Matthew Pilewski
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
Proprietary serum-free medium Optimized for human small airway epithelial cells Current standard for lung epithelial cell growth
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
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.
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
PluriQ Serum Replacement will not significantly affect the proliferation of epithelial cells across multiple passages when used as a supplement for BronchiaLife SAE.
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% Centrifuge BSL-2 Hood Macro/micropipettes Vacuum Cell Counter Penicillin-Streptomycin (PenStrep) Rho kinase (ROCK) inhibitor Trypan Blue Trypsin Neutralizing Solution (TNS)
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.
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.
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.
Total Cells (in millions) 8 7 6 5 4 3 2 1 0 7,200,000 1,400,000 0 2 4 6 Time (Days) Control PluriQ
Number of Cells (in millions) 8 7 6 96% 5 4 3 2 1 0 Control Variable Group PluriQ 87% Viability Total Live Dead
Total Cells (in millions) 8 7 6 5 6,700,000 5,900,000 4 3 2 Control PluriQ 1 0 0 1 2 3 4 5 Time (Days)
Number of Cells (in millions) 8 7 6 96% 93% 5 4 3 2 Total Live Dead 1 0 Control Variable Group PluriQ Viability
Total Cells (in millions) 3.5 3 3,050,000 2.5 2 1.5 1 0.5 0 P-value after 4 days: 0.00014 1,100,000 173,333.33 P-value after 8 days: 0.0000003 162,500 0 2 4 6 8 10 Time (Days) Control PluriQ
Number of Cells (in millions) 1.2 1 97.7% 0.8 0.6 0.4 0.2 80.75% Total Live Dead 0 Control Variable Group PluriQ Viability
Number of Cells (in millions) 3.5 3 2.5 90.25% 2 1.5 1 Total Live Dead 0.5 0 Control Variable Group 70.5% PluriQ Viability
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.
Limited accessibility to PluriQ prevented high replication Possibility that rejection of PluriQ for proliferation was caused by specific cell line
Study differentiation potential using oxygen filter-plating and voltmeter Study potential plasticity of cells moved from PluriQ media back into regular BronchiaLife SAE.
P1: T-75 Total Live Dead Viability Control 7200000 6800000 280000 96% PluriQ 1440000 1240000 200000 87% P3: T-25 after 8 days Control 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% P2: T-50 Control 6700000 6500000 30000 96% PluriQ 5900000 5500000 40000 93% P3: T-25 after 4 days Control 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%