and tissue-based products

Protocol for Thawing Cryopreserved Hepatocytes

Product Instruction

cell

The following procedure may be carried out in a biosafety containment hood to reduce the risk of contamination
and also minimize contact with potentially biohazardous material.

Step-wise Procedure for Thawing Kits (K8000, K8100, K8500 & K8800)

This procedure describes the steps required for the isolation of hepatocytes using XenoTech’s Hepatocyte
Isolation Kits.

Kit Components:
OptiThaw Hepatocyte Media
OptiCount: 50 µL Trypan Blue and 400 µL of 1xPBS1) Warm the OptiThaw media to 37 ± 1°C in water bath before use (typically takes ~15-20 minutes).
2) Remove the cryotube from the LN2 storage unit and immediately place in a 37 ± 1°C water bath for~80 seconds until the frozen cell pellet can move freely when the cryotube is inverted. Do not over-thaw.
Note: For CryostaX™ pools, the contents of the vial can be immediately dispensed into the pre-warmed
OptiThaw Hepatocyte Media. Do not thaw CryostaX™ in a water bath. Once the frozen pellets aretransferred to OptiThaw, gently invert the tube until all of the pellets have melted.
3) Transfer the frozen pellet from the cryotube into OptiThaw Hepatocyte Media. Rinse each cryotube
with 1.5 mL of OptiThaw. Pour this rinse back into the OptiThaw tube and gently invert until fully melted.
4) Centrifuge at 100 x g for 5 minutes at room temperature or 2-8°C.
• Aspirate and discard the supernatant fluid without disturbing the cell pellet.
5) Resuspend the cell pellet(s) with K8200 OptiPlate Hepatocyte Media (for plating hepatocytes) orK8400 OptiIncubate Hepatocyte Media (for suspension incubations), be careful not to over-dilute thecells based on final target cell concentration.
DO NOT VORTEX.

• Remove 50 µL of the homogenous cell suspension and dispense the 50 µL aliquot into theOptiCount tube.
• Mix gently. Cell viability can now be assessed by placing an aliquot from the counting tube on ahemacytometer and counting the dead (blue) cells and viable cell number.

6) Measure the volume of the cell suspension and q.s. to the desired concentration.

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Protocol for Thawing Cryopreserved Hepatocytes (Instruction)

Cryopreserved Hepatocyte Sample Preparation Worksheet
This worksheet may be used to record information during the preparation of your hepatocyte sample. Prepare
additional copies of this sheet as needed.

Hepatocyte Sample Identification# Vials Thawed
Sample ID(Species/Lot Number)

Date of hepatocyte isolation:

1.5 mL

Trypan Blue Cell Count Analysis
A trypan blue exclusion analysis should be performed (step 5 in the thawing protocol) following
re-suspension of the initial cell pellet.
Cells CountedLiveA% Viability[A/(A+B)] x100DeadBDilution
1factorHemacytometer2factorVolume of3sample

CDE

Number ofviable
4
hepatocytes

Final cell
5
concentration

10,00010,0001. The dilution factor will equal 10 if a 50 µL aliquot of the cell suspension was dispensed into XenoTech’s hepatocyte isolation OptiCount tube forsubsequent counting. If an alternate means of dilution was used, a different dilution factor should be calculated.
2. The hemacytometer factor will typically equal 10,000. For more information consult your hemacytometer manufacturer.
3. Volume of the sample indicates the total volume of the cell suspension from which the counting aliquot was removed.
4. The number of viable hepatocytes may be calculated from the following equation:
A × C × D × E
 quadrants

where “quadrants” equals the number of quadrants counted on the hemacytometer.

5. The desired concentration should be determined based on the specific requirements of your experimental design.

Sample Dilution
Use the following table to calculate the final volume needed to reach the desired cell concentration# of viable hepatocytes(determined above)

Desired cellconcentration for use

Final volume

Volume of media to add
to reach desired conc.

FGHI

H = F/GI = H - Volume of sample

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Protocol for Thawing Cryopreserved Hepatocytes (Instruction)

Tips for Working with Hepatocytes• Thaw time is critical; over-thawing cryopreserved hepatocytes will result in low yield and viability. The solid,frozen pellet should be transferred directly into the OptiThaw tube.
• For best results ensure level of water in the water bath is above the highest frozen point in the vial.
• When aspirating supernatant, keep tip of the aspirator at the highest level of media to ensure any cell debris
is removed before reaching the viable cell pellet.
• XenoTech does not recommend pouring off supernatant, due to the high risk of losing the viable cell pellet
during the pour process.
• Never vortex or vigorously resuspend the hepatocytes. A gentle rocking motion is recommended.
• We recommend performing two Trypan blue counts after centrifugation for verification of yield and viability.
• One hepatocyte isolation kit can be used to thaw up to 3 vials.

