![Page 1: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/1.jpg)
Optimization of Human Airway Cell
ProliferationMatthew Pilewski
![Page 2: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/2.jpg)
Epithelial Cells – cells lining the trachea
Epithelium – layer of epithelial cells in the tissue
Many epithelial cell types exist on apical surface
Epithelium form tight junctions that keep airway sealed
Cells are harvested from human lung after lung transplant
Respiratory Epithelium
![Page 3: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/3.jpg)
Proprietary serum-free medium
Optimized for human small airway epithelial cells
Current standard for lung epithelial cell growth
LifeLine BronchiaLife SAE
![Page 4: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/4.jpg)
Proprietary serum-free supplement Used to grow and maintain of hESCs and
iPSCs “maintains healthy cell morphology and
normal karyotype for extended passages in culture”
PluriQ Serum Replacement
![Page 5: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/5.jpg)
The current in vitro culture methods for human lung epithelium growth do not allow for sustainable proliferation of cells for longer than an average of 3-5 culture passages.
Problem
![Page 6: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/6.jpg)
To determine if lung epithelial cells will grow more effectively if PluriQ Serum Replacement is added.
Speed of proliferation Ability to maintain proliferation
after several passages
Purpose
![Page 7: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/7.jpg)
PluriQ Serum Replacement will not significantly affect the proliferation of epithelial cells across multiple passages when used as a supplement for BronchiaLife SAE.
Null Hypothesis
![Page 8: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/8.jpg)
PluriQ Serum Replacement BronchiaLife SAE DMEM-F12 37°C Incubator lung epithelial cells HBE060 T-75/T-50/T-25 culture flasks with
collagen coating Trypsin 0.025% Trypsin Neutralizing Solution (TNS)
Materials Centrifuge BSL-2 Hood Macro/micropipettes Vacuum Cell Counter Penicillin-Streptomycin
(PenStrep) Rho kinase (ROCK)
inhibitor Trypan Blue
![Page 9: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/9.jpg)
1. Lung epithelial cells were isolated from human lung tissue and suspended in DMEM-F12 solution.
2. 100 mL solution of variable media containing 2% PluriQ Serum Replacement was made using 2 mL PluriQ, 1 mL PenStrep, 97 mL BronchiaLife SAE, and 100 uL ROCK inhibitor.
3. 4,000,000 epithelial cells were seeded into two collagen coated T-75 flasks, one flask containing 15mL of BronchiaLife SAE and one containing 15mL of PluriQ media.
4. Flasks incubated in 37C incubator overnight.5. Media in each flask was removed and replaced with
15mL of fresh media every 24 hours for 5 days.
Procedures
![Page 10: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/10.jpg)
9. After 5 days, media was removed and cells were trypsinized using 10mL of trypsin to remove cells from flask.
10. After 10 minutes, 10mL TNS was added to stop trypsinization.
11. Cells removed from flasks, centrifuged, resuspended in 2mL of respective media, and counted.
12. 400,000 cells seeded into two T-50 flasks, each fed with 10 mL of media.
13. Cells incubated overnight and fed with fresh media every 24 hours.
14. After 4 days, cells were trypsinized using 5mL of trypsin and 5 mL of TNS.
15. Cells removed, centrifuged, and resuspended in 2mL of respective media.
16. 10uL aliquot taken for cell count.
Procedures
![Page 11: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/11.jpg)
17. 100,000 cells were seeded in each of 7 T-25 flasks for the control and 7 flasks for the PluriQ.
18. Cells were incubated overnight and fed daily with 4mL of fresh media.
19. After 3 days, 3 control flasks and 3 PluriQ flasks were trypsinized, centrifuged, and resuspended.
20. 10uL aliquot taken from each flask and cell count was taken.
