Epithelial Cells cells lining the trachea Epithelium layer of epithelial cells in the tissue Many epithelial cell types exist on apical surface

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1 Matthew Pilewski

2 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

3 Proprietary serum-free medium Optimized for human small airway epithelial cells Current standard for lung epithelial cell growth

4 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

5 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.

6 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

7 PluriQ Serum Replacement will not significantly affect the proliferation of epithelial cells across multiple passages when used as a supplement for BronchiaLife SAE.

8 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)

9 1. Lung epithelial cells were isolated from human lung tissue and suspended in DMEM-F12 solution 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.

10 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 ,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 uL aliquot taken for cell count.

11 ,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 uL 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.

12 Total Cells (in millions) ,200,000 1,400, Time (Days) Control PluriQ

13 Number of Cells (in millions) % Control Variable Group PluriQ 87% Viability Total Live Dead

14 Total Cells (in millions) ,700,000 5,900, Control PluriQ Time (Days)

15 Number of Cells (in millions) % 93% Total Live Dead 1 0 Control Variable Group PluriQ Viability

16 Total Cells (in millions) ,050, P-value after 4 days: ,100, , P-value after 8 days: , Time (Days) Control PluriQ

17 Number of Cells (in millions) % % Total Live Dead 0 Control Variable Group PluriQ Viability

18 Number of Cells (in millions) % Total Live Dead Control Variable Group 70.5% PluriQ Viability

19 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.

20 Limited accessibility to PluriQ prevented high replication Possibility that rejection of PluriQ for proliferation was caused by specific cell line

21 Study differentiation potential using oxygen filter-plating and voltmeter Study potential plasticity of cells moved from PluriQ media back into regular BronchiaLife SAE.

22 P1: T-75 Total Live Dead Viability Control % PluriQ % P3: T-25 after 8 days Control % % % % PluriQ % % % % P2: T-50 Control % PluriQ % P3: T-25 after 4 days Control % % % PluriQ % % %