Hepatosight -SUser GuideHepatosight® -S User Guide

Contents
1. Product Information............................................................................................................. 2
Unpacking & Handling ..................................................................................................... 2
Components & Description ............................................................................................. 3
Safety Precaution & User Notice ..................................................................................... 5
2. Introduction .......................................................................................................................... 6
3. Preparing for Cell Culture .................................................................................................... 7
Required Equipment and Consumables (Not Provided) .............................................. 7
Preparing Hepatosight® -S Media .................................................................................... 8
Preparing Cell Culture Surfaces ...................................................................................... 9
4. Hepatosight® -S Thawing and Plating ............................................................................... 10Thawing ........................................................................................................................... 10Plating .............................................................................................................................. 12
5. Hepatosight® -S Maintenance ............................................................................................ 13

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1. Product Information
Unpacking & HandlingUpon receiving the shipment of Hepatosight® -S, check whether all temperaturesensitive components are correctly stored. If this is not the case, please contact oursupport team immediately.
Immediately transfer each of the components to the appropriate storage conditions.
Please check the catalog number, lot number, and expiry date. The basal media
expiration date is the shortest (date of expiration on label) so experiments should beplanned accordingly.
The Hepatosight® -S should be handled by technically qualified individuals complying
with good laboratory practices, applicable laboratory regulations, and the MSDS.
Following the User Guide herein is recommended for best results.
The Hepatosight® -S is intended for research use only, not intended for any type of use
in animals or humans.

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Components & Description
COMPONENTS

CAT#STORAGE ON ARRIVAL

Hepatosight® -S Hepatocytes
Cryopreserved, frozen vial
>5 million cells>10 million cells

H-001
H-002

Hepatosight® -S Media
Maintenance medium
70 ml
100 ml

HM-001
HM-002

0.7 ml (100X)
1.2 ml (100X)

HS-001
HS-002

Hepatosight® -S MaintenanceSupplement

Liquid Nitrogen

4°C

-20°C

Hepatosight® -S User Guide1
Certificate of Analysis (CoA)MSDS2
1 Also available online at www.nexel.co.kr
2 Enclosed with shipping documents.

Should any of the above components be missing from your shipment, please contact
us at NEXEL Co., Ltd. or the distributor in your country upon which our support teamwill provide the necessary assistance.

Hepatosight® -S Hepatocytes
Cell Type

Human Induced Pluripotent Stem Cell (hiPSC) derived Hepatocytes

Cell Line of Origin

hiPSC cell line reprogrammed from commercially available normal
donor fibroblast cell line

Quality Control

Please refer to the CoA for lot-specific information.
Virus clearance & STR analysis data is available upon request.

Hepatosight® -S Advanced Media & Media Supplements
The Hepatosight® -S Medium & Supplements need to be combined to make theHepatosight® -S Maintenance Media, after which it should be used within 1 month. DO
NOT FREEZE the Hepatosight® -S Maintenance Media, but aliquot into smaller quantities
for best results.

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The Hepatosight® -S Maintenance Media are serum-free. For additional information on
the composition, please contact our technical support team.
The Hepatosight® -S Maintenance Media are antifungal free as they are not necessary if
proper conditions are kept. NEXEL does not recommend the use of such agents for
accurate results, but they should be used if aseptic cell culture conditions are notpossible.

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Safety Precaution & User Notice

Biosafety Level: 1
For research use only, not intended for any type of use in animal or humans. Appropriate
safety procedures should always be used with this material. Please refer to the MSDS for
detailed instructions.

User Notice & Restrictions:User may use the Product (Hepatosight® -S) for internal research including but notlimited to screening potential drug compounds for efficacy and safety, and for theprovision of such services to third parties. No other right is granted to User whether
expressly, by implication, by estoppel or otherwise. In particular, the purchase of theProduct does not include nor carry any right or license to use, develop or otherwise
exploit the Product commercially, and no rights are conveyed to User to use theProduct for any other purpose.
User agrees to use the Product in compliance with all applicable statutes andregulations, but not to use the Product for any administration or application tohumans. Moreover, User agrees not to use the Product in human subjects for humanclinical use for therapeutic, diagnostic or prophylactic purposes, or in animals forveterinary use for therapeutic, diagnostic or prophylactic purposes, including but notlimited to clinical applications, cell therapy, transplantation, and/or regenerative
medicine without an appropriate license.
In the case that User transfers Product to a third party, User shall convey the UserRestrictions set forth herein to such third party.

