The overall goal of this protocol is to isolate and characterize red adipose tissue, dried mesenchymal stem cells and differentiate them towards insulin-producing cells using simple methods. Mesenchymal stem cells have found the ways in the field of genetics. They can be isolated from various sources such as bone marrow, adipose tissue and perinatal tissue.
They accept excellent cultural characteristics. They are multi potent and can potentially treat various disease. Adipose tissue provides a rich source of mesenchymal stem cells.
These adipose MSCs can be isolated from lipo aspirates easily with high yield compared to other sources like bone marrow. And they can provide a good source for both autologous and allogenic transplantation. In this video are presenting a simple protocol for isolation and characterization of adipose mesenchymal stem cells from rat epididymal fat.
This procedure is a simple highly efficient method for isolation. After isolation, we will endure the differentiation of these adipose mesenchymal stem cells into insulin-producing cells using a simple, relatively short protocol. We will start with isolation of adipose mesenchymal stem cells by collagenous digestion of epididymal fat to obtain stronger vascular fraction.
Then mesenchymal stem cells will be characterized by their plastic adherence, as well as expression of CD markers and differentiation into multiple mesodermal lineages. Anaesthise the animal, then euthanize using cervical dislocation. Spray all over the body with 70%ethanol and start with excision of the skin and the muscles at lower part of the abdomen.
Then expose the testes with the epididymal fat. Gently cut the epididymal fat. Be careful not to cut the blood vessels with the fats.
In a bio safety cabinet, cut the fat tissue into small pieces using forceps and scalpel. Transfer the adipose tissue into centrifuge tube containing 10 milliliters sterile PBS. Start washing by tilting the Falcon tube for 45 seconds.
Then keep the tube standing for five minutes to separate into two layers. Remove the infranated PBS using a 10 milliliter syringe. Repeat this washing steps four to five times till PBS is clear.
After washing, re-suspend adipose tissue in collagenous solution, then incubate in shaking water bath until the tissue is almost homogeneous. Afterwards, vortex the tissue collagenous mixture for 15 seconds, then centrifuge afterwards for five minutes. After centrifugation, you will have three layers.
Carefully discard the supernatant over the stromal vascular fraction. First discard the oil layer at the top carefully, followed by the echos layer without disturbing the stromal vascular fraction pellet. Re-suspend the Stromal vascular fraction in sterile BSA and re-centrifuge for five minutes.
Whole centrifugation, place complete DMEM F-12 media in 25 centimeter square flask. After centrifugation, discard the supernatant and re-suspend the stromal vascular pellet in complete DMEM F-12 media. And transfer to the culture flask.
Place in 37 degree 5%carbon dioxide incubator. Next day, remove unattached cells and replace the media with fresh media. Starting from next day of isolation, The fibroblast-like cells will start to appear.
Then gradually as time passes they will increase in number and size. By passaging, cells will become more homogenous. These cells exhibit all mesenchymal stem cell characterization criteria.
These cells were differentiated towards insulin-producing cells using this simple protocol containing nicotinamide beta mercaptoethanol and Exendin-4. Throughout the differentiation steps, the cells start to lose their fibroblastic shape and I think cluster like morphology. When used insulin-producing cells show positive crimson red disithone staining, then used insulin-producing cells express various beta cell markers like Pdx-1, mafA and insulin.
In addition, induced insulin-producing cells secrete higher levels of insulin in the supernatant when challenged with higher glucose concentration. We provided a detailed, stepwise protocol for isolation and characterization of adipose mesenchymal stem cells. We also provide here a relatively short, simple and efficient protocol for differentiation of adipose mesenchymal stem cells into insulin-producing cells.
This provides a gateway for researcher to enter the field of mesenchymal stem cells and study their differentiation into insulin-producing cells.
