Effects of plating density and culture time on bone marrow stromal cell characteristics.
OBJECTIVE
Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations.


METHODS
Rat MSC were plated at 20, 200, and 2000 cells/cm(2) and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations.


RESULTS
We found optimal cell growth at a plating density of 200 cells/cm(2). Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential.


CONCLUSIONS
Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.
Neuhuber
Birgit
B
Drexel University College of Medicine, Department of Neurobiology and Anatomy, Philadelphia, PA, USA. bneuhuber@drexelmed.edu
Swanger
Sharon A
SA
Howard
Linda
L
Mackay
Alastair
A
Fischer
Itzhak
I
D017667:Adipocytes / Q000166:cytology; D050156:Adipogenesis; D053687:Adult Stem Cells / Q000166:cytology*; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002455:Cell Division; D019070:Cell Lineage; D002478:Cells, Cultured / Q000166:cytology; D019902:Chondrocytes / Q000166:cytology; D020219:Chondrogenesis; D002999:Clone Cells / Q000166:cytology; D039902:Multipotent Stem Cells / Q000166:cytology*; D010011:Osteocytes / Q000166:cytology; D010012:Osteogenesis; D051381:Rats; D011916:Rats, Inbred F344; D017154:Stromal Cells / Q000166:cytology*; D013997:Time Factors