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2. Introduction
NEXEL Co., Ltd. strives to provide high quality human Hepatocytes derived from induced
pluripotent stem (iPS) cells using optimized proprietary protocols. The Hepatosight® -S is ahighly pure and functionally active population of cells, ensuring researchers get a reliable
product. Thus, NEXEL hopes to help the advance of science in tissue-specific research, toxicity
screening, and drug discovery.
This User Guide will help you seed the Hepatosight® -S at the appropriate densities to createhomogeneous layers of Hepatocytes appropriate for a variety of applications related to themetabolic activities such as albumin secretion, glycogen production, or CYP activity. However,please keep in mind that the best individual results will be obtained by close observation, care,and optimization from the user.

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3. Preparing for Cell Culture
Required Equipment and Consumables (Not Provided)
ITEM
Coating Material
Matrigel

CAT#VENDOR354234

Corning

Typical Cell Culture Equipment
Liquid Nitrogen Storage Tank
37°C Water Bath
Tabletop Centrifuge
Biological Safety Cabinet with UV Lamp
Hemocytometer or Automated Cell Counter
Phase Contrast Microscope
Pipettes
Cell Culture Incubator

Typical Cell Culture Consumables
Centrifuge Tubes
Cell Culture Plates
Pipette Tips
Trypan Blue
Phosphate Buffered Saline (PBS)

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Preparing Hepatosight® -S Media
1. Thaw the Hepatosight® -S Media and Supplements by placing them at 4°C 24 hours prior touse.
2. In a biosafety cabinet, thaw and add the Hepatosight® -S Maintenance Supplement (100X)
to the thawed medium to make corresponding Media. Store at 4°C for up to 1 month. DO
NOT FREEZE Hepatosight® -S Maintenance Media.
To avoid oxidation of the media due to air contact and repeatedwarming/opening, it is recommended to aliquot the media into quantities
enough for 2~3 media changes.
3. Hepatosight® -S Maintenance Media should be prepared immediately before plating thecells for best results.

MEDIA TYPEHepatosight® -S Maintenance MediaCOMPONENTS
Hepatosight® -S Maintenance medium
Hepatosight® -S Maintenance Supplement (100X)

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Preparing Cell Culture Surfaces
1. Calculate the amount of coating media required using the following table as a reference.

CELL CULTURE PLATE
COATING VOLUME

6-well(9.6 cm2)
1 ml

12-well(3.8 cm2)
500 µ l24-well(1.9 cm2)
300 µ l48-well(1.0 cm2)
200 µ l96-well(0.33 cm2)
100 µ l2. Prepare the coating solution to working concentration immediately before use as
described in the following table. Coating solution can be kept at 4°C for a short period oftime but such is not recommended.

COATING TYPESTOCK CONCENTRATION

WORKING
CONCENTRATION

MatrigelNA, 100X

1X (15 µ l/ml)

Collagen I is also a viable option to be used as coating material. If Collagen I is used,make sure to thoroughly wash the cell culture plate surface before seeding.
3. Pipette the correct amount of coating solution to each well you intend to use.
4. Gently swirl the plate and check whether all wells are completely covered.

5. Incubate at 37°C CO2 Incubator for more than 1 hour.

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4. Hepatosight® -S Thawing and Plating
Thawing
The Hepatosight® -S can be thawed using typical cell culture thawing protocols. Here, we
present NEXEL’s optimized protocol and recommend our users to follow the instructions tomaximize results. We strongly recommend thawing 1 vial at a time to minimize cell exposure
to liquid DMSO.

1. Calculate the amount of Hepatosight® -S Maintenance Media required. For each vial, 10 mlof Maintenance Media is required for resuspending the vial. The amount of MaintenanceMedia required can be calculated by the number of wells; recommendations for different
cell culture plates can be found below.

