human airway epithelial cells ci-huaec · 2018. 11. 15. · developed for the in vitro cultivation...

InSCREENeX GmbH D-38124 Braunschweig │ Inhoffenstraße 7 [email protected] www.inscreenex.com

Managing Directors Dr. Roland Schucht │Dr. Tobias May HRB 202375, Amtsgericht Braunschweig VAT No: DE268885989

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Content

CI-huAEC .................................................................................................................................................................................... 2

Cell Culture Media & Supplements .............................................................................................................................................. 2

Preparing the Medium for Use and Storage ............................................................................................................................ 2

Getting the cells started after delivery ..................................................................................................................................... 2

Protocol for cultivating CI-huAEC ................................................................................................................................................ 2

Table 1: Recommended Volumes of Coating Solution .................................................................................................... 2

Coating of cell culture plastic ware surfaces ........................................................................................................................... 2

Splitting Routine/Maintenance of the cells .............................................................................................................................. 2

Table 2: Recommended Volumes of Medium/PBS/TE .................................................................................................... 2

Freezing/Thawing of the cells ................................................................................................................................................. 2

TEER-Assay........................................................................................................................................................................... 2

Rebuilding Nature. Rebuilding Life.

Human Airway Epithelial Cells

CI-huAEC

Visit our homepage: www.inscreenex.com



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Product Name Cat. No. Size Price

huAEC-Medium, (ready-to-use)

Consists of basal medium and basal supplements (INS-ME 1013BS)

INS-ME-1013-100ml INS-ME-1013-500ml

100 ml 500 ml

€ 75.00 € 290.00

Freezing medium INS-SU-1004 30 ml € 29.20

huAEC Coating solution (ready-to-use), suitable for cell culture

INS-SU-1018-20ml INS-SU-1018-100ml

20 ml 5x20 ml

€ 40.00 € 160.00


CI-huAEC

Cell Culture Media & Supplements

Recommended for

CI-huAEC (Cat.No. INS-CI-1011)

Human airway epithelial cells

Product Description

InSCREENeX huAEC Medium is

developed for the in vitro cultivation of

immortalized human airway epithelial

cell line CI-huAEC

(Cat.No. INS-CI-1011).

Quality Control

The medium is subjected to quality control

tests and checked for growth

characteristics of the immortalized human

airway epithelial cell line CI-huAEC.

In addition, all media have been tested for

the absence of microbial contaminants like

fungi, bacteria or mycoplasma.

Intended Use

The products are for in vitro use only

and not for diagnostic or therapeutic

procedures.



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The medium and supplements are delivered cooled (@ 2 to 8°C).

Store the medium @ 4 to 8°C in the dark.

The supplements should be frozen @ -20°C after arrival.

It is recommended to aliquot the supplements before and keep them

frozen @ -20°C until use.

If stored properly, the products are stable until the expiry date.

To give the cultivation media thaw the supplements at 15°C to 25°C

and apply the solution to the basal medium (e.g. for the preparation

of 100 ml complete medium add 6 ml of the Basal Supplements to

the Basal Medium).

The complete medium is stable for approximately one month when

stored at 4°C.

Preparing the Medium for Use and Storage


Getting the cells started after delivery

The CI-huAEC line is being delivered on dry ice. We recommend to thaw the cells on a T25

flask. After two days the medium should be changed.

As soon as the cells reached adequate confluency (usually 4-5 days after thawing), the cells

should be splitted (see protocol at page [5]).

By starting the culture, the splitting ratio should not exceed 1 : 2.

In the course of cultivation (after ~2 weeks) the splitting ratio can be extended to 1 : 4 or 1 : 5.



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Protocol for cultivating CI-huAEC

Coating of cell culture plastic ware surfaces

Material

huAEC Coating solution (Cat.No. INS-SU-1018-20ml; Cat.No. INS-SU-1018-100ml)

PBS

Cell culture plastic ware

Preparation

Cover the cell culture dish with the coating solution (see table 1 below for the required volume).

Incubate the cell culture dish for at least 120 min (up to overnight) at 37°C in the incubator.

Aspirate Coating Solution.

Wash the coated cell culture dish once with PBS.

Add cells and media to the coated plate shortly after aspiration of the Coating Solution.

Rebuilding Nature. Rebuilding Life. Rebuilding Nature. Rebuilding Life.

