Proliferative Study of AdiposeDerived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11 Background Information Stem cells Undifferentiated cells Pluripotent Mature into specialized cells ID: 772534
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Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11
Background Information Stem cells Undifferentiated cells Pluripotent Mature into specialized cells Embryonic vs. Adult Embryonic Controversial (destruction of embryo) Adult Non-controversial Self-renewing Great therapeutic potential
Background Information Adipose-derived stem cells (ASCs) Isolated from human adipose tissue (fat) Abundant - Ideal source for regenerative medicine use Plastic surgery LiposuctionHuman ASCs BoneCartilageMuscleFat
Introduction ASCs derived from: Abdominal superficial fat Subcutaneous Above abdominal muscles Omental fatOmentumDeep in abdomenWhich depot is better for clinical applications?
Purpose Anecdotal evidence suggests omental fat stem cells have a greater proliferative capacity than those derived from other depotsGoal of this study is to confirm or deny this theory
Hypothesis Null hypothesis Stem cells derived from omental fat will have an equal proliferative capacity as stem cells derived from abdominal superficial fat
Key Materials CyQUANT® Cell Proliferation Assay Kit Hemacytometer Human abdominal superficial fat Human omental fatSpectraFluor fluorescence reader
Isolation & Expansion Fat tissue from plastic surgeon 2 patients67F & 52FAbdominal & Omental depotsASCs isolated from fat into ASC growth mediaASCs culturedIncubator @ 37°C 1-2 weeks
Initial Count ASCs: Rinsed in phosphate buffered saline (PBS) w/ ethylenediaminetetraacetic acid (EDTA) to disengage clumped cells Suspended w/ trypsin Centrifuged to form pellet Supernatant (w/ trypsin) removedMedia added to pelletASCs vortexed to re-suspendStained w/ Trypan blueCounted using hemacytometer Pellet w/ set # of cells frozen for each depot
Proliferation ASCs for each patient & depot incubated at 3 time points at 3 seating densities in triplicate 24, 48, 96 hrs 2.5k, 10k, 40k cells At each time point Media aspiratedASCs rinsed in PBSPlate frozen to lyse cell membranesDNA preserved
67F Omental – 24hrs 100x
67F Abd. Sup. – 24hrs 100x
67F Omental – 48hrs 100x
67F Abd. Sup. – 48hrs 100x
CyQUANT® Assay Frozen pellet used to create standard curve Cells added from suspended pellet to 16 well plate w/ increasing cell #s Cells thawed & lysed w/ buffer Green fluorescent dye, CyQUANT® GR dye, added to cellsExhibits strong fluorescence enhancement when bound to cellular nucleic acidsStd. curve plates read in SpectraFluor fluorescence readerCyQUANT® GR dye & cell-lysis buffer added to patient cellsFluorescence read in SpectraFluor Readings compared to std. curve to get cell count
DataAverage Cell Counts 2,500 cells Omental Sup. Abd. p-value 24 hours3810.781745.75 0.095 48 hours 4357.09 4685.33 0.837 96 hours 9937.68 11155.51 0.709 10,000 cells Omental Sup. Abd. p-value 24 hours 7895.20 6520.62 0.423 48 hours 10770.66 9981.24 0.662 96 hours 29716.99 45901.66 0.251 40,000 cells Omental Sup. Abd. p-value 24 hours 18669.17 9168.58 0.098 48 hours 15231.59 15316.03 0.938 96 hours 31352.90 35671.46 0.736
DataAverage Cell Counts
DataAverage Cell Counts
DataAverage Cell Counts
Data Analysis/Conclusion Trends show abdominal superficial cells proliferate faster P-values of the t-tests state data is statistically insignificantp-value > 0.05Insufficient evidence to reject the null hypothesis
Possible Sources of Error Sample size Standard curve Initial count w/ hemacytometer Calibration of equipment SpectraFluorPipettes
Future Research Further superficial abdominal vs. omental assays include: Differentiation Adipogenesis (fat)Chondrogenesis (cartilage)Apoptosis (PCD)