CELL CULTURE PLATE
PLATING VOLUME

6-well(9.6 cm2)
2 ml

12-well(3.8 cm2)
1 ml

24-well(1.9 cm2)
500 µ l48-well(1.0 cm2)
300 µ l96-well(0.33 cm2)
200 µ l2. Warm the Maintenance Media at Room Temperature (RT, 25°C) for at least 30 mins. For
each vial to thaw, aliquot 8 ml of Maintenance Media in a 15 ml centrifuge tube.
3. Retrieve the Hepatosight® -S vial(s) from the liquid nitrogen storage tank.
4. Submerge the vial(s) 2/3 in a 37°C water bath so that the mouth of the vial does not comein contact with the water. Constantly check how much has thawed and once ~20% remain(~3 mins), spray the vial(s) with 70% Et-OH, wipe and place it in your biosafety cabinet.
Ideally, the vial(s) should have completely thawed exactly when you start step 5.
5. Open the vial(s) and transfer the contents (~1 ml) using a 1 ml pipette to the aliquoted 8ml of Maintenance Media dropwise while gently swirling the tube.
Dropwise pipetting while gently swirling the tube minimizes osmotic shock andmaximizes mixing, which ensures high viability. Drops will remain on the surface for~1 second and then drop towards the bottom of the tube (visible due to the DMSO
content). For dropwise pipetting, simply pipette slowly into the air ~1 cm above themedia surface. It should take approximately 1 min per 1 ml.
6. Use 1 ml of Hepatosight® -S Maintenance Media to gently rinse the emptied vial andtransfer dropwise to the centrifuge tube containing the cells from step 5 while gently
swirling the tube.
7. Centrifuge the suspended cells at 180 x g for 3 minutes at room temperature.
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8. Carefully discard the supernatant.
9. Resuspend the cells gently using 1 ml of Maintenance Media and check the cellconcentration using a hematocytometer or cell counter and Trypan Blue. Immediately
move on to the Plating section.
Avoid rigorous pipetting of the cells to maximize viability. Single cell resuspensionof the Hepatosight® -S during thawing should easily be achieved by gently pipetting
3~4 times.

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Plating
NEXEL recommends seeding the Hepatosight® -S at a density of ~250,000 cells/cm2 for moststandard applications. Application specific protocols are available upon request. Best results
are obtained by the User’s own optimization, for which NEXEL will try to provide as muchassistance as possible.
1. Calculate the volume of Maintenance Media and cells required to match the correct
density for the culture platform of choice. Below is a table with cell numbers.

CELL CULTURE PLATE

6-well(9.6 cm2)

12-well(3.8 cm2)

24-well(1.9 cm2)

48-well(1.0 cm2)

96-well(0.33 cm2)

PLATING VOLUME

2 ml

1 ml

500 µ l300 µ l200 µ lCELL NUMBER

2,500,000

900,000

450,000

250,000

100,000

Well area (cm2) can vary between different vendors, please check with yourproviders for exact calculations.

2. Combine the volumes as calculated above.
3. Remove the coating solution in the cell culture plates. Avoid drying out the coated wells asmuch as possible.
4. Gently mix by pipetting and evenly distribute the appropriate volumes of cells withMaintenance Media.
5. Move the cell culture plate to the incubator, shake the plate in perpendicular directions toevenly distribute the cells for attachment.
6. The day after, perform the first media change with Maintenance Media (Hepatosight® -S
Maintenance Step 1).

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5. Hepatosight® -S MaintenanceStarting 24 hours after plating the cells, the media needs to be changed every two days.
Ideally, the media should be changed at 48-hour intervals. When performing functional assays
on the cells, we recommend changing the media on the morning of the experiment to ensure
there are enough nutrients for the cells and to deliver the desired drug concentration.
1. Warm the correct volume of Hepatosight® -S Maintenance Media at room temperature forat least 20 minutes.
2. Perform a media change with the newly warmed media in a biosafety cabinet. Pipette
softly onto the cell culture plate walls to avoid any damage to the cell culture.
24 hours after plating, replace a full volume of existing Media with MaintenanceMedia. To remove the spent media, aspirate the media using a pipette without tilting
the plate. Leave a small amount of media so that the cells do not come in contact withair. Then gently add appropriate volume of pre-warmed Maintenance Media.
After the initial full-media change, perform full-media changes on 48-hour interval.
While performing media changes, it is critical to keep the plate flat on a surface.
Tilting the plate during half-media changes can cause the cells to shift position andpotentially induce aggregation towards the tilted direction.
3. Place the plate back in the incubator.
4. Repeat 1 to 3 every 2 days.
We recommend performing any planned assays with the Hepatosight® -S from Day 10
onwards.

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