MC3T3-E1 osteoblastic cells (Sudo et al. 1983
) were generously contributed by Rajaram Gopalakrishnan, BDS, Ph.D. (University of Minnesota; Minneapolis, MN, USA). Passage-20 cells were cultured to confluence in Earle’s-modified MEM supplemented with Penicillin-Streptomycin, L-glutamine, and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). Cells were then removed from the tissue culture flasks by trypsinization, counted by hemocytometer, and added to 1 gm of hydrated Cytodex-3 microcarrier beads (Amersham Pharmacia Biotech; Piscataway, NJ, USA) in culture media. The bead/cell suspension was stirred and allowed to settle several times over the course of two hours in order to facilitate cell attachment to bead surfaces. Bead/cell suspensions were then stirred at a low rpm by a magnetic stir-plate in a tissue culture incubator for approximately 1 week. Cell proliferation was periodically assessed by examination of a small sample of beads under an inverted microscope.
When cell proliferation had progressed so that 5–10 cells were attached per bead, an aliquot of 5 ml of bead/cell suspension was transferred into three sterile polystyrene wells cut from a 24-well tissue culture plate. Culture media was used to top-off each vial, yielding a bead volume that was approximately 30% of the media volume contained in the well. The wells were then inserted into a vertically-configured jig designed to insert into the magnet’s bore ().
(A) Scanning electron micrograph of 200 μm diameter Cytodex-3 bead with varying degrees of MC3T3 cell coverage.
Following 48 hours of culture within the magnet, wells were removed and beads immediately placed in Tri-Reagent RNA isolation reagent (MRC, Inc, Cincinnati, OH, USA). This solution separates total cellular RNA from protein and DNA by phenol and guanidine thiocyanate (as described by Chomczynski and Sacchi 1987
). Following isolation, total RNA was purified through a silica membrane column (RNeasy; Qiagen Corp., Valencia, CA, USA), and quantified by spectrophotometry. Five micrograms of total RNA collected from each experimental condition were amplified by a MessageAmp II kit (Ambion, Inc., Austin, TX, USA). Briefly, this kit reverse transcribes the RNA to a first strand cDNA primed with a T7 oligo primer. This is then incubated with DNA polymerase in order to synthesize double stranded DNA (dsDNA), which in turn serves as a template for transcription. Following purification and the removal of RNA fragments, biotinylated RNA was amplified via transcription of the dsDNA in the presence of biotinylated UTP and CTP. The amplified RNA was purified, quantified by spectrophotometer, and quality checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Amplified RNA (aRNA) was fragmented by a tris/magnesium/potassium acetates solution and 20 μg aRNA was loaded onto an Affymetrix Mouse Genome 430A 2.0 array chip (Santa Clara, CA, USA). This array chip contains 22,600 25-mer probe sets representing over 14,500 well-substantiated mouse genes. Hybridization and image scanning were carried out at the microarray core facility. The defective area, corner noise, outlier area, signal intensity, and 3′:5′ ratio of the β-actin and GAPD (glyceraldehyde-3-phosphate dehydrogenase) housekeeping genes on the array were assessed for quality ensuring by Expressionist Pro4.5 (GeneData AG, CH-4016 Basel, Switzerland) Refiner module. The housekeeping genes are usually consistently expressed in different conditions to keep cells alive. A 3′:5′ ratio close to one indicates the mRNA is near intact since mRNA degrades from its 5′ end. The probe level signals of each chip were condensed into probeset expression values by RMA algorithm (Irizarry et al. 2003
The summarized data were scaled up to median of 100. The genes whose values are less than 50 in all samples were filtered out. Genes that changed more than three fold were tabulated in the sample comparisons and examined with respect to molecular function groups of these differentially expressed genes.
Based on it appears that cells exposed to a net gravitational force of 0 or 2 g show a higher level of gene expression relative to cells exposed to a gravitational force of 1 g. A net gravitational force other than 1 g causes the cells to respond to this stress by exhibiting a higher level of gene expression. This has been seen for cells cultured in earth orbit (Carmeleit et al. 1997
; Harris et al. 2000
) or those exposed to hyper gravity (Hatton et al. 2003
). Cells grown at the 0 g and 2 g magnet locations experience identical magnetic forces whereas cells grown in the magnet center do not experience a magnetic force because the spatial field gradient is negligible. It is also notable that samples at 0 g and 2 g magnet locations have a similar differential gene expression profile. One explanation is that since these cells are experiencing identical magnetic forces and that these forces are stressing the cells more than gravitational forces do. It is possible that intracellular forces occur due to intracellular organelles having different magnetic susceptibilities and thereby different net magnetic forces. This may manifest itself physiologically as internal stress or strain within the cell causing similar gene expression for cells experiencing 0 g and 2 g net forces. It is therefore possible there are magnetic effects that mask more subtle gravitational loading effects. To better understand what is occurring, it is necessary to study the expression level of specific biochemical markers.
Fig. 3 Pairwise comparison of gene expressions in different gravity conditions. Both X-axis and Y-axis represent gene expression levels. The diagonal symmetric distribution of the gene expression values indicates most genes were not differentially expressed. (more ...)
Previous investigations of hyper and hypo gravity effects on cells or organisms have a demonstrable outcome. The fact that both lower and higher net gravitational fields affect gene expression is supported by comparing 2 g to 0 g gravity conditions. A small number of genes (195 genes), are different in this comparison leading one to conclude that these cells may be experiencing similar stresses.
While there are a relatively small number of genes differentially regulated when comparing the 0 g and 2 g expression data, it is critical to identify and evaluate the specific genes expressed. We found that the regulation of a number of genes are similiarly impacted by both 0 g and 1 g including 1,425 up-regulated genes and 101 down-regulated. Some differences were apparent when comparing cells grown in 0 g and 2 g; 845 genes were specifically up-regulated in 0 g and 34 genes down-regulated, while 102 genes were up-regulated and 365 down-regulated in 2 g.
shows that cells grown at 0 g (12.5 T) exhibit 2,270 genes up-regulated relative to cells at 1 g (17 T) whereas cells grown at 2 g (12.5 T) respond by up-regulating 1,527 genes relative to cells at 1g (17 T). The Venn diagram in shows that 1,425 of the same genes from 0 g and 2 g were up-regulated. Fewer genes were down-regulated when comparing 0 g to 1 g, 135 genes, and 2 g to 1 g, 466 genes, where 101 genes were common to both 0 g and 2 g. However, this information does not define the functionality of the genes affected by gravitational stressors.
Genes that show a greater than threefold change in up regulation or down regulation
Venn diagrams showing up regulating and down regulating of genes under different net gravitational loads
For functional gene analysis, gene probe set ID numbers were imported into the Ingenuity Pathway Analysis software (Ingenuity Systems, Inc., Redwood city, CA, USA). The identified genes were related to functional groups by Fishers’ T test. The more significantly affected genes are identified in and . The P values stand for how likely these genes belong to a function group by random chance alone. The smaller the P value, the more significant the identification of a particular gene. The most significant cell response to either hypo- or hyper-gravity is an increase in genes related to cell proliferation and cell death. These are obviously conflicting cell responses whereas one may be attributed to hypo-gravity and the other to hyper-gravity or a combination of gravitational and magnetic stresses. More experiments are necessary to better interpret these results.
Most significant functional groups of up-regulated genes from cells experiencing 0 g or 2 g net force compared to 1 g
Most significant functional groups of down-regulated genes from cells experiencing 0 g or 2 g net force compared to 1 g