I am working on a project that requires stem cell transplant. I need some ideas or a link that I can follow on how to go about with the procedure
Hello Isaac.
The most easy way to familiarize the morphology and stem cell properties in the fields of adult stem cells is to study the appropriate procedure and isolation of stem cells present in bone marrow as a representative mesenchymal stem cells (MSCs).
Study the contents in the links.
Article A protocol for isolation and culture of mesenchymal stem cel...
http://www.scielo.br/pdf/%0D/aob/v14n1/en_a04v14n1.pdf
https://www.youtube.com/watch?v=Dnvq_wMZ2fI
Best regards.
Marrow stromal cells were obtained from about six-week old male rats . MSCs were flushed from the tibiae and femora according to the protocol of Ishaug. Cells were centrifuged at 1,200 rpm for 5
min. The resulting cell pellet was resuspended in basal medium consisting of DMEM, 10% fetal bovine serum, 1% penicillin/streptomycin, and 50 μ g/ml ascorbic acid. Cells were seeded in T75 flasks and cultured at 37°C and 5% CO2. Cells were allowed to adhere to the substrate for three days. The flasks were rinsed twice with phosphate-buffered saline (PBS) in order to remove non-adherent cells.
In the experiments conducted to determine the influence of different growth factors and combinations of inducers, adherent cells were exposed to either basal medium, or medium supplemented with growth factors from day 3 after the cell isolation until the end of the culture, that is, until the end of the differentiation period.
collect the adipose tissue form the subcutaneous gluteal fat of dogs.
wash thoroughly with PBS
mince the fat properly
Mix the mince tissue with collagenase type I (1 mg/mL; Sigma-
Aldrich) and incubate for 2 h at 37C.
strain the suspension through a filter (mesh).
centrifuge 300 g for 10 min
Resuspend the pellete (i.e., the stromal vascular fraction).
spread in the plate and incubate overnight with medium containing 10% fetal bovine serum (FBS, Gibco BRL) at 37C with 5% humidified CO2.
wash the plates after 24 hrs to remove the residues of blood cells and tissues.
cells are attached to the surface, then change media until the cells reach full confluence.
The procedure is almost same for adipose derived stem cells from other animals as well.
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