Treatment of graft-versus-host disease
a technology of graft and host disease, applied in the direction of skeletal/connective tissue cells, immunological disorders, peptide/protein ingredients, etc., can solve the problems of patient death, agvhd requires a different approach, and liver function problems
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example 1
Separation of Mesenchymal or Marrow Stem Cells Using Subfractionation Culturing Method
[0079]After applying local anesthesia to a section of the marrow provider's (mother of treatment target chronic graft-versus-host disease patient) hip, bone marrow was extracted by inserting an injection needle into the hip bone. 15 ml of DMEM (Dulbecco's modified Eagle's Medium, GIBCO-BRL, Life-technologies, MD, USA) which included 20% FBS and 1% penicillin / streptomycin and 2 ml of the marrow extracted from the above mentioned marrow provider was put into a 100 mm culture container and cultivated for 2 hours in a 37° C., 5% CO2 cell cultivator. After cultivation, the culture container was slightly leaned so that the cells attached to the bottom would not fall out and the maximum amount of the upper layer culture liquid in the culture container was moved to a new container.
[0080]After repeating the same procedure once more the culture liquid that was taken was moved to a culture container (Becton D...
example 2
Verification of Separated Mesenchymal or Marrow Stem Cells
example 2.1
Analysis of Mesenchymal or Marrow Stem Cell Characteristics Using Flow Cytometry
[0082]In order to verify whether the cMSC-15 cells that were separated from the marrow using the method in the above Example 1 were mesenchymal or marrow stem cells, a flow cytometry (BD Biosciences) was used to find out if cell surface antigens with stem cell characteristics existed.
[0083]Stem cells that were successively cultivated for 6 to 7 days in a 75 cm2 culture container was treated with 0.25% trypsin and the cells were gathered. The cells were washed 2 times with a 1×PBS / 0.4% BSA to remove trypsin as well as culture liquids. The cells were collected using centrifugal separation and after measuring the number of cells, 1×106 cells were gathered in a 1.5 ml tube and blocked for 1 hour in room temperature using goat serum (Vector). After the completion of blocking, the cells were washed 2 times with a 1×PBS / 0.4% BSA and treated with a phycoerythrin (PE) attached anti-CD14, CD29, CD31, CD34, CD44, C...
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