proliferative study of adipose-derived stem cells jay sehgal north allegheny senior high school...
TRANSCRIPT
![Page 1: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/1.jpg)
Proliferative Study of Adipose-Derived Stem
Cells
Jay SehgalNorth Allegheny Senior
High SchoolGrade 11
![Page 2: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/2.jpg)
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
![Page 3: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/3.jpg)
Background Information
Adipose-derived stem cells (ASCs) Isolated from human adipose tissue (fat)
Abundant - Ideal source for regenerative medicine use
Plastic surgery Liposuction
Human ASCs Bone Cartilage Muscle Fat
![Page 4: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/4.jpg)
Introduction
ASCs derived from: Abdominal superficial fat
Subcutaneous Above abdominal muscles
Omental fat Omentum Deep in abdomen
Which depot is better for clinical applications?
![Page 5: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/5.jpg)
Purpose
Anecdotal evidence suggests omental fat stem cells have a greater proliferative capacity than those derived from other depots
Goal of this study is to confirm or deny this theory
![Page 6: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/6.jpg)
Hypothesis
Null hypothesis Stem cells derived from omental
fat will have an equal proliferative capacity as stem cells derived from abdominal superficial fat
![Page 7: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/7.jpg)
Key Materials
CyQUANT® Cell Proliferation Assay Kit
Hemacytometer Human abdominal superficial fat Human omental fat SpectraFluor fluorescence reader
![Page 8: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/8.jpg)
Isolation & Expansion
Fat tissue from plastic surgeon 2 patients
67F & 52F Abdominal & Omental depots
ASCs isolated from fat into ASC growth media
ASCs cultured Incubator @ 37°C 1-2 weeks
![Page 9: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/9.jpg)
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) removed Media added to pellet
ASCs vortexed to re-suspend Stained w/ Trypan blue Counted using hemacytometer
Pellet w/ set # of cells frozen for each depot
![Page 10: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/10.jpg)
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 aspirated ASCs rinsed in PBS Plate frozen to lyse cell membranes
DNA preserved
![Page 11: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/11.jpg)
67F Omental – 24hrs
100x
![Page 12: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/12.jpg)
67F Abd. Sup. – 24hrs
100x
![Page 13: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/13.jpg)
67F Omental – 48hrs
100x
![Page 14: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/14.jpg)
67F Abd. Sup. – 48hrs
100x
![Page 15: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/15.jpg)
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 cells Exhibits strong fluorescence enhancement
when bound to cellular nucleic acids Std. curve plates read in SpectraFluor
fluorescence reader CyQUANT® GR dye & cell-lysis buffer
added to patient cells Fluorescence read in SpectraFluor Readings compared to std. curve to get
cell count
![Page 16: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/16.jpg)
DataAverage Cell Counts
2,500 cells Omental Sup. Abd. p-value
24 hours3810.78 1745.75 0.095
48 hours4357.09 4685.33 0.837
96 hours9937.68 11155.51 0.709
10,000 cells Omental Sup. Abd. p-value
24 hours7895.20 6520.62 0.423
48 hours10770.66 9981.24 0.662
96 hours29716.99 45901.66 0.251
40,000 cells Omental Sup. Abd. p-value
24 hours18669.17 9168.58 0.098
48 hours15231.59 15316.03 0.938
96 hours31352.90 35671.46 0.736
![Page 17: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/17.jpg)
DataAverage Cell Counts
20 30 40 50 60 70 80 90 1000
2000
4000
6000
8000
10000
12000
2,500 cells
OmentalSup. Abd.
time (hrs)
# of
cel
ls
![Page 18: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/18.jpg)
DataAverage Cell Counts
20 30 40 50 60 70 80 90 1000
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
10,000 cells
OmentalSup. Abd.
time (hrs)
# of
cel
ls
![Page 19: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/19.jpg)
DataAverage Cell Counts
20 30 40 50 60 70 80 90 1000
5000
10000
15000
20000
25000
30000
35000
40000
40,000 cells
OmentalSup. Abd.
time (hrs)
# of
cel
ls
![Page 20: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/20.jpg)
Data Analysis/Conclusion
Trends show abdominal superficial cells proliferate faster
P-values of the t-tests state data is statistically insignificant p-value > 0.05
Insufficient evidence to reject the null hypothesis
![Page 21: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/21.jpg)
Possible Sources of Error
Sample size Standard curve Initial count w/ hemacytometer Calibration of equipment
SpectraFluor Pipettes
![Page 22: Proliferative Study of Adipose-Derived Stem Cells Jay Sehgal North Allegheny Senior High School Grade 11](https://reader035.vdocuments.site/reader035/viewer/2022062322/56649ccc5503460f94996b22/html5/thumbnails/22.jpg)
Future Research
Further superficial abdominal vs. omental assays include: Differentiation
Adipogenesis (fat) Chondrogenesis (cartilage)
Apoptosis (PCD)