Optimization of Human Airway Cell

ProliferationMatthew 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

Respiratory Epithelium

Proprietary serum-free medium

Optimized for human small airway epithelial cells

Current standard for lung epithelial cell growth

LifeLine BronchiaLife SAE

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Limited accessibility to PluriQ prevented high replication

Possibility that rejection of PluriQ for proliferation was caused by specific cell line

Limitations

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

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%