Plastic ware Area in cm2 Coating Solution

T75 75 2,5 ml

T25 25 1,4 ml

6 well 9 0,7 ml

12 well 4 0,25 ml

24 well 2 0,1 ml

96 well 0,32 0,05 ml

Table 1: Recommended Volumes of Coating Solution



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Plastic ware Area in cm2 Culture Medium PBS TE

T75 75 8-10 ml 8-10 ml 2 ml

T25 25 4-5 ml 4-5 ml 1 ml

6 well 9 1,5-2 ml 1,5-2 ml 0,5 ml

12 well 4 1 ml 1 ml 0,2 ml

24 well 2 0,5 ml 0,5 ml 0,2 ml

96 well 0,32 0,1 ml 0,1 ml 0,05 ml

Splitting Routine/Maintenance of the cells

Material

huAEC Culture medium (Cat.No. INS-ME-1013)

PBS

Cell culture plastic ware

Trypsin/EDTA solution (TE)

Sterile Pasteur Pipettes

Preparation

Check the status of the cells microscopically

If the cells are 80-90 % confluent, split the cells

Splitting (see Table 2 for corresponding volumes of Medium/PBS/TE)

Aspirate the cultivation media of the cells with a sterile Pasteur Pipette.

Wash the cells once with PBS.

Aspirate the PBS.

Add TE to the cells.

Incubate the cells with TE at room temperature or at 37°C until the cells start to detach (check

microscopically).

Resuspend the cells with medium or PBS.

Transfer an aliquot of the cell solution to a new cell culture dish/flask.

Add cultivation medium to the cells.

Cultivate the cells at 37°C.

Table 2: Recommended Volumes of Medium/PBS/TE



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Freezing/Thawing of the cells

Material

Freezing medium (Cat.No. INS-SU-1004)

PBS

FBS

Trypsin/EDTA (TE)

15 ml plastic tube

Vials suitable for freezing in liquid nitrogen

Preparation

Freezing

Grow the cells to 90 % confluence.

Wash the cells with PBS.

Trypsinize the cells with TE.

Resuspend the cells with 5 ml of PBS, containing 2% FBS.

Transfer cell suspension in 15 ml plastic tube.

Spin-down cells @200g for 5 min.

Aspirate supernatant.

Resuspend cell pellet with freezing medium (cell concentration 1x106 cells per ml).

Transfer cell suspension in “freezing” vial.

Place vials into Mr. Frosty or comparable devices (to slowly cool down vial).

Place Mr. Frosty in -70°C overnight.

After 24 h transfer vial from -70°C to liquid nitrogen tank for long term storage.

Thawing

Pipette 4 ml medium in a 15 ml plastic tube.

Quickly thaw vial in preheated water bath (@37°C).

Transfer thawed cell suspension to 15 ml plastic tube containing 4 ml medium.

Spin-down cells @200g for 5 min.

Aspirate supernatant.

Resuspend cell pellet with cultivation medium.

Transfer cells in desired cultivation device (recommended is T25 flask or 6 well).




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Material

World Precision Instruments – EVOMX (or comparable instrument for measuring resistance)

Electrode set STX

Transwell Costar Cat.No. 3470 (0,4 µm pore size; 6,5 mm insert for 24 well plates)

Two 15 ml plastic tubes

Ethanol

PBS

huAEC Culture medium (Cat.No. INS-ME-1013; INS-ME-1014)

huAEC Coating solution (Cat.No. INS-SU-1018; INS-SU-1019)

Sterile Pasteur pipettes

100-1000 µl pipette tips

Plating the cells

Pipet 100 µl coating solution to the center of the transwell.

Coat transwell for 2h @ 37°C.

! Important: Include a mock transwell with just coating and culture media.

Carefully remove Coating Solution with Pasteur pipette from transwell.

Plate cell solution (1x105 cells per transwell in 200 µl Culture Medium) into upper compartment of the

transwell. ! Important: Do not exceed 200 µl as the cell solution could spill over to the lower compartment.

Pipet 450 µl Culture Medium through the opening in the lower compartment of the transwell.

Cultivate the cells @ 37°C, 5 % CO2.

TEER-Assay



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Recording TEER values

On day of TEER measurement renew culture media.

! Important. To avoid lifting of the cells stick to the order of the next steps.

1. Aspirate the medium from the lower compartment.

2. Aspirate the medium from the upper compartment.

3. Add 200 µl fresh medium to the upper compartment.

4. Add 400 µl fresh medium to the lower compartment.

Cultivate the cells for 4h to 6h @ 37°C.

Place EVOMX and electrode under laminar flow bench.

Fill ~10 ml PBS in one 15 ml plastic tube under laminar flow bench.

Fill ~10 ml Ethanol in one 15 ml plastic tube under laminar flow bench.

Connect electrode to EVOMX and place electrode in 15 ml tube containing Ethanol.

Make sure that on the EVOMX “R” is measured and the “Mode R” is switched.

In the beginning of the culture the TEER values should be measured in the 2000 Ω range.

Put cells under laminar flow.

Wash the electrode in PBS to remove the Ethanol.

Switch on EVOMX.

Carefully put the electrode into the transwell.

Make sure that the longer electrode is put through the opening of the transwell to the lower

compartment of the transwell.

Record the measurement.

After the experiment place the electrode in

Ethanol for 20 min.

Cultivate the cells for a longer time period if

desired.

Figure 1. TEER-Assay of CI-huAEC lines over time.


human airway epithelial cells ci-huaec · 2018. 11. 15. · developed for the in vitro cultivation...

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