Document [original]

Hollow fibres integrated in a microfluidic cell culture system

C. Winkelmann1, Y. Luo2, A. Lode2, M. Gelinsky2, U. Marx3, F. Sonntag1

1Fraunhofer Institute for Material and Beam Technology IWS, Dresden, Germany;

claudia.winkelmann@iws.fraunhofer.de

2 Technische Universität Dresden, Centre for Translational Bone, Joint and Soft Tissue Research, Dresden, Germany

3 Technical University Berlin, Institute of Biotechnology, Berlin, Germany

Abstract

For in vitro drug screening a modified perfusion micro-bioreactor system with integrated hollow fibres will be demon-

strated. This biocompatible system consists of an integrated closed flow circuit that includes reservoirs and pneumatic

micro pumps. Additional optical online-monitoring devices allow the observation during the cell cultivation. The em-

bedded hollow fibre system which acts as cell carrier consists of a biodegradable biopolymer. One option to fabricate

such 3D structures is the technology of Organ Printing which is realised by an adapted rapid prototyping system entitled

3D Scaffold Printer. With this device specimens consisting of tubes with a diameter smaller than 2 mm can be prepared.

It should be possible to cultivate the hollow fibres inside and outside with different kinds of cells and therefore generate

models of complex tissues.

1 Introduction

Organ typical tissue cultures have been already established

in limited fields of regenerative medicine. Artificial skin

tissues, cartilage and bone implants could be developed

successfully in the last years. The development of such

complex three-dimensional tissue cultures is becoming

also more and more important for the pharmaceutical and

biochemical industry as a substitute for animal experi-

ments. Such an in vitro substance testing requires the as-

similative interaction of different cells or tissues within a

common cycle in analogy of the human body [1]. There-

fore it was our issue to develop a biomimetic 3D tissue

culture with an artificial but functional blood vessel sys-

tem. It should generate a biocompatible and complex

three-dimensional microenvironment that supports the

growth of tissue cells. A possibility to realise a biofunc-

tional vascular system is the integration of several hollow

fibres into the cell carrier [2]. They have to be integrated

and connected to a dynamic micro-bioreactor system to

ensure the supply of the artificial tissue with oxygen and

nutrient. The main challenge of this study was the connec-

tion between the biological and technological system [1].

2 Methods

2.1 Perfusion micro-bioreactor system

The bioreactor system is based on the established Multi-

Organ-Chip platform, developed in cooperation between

Fraunhofer IWS and the Institute of Biotechnology of

Technical University of Berlin. This platform is shown in

Image 1.

Image 1 Multi-Organ-Chip on microscope

The flow is pneumatically realised and is regulated by an

automatic control module. The fluidic parameters can be

adjusted by the software system.

With this system it is possible to cultivate different kinds

of tissues simultaneously due to the integration of different

cell culture chambers, fluidic reservoirs, valve inlets and

micro-actors like an automatic pneumatic pump system

(Image 2). Over a closed flow circuit that rebuilds a micro-

environment the cells can interact with each other [1].

Currently, it includes several replaceable cell inserts for

cultivation. But due to large diffusions paths it is difficult

to ensure the supply of oxygen and nutrient despite the

utilisation of a perfusion system.

A solution for the limitation might be the integration of a

hollow fibres system.

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Image 2 Schematic representation of the layout of a self

contained Multi-Organ-Chip

2.2 3D Scaffold Printer

The 3D Scaffold Printer (Image 3) is an innovative tool

for creation of open porous cell carriers and scaffolds.

The device consists of a 3 channel dispensing module,

flexible PC software and a high-precision XYZ robotic

with a travel range in A3 size. It is possible to structure

media variety of biological (biopolymer) or synthetic

pasty materials into thin strands [3].

Repeating this strands and arranging them during the

process allows the creation of individual porous systems

as cell carrier for the three-dimensional cell cultivation.

Up to three different components can be dispensed. An

accurate XY offset compensation is given by individual

lowering of each cartridge. The exact trajectory of each

cartridge is monitored by a sensor system.

Image 3 3D Scaffold Printer

The 3D Scaffold Printer was developed by the company

GeSiM mbH (Großerkmannsdorf) and the dosing module

is an in-house development by Fraunhofer IWS.

