of medical technology and particularly discloses a preparation method and an application of normal stem cells separated and cultured from urine of mitochondria mt3243AG mutation groups. The preparation method is characterized by comprising the following steps: S1, stem cell extraction; S2, stem cell culture; and S3, stem cell experiment. The preparationmethod and the application have the advantages that noninvasive acquisition can be realized regardless of sex, age and health condition of patients. Besides, as primary cells, stem cells reserve basic properties of original cells, stem cells are separated and extracted from urine of mutation patients, non-mutation urine stem cells are separated from cells by monoclone and have potentials of differentiating into non-mutation normal bone cells and normal fat cells, and theoretical basis and technical support are provided for autologous stem cell tretament for patients.

Description

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Claims

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Application Information

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Owner SHANGHAI SIXTH PEOPLES HOSPITAL
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