Kasper C., van Griensven M., P?rtner R. (Eds.) Bioreactor Systems for Tissue Engineering II: Strategies for the Expansion and Directed Differentiation of Stem Cells

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can also have an impact on the phenotype and be seriously misleading but this

problem can be solved by analyzing iPSCs from different affected individuals as

well as unaffected controls (ideally from the same family to eliminate the effects of

a different genetic background). It is also remarkable that, following the success of

iPSC derivation, cells resembling neurons have been produced directly from mouse

ﬁbroblasts by overexpressing neural enriched transcription factors [90]. These

neurons display electrical activity and can form synapses, but it is unclear yet

whether they bear extensive neuron-like epigenetic remodeling and the ﬁbroblast

genetic program has been effectively shut down. In the near future we may see other

examples of direct transdifferentiation (Fig. 1), and if the transformation is accurate

this may end up being more practical than the uphill differentiation into iPSCs and

then downhill into speciﬁc lineages. In any case, this experiment is a clear indica-

tion that iPSC technology is forcing us to think of cell fate as a navigable condition

rather than a ﬁxed state, and this directly or indirectly will likely inﬂuence how we

perceive human physiology and disease.

Acknowledgments Work in the authors’ laboratories, Chinese Academy of Sciences, National

Natural Science Foundation of China, Ministry of Science and Technology 973 program, National

High Technology Research and Development Program of China, Bureau of Science and Technol-

ogy of Guangzhou Municipality.

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Induced Pluripotent Stem Cell Technology in Regenerative Medicine and Biology 141

Adv Biochem Engin/Biotechnol (2010) 123: 143–162

DOI: 10.1007/10_2009_25

Springer-Verlag Berlin Heidelberg 2010

Published online: 21 January 2010

Production Process for Stem Cell

Based Therapeutic Implants: Expansion

of the Production Cell Line and Cultivation

of Encapsulated Cells

C. Weber, S. Pohl, R. Poertner, Pablo Pino-Grace, D. Freimark,

C. Wallrapp, P. Geigle, and P. Czermak

Abstract Cell based therapy promises the treatment of many diseases like diabetes

mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them

against host-vs-graft reactions and thus enables the usage of allogenic cell lines for

the manufacturing of cell therapeutic implants. The production process of such

implants consists mainly of the three steps expansion of the cells, encapsulation of

the cells, and cultivation of the encapsulated cells in order to increase their vitality

and thus quality. This chapter deals with the development of ﬁxed-bed bioreactor-

based cultivation procedures used in the ﬁrst and third step of production. The

bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on

non-porous glass spheres, which support cell growth and harvesting with high yield

and vitality. The cultivation process for the spherical cell based implants leads to an

increase of vitality and additionally enables the application of a medium-based

differentiation protocol.

Keywords Cell therapy, Mesenchymal stem cells, Encapsulation, Fixed bed

bioreactor, Glass carrier

C. Weber, S. Pohl, P. Pino-Grace, D. Freimark, and P. Czermak

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

Giessen, Germany

R. Poertner

Institute of Bioprocess and Biosystem Technology, University of Hamburg-Harburg, Hamburg,

Germany

C. Wallrapp and P. Geigle

CellMed AG, Alzenau, Germany

P. Czermak (*)

Department of Chemical Engineering, Kansas State University, Manhattan, KS, USA

e-mail: peter.czermak@tg.fh-giessen.de

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

2 Expansion of hMSC-TERT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

2.1 Reactor System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

2.2 Expansion of hMSC-TERT on a Laboratory Scale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

2.3 Theoretical Scale Up of the hMSC-TERT Expansion Process . . . . . . . . . . . . . . . . . . . . . . 156

3 Cultivation of Encapsulated Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

3.1 The Reactor System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

3.2 Cultivation of Encapsulated Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

3.3 Theoretical Scale Up of the Cell Bead Cultivation Process . . . . . . . . . . . . . . . . . . . . . . . . . 160

3.4 Conclusion and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

1 Introduction

Cell based therapy can be deﬁned as the implantation of autologous, allogenic, or

xenogenic cells for the replacement of damaged cells or organ functions. Either the

implanted cells are able to assume this function by themselves or they stimulate

other cells, which then can cause a regeneration of the tissue or organ function.