2.3 Material

In cooperation with the Centre for Translational Bone,

Joint and Soft Tissue Research of TU Dresden an applica-

tion-specific cell-matrix-composite based on alginate hy-

drogels was developed. To cross-link the material an

aqueous solution of calcium chloride was applied. Pre-

liminary tests have shown the suitability of this material

combination. Using pre-sterilised materials and working

under sterile conditions allows the simultaneous embed-

ding of living cells during scaffold production.

3 Results

3.1 Adjusted perfusion micro-bioreactor

system

For integration of the hollow fibres into the micro perfu-

sion bioreactor the cell culture system had to be adjusted to

the new requirements.

Image 4 Schematic representation of the adjusted fluidic

system

For first experiments the fluidic system has been equipped

with only one cell culture chamber (Image 4). Its structure

was modified to include the hollow fibres. Additionally,

the design of the fluidic channels must be specifically en-

gineered to enable the circulation of cell culture media

through the whole system.

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The first version of the adjusted perfusion micro-bioreactor

system was manufactured successfully (Image 5).

Image 5 Adjusted perfusion micro-bioreactor system

3.2 Adjusting of the 3D Scaffold Printer

dosing module

By using the 3D Scaffold Printer technology it should

also be possible to create hollow fibres. For this it was

necessary to adjust the dosing module to the new re-

quirements. (Image 6)

Image 6 Dosing module of the 3D Scaffold Printer

Therefore two cartridges were connected with each other

over an engineered dispensing needle system to enable a

dual extrusion. One of the needles is fixed inside the other

and acts as spacer and has to be exactly aligned for the

creation of hollow fibres. Therefore both needles defined

the inside and outside diameter of the structure according

to the printing materials.

Image 7 Schematic representation of the adjusted dosing

module

In a printing process (Image 7) the first cartridge is filled

with cell carrier material (e. g. a biopolymer paste) which

is extruded by applying a defined pressure. The other car-

tridge is containing a cross linking agent to cure the cell

carrier medium. Since both printing materials usually

have different viscosities and therefore different rheologi-

cal properties, the dosing module was redesigned to gen-

erate different pressures in one printing process. More-

over, by using media with a low viscosity it is necessary

to attach a negative pressure on the cartridges to prevent

uncontrolled leakage of the media. The regulation of

switching between pressure and vacuum has to be ar-

ranged by the control module. Additionally, both car-

tridges are controlled in parallel to the dosing process and

the trajectory over this module to ensure a continuous

structure production.

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3.3 Plotted hollow fibres

In response to the adjustment of the dosing module the

first hollow fibres based on the alginate cell-matrix-

composite could be produced successfully by using the 3D

Scaffold Printer technology (Image 8 and 9).

Image 8 Cross section of a plotted hollow fibre

Image 9 Top view of plotted hollow fibres

First produced hollow fibres could be realised with a

minimal outside diameter of ca. 720 µm and an inside di-

ameter of ca. 250 µm by using a dosing needle with an in-

side diameter of 840 µm and an integrated thorn with an

outside diameter of 520 µm.

The produced hollow fibres could also be successfully

integrated into the newly developed bioreactor system.

4 Conclusion

With the novel technology platform a new powerful tool

for research and development in the area of tissue engi-

neering, drug screening and cytotoxicity tests will be

available.

The next item is the assessment of the suitability and

steadiness of the hollow fibres during perfused cell culture

conditions. Moreover, studies will be continued related to

flow behaviour and diffusion properties as a function of

pressure applied to the fluidic system. To expand the ap-

plicability of the system the results shall also be transferred

to other biopolymers.

Acknowledgements

The authors gratefully acknowledge the financial support

by the European Union, the Federal State of Saxony and

the ESF (European Social Fond).

5 References

[1] Sonntag, F., Gruchow, M., Wagner, I., Lindner, G.,

Marx, U.: Miniaturisierte humane organtypische Zell-

und Gewebekulturen. BIOspektrum 2011

[2] Meyer, W., Engelhardt, S., Novosel, E., Elling, B.,

Wegener, M., Krüger, H.: Soft Polymers for Building

up Small and Smallest Blood Supplying Systems by

Stereolithography. J. Funct. Biomater. 2012, 3, 257-

268

[3] A. Lode, K. Meißner, Y. Luo, F. Sonntag, S. Glorius,

B. Nies, C. Vater, F. Despang, Th. Hanke, M. Ge-

linsky: Fabrication of porous scaffolds with defined

inner and outer morphology by 3D plotting of a pasty

calcium phosphate bone cement. J. Tissue Eng. Reg.

Med. 2012 (accepted)

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