Effects of plating density and culture time on bone marrow stromal cell characteristics

Abstract

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.Conclusion-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. Keywordsadult stem cells; heterogeneity; variability; differentiation potential; expansion Bone marrow stromal cells (MSC) represent a heterogeneous population derived from the nonblood forming fraction of bone marrow that regulates hematopoietic cell development. In vitro, adult mesenchymal stem cells resident in this bone marrow fraction differentiate into bone, cartilage and fat (1). Cultured MSC have also been shown to regenerate cardiac (2) and Correspondence: Birgit Neuhuber, Ph.D, Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, Phone: (215) Fax: (215) 843-9803, E-mail: bneuhuber@drexelmed.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In this study, we cultured rat MSC for which no effort was made to limit heterogene...

Concise Review: Optimizing Expansion of Bone Marrow Mesenchymal Stem/Stromal Cells for Clinical Applications

“…Although it has been established that MSCs succumb to senescence during extensive in vitro culture, the optimal cell density for growth is unclear. Using rat MSCs, Neuhuber et al found optimal cell growth when plating at 200 cells per cm 2 compared with 20 cells or 2,000 cells per cm 2 [48]. However, other studies suggest even lower plating densities (~1.5-200 cells per cm 2 ) favor proliferation [25,49,50].…”

“…Multilineage potential is universally used by the community as a way to identify an MSC [4] as well as to quantify progenitor potency. Cryopreservation [16], density gradient isolation [52], and seeding density [48] do not yield appreciable effects on MSC multilineage potential. Despite some disparities [14,15], osteogenic potential is the most commonly retained lineage after passage 5 [13, 16-18, 48, 53] ( Table 1).…”

Isolation, Culture and Characterization of New Zealand White Rabbit Mesenchymal Stem Cells Derived from Bone Marrow

“…Morphological heterogeneity in MSCs is generally associated with the presence of cells at different differentiation levels and not with the distinct cell culture subtype (Docheva et al, 2008). Besides, large flat MSC is described as mature senescent cell with low mitotic potential (Neuhuber et al, 2008;Fu et al, 2012). Proliferation rate in mesenchymal stem cells is very high and one MSC can proliferate to 2×106 cells in a single passage in rabbits (Lapi et al, 2008).…”

Effects of human placental serum on proliferation and morphology of human adipose tissue-derived stem cells

“…47 FGF-2, among placental growth factors, is known to have a most powerful effect on MSCs proliferation. 35 Fortunately, this growth factor seems to be in both fetal and maternal circulations during normal pregnancies. 47,48 On the other hand, there might be a correlation between the type of growth factor isoforms in serum and the types of their receptors.…”

“…According to previous studies, three major sub-populations were defined: (a) MSCs with a spindle shape cells, (b) large flat cells and (c) triangle cells. [34][35][36][37] A fourth sub-population as mitotic or round cells was also included. To assess effects of different sera on cell morphology, ADSCs were cultured on coverslips.…”