10.1007_978-3-540-89208-3_546

7/27/2019 10.1007_978-3-540-89208-3_546

http://slidepdf.com/reader/full/101007978-3-540-89208-3546 1/4

J. Vander Sloten, P. Verdonck, M. Nyssen, J. Haueisen (Eds.): ECIFMBE 2008, IFMBE Proceedings 22, pp. 2277–2280, 2008

www.springerlink.com © Springer-Verlag Berlin Heidelberg 2009

Development of a Production Process for Stem Cell Based Cell Therapeutic

Implants Using Disposable Bioreactor Systems

C. Weber 1, S. Pohl1, R. Poertner 2, C. Wallrapp3, P. Geigle3, and P. Czermak 1,4

1 Institute of Biopharmaceutical Technology, University of Applied Sciences Giessen-Friedberg, Giessen-Germany2

Institute of Bioprocess and Biosystem Technology, University of Hamburg-Harburg, Hamburg, Germany3CellMed AG, Alzenau, Germany4Department of Chemical Engineering, Kansas State University, Manhattan KS-USA

Abstract — Cell therapy enables the treatment of various

diseases like diabetes mellitus or stroke. The basis of many cell

implants are mesenchymal stem cells, which have to be

multiplied and harvested with high viability prior to use. For

that purpose bioreactor systems are required, which allow the

cultivation of the cells under GMP-conforming conditions.

Here a fixed bed cultivation system for the expansion and

differentiation of a hMSC production cell line on the basis of

commercially available polymeric or glass syringes is

introduced.

Keywords — hMSC-TERT, fixed bed bioreactor, harvesting

I. I NTRODUCTION

Cell therapy is a promising method for treating manydiseases like stroke or diabetes mellitus [1]. The CellMedAG (Alzenau, Germany) is developing implantable celltherapeutically systems on the basis of genetically modifiedand alginate-encapsulated human mesenchymal stem cells[2]. The product CellBead-Neuro© for example secretesGLP-1, a protein with anti apoptotic activity, which reduces

the damaging of brain tissue after a stroke. The CellBeads- Neuro

©can be implanted behind the blood-brain barrier

using a syringe with canula. The production process of CellBeads

©, representative for encapsulated cell implants

consist mainly of the steps of expansion of the productioncell line, encapsulation, and differentiation of the implants.A fixed bed system on the basis of commercially available

syringes has been described by Weber et al. [3]. This systemwas modified for the purpose of multiplying hMSC-TERT.

II. MATERIALS AND METHODS

A. The Reactor System

The core of the cultivation system is the fixed bed reactor

which consists of a single-use plastic or glass syringe. Theoriginal syringe piston is replaced by a specially milled piston, which enables the perfusion of the reactor (Fig. 2 andFig. 4). A schematic of the experimental setup is shown inFig. 3. Two chambers consisting of opto-electronical oxygenmini sensors (PreSens, Regensburg, Germany) inserted into

glass tubes enable the non-invasive monitoring of thecultivation status by measuring the oxygen concentration in

the medium in- and outlet (Fig. 4). The fixed bed reactor andthe conditioning vessel are placed into an incubator whilethe refueling section can be stored at 4°C.

Fig. 2 Schematic of the syringe with piston and a photograph of a piston prototype

500 μm500 μm

Fig. 1 Light microscopical image of CellBeads©

Syringe

MediumChannel

Seals

Syringe

MediumChannel

Seals

a b

7/27/2019 10.1007_978-3-540-89208-3_546


2278 C. Weber, S. Pohl, R. Poertner, C. Wallrapp, P. Geigle, and P. Czermak

_______________________________________________________________

IFMBE Proceedings Vol. 22

_________________________________________________________________

Fig. 3 Schematic of the fixed bed reactor system and auxiliary units

Fig. 4 Fixed bed reactor (3 ml) for the Cellbead© differentiation (a) andexpansion of hMSC-TERT (b)

B. Cultivation of hMSC-TERT in 6-Well Cell Culture Plateson Various Carriers

Potential packing materials for fixed bed cultivation of hMSC-TERT were tested in 6-well cell culture plates

(Tab.1) The carriers were inoculated with 5000 cells/cm2

and incubated in a humidified incubator (37°C, 5% CO2).EMEM + 10% FCS was used as the medium. The cell den-

sity was determined by disintegration of the cells by citricacid, staining of the nuclei with crystal violet and countingthe nuclei by using a Neubauer-hematocytometer.

