Guide to spheroid formation using Verified forSpheroids™ Human Hepatocytes
Maureen Bunger; Lonza Walkersville, Inc., Walkersville, MD, USA

Guide to spheroid formation
using Verified for Spheroids™
Human Hepatocytes
Use this protocol for spheroid formation with our Verified for Spheroids™ HumanHepatocytes to support your next steps towards more physiologically relevant liver cell
culture systems.

Introduction
The liver is the body’s main site of metabolism of most small moleculedrugs and consequently, in vitro models that accurately replicate liverfunction are highly desirable. It has recently been shown that primary
hepatocytes, the main functional cells of the liver, can self-assemble
into small spheroids when cells are placed into low-adherence platesor hanging liquid drops.1 The formation of these spheroids improves thein-vivo-like response and increases the life span of the hepatocytes.
In this Technical Note, we describe the development of a simple
spheroid formation protocol using our Verified for Spheroids™ Human
Hepatocytes, to support researchers desire to implement hepatocyte
spheroids as a routine model for in vitro metabolism, disease modeling,and toxicity testing.

Protocol1. To formulate HCM™ Hepatocyte Culture Medium, transfer thecontents of the HCM™ SingleQuots™ Kit to HBM™ Basal Medium with apipette, and rinse each vial with medium. Store at 4°C for up to1 month.
2. Make complete Spheroid Formation Medium (SFM) by adding thefollowing supplements to HCM™ Medium: FBS (final 20%) and HEPES
Buffer (final 25mM). Store at 4°C for up to 1 month.

Materials
1. Primary Verified for Spheroids™ Human Hepatocytes (Lonza, cat. no.
HUCPI or HUCPG)
2. HCM™ Bullet™ Kit (Lonza, cat. no. CC-3198)
3. Fetal Bovine Serum (FBS, Hyclone, cat. no. SH30071.03)
4. 1M HEPES Buffer (Lonza, cat. no. 12509079)
5. Human Cryopreserved Hepatocyte Thawing Medium (Lonza,cat. no. MCHT50)
6. 96-well Corning U-bottom ultra-low attachment (ULA, Sigma,cat. no. CLS7007)

3. Thaw Verified for Spheroids™ Human Hepatocytes in thawing
medium and centrifuge at speeds and times described in ourProtocol. https://lonza.picturepark.com/Go/ACl6hFB1/D/29887/1

FBS concentration

0%

Note: We tested the effect of various levels of serum addition to themedia on spheroid formation. Results indicate that spheroids requirea minimum of 10% serum to form, but 20% serum produces the most
optimal spheroids (Figure 1).

Note:Each batch of Lonza’s HUCPI and HUCPG are characterized for spheroidformation. Look for the Verified for Spheroids™ Specification in batch
certificates or visit product web pages for up-to-date inventory.

2%10%20%Day 11

Figure 1
At least 10% serum is required for spheroid formation. 3,000 primary human hepatocytes seeded in ULA U-bottom plates with serum at concentrations ranging form 0 – 20%.
Optimal results are obtained when 20% serum is included in spheroid formation medium.

Guide to spheroid formation using Verified forSpheroids™ Human Hepatocytes
Maureen Bunger; Lonza Walkersville, Inc., Walkersville, MD, USA

4. Resuspend cells in 3 – 8 mL SFM and count cells using trypan blue
and a hemocytometer.
5. Adjust cells to a final concentration of 1.0 x 104 – 3.0 x 104 cells/mL
in SFM depending on the size of spheroid desired.
Note: We tested seeding between 1,000 – 5,000 cells per well. While
all seeding densities produced spheroids, increasing the number ofcells/well resulted in larger spheroids with cores beyond 500 µM. This
enlarged size increases the risk for necrotic cores and therefore, we
recommend using less than 3,000 cells/well when forming spheroids(Figure 2).
Cell no.

Day 7

1,000

500 μm

3,000

5,000

500 μm500 μmAdditional studies can be found in a separate White Paper, https://lonza.
picturepark.com/Go/dfhpfzvf/D/34835/1 that show certain hepatocyte
lots can last in spheroid culture up to 28 days with continued high-level
Cytochrome P450 activity. These efforts support the potential use ofour Verified for Spheroids™ Human Hepatocytes for extended toxicology
and DMPK studies and reduce the need for researchers to test different
donors for spheroid formation.
References
Bell CC, Hendriks DF, Moro SM, Ellis E, Walsh J, Renblom A, Puigvert LF, Dankers AC, Jacobs F,Snoeys J, Sison-Young RL. Characterization of primary human hepatocyte spheroids as amodel system for drug-induced liver injury, liver function and disease. Scientific reports.
2016 May 4;6:25187.
1Figure 2
Spheroids of different size. 1,000 to 5,000 primary human hepatocytes cultured in ULA
U-bottom plates on day 7 after second media change.
Spheroids reach size of 500 µM when 3,000 cells are used for spheroid formation.

Contact information
6. To begin spheroid formation, add 100 µL cell suspension to wells ofa U-bottom ULA 96-well plate and place in incubator at 37°C 5% CO2.
Incubate undisturbed for 120 – 168 hours.
Note: For extended cultures of spheroids, we recommend filling allouter edge wells with medium only to assist with evaporation issues.
Any disturbance of the plates or mishandling during incubation can
negatively affect spheroid formation.
7. Once spheroids have formed, remove 50% of medium and add freshcomplete HCM™ Medium every 2 to 3 days. More frequent media
changes can facilitate faster removal of remaining serum usedduring spheroid formation.
Our Verified for Spheroids™ Human Hepatocytes are plateable humanhepatocytes that are already qualified as general purpose (cat. no.
HUCPG) or Interaction Qualified (cat. no. HUCPI) and further tested forforming a tight spheroid by 7 days in culture. In our extended studies, it
was observed that there is some donor-to-donor variability in both theformation rate and longevity of hepatocyte spheroids.

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Lonza Walkersville, Inc. – Walkersville, MD 21793
For research use only. Not for use in diagnostic procedures.
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