in vitro permeability study - courses · 2017-09-25 · in vitro permeability study yan-ru lou, phd...

www.helsinki.fi/yliopisto

In vitro permeability studyYan-Ru Lou, PhD

Adjunct Professor (Docent)Division of Pharmaceutical Biosciences, Faculty of Pharmacy,

University of Helsinki

25.9.2017

25.9.2017 1Faculty of Pharmacy/ Yan-Ru Lou


Responsible teacher in the practical:Yan-Ru Lou

Checking the reports: Yan-Ru Lou

25.9.2017Farmasian tiedekunta / Yan-Ru Lou 2


•Preventing contamination•Aseptic techniques: do not touch a sterile thing to anonsterile surface; do not open a cell culture dish inunclean air•Culture one cell type at a time

•Be gentle to cells•Check cells regularly under microscope

25.9.2017 3

Cell culture

Faculty of Pharmacy/ Yan-Ru Lou

www.helsinki.fi/yliopisto 25.9.2017 4

Cell counting

Count the cells within the large square and those crossing the edge on two out of thefour sidesThe volume of 1 large corner square = 10-4 mlCount 4 large corner squares and calculate the averageIf the average is 50, the dilution ratio is 1:2, the cell density in your cell solution is 50 x 2x 104 = 106 cells/ml



MDCKII cells

MDCKII cells, Madin-Darby canine kidney cells (Canis familiaris; dog)



Culture of MDCKII cells

The cells grow in a few days to cover 80% of the surface area of a cell culture flaskand they can be subcultured into new flasks or to permeable cell culture inserts

Wash the cells with PBS and thenincubate with TrypLE

Growth conditions:Dulbecco´s Modified Eagle medium (DMEM),supplemented with 10% serum,penicillin-streptomycinand L-glutamine.+37ºC, 95% relative humidity, 5% CO2medium changed three times per week


Add the medium and centrifuge.Suspend the cell pellet in the mediumand calculate cell number. Split thecells into new flasks


Transwell systems used in thepermeability study



Culture of MDCKII cells on thepermeable supports

4.75 x 105 MDCKII cells are seeded onto12-well plate polyester membrane inserts

Cell attachmenttakes about 1 day

Cell monolayer covers thesurface area of the transwell

Permeability study6 days after seeding



Transwell

Nature Protocols 4, - 662 - 673 (2009): Modified figure



Permeability experiment using luciferyellow and propranolol as referencecompounds, paracetamol as a testcompound

•Lucifer yellow has low permeability --- used toassess the integrity of the cell monolayer•Propranolol has high permeability --- used as ahighly permeable reference drug•HBSS-Hepes buffer, pH 7.4•Drug concentrations:lucifer yellow and paracetamol at 250 µMPropranolol at 50 µM•Drug transport direction A → B



Cell seeding in inserts in a 12-well plate 5.10.2017

Nocells

A

C

B

1 432

cells

cells

cells

cellscells cells

cells cells cellscells

cells

To be used forpropranololtest

To be used forparacetamoltest

To be used forlucifer yellowtest



Medium change on Friday,Monday and Tuesday

6, 9, and 10.10.2017Supervisors will change medium on weekends



Preparation of the permeabilityexperiment

25.9.2017 13

10.10.2017

After medium change on Tuesday morning 10.10,each student prepares and labels these in the biglab 2060a. They can be found on the table in frontof the fume hood:

•Seven 12-well plates•15-ml tubes for standard dilutions•Eppendorf tubes for samples•One black and one white 96-well platesKeep them in your own work space on thosetables reserved for teaching



Permeation study day

25.9.2017 14

Morning:Prepare serial dilutions for three standard curves

Prepare and perform permeability test of propranolol

Lunch breakAfternoon:

Prepare and perform permeability tests of lucifer yellow and paracetamol

Add samples into two 96-well plates

Measure lucifer yellow samples

Send your UPLC sample layout (the Excel form) to Timo by email([email protected])


11.10.2017


Preparation of the permeabilityexperiment

25.9.2017 15

11.10.2017

In the morning, preparing test solutionsü Ready 500 ml transport buffer: HBSS-10 mM Hepes (pH 7,4)ü Prepare test solutions:1. 250 µM lucifer yellow in transport buffer (in dark)2. 50 µM propranolol in transport buffer3. 250 µM paracetamol in transport bufferü Warming transport buffer at 37˚C water bath



Preparation of serial dilutions forstandard curves

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Everyone makes 3 standard curves:lucifer yellow, propranolol, and paracetamol

For example:


Name of solution Amount of 100 µM Pa(ml)

Amount of TransportBuffer (ml)

Paconcentration

(µM)