Examples are the treatment o f diabetes mellitus with insulin- and GLP-1-secreting

cells or Parkinson disease with dopamine-secreting cells as well as the implantation

of mesenchymal stem cells for rege neration of heart, cartilage, or bone tissue [1–6].

The implantation of allogenic (or xenogenic) cells demands a protection against

host-vs-graft reactions which can be achieved by a suppression of the recipient’s

immune system or by an encapsulation of the transplanted cells.

Promising, for the treatment of many endocrine or degenerative diseases are

microencapsulated stem cells, for example the CellBead

system developed by the

CellMed AG (Alzenau, Germany). These cell beads consist of an inner cell contain-

ing core which is surrounded by an alginate capsule (Fig. 1). Each cell bead

contains about 2,000–3,000 cells in a non-proliferating state. The diameter of the

cell containing core bead is about 400 mm and the diameter of the alginate capsule

between 500 and 600 mm.

Core bead: 2000 – 3000 cells

Alginate capsule

Fig. 1 Light-microscopic image of cell beads

144 C. Weber et al.

The allogenic production cell line is based on human mesenchymal stem cells,

which are transfected with the gene of the catalytic subunit of human telomerase.

The telomerase activity counteracts the shortening of the telomeres after each cell

cycle, which indicates a transformation of the cells to a permanent cell line [7].

Dependent on the designated use the cells are genetically modiﬁed with additional

therapeutic genes.

The cell beads can be implanted by injection using a sterile syringe. At the

implantation site the cells are provided with nutrients by diffusion through the capsule

and in turn release therapeutic molecules. Antigens and components of the host

immune system cannot penetrate through the capsule (Fig. 2). The advantages of

this therapeutical system are a continuous drug delivery and, due to the local applica-

tion, high drug concentrations at the point of implantation. Furthermore, a production

in stock is possible since a cell line is used.

The implantation of this cell therapeutic system behind the blood-brain barrier

makes them suitable for the treatment of, e.g., stroke. Clinical phase 1 studies with

GLP-1-transfected cells revealed a reduced loss of neuronal cells in a controlled

cortical impact rat model due to the implantation of GLP-1 secreting cell beads

[8]. Originally, intestinal cells secrete GLP-1in response to food intake. GLP-1

enhances insulin secretion of beta-cells as well as promoting insulin sensitivity.

Since GLP-1 receptors are also expressed throughout the mammalian brain, the

implantation of GLP-1 secreting cell beads into the brain may cause neurotrophic

and neuroprotective effects.

The production process of cell based implants consists of three steps, whereas

this chapter is focused on the ﬁrst and third production steps (Fig. 3).

Due to the fact that the cells themselves are the product, the requirements of the

stem cell expansion process differ from a common mammalian cell culture where,

for example, a virus or protein is the product.

The cultivation and harvesting procedure for the expansion of the stem cell

based production cell line should be very gentle in order to obtain high yields and

vitalities of harvested cells. Th e used carrier should be non-porous which eases the

separation of detached cells from the carrier.

The cell bead cultivation process aims to increase the vitality of the encapsulated

cells. A decrease of the cell vitality of the implants may be caused by the harvesting

Nutrients, oxygen,

therapeutic molecules

Antibodies, complement,

Macrophages, ...

Antigens

Alginate capsule

Core bead

Fig. 2 The alginate capsule of a cell bead acts as a semipermeable membrane

Production Process for Stem Cell Based Therapeutic Implants 145

and encapsulation proce dure. Apoptotic cells will be decomposed during this

cultivation.

An optional differentiation of the cell beads may support cell survival at the

transplantation place. Furthermore, a differentiati on may induce the expression of

therapeutic molecules.

Here we introduce two ﬁxed-bed bioreactor systems, one for the expansion of

the adherent production cell line and the other for the cultivation of the cell beads.

As an example, the application of an adipogenic differentiation protocol was

investigated.

Both ﬁxed-bed systems have to be transferred into a GMP-process. Therefore

some requirements are addressed to the reactor systems. For maintaining sterility

during the process they have to be designed as a closed system with the possibility

of online monitoring. Furthermore, automation of the process should be enabled to

reduce human error and labor input. A simple design of the reactor system and its

peripheral components beneﬁts the man ufacturing of disposables, which reduces

extensive cleaning procedures and documentation effort. Figure 3 gives an over-

view about the cell bead production process and its requirements.