C. Cultivation of hMSC-TERT in the Fixed-Bed Reactor System

Two cultivations were performed either with 2mm borosilicate glass spheres (1100 cm

2/reactor) or BioNocII

(9600cm2/reactor) in 50 ml glass syringes (60 ml fixed bedvolume). The bioreactor system was inoculated after autoclaving with 5000 cells/cm2. For that purpose the

reactor was filled with cell suspension and incubated for two hours without perfusion. The medium (500 ml EMEM+ 10% FCS) was aerated by surface aeration in theconditioning vessel. Daily measurement of the glucoseconcentration combined with the oxygen monitoringenabled the calculation of the cell number. The cell number

was also determined after the cultivation by staining of thenuclei as described above. The cultivation was stoppedwhen the oxygen concentration in the outflow of the reactor reached 12% of air saturation.

7/27/2019 10.1007_978-3-540-89208-3_546


Development of a Production Process for Stem Cell Based Cell Therapeutic Implants ... 2279

_______________________________________________________________


_________________________________________________________________

D. Harvesting of hMSC-TERT

The investigations were performed in 3ml-reactors. Thevitality was determined via the trypan blue exclusion

method. The harvest procedure was performed in two ways.1. Rinsing of the system with phosphate buffered saline

and filling the fixed bed with Accutase or Trypsin

(2.5mg ml-1) solution and incubating for 20 minutes.2. Rinsing of the system with phosphate buffered saline

and than perfusing the fixed bed with 20 ml Accutase

or Trypsin solution for 10 minutes at a superficialvelocity of 1.3 x 10-3 m s-1.

III. R ESULTS AND DISCUSSION

A. Cultivation of hMSC-TERT in 6-Well Cell Culture Platesand in the Fixed Bed Reactor System

The cells showed no adhesion to the soda-lime glass

spheres (Tab.1). Growth rates during the exponential phaseof 0.34 - 0.37 d

-1resulted from the cultivation on BioNocII

and borosilicate glass spheres in 6-well cell culture plates.The surface specific cell density was about six times higher for borosilicate glass spheres compared to BioNocII but ithas to be noted that BioNocII offers a greater volumesurface area per volume of packing.

Tab.1 Results of cultivation of hMSC-TERT on different carriers

in cell culture plates. BSGS: Borosilicate glass spheres, SLGS:Soda-lime glass spheres

Carrier Growth Rate

[d-1]

Maximum Cell

Density [cm-2]

Specific

Surface Area

BioNocII 0,34 ± 0,03 (6,1 ± 0,3) x 104 2400

[cm² g-1]

2 mmBSGS

0,37 ± 0,01 (35 ± 1,6) x 104

2 mmSLGS

- -

18,3[cm2 cm-3]

The growth rates in the fixed bed reactor were 0.43(borosilicate glass spheres) and 0.46 d-1 (BioNocII) and

were about 25% higher compared to the cultivations in 6-well cell culture plates, which can be rationalized by a better nutrient supply due to the medium flow (Fig. 5, Fig. 6). The

inoculation procedure resulted for BioNocII in a low yieldof adhesive cells. This procedure has to be improved toreduce the amount of inoculum. The oxygen transfer rateusing surface aeration only is too low and thus themaximum cell density could not be reached in the reactor

system. A promising method using a disposable membrane

dialyser with a high volume specific contact area is beingtested at present.

