Dear colleagues,
I am learning to culture mouse bone marrow derived MSC.
I dissected long bone from mouse and took out the marrow followed by red blood lysis by using ACK buffer. I plated the cells in 6 well maintained in DMEM + 10% FBS + PenStrep. I changed the media 24 hour after plating and after 2~3 days I can see few cells attached. After 6 days I passaged using Trypsin-EDTA using 1:2 split ratio.
I hope my question is not too silly to ask but I would really appreciate if any of you can point me to the right direction. I took the picture of my MSC after 1 passage. I noticed there are at least 2 two types of cells in my culture (that I labeled A and B). Which one is the actual MSC? Then what is the other cell?
Why my MSC doesn't exhibit spindle like morphology? Is it because I plated in too low density?
Any feedback would be appreciated. Thank you in advance!!
The cell labeled A might be the good one. The others will not divided. The MSC "like" to proliferate at low density. You will eliminate unwanted cells by passaging another time or a little more.
Hello
Look at this article ( EFFECTS OF PLATING DENSITY AND CULTURE TIME ON BONE MARROW STROMAL CELL CHARACTERISTICS )
I had also experienced the same problem as you were facing. Cell "B" population probably stems from the hematopoietic fraction of the bone marrow. There are three things that can be done to address this: (1) to use a medium (alpha MEM) that favors MSC growth over hematopoietic counterparts; (2) to intermittently (gently) refresh the medium every 8 hour up to 72 hours; (3) when the MSC population gets to 70-80% confluent, trypsinize at room temperature no more than 2 minutes and eliminate the still-adherent cells for subculture.
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