As part of the 64th National conference of the Association of Physiologists and Pharmacologists of India at Manipal Academy of Higher Education, Manipal, a half-day Workshop will be conducted on “ Advancements in Regenerative Medicine – Stem cell research studies”.
Organizing team: School of Regenerative Medicine, MAHE, Bangalore.
- Ms. Siva Priya
- Ms. Sushma
- Ms. Chaitra Venugopal
- Ms. Nistha Kusum Jain
- Mr. Shiva Kumar
- Mr. Krishnamoorty
Duration of Workshop: 6 hours
Importance of the Workshop:
Stem cells have emerged as an interesting field for research in regenerative medicine. Regenerative medicine is an interdisciplinary approach that seeks to repair or replace damaged or diseased human cells or tissues to restore normal function, which holds the promise of revolutionizing patient care in the 21st century.
Under graduate, post-graduate students, Researchers, Doctoral students, Early career Faculty, Scientists, Physicians with special interest in stem cell biology and translational research
The workshop aims to demonstrate and give hands-on interactive experience on isolation of bone marrow mesenchymal stem cells from adult mouse, Seeding and expansion of mouse bone marrow mesenchymal stem cells, characterization of mouse bone marrow mesenchymal stem cells and cryopreservation. The deliberations will focus on demonstrating the utility of stem cell culture, expansion and characterization techniques in both clinical and research landscapes.
Techniques and modalities covered:
|Domain||Parameter measured||Technique used|
|Isolation of bone marrow mesenchymal stem cells from adult mouse||Isolation of BM-MSC from||Femurs from the mouse will be removed and stripped of all the connective tissue and then washed in sterile PBS containing 100 U/ml of penicillin. The end of the tibia and femur will be clipped to expose the bone marrow. A needle will be inserted at one end of the bone and the cells will be flushed out several times with culture media using a 5 ml syringe. Culture media consisted of DMEM knock-out (Invitrogen, Carlsbad, California, USA) media with 10% FBS (Hi-Media, Telangana, India), 100 U/ml penicillin – streptomycin (Invitrogen, Carlsbad, California, USA), 1% L-glutamine (Invitrogen Carlsbad, California, USA). The culture media containing cells will be collected into a falcon tube and centrifuged at 1000 rpm for 10 minutes. The supernatant will be discarded and the pellets will be re-suspended in culture media.|
|Seeding and Expansion of mouse bone marrow mesenchymal stem cells||In vitro BM-MSC seeding and Culture expansion of BM-MSC||First, we will demonstrate how to seed BM-MSC in tissue culture plate for the participants. The cells from each bone marrow will be plated in a 35 mm tissue culture dish. One week following initial seeding, the cells will be sub-cultured (expansion) by trypsinising with 0.25% trypsin for 2 minutes at 37 degree C. The trypsin will be neutralized by adding culture media and the cell suspension thus obtained will be centrifuged at 1500 rpm for 5 minutes following which cells will be re-plated in 25 cm2 flasks. The culture media will be replaced every 2 days and once the cells reached confluency, the cells will either frozen or further expanded. As the cells takes one weeks to reach confluency, pre-cultured BM-MSC will be used to demonstrate trypsin treatment and also to show the morphological features under the microscope.|
|Characterization of mouse bone marrow mesenchymal stem cells||
||Performance of conventional RT-PCR will be demonstrated to evaluate the expression of mesenchymal stem cell-surface antigens (CD 90, CD105 and CD 73) and for the absence of hematopoietic markers (CD 45, CD 34) in rat BM-MSC. GAPDH will be used as a control.
Tri-lineage (adipocyte/ chondrocyte/osteocyte) differentiated and differentiated BM-MSC will be demonstrated under the light microscope.