Pa1 7.5 2.5 75

Pa2 3.75 6.25 37.5

Pa3 0.75 9.25 7.5

Pa4 0.15 9.85 1.5

Pa5 0.075 9.925 0.75

Pa6 0.015 9.985 0.15

Pa7 0.005 9.995 0.05

11.10.2017


Propranolol permeability test

25.9.2017 17

Nocells

A

C

B

1 432

cells

cells

cells

cellscells cells

cells

To be used for propranolol test

To be used for paracetamol test

To be used for lucifer yellow test

A

C

B

1 432

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

Propranolol

Propranolol-5min

Propranolol-10min

Propranolol-15min

1 2 3 4

5 6 7 8

9 10 11 12

cells cells cellscells

Empty a insert,transfer it to the new well

Start the timer

Keep 30 s intervalbetween additionsof test solution

0.5ml 0.5ml0.5mlAdd propranololtest solution

0.5ml


Lucifer yellow and paracetamolpermeability test

A

C

B

1 432

Nocells

A

C

B

1 432

To be used for propranolol test

To be used for paracetamol test

To be used for lucifer yellow test

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

1.5 mlbuffer

30 min

Lucifer yellow-30min

Paracetamol-30min

21 22 23

24 25 26 27

Empty a insert,transfer it to the new well

Start the timer

Keep 30 s intervalbetween additions

cells

cells

cells

cellscells cells

cells

0.5ml 0.5ml

0.5ml 0.5ml 0.5ml

Add luciferyellow testsolution

0.5ml

0.5mlAddparacetamoltest solution


TEER measurement

25.9.2017 19

STX2 electrode

Touchingthe bottomof the well

WRONG WRONG


Varioskan

25.9.2017 20

Lucifer yellow is measured by fluorometry Varioskan Flash(Thermo Fisher Scientific)Excitation 425 nm and Emission 538 nm200 µl of samples in a black 96-well plate



UPLC

25.9.2017 21

•Propranolol and paracetamol•200 µl of samples in an UPLC white 96-well plate•Remember to label your plate with your name anddate.•Fill in the UPLC plate map (an Excel form isavailable).



Supervisors

25.9.2017 22

Cell work supervisors Analysissupervisor

Yan-Ru

Mariia

Patrick

Heikki

Petter

Alma Timo


Jasmi


5.10Thu

6.10Fri

7.10Sat

8.10Sun

9.10Mon

10.10Tue

11.10Wed

Cell seeding Medium change Medium change Medium change Medium change Permeation study

8:15-10:15MB, JK

Hanna, Emilia

9:00-10:00MB, JK

Hanna, Ida

Saturday eveningYRL

9:00-10:00PS

Sampo, Noora

9:00-10:00PS

Sampo, Noora

8:00-18:00AK 9-18PL 8-12MB 8-12YRL 8-12JK 12-1810:15-12:15

MB, JKNoora, Heli

10:00-11:00MB, JK

Heli, Sampo

10:00-11:00AK

Emilia, Ida

10:00-11:00AK

Hanna, Heli

12:15-14:15PS, HS

Ida

11:00-12:00HS, YRL

Emilia

11:00-12:00AK

Hanna, Heli

11:00-12:00YRL

Emilia, Ida

14:15-16:15PS

Sampo

12:00-13:00YRL

12:00-13:00MB

12:00-13:00MB

5-11.10.2017

25.9.2017 23

Teachers:YRL: Yan-RuMB: MariiaHS: HeikkiPS: PetterPL: PatrickAK: AlmaJK: Jasmi

7 students:4.10: add medium to transwells by JK4.10: prepare cell lab by MB4.10: prepare permeation lab by PS4.10: prepare transport buffer by YRL

www.helsinki.fi/yliopisto 25.9.2017 24Faculty of Pharmacy/ Yan-Ru Lou

Data processing and results

• Raw data, three standard curves, and original calculationsare presented in Microsoft Excel documents.

• Plot the accumulated test compound in the basolateralchamber versus time in Microsoft Excel documents.

• The average TEER values per insert before and after drugtest are presented in the Appendix of the Microsoft Wordreport.

• Papp per insert is calculated and their average and SD arepresented in Microsoft Word report.

• Mass balance is presented in Microsoft Word report.


Report is written in English using Microsoft Word.1. Title2. Author’s name3. Introduction: 1-2 pages including the background and the aim of the study4. Materials and Methods: no more than 3 pages5. Results: present only the calculated results, includingqPapp of each transwell in figuresqthe average Papp and standard deviations in a tableqmass balance of each transwell in a table

6. Discussion: interpret the results, explain the possible reasons for the wired results, compareobtained data with the literature data, improvement of the study, future perspective

7. Conclusion:8. References:9. Appendix:

q average TEER value per insert before and after tests in a table

25.9.2017 25Faculty of Pharmacy/ Yan-Ru Lou

Report instruction


Rules

25.9.2017 26

•Be on time. If you miss or come late for more than 30 min to any session withoutgood reason, you will not get credit.•For permeation study:

Bring your own pen, calculator, the handbook, and memory USB stickBring your own lab coats

•For cell lab work:Wear non-high heel shoesTie up your long hair

•Dealines for submission of your report in Word document and Excel raw data withcalculations: submit your report to Yan-Ru Lou ([email protected])

27.10 Friday at 23:59



Ø Page 18, first table, Pa4, the amount of transport buffer should be9.85 ml, not 9.855.

Ø Page 18, 2) Standard dilutions of Lucifer yellow (LY) for standardcurve:

20 µM LY = 10 µl of 20 mM LY stock + 9.99 ml of Transport BufferØ Page 19, 2.5. Centrifuge the cell suspension at 1000 rpm 200 rcf for

5 minutesØ Page 19, 2.7. Count the cells by using 0.1% Erythrosine B-dyeØ Page 20, step 1.3. Wash the cell monolayers with prewarmed (37°C)

transport buffer by adding 0.5 ml buffer to the apical side and 1.5 mlto the basolateral side of the monolayers.

Ø Page 20, step 2.7. Once you finish the measurements, wash theelectrodes thoroughly first with MilliQ-H2O and then with 70% EtOH.

Ø Page 26, Papp = dM/dt * 1/[A * (C1-C2)]

25.9.2017 27

Errors to be corrected in thehandbook


in vitro permeability study - courses · 2017-09-25 · in vitro permeability study yan-ru lou, phd...

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