Primary Human Suspension Hepatocytes Culture Protocol

• Primary Human Suspension Hepatocytes
• Primary Human Suspension Hepatocytes Culture Materials
• Suspension Hepatocytes Growth Protocol
• Primary Suspension Hepatocyte Growth Results

Primary Human Suspension Hepatocytes

Our extensive collection of normal human primary liver cells includes characterized suspension Hepatocytes, which can be used to model the function of Hepatocytes in vitro. Suspension cells in culture float in media and don’t form a monolayer on the culture surface. We currently offer two options for the characterized suspension Hepatocytes: the mixed gender pool and the single donor. Our pooled suspension Hepatocytes are normalized for cell number to ensure that each donor contributes an equal contribution to the pool. Our single donor human suspension consists of a homogenous population of Hepatocytes and it is offered in various pack sizes. These primary suspension Hepatocytes are ideal for the studies of enzyme induction, toxicity, drug screening, transporter efflux activity, and potential drug-drug interactions.

Our primary Hepatocytes collection are isolated from whole human livers, with each donor providing documented consent for research use of non-transplantable organs or tissues. The suspension Hepatocytes cells are cryopreserved for ease of use and undergo testing for enzyme profiling data and ensuring a post-thaw viability of ≥70%.

In this protocol, we demonstrate how to thaw and grow primary Hepatocytes in suspension culture.

Primary Human Suspension Hepatocytes Culture Materials

• Human Characterized Suspension Hepatocytes 20-Donor Mixed Gender Pool (HLP120-5M) or 10-Donor Mixed Gender Pool (HLP110-5M) or Normal Human Characterized Suspension Hepatocytes (HLP101). Note: Upon receipt, immediately store cryovial(s) in liquid nitrogen.
• 50ml Centrifuge Tube
• Tissue culture treated multiwell plates
• 1X Hepatocyte Plating Medium (HPM) for Human Hepatocytes

Components	Catalog Number	Working Stock	Final Dilution	Final Conc.	Final Volume (mL)
DMEM. High Glucose	D1145-500ML			1x	464
FBS	ES009-M			5%	25
Dexamethasone	D4902-25MG	2mM	2,000x	1µM	0.25
Insulin	I9278-5ML	10mg/mL	2,500x	4µg/mL	0.2
Gentamicin	G1272	10mg/mL	1,000x	10µg/mL	0.5
L-glutamine	TMS-002-C	200mM	100x	2mM, 1x	5
NEAA	TMS-001-C	10mM	100x	0.1mM, 1x	5
				Total volume	500

1X Maintenance Medium for Human Hepatocytes

Note: For Dexamethasone, dissolve 25 mg into 32 mL of ethanol (100%) to make 2mM stock. For Linoleic acid, dissolve 100mg into 2mL of ethanol (100%) to make 50mg/mL stock.

Components	Catalog Number	Working Stock	Final Dilution	Final Conc.	Final Volume (mL)
Williams E	W1878-500ML			1x	466.4
Human Insulin	I9278-5ML	10mg/mL	1,600x	6.25 µg/mL	0.313
Human Transferrin	T0665-50MG	1mg/mL	160x	6.25 µg/mL	3.125
Selenium	S9133-1MG	0.1mg/mL	16,000x	6.25 ng/mL	0.031
Dexamethasone	D4902-25MG	2mM	20,000x	0.1µM	0.025
HEPES	H0887-20ML	1M	66.6x	15mM	7.5
BSA, Fatty acid free	A8806-1G	50mg/mL	40x	1.25mg/mL	12.5
Linoleic Acid	L1012-100MG	50mg/mL	9,346x	5.35 µg/mL	0.053
L-Glutamine	TMS-002-C	200mM	50x	4mM	10
Gentamicin	G1272	10mg/mL	5,000x	2ug/mL	0.100
				Total volume	500

Suspension Hepatocytes Growth Protocol

The protocol was performed within a Class II laminar flow biohood unless otherwise specified. Incubators were set to 37^oC and 5% CO₂. PPE such as safety glasses, gloves, and lab coat should be worn.