0,0E+00

5,0E+04

1,0E+05

1,5E+05

2,0E+05

2,5E+05

3,0E+05

3,5E+05

4,0E+05

0 50 100 150 200 250 300

Time [h]

C e l l d e n s i t y [ c m - 2 ]

0

20

40

60

80

100

120

O x y g e n s a t u r a t i o n [ %

]

Cell density, calculated from oxygen consumption

Cell density, counted after cultivationCell density, 6-well cell culture plates

Cell density, calculated from glucose consumption

Oxygen,inlet

Oxygen, outlet

Fig. 5 Cultivation of hMSC in the fixed bed reactor on borosilicate glass

spheres

0,0E+00

1,0E+04

2,0E+04

3,0E+04

4,0E+04

5,0E+04

6,0E+04

7,0E+04

0 50 100 150 200 250 300

Time [h]

C e l l d e n s i t y [ c m - 2 ]

0

20

40

60

80

100

120

O x y g e n s a t u r a t i o

n [ % ]

Cell density, calculated from oxygen consumption

Cell density, calculated from glucose consumption

Cell density, counted after cu ltivationCell density, 6-well cell culture plates

Oxygen, inlet

Oxygen, outlet

Fig. 6 Cultivation of hMSC in the fixed bed reactor on BioNocII

7/27/2019 10.1007_978-3-540-89208-3_546


2280 C. Weber, S. Pohl, R. Poertner, C. Wallrapp, P. Geigle, and P. Czermak

_______________________________________________________________


_________________________________________________________________

B. Harvesting of hMSC-TERT

The application of Accutase combined with the perfusionof the system with that enzyme solution led to the highest

yield of harvested cells (BioNocII: 47%, borosilicate glassspheres: 92%) and vitalities of 100% (Fig. 7).

Fig. 7 Yield and Viability at harvest of hMSC(a: BioNocII; b: borosilicate glass spheres)

IV. CONCLUSION

Assuming that the same maximum cell densities as thosein 6-well cell culture plates are achievable in the fixed bedreactor, the system volume specific yields after theharvesting procedure would be expected to be 5.9 x 106

(borosilicate glass) and 5.2 x 106 (BioNocII) cells per ml

reactor volume. An optimization of the harvesting procedure may lead to a further increase of the volume

specific yield with BioNocII.The advantages of the system proposed here are simple

automation and process monitoring as well as a high yieldof harvested cells separated from the carrier. Furthermoreall tubes and vessels may be manufactured as pre-packagedand gamma sterilized disposables.

The cultivation and differentiation of the CellBeads© in

the syringe based reactor offer the use of the syringe basedreactor as an implantation tool, which avoids contaminationduring the critical transfer step. Overall, this system offers agood foundation for the development of a GMP-conforming production process for encapsulated cell based implants.

ACKNOWLEDGMENT

The authors would like to thank the Federal Ministry of

Economics and Technology of Germany for financial

support (KF0143002UL5) as well as the CellMed AG for provision of the hMSC-TERT and CellBeads

©.

R EFERENCES

1. Zimmermann H, Shirley SG, Zimmermann U. (2007) Alginate-based

encapsulation of cells: past, present, and future. Curr Diab Rep.7:314-320

2. Zimmermann U, Cramer H, Jork A, et al. (2001) Microencapsulation-

based cell therapy. Biotech-nology, 10:547-5713. Weber C, Pohl S, Czermak P, et al. (2007) Cultivation and

differentiation of encapsulated hMSC-tert in a new developed small

scale syringe like fixed bed reactor. Open Biomed. Eng. J. 1:64-70

4. Weber C, Pohl S, Czermak P, et al. (2007) Expansion and harvesting

of hMSC-TERT. Open Biomed. Eng. J. 1:38-46

Address of the corresponding author:

Peter Czermak

Institute of Biopharmaceutical Technology

University of Apllied Sciences Giessen-FriedbergWiesenstrasse 14

Giessen, 35390

Germany [email protected]

0

20

40

60

80

100

3 ml

Accutase

20 ml

Accutase,

perfusion

3 ml Trypsin 20 ml

Trypsin,

perfusion

Y i e l d [ % ]

0

20

40

60

80

100

V i a b i l i t y [ % ] .

Yield Viability

0

10

20

30

40

50

60

Y i e l d [ % ]

0

20

40

60

80

100

V i a b i l i t y [ % ]

a)

b)

10.1007_978-3-540-89208-3_546

Documents