Phoenix Bio

PXB-cells

What are PXB-cells®?

PXB-cells® are ~95% pure fresh human hepatocytes isolated from PXB-mouse®

PXB-cells - fresh human hepatocytes isolated from PXB-mouse

Why use PXB-cells®?

PXB-cells® are used for in vitro studies in drug metabolism, enzyme induction/inhibition, safety assessment, transporter evaluation, anti-HBV therapeutics efficacy testing and other studies which require the use of primary human hepatocytes (PHH)

  • Stable supply on demand of ​PXB-cells® allows reproducible experiments with the same source of donor hepatocytes
  • High human-specific enzymes and transporters activity in PXB-cells® can be maintained for a long term (over three weeks)
  • High cell adhesion allows using PXB-cells® as planar or spheroid culture
  • Hepatitis B virus (HBV) can persistently infect PXB-cells®
  • Direct comparison between in vitro and in vivo data is possible, as PXB-cells® and PXB-mouse® have the same source of donor hepatocytes​

Cell morphology

  • Good adhesion
  • Long-term culture is possible (Day 100 culture results are available)

Culture on collagen coated plate

DAY 2

DAY 8

DAY 24

DAY 47

PXB-cells® show human-type enzyme activity

CYP3A activity in PXB-cells 

Measured enzyme Enzyme activity Matrix Processing time Metabolites
CYP3A Midazolam 1’-hydroxylase Midazolam (10 µM) 2 hours 1’-hydroxymidazolam

Measurement by HPLC

CYP3A activity in PXB-cells -21days

Figure shows the comparison of CYP3A activity of commercially sourced donor hepatocytes and PXB-cells® produced with the same source of hepatocytes. PXB-cells® show equivalent to or better CYP3A activity than the original human hepatocytes for 21 days

Gene expression of drug metabolizing enzymes in PXB-cells®

hCYP2C9 activity in PXB-cells CYP2C9: The expression level of human CYP2C9 is lower than immediately after isolation (before plating), but it is maintained for 21 days

hCYP3A4 activity in PXB-cells CYP3A4: Human CYP3A4 expression decreased immediately after plating, but later it is maintained at higher level than immediately after isolation

UGT1A1: Human UGT1A1 maintains 21 days of expression equivalent to or better than immediately after isolation

OATP1B1: The expression of human OATP1B1 is maintained for 21 days at lower than the initial post-isolation level (before plating)

BSEP: Human BSEP is reduced immediately after separation, but it is maintained for 21 days

MRP2: Human MRP2 maintains the same or higher expression level for 21 days

PXB-cells® Supply Process

PXB-cells supply process

How are PXB-cells delivered to clients?

PXB-cells are delivered by the following plate formats under temperature-controlled conditions (15-25 degrees Celsius).

24-well Plate: 1.0 x 10^7 cells per plate
96-well Plate: 6.5 x 10^6 cells per plate
T75 Flask*: 1.6 x 10^7 cells per flask

* Please note that you can re-plate PXB-cells to your preferred formats from T75 Flasks. We will provide a protocol for the re-plating procedures upon request.

To maintain optimal culture conditions PXB cells should be kept in dHCGM media. We provide dHCGM media (-FBS) in the following formats under frozen conditions:

dHCGM Medium (- FBS) 100 mL/bottle
dHCGM Medium (- FBS) 250 mL/bottle

* Please note that we can provide a protocol for the preparation of the dHCGM medium upon request.

Case Studies using PXB-cells

Case Study I: Mitochondrial Toxicity with PXB-cells

Case Study I: Mitochondrial Toxicity with PXB-cells

Ikeyama, Y. et al. Toxicol. in Vitro 2020; 65: 104785.

  • The mitochondrial function of a few lots of cryopreserved human hepatocytes (CHH) and PXB-cells® were directly compared
  • PXB-cells® outperformed CHH lots in terms of energy metabolism and mitochondrial function

Case Study II: PXB-cells® in metabolite identification

Case study with PXB-cells - metabolite identification

Kanamori T. et al, Forensic Toxicol. 2018; 36(2): 467-475

PXB-cells® were used as a model in vitro system for the prediction of the in vivo metabolism of new drugs of abuse.
A powerful synthetic opioid fentanyl and its analogue acetylfentanyl were studied. Major and minor metabolites were detected with PXB-cells®. The metabolite profiles in PXB-cells® was consistent with the in vivo metabolite profiles of drug users reported previously.

Overall, higher amounts of metabolites were formed in PXB-cells® as compared to another source of commercially available primary human hepatocytes (PHH). Some metabolites were detected only in the medium of PXB-cells®. The difference is attributed to the faster decrease in the activity of metabolizing enzymes in PHH versus PXB-cells® after the start of incubation.

Metabolites identified with PXB-cells

Metabolites identified with PXB-cells -case study

Methods

Seeding was performed at PhoenixBio (Japan) facility. 24-well plates were delivered to study facility 2 days later (at Day 0). Drug was added at Day 4 or Day 11 at a final concentration of 10 µM. PXB-cells® were cultured with the drug for 24-48 hours. Media were collected and analyzed by liquid chromatography/ mass spectrometry (LC/MS). The performance of PXB-cells® was compared to the regularly purchased primary human hepatocytes (PHH).