Culture, Characterization and Differentiation Potential of Rat Bone Marrow derived Mesenchymal Stem Cells

Abstract

Abbreviations: rBM-MSC, rat bone marrow derived mesenchymal stem cells; CFU-F, colony forming unit-fibroblast; DPBS, dulbecco's phosphate buffer saline; DMEM, dulbecco's modified eagles medium; RT PCR, reverse transcription polymerase chain reaction; RNA, ribonucleic acid IntroductionBone marrow hosts variety of tissues including hematopoietic lineage cells and mesenchymal stem cells (MSCs). The hematopoietic cells are the major source of the blood cells in the adult body which are regulated within the microenvironment of the stromal cells of the bone marrow.1,2 MSCs are multipotent cells and can be differentiate into various cell lineage depend upon their environment and culture condition in which they are kept. Rat bone marrow derived stem cells (rBM-MSCs) have long term self renewing and capabilities of pluripotency including osteoblast, adipocytes, chondrocytes, tenocytes, muscle cells and neurogenic cells, which make them ideal source of stem cells for regeneration of injured tissue. [3][4][5] Bone marrow derived mesenchymal stem cells have been isolated from different species. Report says that each species having different culturing and growing capacity. 6 For example human bone marrow derived stem cells are easy to harvest and maintain in culture condition where as rat MSCs is more difficult compared to other species. 7,8Technical difficulties in isolation limited the number of animal experiment and its required animal transplantation model for pre clinical studies.9 Density gradient centrifugation, 10 plastic adherence 11 and immunomagnetic selection 12,13 like various methods are available for isolation of MSCs from bone marrow. No appropriate methods are available for selection of suitable cells. Each method has their own pros and cons. Therefore this study combined density gradient centrifugation and plastic adherence for easy and reliable method for selection of suitable cells. Materials and methods Collection of bone marrowFour male wistar rats aged 8-14weeks old were prepared for collection of bone marrow. All procedure was undertaken after getting prior approval from Institutional Animal Ethics Committee (IAEC). Briefly after euthanasia, animals back side were cleanly shaved and prepared aseptically. The skin incision was made on the lateral aspect of thigh and both femur and tibia from one rat were taken after stripping out adherent muscles of the knee end. The collected bone was taken to laminar hood and placed in Petri dish containing DMEM (Dulbecco's Modified Eagles Medium) media with antibiotics. Both the metaphyseal region of femur was cut with scissors and needle was inserted into the bone to aspirate the content by flushing with 1ml of complete media. The cell suspension was centrifuged at 980rpm for 5min to concentrate the cells. The cell pellets were resuspended with 5ml of complete DMEM medium then layered over HISTOPAQUE -1077(Sigma) and centrifuged at 2500rpm for 30minutes. Mononuclear cells were collected from the interface by gradient centrifugation and washed with Ca+ and Mg+ free ...

Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists

“…Flow cytometry analysis was performed to determine whether the preconditioning protocols with LPS or Poly(I:C) would change the expression of key cell surface markers used for the identification of the MSC phenotype. Cells were treated with LPS or Poly(I:C) or kept in control medium without stimulation for 1 h, washed, and allowed to grow for an additional 23 h. While CD90 and CD29, two MSC markers [38, 39], were expressed by more than 98% of the cells in all conditions (Figure 3), less than 4.12% of the cells expressed the hematopoietic lineage markers CD45 and CD34 in all experimental groups (Figure 3). In line with these results, we observed that the morphology of MSCs was not changed 23 h after the removal of LPS or Poly(I:C), as assessed by staining the cells with phalloidin for F-actin labeling (Figure 4(b)).…”

Behavior and biocompatibility of rabbit bone marrow mesenchymal stem cells with bacterial cellulose membrane

“…Any excess stain was washed away with distilled water. The colonies were observed and macroscopically counted on the 24-well plates ( Paramasivam et al, 2016 ).…”

Effect of PDGF-B Gene-Activated Acellular Matrix and Mesenchymal Stem Cell Transplantation on Full Thickness Skin Burn Wound in Rat Model

“…Bone marrow was obtained from femur and tibia of adult wistar rat. Isolation, expansion, characterization and differentiation studies were performed as per the protocol developed in our lab [21,22]. The 3rd passage rat bone marrow mesenchymal stem cells (rBMSC) were used after attaining 40-80% confluence.…”