Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor

摘要

This paper describes a microquantitative method for glucose determination in situ of living cells in real-time.In this noveltechnique adherent cells are cultured onto microcarrier beads and packed into a renewable microcolumn within a microsequential injectionlab-on-valve system (mu-SI-LOV).Glucose sensing is performed through the use of a two-step,NAD-linked enzymatic process.The course of the assay is monitored in real-time,by absorbance of NADH at 340 nm.The microsequential assay based on plugozzle design has a linear dynamic range for glucose of 0.1 to 5.6 mM.The design of the (muSI-LOV) system allows the assay to be carriedout using only 40 muL of the enzyme reagent and 3 muL of sample.The techniqe was tested on a murine hepatocyte cell line (TABX2S)adhered to Cytopore beads.Rapid cellular glucose consumption,in this technique,is facilitated by a high cell density,which allows a large number of cells (10~4-10~5) to be rtained in a very small volume (3 muL).In turn,this cell density results in the rapid depletion of glucose from the cellmediumover short time periods (<2 min).In conjunction with the assay development,the assay development,the plugozzle design and its ramifications on mixing in generalare presented and discussed.