A novel application of Bes-Gluconate-Polyethylene Glycol (BGP) preservation solution to maintain isolated cells for bioartificial devices. Functionality/viability studies

TOMATIS, C; BERCA, C; RODRIGUEZ J.V.; GUIBERT E.E.; MARÍA C. ROBERT

Maresias, San Pablo

Congreso; 14°SLABO- 5°OBI; 2017

SLABO

A bioartificial device needs suitable cells to perform their function. Those cells are usually cold preserved to wait until they are necessary. Despite the need for proper characterization of the cells to be introduced in the bioartificial device, there is currently no universal technique to evaluate their functionality. Usually, a combination of tests must been used to declare live one cell population. The main objet of this work was to evaluate hypothermic preservation of rat isolated liver cells and a Neuro-2a (N2a) neuronal cell line in a novel BGP-cold storage (BGP-CS) solution, developed by our group for organ preservation (Pascucci F. et al., Cryobiology 2012). Those cells were chosen due to their possible application in transplant or bioartificial device. Different types of viability/functionality tests were selected. Membrane permeability was evaluated by Trypan blue exclusion test (TB) and propidium iodide/Hoechst (Pi/H) staining. To study cellular metabolism, cleavage of fluorescein diacetate (FDA), uptake of glucose by its analog 2-NBDG and MTT metabolization were used alone or in combination with Pi counterstaining. Liver cells were isolated by collagenase perfusion of rat livers and resuspended in Krebs-Heinseleit solution. As a control freshly isolated cells (FIC) were rewarmed (1h, 37°C). N2a cells were plated and cultured for 48h in Minimun Essential Medium (MEM) supplemented with 10% inactivated Fetal Bovine Serum (FBS). Both cell types were preserved for 24h at 4°C in BGP-CS and culture medium, after that the cells were rewarmed. Once the conditions for each test and cell type were set, the effect of BGP-CS on hypothermic preservation was evaluated. In case of hepatocytes, preliminary results of Pi/H and FDA/Pi tests showed that cell viability/functionality after hyporthermic preservation using William´s medium E was lower respect to FIC and to hypothermic preservation in BGP-CS (p