"Dynamic in-vitro organ models of metabolism" Arti - - PowerPoint PPT Presentation

"Dynamic in-vitro organ models of metabolism"

Arti Ahluwalia Multi Dimensional in vitro Models Group Centro di Ricerca “E.Piaggio” University of Pisa

100 years on, what’s missing?

• 1. Cells do not live on 2D plates….
• 2. Stationary conditions do not exist in-vivo….But in current

in vitro systems kinetics are overlooked, usually the steady state behaviour is observed (eg 24 hour responses).

• 3. Cells do not live in isolation but are surrounded by a

complex network of signals relayed by the bloodstream….

Why do we need better ivms?

Homer, a 2D static culture system is too simple to mimic the native environment… It doesn’t matter Peter, it’s SIMPLE!

To study systemic responses in a piecewise manner To understand cross-talk and multiple pathway mechanisms Models for drug testing and for evaluating systemic side effects To develop disease models To develop personalized therapy based on gene and disease specific models To reduce animal testing by developing more reliable and realistic human models

Our approach : Organomics

Several modular organ models can be connected in series or parallel through an inter-connected culture system, reproducing physiologically relevant multi-

• rgan

models for

• rganomics

(study

• f
• rgan

crosstalk)

Wiki/organomics

Nutrient Metabolism

Hi fat! How are you? Too much fried food last night Hey guys do your job! I can’t handle the FFA’s so fast Oh the shear stress of it all!

Can we recreate this circuitry in-vitro?

Can we recreate metabolic homeostatsis and simulate effects of nutrient imbalance in vitro?

Basic Design Rules

• Select relevant cell/tissue types
• Assemble in 3D scaffolds if appropriate
• Select appropriate culture media
• Scale cell numbers by rational and iterative

design

• Design connectable bioreators
• Optimise fluid residence times and flow rates
• Select appropriate controls and end points
• Build circuit complexity one tissue @ a time

Mazzei et al, 2010, Biotechnology & Bioeng. , Vozzi et al., Biotceh & Bioeng. 2011, Vozzi et al Tissue Eng Part A. 2009 , Guzzardi et al. Tissue Eng 2010,

Modular Bioreactor chambers (MmCB)

dynamic

Vinci et al. Biotech. J, 2009, 2010, 2011,

Hepatocytes remain well differentiated and functional

3D dynamic Higher density Increased vitality Increased metabolic competence

Effect of flow on detoxification genes

Effect of flow rate

Endothelial cells

control LFC Human endothelium

Laminar flow module

FLOW brings about Elongation CSK rearrangement NO increases Endothelin decreases Vozzi et al. Biotech & Bioeng. 20111

Equivalent circuit for nutrient metabolism

Systems Analysis Bottom-up engineering approach

Visceral circuit

Down scaled and reduced to essential features of central metabolism

The new modular system

LFC , monolayer of endothelial cells Partially digested adipose tissue Hepatocytes on microfabricated 3D scaffold Cell ratios are scaled using allometry

ONE-WAY (individual cultures)

Albumin Glucose Urea FFA TGA Glycerol Alanine Lactate IL6 E-Selectin Adipnectin TFG β CRP Iori et al. Plos One, 2012

TWO WAY (Endothelium +Adipose tissue) ×Hyperlipidemia, hypogly- cemia THREE WAY (Endothelium +Adipose tissue+hepatocytes) GLUCOSE and LIPID HOMOEOSTASIS

TWO-WAY (increase cross-talk) THREE-WAY (add hepatocytes as regulators)

Clincal observations

• Chronic Hyperglycemia is linked to insulin resistance
• Adipogenic cytokines (e.g. IL6) implicated in

endothelial dysfunction (E-selectin is a precocious marker of EC stress ) fasting state, normoglycemia, no Insulin post absorptive resting state, normoglycemia, low Ins diabetes type 1, hyperglycemia, no Insulin diabetes type 2, hyperglycemia, low Insulin

Challenged Model

Summary: challenged 3-WAY model

FFA IL-6 E-selectin Glucose

• 50

50 150 250 350 Fasting Post absorptive Diabetes 1 Diabetes 2

% variation Systemic and endothelial inflammation in the presence of high glucose. Modulated by insulin

Obesity in vitro

To investigate the effect of increasing adiposity on maintainence of homeostasis and systemic inflammation in-vitro 35% 12% 25%

Preliminary results

Control 12% 25% 35% LPS HUVEC vWF expression with increased adiposity

• All 3 cell types are necessary to obtain

metabolite regulation

• Hepatocytes are the master regulator

IN HYPERGLYCEMIC CONDITIONS

• Dysregulation of metabolites
• Endothelial specific stress @high glucose conc.
• Systemic stress is significantly increased at high

glucose concentrations only when insulin is absent IN PRESENCE OF EXCESS ADIPOSITY

• Preliminary tests on 2 way cultures: endothelial

stress increases with adiposity

Main Results

Thanks

THANK YOU!

Thanks

• A. Tirella, T. Sbrana, M. La

Marca, S. Giusti, G. Mattei, D. Cei, V. Di Patria,, B. Vinci, D. Mazzei, N. Ucciferri

• E. Iori & A.Avogaro (Univ of

Padova) F.Vozzi, C. Domenici (CNR)