Thawing and Plating Suspension Hepatocytes

1. Fill a 50mL centrifuge tube with 45mL of 4^oC Hepatocyte Plating Medium (HPM).
2. Remove hepatocyte vial(s) from storage tank or freezer.
3. Immerse the vial up to the cap into the 37^oC water bath. Be careful not to completely submerge the cap.
4. Gently shake the vial and shake until the ice pellet has melted to the point of a small spindle.
   Note: Do not fully thaw the cell suspension. It will take approximately 90 – 120 seconds.
5. Remove the vial from the water bath. Spray with 70% IPA or wipe down with a 70% IPA wipe and transfer into the bio safety cabinet.
6. Pour the contents of the vial into the 50mL conical containing HPM.
7. Remove 1mL of this medium and hepatocyte suspension using a pipette and place into the vial, ensuring collection of any remaining cells. Place in conical.
8. Invert the conical gently 3 to 4 times to ensure resuspension of the hepatocytes.
9. Centrifuge the conical at 100xg for 10 minutes.
10. Aspirate the supernatant.
11. Resuspend the cell pellet in 3-4mL of 37^oC Hepatocyte Maintenance media. Gently resuspend the pellet by slowly rocking the 50ml conical back and forth, allowing the media to repeatedly wash over the pellet until fully resuspended.
12. Calculate the total cell count and cell viability using the Trypan Blue Exclusion method:

    a. Prepare a 1:10 dilution by combining 400µL of media with 50µL of Trypan Blue, then add 50µL of well-mixed cell suspension.

    b. 1:5 = 350µL media + 50µL trypan + 100µL of cell suspension.

    c. 1:2 = 200µL media + 50L µof trypan + 250µL of cell suspension.

    i. Calculate cell viability percentage: {Live Cells/(Live + Dead Cells)}*100
    ii. Calculate cell yield per mL: (Total Live Cells/Number of Squares Counted)*10,000*Dilution Factor

13. Add additional Hepatocyte Maintenance Medium to reach the desired cell concentration for experimental design.

Primary Suspension Hepatocyte Growth Results

The primary hepatocytes grown in suspension (pooled and non-pooled samples) were tested for viability using the Phase I (CYP) and II (UGT, SULT) metabolism activity via ECOD assay, which measures the rate of clearance of 7-ethoxycoumarin and the rate of formation of metabolites 7-hydroxycoumarin sulfate (7-HCS) and 7-hydroxycoumarin glucuronide (7-HCG). Hepatocytes in suspension were incubated for 60 min at 37^oC under 5% CO₂ and saturated humidity with substrates as indicated the table shown below; samples were terminated with 3x acetonitrile containing the analytical internal standard. Metabolites were analyzed by LC-MS/MS.

Table 1. Example of donor demographics of 20 pooled hepatocytes for primary pooled suspension hepatocytes.

Lot #	Gender	Ethnicity	Age	BMI	Serology Testing (HIV/HBV/HCV)
ETB	Male	AA	38	24.1	All Negative
004	Male	Caucasian	36	23.7	All Negative
EXW	Male	Hispanic	50	31.9	All Negative
GBT	Female	Caucasian	37	20.7	All Negative
JYN	Female	Caucasian	49	34.4	All Negative
XLD	Male	AA	20	21	All Negative
JDL	Male	Caucasian	54	36.8	All Negative
RVS	Male	Caucasian	57	23.2	All Negative
CZW	Female	Caucasian	39	24.4	All Negative
DEC	Male	Hispanic	53	30.9	All Negative
XMN	Female	Caucasian	19	36.6	All Negative
003	Female	Caucasian	4	14.9	All Negative
OSZ	Male	Asian	55	20.4	All Negative
ZMC	Female	AA	38	21.9	All Negative
WAP	Male	Caucasian	37	34.4	All Negative
BRX	Male	Caucasian	45	29.1	All Negative
BEI	Male	Hispanic	47	25.8	All Negative
CGM	Male	Caucasian	5	16.4	All Negative
HUB	Male	Asian (Indian)	31	28.2	All Negative
ZNE	Female	Caucasian	55	28.5	All Negative

Table 2. Example results of Phase I (CYP) and II (UGT, SULT) metabolism activity for pooled suspension hepatocytes.

	Substrate	Final (M)	Results (pmole/million cells/min)
CYP1A2	Phenacetin	100	54.6
CYP2A6	Coumarin	50	113
CYP2B6	Bupropion	500	30.3
CYP2C9	Diclofenac	25	355
CYP2D6	Dextromethorphan	15	33.4
CYP2E1	Chlorzoxazone	250	28.3
CYP3A4	Testosterone	200	244
CYP3A4	Midazolam	20	157
CYP2C8	Amodiaquine	20	389
CYP2C19	Mephenytoin	250	7.29
General	7-EC	100	28.6
UGT	7-HC	100	108
SULT	7-HC	100	15.4

Table 3. Example results of Phase I (CYP) and II (UGT, SULT) metabolism activity via ECOD assay for primary suspension hepatocytes.

Enzyme	pmole/min/million cells
CYP1A2	131.50
CYP2A6	10.48
CYP2B6	114.50
CYP2C8	48.00
CYP2C9	111.33
CYP2C19	8.03
CYP2D6	38.33
CYP2E1	106.00
CYP3A4 (Midazolam)	73.33
CYP3A4 (Testosterone)	152.00
GEN	12.13
SULT	4.18
UGT	543.33
AO	64.33