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.

Materials and 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.

Figure 1. Experimental design

Figure 2. MSC cultures contain morphologically distinct cell populations

Micrographs depict MSC harvested at one time from the same rat. (A–C) A subpopulation of MSC exhibit elongated, spindle-like shapes. (D–F) A second subpopulation of MSC has flattened polygonal morphologies with short or no processes.

Figure 3. MSC plating density effects growth kinetics across multiple passages

MSC were plated at 20, 200 and 2000 cells/cm², passaged at 50% confluency and counted at each passage in order to analyze the effect of plating density on rate of proliferation. A) Cumulative PDL and doubling times were calculated. B) Micrographs depict growth patterns of MSC plated at 20 cells/cm² (top), 200 cells/cm² (middle) and 2000 cells/cm² (bottom). Cultures plated at high density were spread evenly throughout the dish, whereas cells plated at lower densities grow in colonies. Pictures are representative of cultures one day after plating (left column), mid-way through culture (center column) and on the day of passaging at 50% confluency (right column).

Figure 4. Growth kinetics of heterogeneous cultures and cloned spindle-shaped and flat cultures

Heterogeneous MSC from passage 5, and cloned populations of spindle-shaped and flat MSC were plated at 20, 200 and 2000 cells/cm², expanded for 14 days and counted in duplicate on each day to determine growth rate. Cumulative PDL and doubling times were calculated for heterogeneous MSC (A), spindle-shaped clones (B) and flat clones (C).

Figure 5. Proportion of MSC with flat morphology increases over time in culture regardless of plating density

MSC were plated at 20, 200 and 2000 cells/cm², counted daily until confluent and classified as spindle-shaped or flat. Data is presented as percentage of total MSC counted on day one after plating (d1), on the day cultures reached 50% confluency (d-50%) and on the day after cultures reached 100% confluency (d-100%). While percentages vary between conditions, the proportion of MSC with flat morphology in heterogeneous cultures increases within one passage over time in culture, as well as with increased passage number. The time elapsed from seeding to cultures achieving 50% or 100% confluency varied according to plating conditions.

Figure 6. Chondrogenic and adipogenic potential is limited in MSC with flat morphology, but is unaffected by plating density

MSC plated at different densities as well as cloned spindle-shaped and flat populations were subjected to chondrogenesis (A–E) and adipogenesis (F–J) assays. MSC plated at 20 (A), 200 (B) and 2000 cells/cm² (C) demonstrated high levels of chondrogenesis evident by medium or large size pellets and positive Safranin O staining. Spindle-shaped MSC clones also successfully differentiated in the chondrogenic assay as indicated by the large pellet with positive Safranin O staining (D), whereas the flat MSC population did not differentiate under chondrogenic conditions (E). MSC plated at 20 cells/cm² showed no Oil Red O staining in the adipogenesis assay (F), but this may have been due to a limitation of the assay in culture with low cell density. MSC plated at 200 (G) and 2000 cells/cm² (H) were positive for Oil Red O staining, indicating adipogenic differentiation. The cloned population of spindle-shaped MSC was positive for Oil Red O staining (I); however, cloned flat MSC showed no evidence of adipogenic differentiation (J).