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Primary Human Suspension Hepatocytes Culture Protocol

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

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

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.

Suspension Hepatocytes Growth Protocol

The protocol was performed within a Class II laminar flow biohood unless otherwise specified. Incubators were set to 37oC 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 4oC Hepatocyte Plating Medium (HPM).
  2. Remove hepatocyte vial(s) from storage tank or freezer.
  3. Immerse the vial up to the cap into the 37oC 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 37oC 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 37oC 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.

 

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 1. Example of donor demographics of 20 pooled hepatocytes for primary 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 2. Example results of Phase I (CYP) and II (UGT, SULT) metabolism activity for pooled 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

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