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Protocol for Thawing Cryopreserved Hepatocytes (Instruction)

Suspension Incubations
Materials Needed:• Flat-bottomed, uncoated incubation vessel• Static or shaking incubator temperature: 37°C, Atmosphere: Rh (95%), CO2 (5%), for long-term incubations ahumidified incubator is recommended• OptiIncubate Hepatocyte Media (K8400)
Typical final cell concentration
per incubation

1 x 106 cells per mL

Flat-bottom incubation vessel

6-, 12-, 24-, 48-, 96- well

Typical incubation times

0, 30, 60, 90, 120, 240 min

Incubation media selection:
For long-term incubations ( > 2 hours), OptiIncubate Hepatocyte Media (K8400) is recommended to maintain ahigher viability throughout the incubation time.
If the test compound may be susceptible to protein binding, a buffer such as KHB is recommended because it does
not contain any protein supplementation.

Krebs Henseleit Buffer (KHB)
pH 7.4 no nutritional supplementation (Sigma)

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Protocol for Thawing Cryopreserved Hepatocytes (Instruction)

Protocol for Plating and Culturing Hepatocytes
Materials Needed:•••••

BioCoat™ Collagen I Cellware (BD Biosciences) or equivalent
Cell Culture Incubator Temperature: 37°C Atmosphere: Rh (95%), CO2 (5%)
OptiCulture Media and Pen/Strep (K8300)**日本では、K8300は、培地K8300Mと抗生剤PS100を別製品として販売しております。
OptiPlate Hepatocyte Media (K8200)
OptiMatrix Hepatocyte Overlay (K8600/K8650)*K8300 comes with a supplemental vial of Pen/Strep. Add the entire contents of the vial to the bottle of media and update the expiration
date to one month from the date of the addition.

Procedure1) Thaw hepatocytes as stated earlier in the protocol.
2) Dilute the hepatocyte suspension to the desired concentration with OptiPlate media. The table below
shows a range of recommended seeding densities for each species. See the lot specific data sheet for aparticular lot’s recommended seeding density.
3) Add appropriate volume of cell suspension to each well. The table below provides recommended seeding
volumes for the various species and plating formats.
6-Well Format
Species
HumanRat
MonkeyMouse

12-Well Format

24-Well Format

48-Well Format96-Well Format*

Recommended
Recommended
Recommended
Recommended
Recommended
Recommended
Recommended
Recommended
Recommended
Recommended
Seeding Density Seeding/Feeding Seeding Density Seeding/Feeding Seeding Density Seeding/Feeding Seeding Density Seeding/Feeding Seeding Density Seeding/Feeding(million cells/mL) Volume Per Well (million cells/mL) Volume Per Well (million cells/mL) Volume Per Well (million cells/mL) Volume Per Well (million cells/mL) Volume Per Well
1.0 - 1.6
1.2 - 1.4
1.4 - 2.2
0.4 - 0.61.7 mL
1.7 mL
1.7 mL
1.7 mL

1.0 - 1.6
1.2 - 1.4
1.4 - 2.2
0.4 - 0.6650 µL
650 µL
650 µL
650 µL

1.0 - 1.6
1.2 - 1.4
1.4 - 2.2
0.4 - 0.6

330 µL
330 µL
330 µL
330 µL

0.75
1.2 - 1.4
1.4 - 2.2
0.4 - 0.6200 µL150 µL150 µL150 µL

0.75
0.6 - 0.7
1.4 - 2.2
0.4 - 0.675 µL
75 µL
50 µL
50 µL

* Do not swirl 96-well plates to distribute cells.

4) Place the seeded culture vessel in the 37°C incubator and swirl in a figure 8 pattern to evenly distributethe cell suspension. Do not swirl 96-well plates.
5) Allow cells to attach for 2-4 hours in a 37°C humidified CO2 static incubator. Check attachment every
hour until sufficient confluency is achieved (the hepatocytes will flatten out over time to fill in the majority ofthe gaps).
6) After the attachment period, swirl the culture vessel (to suspend the unattached cells) and aspirate media
containing non-attached cells.
7) Add appropriate volume of 2-8°C OptiCulture Media solution (with or without OptiMatrix) to each well orplate and return dishes to incubator.
• OptiMatrix, as used for overlay, should be diluted to 0.25 mg/mL in the OptiCulture Media.
• To achieve maximum confluency when working with rat hepatocytes, DO NOT include OptiMatrix in theHepatocyte Culture Media at the 2-4 hour time point media change. OptiMatrix overlay should be performed
18-24 hours post seeding.

8) Every 24 hours, the media should be aspirated and replaced with OptiCulture Media warmed to 37°C.
9) Dosing with compound can begin after the hepatocytes have been in culture for 24-48 hours.
• Media should be aspirated and replaced with fresh dosing solution (OptiCulture Media and test compound)at 37°C every 24 hours.
• Cultures can be maintained for 6 to 7 days.

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