2 Expansion of hMSC-TERT

2.1 Reactor System

Many disposable reactor systems used in the cultivation of adherent animal cells

like T-ﬂasks, roller bottles, wave reactors, stirred-tank reactors, or spinner ﬂasks are

established, but they show more or less drawbacks. T-ﬂasks and their cognate

1. Expansion of hMSC-TERT

2. Encapsulation of the cells

3. Cultivation of the CellBeads®

– Gentle cultivation and

harvesting procedure

High vitality

– Increasing of the vitality

– Differentiation of the cells

Transfer into GMP process

Closed systems

Online monitoring

Automation

Disposable

Fig. 3 Steps and requirements of the cell bead production process

146 C. Weber et al.

systems are labor consuming in operation, or intricate in automation. Suspension

reactors in combination with a microcarrier have the drawback that a special system

for the separation of the enzymatically detached cells from the carrier is necessary.

This counteracts a simple design which is demanded for manufacturing as a

disposable. Therefore a ﬁxed-bed bioreactor system was developed, which is

based on a non-porous carrier. The advantage of a non-porous carrier is that the

adherent cells, located on the carrier surface, can be easily ﬂushed out of the reactor

with the medium ﬂow after enzymatic detachment. A carrier screening has revealed

that non-porous borosilicate glass spheres (BSGS) are most suitable with respect to

the growth behavior, cell harvest, and nutrient supply.

Figure 4 shows a prototype of the ﬁxed-bed reactor. It consists of a glass

cylinder with a stainless steel lid and bottom plate, which are equipped with

hose connectors for the medium inlet and outlet. A funnel shaped insert leads to a

nearly uniform inﬂow. The periphery of the ﬁxed bed consists of some ﬂasks that

act as conditioning, collecting, waste, and storage vessels, the tubing, some pinch

valves, two peristaltic pumps, single use noninvasive oxygen sensors (PreSens –

Precision Sensing GmbH, Regensburg, Germany) for process monitoring, and

sterile air ﬁlters for adjustment of the vessel pressure. All parts except the valves

and the pumps can be designed for production as a disposable, or they are already

available as commercial single use items. A further advantage of this system is a

Conditioning

vessel

Collecting

vessel

Enzyme

solution

IPO2

Incubator

37°C

5% CO

Waste

Medium PBS

Inoculum

4°C

Fig. 4 Fixed-bed reactor system for the expansion of hMSC-TERT

Production Process for Stem Cell Based Therapeutic Implants 147

comfortable automation of the process including the inoculation, culturing, and

harvesting procedures.

The oxygen supply can be provided by surface aeration or in larger systems with

a single use membrane oxygenator. Bubble aeration would have the drawbacks of

foam forming when serum containing medium is used.

The reactor system can be operated in a humidiﬁed incubator at 37



C and 5%

. Larger scales demand special heating strategies for the ﬁxed bed and the

conditioning vessel. Furthermore, a gas mix station is demanded when the medium

is aerated by using membrane oxygenators in order to enrich the feed air with CO

which is necessary for a pH adjustment of sodi um bicarbonate buffered medium.

For determination of growth and consumption kinetics and for scale up calcu-

lations a mathematical model was used that is shown here in a simpliﬁed form

(Fig. 5).

The system is mainly composed of the interacting compartments, the ﬁxed bed,

and the conditioning vesse l. The concentration c of any nutrient component in the

ﬁxed bed depends on time and axial position z and can be expressed by the

convection-consumption equation

e 

¼v 

   q  X

ðtÞ (1)

with the superﬁcial velocity, v, which is deﬁned as the ﬂow velocity in the reactor

tube without the packed bed, the cell speciﬁc consumption rate q of the regarded

nutrient component, the time dependent and volume speciﬁc cell density

ðtÞ¼X

 e

mt

(2)

()

vqXt

d d

×=-×-××

(, )

Fixed bed

Conditioning

vessel

cftz=

()

cft=

Effectiveness

factor

Consumption

rate

Cell densitySuperficial velocity

Porosity

Oxygen supply

Vc×

Fig. 5 Simpliﬁed illustration of the mass balances of the ﬁxed-bed reactor and its associated

conditioning vessel

148 C. Weber et al.