Sandwich ELISA for polymer detection using a mouse monoclonal antibody specific for A1AT polymers (2C1m Ab) and sandwich ELISA (intracellular and extracellular) have been used to test Def-HEP A1ATD (Figure 2).Figure 2: ELISA for polymer and secreted A1AT using antibodies specific for A1AT polymers (2C1m Ab) or all conformers of A1AT.Primary human hepatocytes (PHH) and hepatocellular liver carcinoma cells (Hep G2) are used as internal controls.
Figure 3 a) Relative expression of key hepatocyte maturity markers via Taq Man® gene expression assays (Applied Bio-systems). Recommended cell culture medium: The cells are grown at 5% oxygen in Hepato ZYME-SFM based media (Gibco, Catalog number 17707-021).
The m RNA expression levels of albumin (ALB), HN4Fa, and a1-antitrypsin (A1AT) were quantified in cryopreserved Def-HEP 10 days post thaw (Cryo) versus fresh cells. Defini GEN provides pre-tested and validated supplemental with 1x non-essential amino acids, 1X Pen/Step (optional), 1X Lipid concentrate, 10 mg/ml Insulin and 1µl/ml Transferrin. Just prior to use add 1µl/ml Oncostatin M (OSM) and 1µl/ml Hepatocyte Growth Factor (HGF).
b) ELISA for polymer and secreted A1AT using antibodies specific for A1AT polymers (2C1m Ab) or all conformers of A1AT was also performed to compare fresh cells versus cryopreserved cells. Alpha 1-antitrypsin deficiency (A1ATD) is an autosomal codominant genetic disorder caused by defective production of alpha 1-antitrypsin (A1AT), leading to decreased A1AT activity in the blood and lungs, and deposition of excessive abnormal A1AT protein in liver cells.
Collectively these cell products can oﬀer disease modelling and drug discovery researchers unprecedented tools for elucidating the underlying mechanisms of the disease.
Hepatocytes maturation markers: Def-HEP A1ATD cells display the functional characteristics of primary human hepatocytes (PHH) including albumin and A1AT production (Figure 1), glycogen storage and multiple drug-metabolising CYP450 activities.
Functional test: Alpha 1 Antitrypsin deficiency (A1ATD) is characterised by intracellular accumulation of large polymers of mutant A1AT within hepatocytes.In particular the Z allele results in the intracellular retention of almost 85% of the synthesized A1AT.Def-HEP A1ATD cells are produced from i PSC using the Opti DIFF platform.The cells are generated using i PSC technology and the resulting cells display many of the functional characteristics of primary human cells including albumin production, glycogen storage, and multiple drug-metabolising CYP450 activities.Accordingly, they should be handled in a similar fashion to primary human hepatocytes.The corrected version of the cell line oﬀers the same genetic background with the corrected A1AT gene is also available for purchase.