CELLAPPLICATIONS, INC.

General Instructions for Use
Human Gastrointestinal Epithelial Cells
Instructions apply to Gastric (HGaEpC), Intestinal (HInEpC), Colonic (HCnEpC),
Duodenum (HDuEpC), Ileum (HIlEpC), and Jejunum (HJeEpC) Epithelial Cells
Be sure to wear face protection mask and gloves when retrieving cryovials from the liquid nitrogen storage tank. The dramatic
temperature change from the tank to the room could cause any trapped liquid nitrogen in the cryovials to burst and cause injury.

Open all the packages immediately upon arrival andexamine each component for shipping damage. Notify Cell
Applications, Inc. or your distributor immediately if there
is any problem.

1.

I. STORAGE

2.

A. CRYOPRESERVED VIALS* (732Ga-##, 732In-##,732Cn-##, 732Du-##, 732Je-##)3.

Store the cryovials in a liquid nitrogen storage tank
immediately upon arrival.
B. GI EPITHELIAL CELLMEDIUM (716DC-50)

DEFINED

II. PREPARATION FOR CULTURING

4.
5.

CULTUREStore at -20°C in the dark. The frozen medium is stablefor 6 months. Once thawed, keep at 4°C in the dark anduse within 4 weeks. Freeze-thaw cycles are notrecommended. If necessary, the thawed medium can bealiquoted and refrozen once.
C. GIEpC COATING SOLUTION (025-05)
Store at -20°C. The frozen coating solution is stable for6 months. Once thawed, keep at 4°C and use within 4weeks.

III. CULTURING HGIEpC
A. PREPARING CELL CULTURE
CULTURING HGIEpC
1.

D. GIEpC THAWING MEDIUM (716T-20)
Store at -20°C. The frozen medium is stable for 6months. Once thawed, keep at 4°C and use within 3months.
Reagent not included in the Total Kit:

2.
3.

4.

TEER ASSAY OPYIMIZED GI EPITHELIAL CELLCULTURE MEDIUM (716TA-50)
Store the Culture Medium (716TA-50) at 4°C in thedark immediately upon arrival. Use within 4 weeks ofarrival. If not used immediately, store at -20°C uponarrival. The frozen medium is stable for 6 months. Once
thawed, keep at 4°C and use within 4 weeks. Re-freeze
the culture medium is not recommended.

Make sure the Class II Biological Safety Cabinet, withHEPA filtered laminar airflow, is in proper working
condition.
Clean the Biological Safety Cabinet with 70% alcohol
to ensure it is sterile.
Turn the Biological Safety Cabinet blower on for 10min. before cell culture work.
Make sure all serological pipettes, pipette tips andreagent solutions are sterile.
Follow the standard sterilization technique and safety
rules:
a. Do not pipette with mouth.
b. Always wear gloves and safety glasses whenworking with human cells even though all thestrains have been tested negative for HIV, Hepatitis
B and Hepatitis C.
c. Handle all cell culture work in a sterile hood.

5.

WARES

FOR

Take the GIEpC Thawing Medium and GIEpC Culture
Medium from the refrigerator. Decontaminate the bottlewith 70% alcohol in a sterile hood.
Plan the seeding density in each experiment.
Thaw the GIEpC COATING SOLUTION at 4°C
overnight. Ensure that the thawing medium is coldenough to avoid gelling.
Prepare number of wells of a 48 well plate for culturing
HGIEpC by pipetting 150 l of GIEpC Coating
Solution per well. (See tables for cell numbers andvolume used).
Incubate coated plate at room temperature for at least 30
minutes (do not exceed one hour, so adjust your time
before taking out the cell vial).

Cell Applications Inc (hereinafter CAI) warrants that its products are manufactured with the utmost care and stringent quality control procedures. However, if you
should ever have a problem with the products, we will either replace the products, or in the case we cannot deliver the products, provide you with a refund. Suchwarranty is applicable only when CAI’s cells are used in conjunction with CAI’s medium and subculture reagents, and vice versa.

B. PREPARING MEDIUM FOR CULTURING HGIEpC
1. Bring GIEpC Thawing Medium to room temperature.
Transfer 8 ml of GIEpC Thawing Medium in a 15 mltube.
2. Take out the required volume of Culture Medium
according to the Table on right side and warm up toroom temperature*.

* Do not warm GIEpC Culture Medium to 37oC.
C. THAWING AND PLATING HGIEpC
1.

Remove the cryopreserved vial of HGIEpC from theliquid nitrogen storage tank using proper protection foryour eyes and hands.
2. Turn the vial cap a quarter turn to release any liquidnitrogen that may be trapped in the threads, then retighten the cap and bury the cryovial in dry ice.
3. Thaw the cells quickly by placing the lower half of thevial in a 37°C water bath and watch the vial closely
during the thawing process. This usually takes about 90
sec.
4. Take the vial out of the water bath when only small
amount of ice left in the vial. Do not let cells thaw
completely.
5. Decontaminate the vial exterior with 70% alcohol in asterile Biological Safety Cabinet.
6. Remove the vial cap carefully. Do not touch the rim ofthe cap or the vial.
7. Resuspend the cells in the vial by gently pipetting thecells once with a pre-wet, 1 ml aerosol pipette tip set at950 l. Do not to pipette vigorously as it might cause
foaming.
8. Transfer the cell suspension (1 ml) drop wise to the 15ml conical tube containing 8 ml GIEpC Thawing
Medium prepared in Section III B step 1.
9. Add 1 ml of fresh GIEpC Thawing Medium to thecryovial for wash and add to the 15 ml tube to a final
volume of 10 ml. Mix by gently inverting the tube
couple of times to avoid osmotic shock.
10. Centrifuge cells in the 15 ml conical tube at 200g for 5minutes. Remove supernatant carefully by aspiration
without disturbing the cell pellet. Gently break the pellet
by flickering the tube few times with your finger.
11. Re-suspend HGIEpC at 5 x 105/2.5ml in GIEpC
Culture Medium. Use a pre-wet, 1 ml aerosol pipette
tip to evenly re-suspend the pellet in 1 ml GIEpC
Culture Medium without any visible cell clumps. Thenadd the rest 1.5 ml medium and gently re-suspend again.
12. Aspirate GIEpC Coating Solution from the wells of theplate prepared in Section III A step 5.
13. Seed HGIEpC suspension to the coated wells assuggested in the reference tables. Rock gently to evenly
distribute the cells.
14. Place the plate in a 37oC, 5% CO2 humidified incubator.
For best results, do not disturb the culture for 24 hours
after plating.
15. Carefully change to fresh room temperature GIEpC
Culture Medium after 24 hours by removing the old
medium with 200 ul pipette tips. Do not use suction
and do not let cells dried up.
Copyright© 2020 Cell Applications Inc.
5820 Oberlin Drive, #101
San Diego, CA 92121

16. Change the GIEpC Culture Medium every other day.
17. The HGIEpC monolayer culture should expand inpatches in GIEpC Culture Medium for up to 7 days.
Cells should be near confluent by day 6.
Volume per Well
TCWell
Plate
9648
24

GIEpC
CoatingSolution

GIEpC Suspension in
716DC-50 (Section

0.1 ml0.15 ml0.25 ml

200 µl500 l
1 mL

III C step 11)

Approx. No ofHGIEpC
required forNormalCulture
0.4 x 105
1 x 105
2 x 105

Table 1: Seeding guide of corresponding cell numbers
and working volumes for normal seeding

Tel: (858) 453-0848 Toll-Free: (800) 645-0848 Fax: (858) 453-2862
Email: info@cellapplications.com Web: www.cellapplications.com
201023