Tissue Engineering Aim: Transplant Pharmacological Testing Artificial Organs Basic steps Biopsy Cells extraction Cell seeding Cell Growing Cell monolayer.

Development of a high-throughput bioreactor

system for biomedical applications

Mazzei Daniele Interdepartmental Research Center “E.Piaggio”

Tissue Engineering

Aim: Transplant Pharmacological Testing Artificial Organs

Basic steps• Biopsy• Cells extraction• Cell seeding• Cell Growing• Cell monolayer

High Throughput Screening (HTS)

HTS, is a method used in experimentation , such as drug testing

Is based on a brute-force approach to collect a large amount of experimental data in a short time and with few animals

HTS is achieved nowadays using multi-well equipment that contains cell cultures

?Does it work? The multi-well system presents a

principal problem, the environment discrepancy problem, that affects the relevance of tests

the environment discrepancy problem implies that the collected data are not directly usable in drug tests, because the cell monolayer grown in wells is only a brutal approximation of biological reality

This seems to be a paradox since the multi-well has been the core element of the HTS

Our IdeaTransfor

m a bioreactor

system in a HTS

machine

• Autonomous

• Safe• Usef

ull• Easy

to use

A bioreactor is a system able to maintain a cell culture in acontrolled environment, that mimics a living organism.

The Bioreactors A bioreactor is a more

realistic approximation of in-vivo Physiology.

Bioreactors have not been designed for HTS

We present a new bioreactor, called MCB, able to perform HTS experiment in a in-vivo-like simulated environment for long time

The MCB System

The MCB bioreactor system consists of the following parts:Cell culture chamberMixing chamberElectronic board and

electro-valve systemPeristaltic pump, PCHeating system

The mixing chamber The mixing chamber

allows regulation of pH and Oxygen flow

The medium is perfused with gas according to the measured pH

pH regulation is performed by inlet of two different gases : CO2 and Air

The Control System

The micro controlled board runs the μTNetOS operating system

The goal of this application is to realise an autonomous bioreactor by a real-time control of environmental variables

µTNetOS μTNetOS is a generated

operating system: the system generator takes as

input the description of an XML based protocol,

The system uses cooperative multi-tasking to run concurrent activities

μTNetOS is based on a robotics programming framework called Robotics4.NET

void TimerDoStep(Timer* t) { Heap *h = &t-> priorityQueue; while (h->pos && heap_Top(h) ->val <= GETTIME()) { HeapElement e = heap_Remove(h); int d = e.task->nextState(e.task); if (d) { e.val = GETTIME() + d; heap_Insert(h, e); } } }

Robotics4.Net and the Bioreactors

Bioreactors µTNetOS[Roblet]

PC [Brain

and bodymap]

The framework proposes a software architecture inspired to the architecture of the human nervous system.•The Brain: The core of control system•The Bodymap: A sort of black board used to send and receive messages•The Roblets: The appendix of the system, as the parts of our nervous system, read data from the environment and convert the Brain signal into an action

The High Throughput Bioeactor

Grazie a Robotics4.Net ogni bioreattore sarà visto come un sistema autonomo

Attraverso il PC sarà possbile controllare più bioreattori

Il controllo del sistema rimane però a bordo del bioreattore

Bioreactor

1

• Roblet: Bioreactor_1

Bioreactor 2

• Roblet: Bioreator_2

Bioreactor 3

• Roblet Bioreactor_3

A High Throughput bioreactor experiment, employs many bioreactors with different cell culture chambers

The network connections allows remote monitoring of experiments (through Internet)

A pathology can be simulated in one of the Bioreactor, and the others can be used as control

The user interface The Graphical User

Interface is developed in C#.Net

It is based on a multi tab structure

GUI is used to read data from the bioreactors and to setup the experimental variables

There are tools for sensor calibration

Manual control is possible

Preliminar Controll Results 48h parallel experiment with 4 bioreactorsThe data extracted from the bioreactors during

the experiment shows how the new system is able to correctly control the environmental variables

Preliminar Controll ResultsEndothelial cells (HUVECS) on a laminar flow culture chamber after 48h We can observe the

morphology of the cells, they do not show any type of cell membrane damage

We can use the cells as sensors of the environment, in the micrograph we can observe how the endothelial cells, are oriented with the medium flow and have an elliptical shape similar to their in-vivo morphology.

New Modular MCB Chamber Same dimension of Multiwell Modular connection in serie and

parallel configuration Easy to use Microfluid-dynamic modelled

New Modular MCB Chamber

New Modular MCB Chamber

1mm Inlet and Outlet

1mm inlet , 2mm outlet

Velocity reduced of 0,005 m/s !

New Modular MCB Chamber

How to control pHpH = -log10[H+]

H2O + H2O H3O+(aq) + OH-

(aq)

The culture medium is added with a BufferA buffer is a solution able to stabilized the pH of a liquid

Phosphate buffer H2CO3/HCO3 Is a water solution of Carbonic Acid

and sodium bicarbonate NaHCO3.

Key Points:•The Solubility of Oxygen in water is very Low •The Solubility of Carbon Dioxide in water is very High •Water + O2 pH increase slowly•Water + CO2 pH decrease very fast

How to control pHCO2 + H2O HCO3

- + H+ 4H+ + OH- + O2 2H2O + OH-

•High delay•Only few CO2 mole decrease pH•High risk for cell culture!•CO2 and O2 kinetich are very different

Impossible to use a simple On-Off Controll!Impossible to find a controll transfer function!

ASM Environment Simulator

Developed of an ASM Environment Simulator Simulate the fluid/gas environmentTesting the pH controll algorithm

Arti AhluwaliaGiovanni VozziFederico VozziBruna Vinci Daniele MazzeiFrancesca MontemurroCarmelo De MariaMariangela Guzzardi

Interdepartmental Research Center “E. Piaggio” Bio- Group

http://dionisio.ing.unipi.it