21. Final 8 flasks were trypsinized, centrifuged, resuspended, and counted after 8 total days of incubation.
22. T-test performed on T-25 cell counts to determine significance.
Procedures
![Page 12: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/12.jpg)
Passage 1: T-75 Proliferation(Initial Growth)
0 1 2 3 4 5 60
1
2
3
4
5
6
7
8
ControlPluriQ
Time (Days)
Tota
l C
ells (
in m
illion
s)
1,400,000
7,200,000
![Page 13: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/13.jpg)
Passage 1: T-75 Proliferation(Initial Growth)
Control PluriQ0
1
2
3
4
5
6
7
8
TotalLiveDead
Variable Group
Nu
mb
er
of
Cells (
in m
illion
s) 96%
87%
Viability
![Page 14: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/14.jpg)
Passage 2: T-50 Proliferation
0 0.5 1 1.5 2 2.5 3 3.5 4 4.50
1
2
3
4
5
6
7
8
ControlPluriQ
Time (Days)
Tota
l C
ells (
in m
illion
s)
5,900,000
6,700,000
![Page 15: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/15.jpg)
Control PluriQ0
1
2
3
4
5
6
7
8
TotalLiveDead
Variable Group
Nu
mb
er
of
Cells (
in m
illion
s)
Passage 2: T-50 Proliferation
96%
93%
Viability
![Page 16: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/16.jpg)
Passage 3: T-25 Proliferation
0 1 2 3 4 5 6 7 8 90
0.5
1
1.5
2
2.5
3
3.5
Control
PluriQ
Time (Days)
Tota
l C
ells (
in m
illion
s)
162,500173,333.33
3,050,000
1,100,000
P-value after 4 days: 0.00014
P-value after 8 days: 0.0000003
![Page 17: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/17.jpg)
Control PluriQ0
0.2
0.4
0.6
0.8
1
1.2
TotalLiveDead
Variable Group
Nu
mb
er
of
Cells (
in m
illion
s)
Passage 3: T-25 Proliferation After 4 Days
97.7%
80.75%
Viability
![Page 18: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/18.jpg)
Control PluriQ0
0.5
1
1.5
2
2.5
3
3.5
TotalLiveDead
Variable Group
Nu
mb
er
of
Cells (
in m
illion
s)
Passage 3: T-25 Proliferation After 8 Days
90.25%
70.5%
Viability
![Page 19: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/19.jpg)
The null hypothesis stated that PluriQ would not significantly affect the proliferation of epithelial cells
Null hypothesis rejected: Epithelial cells significantly less proliferation when grown with PluriQ.
Conclusions
![Page 20: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/20.jpg)
Limited accessibility to PluriQ prevented high replication
Possibility that rejection of PluriQ for proliferation was caused by specific cell line
Limitations
![Page 21: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/21.jpg)
Study differentiation potential using oxygen filter-plating and voltmeter
Study potential plasticity of cells moved from PluriQ media back into regular BronchiaLife SAE.
Further Study
![Page 22: Matthew Pilewski. Epithelial Cells – cells lining the trachea Epithelium – layer of epithelial cells in the tissue Many epithelial cell types exist](https://reader031.vdocuments.site/reader031/viewer/2022013105/56649ee75503460f94bf804a/html5/thumbnails/22.jpg)
P1: T-75 Total Live Dead ViabilityControl 7200000 6800000 280000 96%PluriQ 1440000 1240000 200000 87%
P2: T-50Control 6700000 6500000 30000 96%PluriQ 5900000 5500000 40000 93%
P3: T-25 after 4 daysControl 1200000 1200000 30000 98%
1100000 1100000 30000 97%1000000 980000 30000 98%
PluriQ 160000 120000 50000 72%130000 110000 30000 81%230000 170000 70000 72%
P3: T-25 after 8 daysControl 2700000 2500000 210000 92%
3200000 3000000 280000 91%3100000 2800000 370000 88%3200000 2900000 330000 90%
PluriQ 200000 150000 60000 73%170000 120000 50000 70%130000 70000 60000 56%150000 130000 30000 83%